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Presentations, posters, round table notes, and crystallization tips from the Recent Advances in Crystallization Meetings (RAMC).

Search for a presentation, poster, note or tip using an name or keyword, or simply browse below. Still having trouble finding what you want? Contact tech@hrmail.com.

Fine sampling pH to identify optimal crystallization conditions
By: Alexander McPherson
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Membrane protein crystallisation for X-ray free electron laser applications
By: Valerie Panneels
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Crystallization screening using trace fluorescence labeling
By: Marc Pusey
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Distinguishing biologically relevant interfaces from lattice contacts in protein crystals
By: Guido Capitani
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The use of surface plasmon resonance to guide the choice for nucleic acid oligomers for co-crystallization with proteins
By: Claire Stevenson
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Symmetry ideas in protein assembly
By: Todd Yeates
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Crystallizing membrane proteins for structure function studies using lipidic systems
By: Martin Caffrey
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Maximizing crystallization success with microseed matrix screening
By: Galina Obmolova
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The benefits gained from a redesigned crystallisation strategy focused on a high throughput seeding technique
By: Alexey Rak
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Crystallisation Conditions – The good, the bad and those with special challenges
By: Janet Newman
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Biophysical methods to guide protein crystallization
By: Paul Erbel
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Seeding Workshop RAMC
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Random microseeding: A theoretical and practical exploration of seed stability and seeding techniques for successful protein crystallization
By: Patrick Shaw Stewart
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Microseed matrix screening crystallization of antibody fragments and antibody-antigen complexes
By: Galina Obmolova
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Highways, biways and detours: the IspD story
By: Terese Bergfors
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A manual, low cost protein-crystallization plate jig for in-situ diffraction in the home laboratory
By: David Hargreaves
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Crystallizing membrane proteins for structure-function studies using lipidic systems
By: Martin Caffrey
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TTP Labtech
By: TTP Labtech
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By: Rigaku
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Douglas Instruments
By: Douglas Instruments
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Centeo Biosciences
By: Centeo Biosciences
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Commercial Presentation - Avid Nano
By: Avid Nano
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A thermal stability assay that can help estimate the crystallization likelihood of biological samples
By: Jose Marquez
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T6 - Non-conventional methods for protein crystallization: using physical parameters to control the crystal quality for X-ray crystallography
By: Abel Moreno
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T5 - Biophysical methods to guide protein crystallization and inhibitor binding studies
By: Paulus Erbel
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T4 - Crystallization of macromolecular complexes: use of limited proteolysis as a rational tool in construct design
By: Jerome Basquin
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T3 - Microfluidics, crystallization and crystallography
By: Claude Sauter
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T2 - Nanobodies for the structural analysis of GPCR transmembrane signaling
By: Jan Stayeart
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T1 - Why don't protein crystals grow larger? Why can't they grow smaller?
By: Alexander McPherson
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T13 - Automated Screening and Remote Data Collection at the Stanford Synchrotron Radiation Laboratory
By: Aina Cohen
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T12 - Macromolecular Cryocrystallography: Some Opinions about Best Practices
By: J.W. Pflugrath
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T11 - Deconstruction of Drop Volume Ratio/Temperature Optimization Experiments
By: Joseph Luft
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T10 - Integral Membrane Proteins: Strategies for Pre-Crystallization and Crystallization
By: Michael Wiener
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T9 - Microfluidic Hybrid Method for Simultaneous Screening and Optimization Validated with Crystallization of Membrane Proteins
By: Liang Li
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T8 - Protein crystallization in restricted geometry: Old ideas for modern applications
By: Joseph D. Ng
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EP7 - Thermo Scientific’s Rhombix Products for Automated Crystallization
By: Paige Vinson
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EP6 - Alchemist II: Capacity, Reproducibility and Efficiency of a High-throughput Crystallization Screen Preparation System
By: C. Sterling
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EP5 - Recent Advances in DLS and DPI technologies
By: Ulf Nobbmann
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EP4 - Automation Options from Genomic Solutions
By: Sean Rubin
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EP3 - Formulator - The Next Generation Liquid Handler
By: Jeremy Stevenson
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EP2 - Topics in Automatic Protein Crystallization, Including Microseeding and Scaling Up Drop Volumes
By: Patrick Shaw Stewart
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EP1 - Art Robbins Instruments Overview of Protein Crystallization Products: Phoenix, CrysCam and Intelli-Plate
By: David Wright
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T7 - Co-crystallization of Soluble and Membrane Proteins with Designed Ankyrin Repeat Proteins
By: Markus G. Grütter
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T6 - Towards Chaperone-Assisted membrane protein crystallography
By: Serdar Uysal
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T5 - Synergy between High-Throughput and Novel Crystallization Techniques
By: Jonathan M. Hadden
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T4 - Crystallization of Protein - Ligand Complexes — Navigating the hurdles
By: Annie Hassell
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T3 - The importance of nucleation and seeding in protein crystallization
By: Allan D\'Arcy
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T2 - Development of an Alternative Approach to Protein Crystallization
By: Alexander McPherson
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T1 - Fluorescence-based Analytical Crystallization Technologies (FACTs)
By: Mark Pusey
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T15 - Crystal engineering: enhancing protein crystallization by reducing surface conformational entropy
By: Zygmunt Derewenda
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T14 - Inhibition of protein crystallization by evolutionary negative design
By: Ard Louis
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T13 - High throughput crystallization
By: Shane Atwell
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T12 - High-Performance Size Exclusion Chromatography as a Fundamental Tool for the Production of High-Quality Membrane and Soluble Protein Crystals
By: Larry Miercke
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T11 - Refolding of membrane proteins for structural studies
By: Lars Linden
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T10 - robotic system for crystallizing membrane and soluble proteins in lipidic mesophases
By: Martin Caffrey
T9 - A review of seeding methods: Old and New
By: Aengus Mac Sweeney
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T8 - Introduction to Acta Cryst F
By: Howard Einspahr
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T7 - Radiation damage: Why care
By: Elspeth Garman
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T6 - Tricks and improvements in Structural Genomics
By: Chantal Abergel
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T5 - Evaluation of crystallization droplet
By: Christian Betzel
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T4 - Snags with tag
By: Paul Ramage
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T3 - Experimental mapping of protein precipitation diagrams
By: Morten Sommer
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T2 - Origin of a Species: History and observations of one high throughput crystallization laboratory
By: Joe Luft
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T1 - Notes on the problem of identifying detergents for the crystallization of lipophilic proteins: Two recent experiences
By: Alexander McPherson
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T15 - Various strategies used to obtain proteins for crystallization and biostructural studies
By: Alexander McPherson
T14 - Use of protein engineering to enable crystallization and derivatization of the complete extracellular domain of the bc subunit of the IL-3, IL-5 and GM-CSF receptors
By: . G. Young1, S. E. Gustin1, A. P. Church1, J. M. Murphy1,2, S. C. Ford1, D. A. Mann1, I. Walker1, D. M. Woltring1, D. L. Ollis2 and P. D. Carr2
T13 - Crystallization and improved diffraction of an enzyme of therapeutic interest using a combination of rational construct design and crystallization optimization
By: Jeffrey Ohren, Peter Kuffa, Huifen Chen, Amy Delaney, Patrick McConnell, Chunhong Yan, Craig Banotai, David Dudley, Jim Dyer, Eric Fauman, Joseph Loo, Rachel Loo, Anil Mistry, W. Thomas Mueller, Shridhara Murthy, Cindy Spessard, Joseph Warmus, Alexander Pavlovsky, Erli Zhang, Charles Hasemann
T12 - Crystallizing problem proteins, some practical solutions
By: Jonathan M Hadden
T11 - A novel free-mounting system for protein crystals: transformation and improvement of diffraction power by accurately controlled humidity changes
By: 1,2Reiner Kiefersauer, 1Holger Dobbek, 1Manuel E. Than and 1Robert Huber
T10 - Crystal engineering yields cyclophilin D crystals diffracting to 1.5 Å resolution
By: Daniel Schlatter, Ralf Thoma, Armin Ruf, Martine Stihle, Erich Kung, Francis Müller, Edilio Boroni & Michael Hennig
T9 - Seeding for optimization of crystal quality
By: Terese Bergfors
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T8 - Crystallization of kinases: a protein purifier's perspective
By: Paul Ramage
T7 - Rapid production of fusion proteins using the RTS 100/500 systems
By: Jan Stracke, Michael Schraml, Andreas Junger, Dorothee Ambrosius and Martin Lanzendorfer
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T6 - What was learned from 717,312 crystallization experiments
By: George T. DeTitta1, Melissa A. Bianca1, Robert J. Collins1, Ann Marie E. Faust1, Jillian N. Kaczmarek1, Joseph R. Luft1, Nancy A. Urban1, Walter A. Pangborn1, Jennifer R. Wolfley1, Igor Jurisica2, Patrick Rogers2, Gerald Quon2, Janice Glasgow3, Suzanne Fortier3
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T5 - Ribosome crystallization and crystallography
By: Nenad Ban1, P. Nissen2, J. Hansen2, P. B. Moore2 and T. A. Steitz2,3,4
T4 - Structural Genomics and its impact on protein crystallization
By: Neera Borkakoti
T3 - Overcoming some of the challenges in crystallization of kinases
By: Annie Hassell
T2 - Current strategies for membrane protein crystallization
By: Larry Miercke
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T1 - Various strategies used to obtain proteins for crystallization and biostructural studies
By: Dorothee Ambrosius, F. Hesse, M. Lanzendörfer, R. Engh, S. Palme and P. Ruger
T16 - Use of molecular biology to achieve crystals of 1:2 EPO:EPO receptor complex at 1.9A
T15 - Crystal Annealing - Nothing to lose
By: Clare E.M Williams, David M. Lawson, Suzanne M. Mayer, and Laure Delarbre
T14 - Improving the diffraction quality of MTCP1 crystals
By: Irene T. Weber, John Petock, Charles Reed, Zheng-Qing Fu, Yuan-Fang Wang, Garrett C. Du Bois, Sherry P. Song, and Robert W. Harrison
T13 - Pharmaceutical applications for crystalline proteins example: Interferon alfa-2b
By: Paul Reichert
T12 - RNA Crystallization of the Satellite Tobacco Mosaic Virus
By: Joseph D. Ng
T11 - Crystallization of membrane proteins
By: Christian Ostermeier
T10 - Protein engineering: A powerful tool for crystallising biological macromolecules?
By: Glenn E. Dale and Allan D'Arcy
T9 - A new protein folding screen: A rapid and straightforward method to evaluate protein folding conditions
By: N.A. Armstrong, E. Gouaux, G.-Q. Chen, Y. Sun, and A. DeLencastre
T8 - Design of a crystallizable form of RIIß regulatory domain by limited proteolysis
By: D.E. Danley, M.E. Haggan, D. Cunningham, K.F. Fennel, T.A. Pauly, and P. Lemotte
T7 - The use of molecular biological and biochemical techniques for the crystallization of proteins
By: Markus G. Gruetter
T6 - Successful use of dynamic light scattering for optimizing soluble and membrane protein crystallization
By: Larry Miercke, Jolanta Krucinski, and Robert Stroud
T5 - The development of high throughput methods for macromolecular microbatch crystallization
By: Joseph R. Luft1, Melissa A. Bianca1, Lisa M. Owczarczak1, Daniel R. Weeks1, Igor Jurisica2, Patrick Rogers2, Janice Glasgow3, Suzanne Fortier3, and George T. DeTitta1
T4 - Protein crystallography: The fat lady will sing by 2010
T3 - Crystallization screens and limited proteolysis to generate diffraction quality crystals
By: Anna M. Stevens, Jennifer L. Pawlitz, Steve H. Sarfaty, Huey-Sheng Shieh, Roderick A. Stegeman and William C. Stallings
T2 - Crystallisation of proteins of pharmaceutical interest: Criteria, strategy, and modification
By: Allan D'Arcy, Glenn Dale, Martine Stihle, and Brigitte D'Arcy
T1 - Polymers as nucleants under high salt conditions
By: Alexander McPherson
T16 - Hardware for cryocrystallography
By: Elspeth Garman and Mike Pickford
T1 - Polymers as nucleants under high salt conditions
By: Alexander McPherson
T15 - Flash freezing isomorphous xenon or krypton derivatives of protein crystals
By: O. Sauer1, R. Dutzler2, C. Kratky
T14 - Shrinking protein crystals - a way to higher resolution
By: Patrick Cramer
T13 - The Cyberlab C-200: A Fully Automated System for Protein Crystallization Vapour Diffusion Experiments
By: Tom Friedlander
T12 - Intelligent Computational Aids for Crystal Growth
By: John M. Rosenberg1, Patricia A. Wilkosz1, K. Chandrasekhar1, Devika Subramanian2, Daniel Hennessy3 and Bruce Buchanan3
T11 - Lipidic Cubic Phases: A Novel Concept for the Crystallization of Membrane Proteins
By: Ehud M. Landau, Gabriele Rummel, Eva Pebay-Peyroula and Jurg P. Rosenbusch
T10 - Crystallization of the membrane protein cyclooxygenase-2: Beauty is only skin deep
By: Stefania Di Marco, John P. Priestle and Markus G. Grutter
T9 - The Role of Oil in Macromolecular Crystallization
By: Imperial College of Science, Technology & Medicine, London SW7 2BZ, United Kingdom
T8 - Effect of a magnetic field on the protein crystallization
By: Tohoku University, Institute For Materials Research Katahira 2-1-1, Aoba-ku, Sendai 980-77, Japan
T7 - Drop-Drop: Macromolecular crystallization using Micro-Volume Vapor Diffusion
By: George T. DeTitta and Joseph R. Luft
T6 - Control of nucleation by a simple temperature shift method
T5 - Just So Stories on Gal6 and Other Complexes (With apologies to R. Kipling)
By: Julie Rosenbaum1, Troy Messick1, Stephen Albert Johnston2 and Leemor Joshua-Tor1
T4 - Light scattering: a crystal ball for crystallization or just another way to waste a few mg of protein?
By: Terese Bergfors
T3 - HIV Integrase--Improving Crystal Quality Through Molecular Biology Technics
By: Anne M. Hassell and Lisa Shewchuk
T2 - Crystal Engineering: Rationale Design or Rolling Dice?
By: Glenn Dale, Martine Stihle, Brigitte D'Arcy, and Allan D'Arcy
T1 - Hyperthermostable Proteins: A possibility for improved crystallization?
By: Michael Hennig
A ceiling crystallization kit for optimizing the quality of protein crystals
By: Alaa Adawy
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Delivery of compounds in crystallization plates; A new practical approach for hit validation and fragment-based screening by X-ray analysis
By: J.M. Rondeau
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Continuous improvement in a crystallization lab at AstraZeneca
By: Margareta Ek
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Automated protein crystal optimization with TTP Labtech dragonfly
By: Joby Jenkins
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Biophysical and Structural Studies of the ERR-DNA complexes
By: Karima Tazibt
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A case study of the effects of metal ions and TCEP on the crystallization of a cysteine protease
By: Frederic Villard
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Two rings for folding Crystal structure of the mammalian chaperonin CCT in complex with tubulin
By: Ines Munoz
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Insights into the autoinhibition of a kinesin-4 family member
By: Sarah Bianchi
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mosquito Crystal and mosquito LCP
By: Joby Jenkins
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Supramolecular hydrogels for protein crystallization
By: Teresa Conejero Muriel
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Study of the evolution of nuclear receptor ligand binding using Amphioxus as a model
By: Maria Takacs
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Production and Characterization of Reinforced Cross-linked HEWL Crystals (RCLECs)
By: Hernandez-Hernandez Angeles
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A good practice towards effective UV imaging of a crystallization experiment
By: Jian Xu
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Bringing structure into the centrosome de novo structure solution of centriolar coiled coil proteins
By: Sebastian Kraatz
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Tackling crystallization issues of challenging targets - Three case studies from Novartis Basel
By: A Hinninger
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High-Throughput Crystallization at UZH
By: Beat Blattmann
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Towards the structural basis of peptide binding to Bezerra dipeptidyl peptidase 11 of Porphyromonas gingivalis
By: Gustavo Arruda Bezerra
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Characterizing the Structures of Proteins Involved in Lipolysis G0S2 - a Novel Inhibitor of ATGL
By: R Viertlmayr
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P27 - Separation of the different oligomers of the heterologous nucleoprotein from Rift Valley Fever Virus by means of preparative electrophoresis.
By: David Ruiz Carillo
P26 - Understanding your lab thermal environment and managing temperature to improve protein crystals.
By: Gabriela Juarez-Martinez
P25 - Macroseeding - a key to high resolution complex structures.
By: Hongwei Guo
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P24 - Minstrel HT UV: A fully automated and high performance imaging system for protein crystallization with UV fluorescence.
By: Jian Xu
P23 - Make the best of your crystal: Supply of organic solvent to the naked protein crystal improves crystal order and cryo behavior.
By: Reiner Kiefersauer
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P22 - Automated screening of recombinant protein expression for structural studies.
By: Karina Valer
P21 - Thinking outside the box to solve a novel protein structure.
By: Kirsten Kahler
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P20 - Dichroism of a [2Fe:2S] cluster in Ferredoxin 4 from Aquifex aeolicus.
By: Jonas Muhle
P19 - In situ X-ray diffraction screening of crystals and data collection in the home lab.
By: Vernon R. Smith
P18 - What a difference a buffer can make! Optimization and improving crystallization methods of a protease with the air of biophysics and automation.
By: Paris Ward
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P17 - In situ diffraction screening at the crystallization platform at the Swiss Light Source.
By: Christian Stimimann
P16 - Combining in-site proteolysis and microseed matrix screening to promote crystallization of PrPc-nanobody complexes.
By: Romany Abskharon
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P15 - Structural biology and genomics platform in Strasbourg: strategies for the crystallization of macromolecular complexes.
By: Pierre Poussin-Courmontagne
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P14 - Role of protein surface active compounds in protein crystallization.
By: Jindrich Hasek
P13 - Fast optimization: 2-D grid seeding method.
By: Lesley Haire
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P12 - Protein crystallization setup and workflows in the BI structural research group.
By: Protein crystallization setup and workflows in the BI structural research group.
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P11 - Changes in the oligomerization state of human S100A12 depend on zinc-calcium interplay: role of biochemical and biophysical characterization in optimizing crystallization
By: Olga Moroz
P10 - The CrystalHarp - combining in situ diffraction and high pressure freezing in capillaries.
By: Beat Blattmann
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P9 - Structural analysis of MamA, a magnetosome associated protein from two different magnetospirillium species.
By: Natalie Zeytuni
P8 - A fast and fully automated solution for lipidic cubic phase (LCP) screening using Mosquito LCP.
By: Joby Jenkins
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P7 - Mosquito Crystal: Fast, reliable automation of protein crystallization drop set-up.
By: Joby Jenkins
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P6 - Protein crystallization with alternative polymer precipitants.
By: Tereza Skalova
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P5 - Protein crystallization at York, ongoing developments and individual stories.
By: Shirley Roberts
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P4 - Obtaining protein-fragment structures in a fragment based drug discovery campaign.
By: Linda Oster
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P3 - Properties of macromolecular solutions relevant to crystallization: Use of new amphiphilic molecules for soluble and membrane protein crystallization.
By: Francoise Bonnete
P2 - Characterization and crystallization of MID962-1200: A trimeric autotransporter from M. catarrhalis.
By: Mahmudul Hasan
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P1 - Macromolecularc crystallization: Robotics, procedures and innovations.
By: Fabrice Gorrec
P19 - RThe Path to the First Known Crystal Structure of the Plk1 Kinase Domain: How a little contaminant went a long way
By: Simon Low
P18 - Protein Crystallization at York - Ongoing Developments and a Few Personal Stories
By: Shirley Roberts
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P17 - Dynamic Light Scattering: Low Polydispersity related to Crystallization Success of Rice Virus.
By: Ulf Nobbmann
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P16 - MRC-LMB high-throughput protein crystallization system
By: Fabrice Gorrec
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P15 - Methods Testing in High-Throughput Crystallization
By: Sinem Ozyurt
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P14 - L-shaped RNA Domain is Stabilized by Divalent Cations
By: Sergey Dibrov
P13 - Mechanistic Studies of Allosteric Regulation in the Type II Citrate Synthases
By: Nham T. Nguyen
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P12 - On the Path to the Crystal Structure of Cholesteryl Ester Transfer Protein (CETP)
By: Anil Mistry
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P11 - Fundamentals of Water Equilibration Rates used to Improve the Quality of Protein Crystals
By: Pedro M. Martins
P10 - Does protein Stability Correlate with Protein Crystallizability?
By: Liping Wang
P9 - Using a Tetra Detector Array as a Size Exclusion Chromatography Detection Tool for Optimizing Detergent Concentration During Membrane Protein Crystallization
By: Larry Miercke
P8 - Technology Development at the Center for High Throughput Structural Biology (CHTSB)
By: J.R. Luft
P7 - Inching Towards a New Metric
By: Janet Newman
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P6 - The Best of Both Worlds: Attempts to Combine Structural Genomics Approaches with Hypothesis-Driven Research
By: Heidi Schubert
P5 - The Crystal Structure of Human CYP3A4 in Complex with Clotrimazole
By: You-Ai He
P4 - Advancing Technologies to Expedite Crystal to Structure Delivery
By: Neil Grodsky
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P3 - Finding the keys to Crystallization Success: Ligands, Tags, and Crystallization Methods
By: Elizabeth Fry
P2 - The Efficient use of a 1536 High-Throughput Crystallization Screen to Guide Subsequent Optimization
By: Edward Snell
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P1 - Searching for Silver Bullets: An Alternative Strategy for Crystallizing Macromolecules
By: Bob Cudney
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P10 - Refolding of GPCRs and ion channels for structural studies
By: Lars Linden et al
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P9 - Crystallographic study of Thermus thermophilus Initiation factor 2
By: Angelita Simonetti et al
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P8 - SOD-like Proteins: unstructured in solution become ordered in the crystal
By: Manuela Benvenuti et al
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P7 - Structural insights into trehalose biosynthesis
By: Robert Gibson_Gideon Davies
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P6 - Structural Genomics of human targets: Crystallization strategies at SBGP
By: Pierre Poussin-Courmontagne et al
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P5 - Crystallization tips from the York Structural Biology Laboratory
By: Shirley Roberts
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P4 - There is no high throughput without high output
By: Michael Malkowski et al
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P3 - Crystallization of Membrane Proteins Requires Optimal Detergent Concentration, Precipitant concentration, and Use of Additives for Improved Diffraction
By: Joseph OConnell et al
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P2 - A Vapor batch method for volatile organics
By: Lesley Haire
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P1 - Part I: Visualisation & Differentiation of Protein/Salt Crystals Part II: Monitoring Nucleation and Crystal Growth
By: Karsten Dierks et al
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P22 - Gels in crystallization of biological macromolecules: additives with numerous properties
By: Christian Biertumpfel, Jerome Basquin, Claude Sauter & Dietrich Suck
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P21 - A novel free-mounting system for protein crystals: transformation and improvement of diffraction power by accurately controlled humidity changes
By: Reiner Kiefersauer1,2, Holger Dobbek1, Saulius Grazulis1, Manuel E. Than1 and Robert Huber1
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P20 - Rb-E2F peptide structure from highly mosaic crystals using the micro- diffractometer at ESRF
By: Bing Xiao1, Manfred Burghammer2, Anastassis Perrakis2 and Steven J Gamblin1
P19 - Microheterogeneity in the growth of protein crystals
By: Bill R. Thomas
P18 - Pseudosymmetry in crystals built of homodimeric enzyme complexes
By: Hana Petrokova1, Jindrich Hasek1, Jan Dohnalek1, Jarmila Duskova1, Tereza Skalova1 and Eva Buchtelova2
P16 - Large-scale production, purification and crystallization trials of E. coli proteins for structural genomics
By: Younge Li, Véronique Sauve, J. Sivaraman, Gurvan Michel, Joao Barbosa, Robert Larocque, Stephane Raymond, Allan Matte, Joseph Schrag and Mirek Cygler
P15 - Tyrosine regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase: one protein in five different crystal forms
By: Alexander McPherson
P14 - Exploring two variables in vapor diffusion protein crystallization: plate types and crystallization screens
By: Jennifer Pawlitz1, Stephanie Shieh1, Jennifer Sharamitaro1, Eric Sturman2, Mairi Lough3 and Anna Stevens1
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P13 - The crystallization of (membrane) proteins, using a flexible Sparse Matrix Screen
By: J.P. Zeelen
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P12 - Equilibration Study: Using the sitting drop technique, are you crystallizing your protein by vapor diffusion or batch?
By: Elizabeth L. Forsythe and Marc L. Pusey
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P11 - Crystallization and improved diffraction of an enzyme of therapeutic interest using a combination of rational construct design and crystallization optimization
By: Jeffrey Ohren, Peter Kuffa, Huifen Chen, Amy Delaney, Patrick McConnell, Chunhong Yan, Craig Banotai, David Dudley, Jim Dyer, Eric Fauman, Joseph Loo, Rachel Loo, Anil Mistry, W. Thomas Mueller, Shridhara Murthy, Cindy Spessard, Joseph Warmus, Alexander Pavlovsky, Erli Zhang, Charles Hasemann
P10 - The Xenograft Antigen in Complex with the B4 Isolectin of Griffonia simplicifolia Lectin-1
By: Wolfram Tempel1, Leigh Ann Lipscomb, John P. Rose1 and Robert J. Woods1,2
P9 - Experiences in microbatch crystallization
By: Lesley F. Haire, Nishi Vasisht, Miri Hirshberg, J. Li, Ian Taylor and Stephen J. Smerdon
P8 - Protein Crystallization on the International Space Station using CPCF - APCF - PCD
By: Jurgen Stapelmann
P7 - Comparing dynamic light scattering and analytical ultra-centrifugation as tools for protein crystallization
By: Claire Minshull and Jason Breed
P6 - Experiences with the use of the microbatch under oil method of crystallization
By: Nafeesa Noordeen and Sandra W. Cowan-Jacob
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P5 - Characterisation and crystallization of the ligand binding domain of PPARa in complex with a drug
By: Marie Anderson
P4 - Experience with (co-)crystallization of cAMP-dependent protein kinase (PKA)
By: Christine Breitenlechner1, Jochen Kluge1,2, D. Bossemeyer3 and R. A. Engh1,2
P3 - Crystal structure of the V-region of Streptococcus mutans antigen I/II at 2.4 Å resolution suggests a sugar preformed binding site
By: Nathalie Troffer-Charlier(1,2), Joëlle Ogier(1), Dino Moras(2) and Jean Cavarelli(2)
P2 - Crystallization experiments on the photosystem II reaction center from Pisum sativum
By: Ivana Kuta Smatanova
P1 - Rapid production of fusion proteins using the RTS 100/500 systems
By: Jan Stracke, Michael Schräml, Andreas Junger, Dorothee Ambrosius and Martin Lanzendorfer
P30 - Tweaking protein crystals for improved diffraction
By: Jolanta Krucinska, Larry Miercke, and Robert Stroud
P29 - Biological Crystallization Database
By: Jane E. Ladner, Michael Tung, and Gary L. Gilliland
P28 - Crystallization under microgravity conditions of the outer surface (S-layer) glycoprotein of the hyperthermophile Methanothermus fervidus and preliminary crystallographic information
By: Christine Evrard1, Jean-Paul Declercq1, Tony Debaerdemaeker2, and Helmut Konig3
P27 - A fast and inexpensive method for high-throughput crystallization screening
By: Thomas I. Zarembinski and Andrzej Joachimiak
P26 - Co-crystallization and x-ray structure determination of the CYT C2-RC complex from Rhodobacter sphaeroides*
By: Herbert L. Axelrod, Edward C. Abresch, Melvin Y. Okamura, and George Feher
P25 - CRYSTOOL random screening and why it's good for you
By: Brent W. Segelke and Bernhard Rupp
P24 - The crystal structure of truncated human coagulation factor IXa reveals an explanation for its poor amidolytic activity
By: Katrin Sichler1,2, Karl-Peter Hopfner2, Annette Karcher1, Anja Lang1, Erhard Kopetzki1, Robert Huber2, Wolfram Bode2, Richard A. Engh1,2, Hans Brandstetter2
P23 - Proteolytic truncation and microseeding required for growth of single crystals of a protein displaying good DLS behaviour
By: Margaret O'Gara
P22 - The 2D and 3D crystallization of membrane proteins
By: Johan Zeelen and Werner Kühlbrandt
P21 - Solubility studies on the crystals of Xylanase pI 9
By: Sinikka Uotila, Antti Hassinen, Gilles Joucla1 and Kalevi Visuri
P20 - The Thermus thermophilus mini-chaperonin: crystals for different MAD phasing experiments
By: .Dementieva, S.Korolev, M.Walsh1, R.Sanishvili and A.Joachimiak
P19 - Mass spectroscopy as a tool to determine protein homogeneity
By: Armando Villasenor, Kenneth Straub, Michelle Browner
P18 - Lucky numbers: Crystal Screen solutions that produce the most crystals
By: ChiehYing Chang, Kevin Kish, John Sack, and Howard Einspahr
P17 - Preparing targeted crystallization screens
By: Kevin Kish, ChiehYing Chang, Bruce Jacobson, and Howard Einspahr
P16 - Development of a relational database for structure-based drug design
By: B. Yu, S. Gulnik, M. Eissenstat, and J.W. Erickson
P15 - Further crystallization and associated stories from the Structural Biology Laboratory at York
By: Shirley Roberts
P14 - Unconventional crystallisation techniques that have produced high quality protein crystals
By: Jonathan. M. Hadden
P13 - Crystallization of toxin A from Salmonella enterica
By: Galya Obmolova
P12 - Screening organisms as important variable in the crystallization of the tRNA-modifying enzymes TGT and QueA
By: Klaus Reuter, Clemens Grimm, and Ralf Ficner
P11 - Design and use of a crystallization -"Cold Box"
P10 - Effects of hydrostatic pressure on the growth kinetics of tetragonal and orthorhombic lysozyme crystals
By: Gen Sazaki1, Yukiko Nagatoshi1,2, Y. Suzuki3, Satoru Miyashita1, K. Nakajima1,and Hiroshi Komatsu1
P9 - Effects of a magnetic field on the protein crystallization
By: Gen Sazaki1,2, Shin-ichiro Yanagiya1,3, Satoru Miyashita1, Kazuo Nakajima1, Kazuo Watanabe1,2,and Mitsuhiro Motokawa1,2
P8 - Crystallization of membrane proteins in lipidic mesophases: Applicability and a hypothesis for the crystallization mechanism
By: Peter Nollert
P7 - A simple and fast approach to achieve new crystal complexes for diffraction studies
By: Christine B. Luong and Bradley A. Katz
P6 - Obtaining homogeneous preparations of human cytomegalovirus protease for crystallography through Preparative Isoelectric Membrane Electrophoresis (PrIME)
By: Melissa S. Harris1, John A. Shelly2, Donna J. Paddock3, Alfredo G. Tomasselli3, Robert L. Garlick3, Eric T. Baldwin1, and Peter A. Wells2
P5 - Streaking to better crystals: Crystallization of S. aureus Thymidylate Kinase
By: D. Bryan Prince and Timothy E. Benson
P4 - Automated screening for crystallization conditions of proteins
By: Klaus Hellerbrand1, Rick Engh1, and Uwe Jacob2
P3 - Structural studies on the GABA transporter from E. coli
P2 - The incorporation of a non-natural amino acid may help to crystallize a protein and to solve its crystal structure
By: Jean-Paul Declercq and Christine Evrard
P1 - Screening with microbatch: tips and tricks
By: Allan D'Arcy, Martine Stihle, Glenn Dale, Chatana Yuvaniyama, and Brigitte D'Arcy
P16 - More Crystallization Stories from the Protein Structure Group at York
By: Shirley Roberts (on behalf of the group)
P15 - Crystallization of and Preliminary Diffraction Data for Retinol Dehydratase, a Vitamin-Metabolizing Sulfotranserase
By: John Gately Luz*, Marcia Newcomer#, Jochen Buck*
P14 - Screening for crystallization conditions of 10-formyltetra-hydrofolate dehydrogenase and its N-terminal domain
By: Sergey A. Krupenko and Conrad Wagner
P13 - Crystallization and X-ray Structure of Azotobacter vinelandii molybdate-binding protein at 1.2E resolution
By: Clare E.M. Williams, David M. Lawson, Richard N. Pau & Lesley A. Mitchenall
P12 - X-ray Structural analysis of Klebsiella Pneumoniae Nitrogenase Component 1 (Kp1)
By: David M. Lawson, Suzy M. Mayer, S. Mark Roe*, Carol A. Gormal & Barry E. Smith
P11 - A new method to slow vapor diffusion rate in macromolecule crystallization
By: Breed, J. and Zeth, K.
P10 - Crystallization of glucosamine 6-phosphate synthase from E. coli
By: G. Tepliakova (Obmolova)
P9 - A novel capillary-free mounting system for protein crystals: Fine Adjustment of the crystal environment with the possibility of manipulating the crystal quality
By: Reiner Kiefersauer*, Rick Engh*, Takemasa Kawashima**, Robert Huber*
P8 - Hardware for Cryocrystallography
By: Elspeth Garman and Mike Pickford
P7 - Crystallization of fumarate reductase of Shewanella putrefaciens MR-1
By: David Leys, Vincent Vileret, Alexandre Tsapin, Terrance Meyer, Jozef Van Beeumen
P6 - APCF, Advanced Protein Crystallization Facility: Results of Recent Missions - Actual Facility Features
By: J. Stapelman, R. Bosch, L. Potthast, K. Fuhmann*, J. Helliwell**
P5 - Using ligand crystal contacts to produce protein-inhibitor complex crystals
By: Hans E. Parge, Richard Showalter, Laura Pelletier, Peter Dragovich and Susumu Katoh
P4 - A 3D Microscope for Protein Crystal Growth
By: Carlo Ciatto, Thomas Gleichmann, Manfred S. Weiss, Dietmar Schwertner & Rolf Hilgenfeld
P3 - Improved Quantitive Analysis Of Dynamic Light Scattering Experiments
By: Philippe BENAS(*), Bernard LORBER, Philippe DUMAS
P2 - Flipper Devices And Other Tools For Freezing And Storing Of Protein Crystals
By: Christian Oefner, Albert Hoffmann and Allan D'Arcy
P1 - Comparative Assessment of Microgravity- and Earth-Grown Crystals of Thaumatin and Aspartyl-tRNA Synthetase
By: Joseph D. Ng, Bernard Lorber and Richard Giege
What bias can be seen in crystallization screens?

The JCSG has published a study that shows the pH's used and the pI's of the proteins. There are clusters of proteins with similar pIs that are easier to crystallize. However the pH's used are clustered around the pH's that are most often used in crystallization screens. It was commented that even when the data are normalized, there seems to be two crystallizability peaks occurring (see: Slabinski, L. et al. 2007 Protein Science 16:2472-2482.). For proteins <345 aa, there is a crystallization peak for those with a pI of 5-6 and again at a pI of 8-9. For proteins >345 aa, there is only one crystallizability peak, and that is for the proteins with a pI of 6-7. Crystallization screens typically have nominal pH ranges of 4 to 9 (nominal meaning the pH of the buffer is given, NOT the pH of the final solution).

How does the pI of a protein affect its tendency to crystallize?

It was reported that most proteins in the PDB have low pIs. Is this a general tendency? (Is this really true? Most E. coli proteins have a pI around 4.5, but is this true for all the proteins in the PDB?). One person said that when they have proteins with a high pI (>8) that won't crystallize, they use reductive methylation. This reduces the pI and also reduces the solubility of the protein, which is often helpful. One person stated that the pI needs to be determined experimentally, one cannot trust the answer you get from ExPasy and similar web sites as they only calculate the theoretical pI. Others said that a good estimate can be obtained from the sequence. Set up the crystallization experiment far from the pI (because the protein is least soluble at its pI). One person uses low pH for proteins with high pI. It was mentioned that Molecular Dimensions has a screen called ZetaSol, that allows high, neutral or low pH to be used as appropriate, depending on the pI of the protein.

How should we treat our seed stocks?

It was reported that freezing had always worked in several people's hands, in hundreds of cases. It worked even in cases where the crystals were unstable at 4 degrees Celsius. Another person reported that in one case seed crystals were stable at 22 degrees Celsius, but did not work when they were frozen. They seemed to freeze okay, but dissolved when they were defrosted. Two people commented that the potency of seed stocks could be increased by freezing. This was attributed to the freezing breaking up the seed crystals into smaller fragments. Cross-seeding can work when there is homology between the two proteins. However it was reported that they have never seen a case that could not be explained by the addition of chemical additives in the seed stock, e.g., a metal ion. One person reported a case where self-seeding doesn't work, but cross-seeding does (in this case it is the same protein, but different space groups).

When setting up vapor diffusion experiments, should you dispense the reservoir solutions or the protein first?

A show of hands indicated that putting down the protein solution first is far more popular. One person reported they did the opposite, pipetting reservoir first then protein. One person put down the reservoir solution first because it could be viscous, and this order gives the best mixing (but others pointed out that they did not want mixing). One person reported that either order was okay, but that it is essential to be consistent, because the results will be different from the two sequences. In one case, dispensing a peptide first, then ether, gave crystals. Dispensing the ether first then the peptide gave precipitate. It was suggested that dispensing high molecular weight PEG first could result in drying out or the absorbance of water. This implies that PEG should be dispensed first, because protein will almost always dry rather than absorb water! One person reported it was shocking that there is so little agreement, and that there should be well-established Standard Operating Procedures (SOPs) so that outsiders could receive clear guidance. One person said we should look into this as a community. Editor noted what he was reacting to was not this discussion, but the one we had about how to handle the Deep Well blocks. Someone asked how often we should renew our Deep Well blocks (containing the crystallization solutions.) When they stand for any longer period of time, a concentration gradient forms. One person reported using a shaker to try to eliminate the gradient and it does not work. One person said to invert the Deep Well block ten timesx, then centrifuge it a few minutes at very low speed (500 rpm) to avoid droplets on the lid. You don't want to cross-contaminate the wells when you pull the lid or tape sealer off. But there was no good agreement on how to handle this question and then someone said we should establish standards.

What is the best way to carry out dehydration experiments?

There are many good examples of the benefits of dehydration of crystals after growing them. A show of hands showed that a third of the audience has used the method. Some people increased the concentration of PEG by a third. Others used a 4% higher concentration. Extra sodium chloride can be added to harvesting solution. Overnight soaks are also used. One person a technique where drops are dried out by evaporation until a thick glop is formed. Sometimes this causes twin crystals to fall apart. (Acta Cryst. (2005). D61, 1173-1180 [ doi:10.1107/S0907444905019451 ])

Post-crystallization treatments for improving diffraction quality of protein crystals, B. Heras and J. L. Martin Abstract: X-ray crystallography is the most powerful method for determining the three-dimensional structure of biological macromolecules. One of the major obstacles in the process is the production of high-quality crystals for structure determination. All too often, crystals are produced that are of poor quality and are unsuitable for diffraction studies. This review provides a compilation of post-crystallization methods that can convert poorly diffracting crystals into data-quality crystals. Protocols for annealing, dehydration, soaking and cross-linking are outlined and examples of some spectacular changes in crystal quality are provided. The protocols are easily incorporated into the structure-determination pipeline and a practical guide is provided that shows how and when to use the different post-crystallization treatments for improving crystal quality. One person reported they increased PEG concentration from 40% to 70%. This increased resolution from 10Å to 2.8Å. This person then dropped the concentration from 40% to 20%. This caused cracking of the crystals which then rapidly healed to give 2.5Å. This shows that it is hard to make predictions. There was a consensus that annealing often works. It is thought to cause shrinkage of the crystal, which increases the number of hydrogen bonds. Dehydration and increasing PEG concentration often works.

How many conditions should be used to screen a protein? How many repetitions should be included? How many drops should be set up (using multi-drop plates)?

It was recommended 200 conditions (two plates) at two temperatures for an initial screen. However larger screens are also acceptable, but remember that this strategy generates a lot of images to look at. It was noted that large screens already have a lot of duplication in them, with identical or very similar conditions. One person reported larger screens do not generate significantly more hits.

Should high molecular weight proteins be crystallized at lower concentrations (mg/ml) than smaller ones?

Proteins with over 500 amino acids and those with fewer than 100 were said to be more difficult to crystallize (Slabinski, L. et al. 2007 Protein Science 16:2472-2482). It was said that a paper by Tom Peat and Janet Newman showed that high molecular weight proteins are crystallized with lower precipitant concentration on average (Peat et al. Acta Cryst. (2005). D61, 1662-1669).

When should you use 4 degrees Celsius? Should you start at 20 degrees Celsius, then change to 4 degrees Celsius? If so, when?

Start at 20 degrees Celsius and only change to 4 degrees Celsius because you don't want to work in the cold room for optimization! However, at least one person does the opposite of everyone else, starting at 4 degrees Celsius then changes to 20 degrees Celsius. It was reported that data mining has shown that room temperature is the most popular, and also that the trend is towards higher temperatures. Light scattering can tell you whether solubility increases or decreases as the temperature is raised. The recommendation is to use the temperature at which the protein is most soluble. Jose Antonio Marquez' results indicate that one should use a temperature that is roughly 25 degrees Celsius below the melting temperature of the protein as shown in thermal shift (Thermofluor) experiments.

What is the best way to use in situ proteolysis?

Use the Hampton Research Proti-Ace kit, then run a gel to see which proteases work well. If a protease gives good results, then run a screen with protein treated with this protease. Limited proteolysis can work better than in situ because with in situ you may have too many crystallization experiments. Others felt that this is not a problem, and that a combinatorial experiment can work well. Mass spec is another way to find which proteases work well.

How should you go about crystallizing an RNA-protein complex?

Janet Newman has recommended the Granada Crystallization Box with counter-diffusion for this situation. A slight excess of RNA is recommended. RNases can be purified out of the protein sample, but the robots and plates must be kept clean. It's important to map the RNA against the protein to make sure that you use the right length of RNA

What should you do if you have a Cys-rich protein? For example, a protein with 130 amino acids, and 7 or 8 Cys residues?

It was pointed out that there are three commonly used reducing agents in the context of crystallization: Beta-mercaptoethanol (2-mercaptoethanol) is the weakest, DTT (dithiothreitol) is the next strongest and TCEP hydrochloride is the strongest of all. Which one to use will depend on what pH you are working at (DTT is not so stable at pH 8) and what buffer is present (TCEP is not particularly stable in phosphate buffers, especially at neutral pH. Therefore, if TCEP is to be used in PBS buffers, prepare the working solution immediately before use). Read chapter 14 of the 2nd edition of book Protein Crystallization edited by Terese Bergfors, International University Line, 2009. This chapter contains a discussion of which reducing agent to use and when it is contra-indicated. Remember as well that if you have a 20,000 Da protein with 6 free cysteines, then the protein concentration is 1 mM and the free cysteines require at least 6 mM of antioxidant.

How often should you replace commercial screen stocks?

Buy screens in tubes, then transfer the solutions to deep-well blocks and freeze. Others said that freezing could cause precipitation. Replace when used up, roughly once every 6 weeks.

How to crystallize protein in different oligomeric states? Should you crystallize as oligomer or momomer if both forms are available at different concentrations?

Use the oligomer, especially if it is the physiologically-relevant form. Use both, to double your chances. The monomer always has the option to form higher oligomeric forms under the crowded conditions in crystal packing

Should there be another RAMC?

Yes definitely.

Is it worth trying crystal dehydration for tough cases?

Yes, it is worth trying. A nice paper on this method: C. Abergel, Acta Cryst. (2004). D60, 1413-1416 Spectacular improvement of X-ray diffraction through fast desiccation of protein crystals.

Is glutaraldehyde cross-linking of crystals an effective way to protect them during soaking and/or cryopreservation?

Yes, it can work, " incredibly well". The best available reference for the method is a paper by Carol Lusty, J. Appl. Cryst. (1999). 32, 106-112 A gentle vapor-diffusion technique for cross-linking of protein crystals for cryocrystallography.

It was pointed out that this method is (like so many others) very protein crystal- dependent. Some protein crystals will effectively cross-link in 5 to 10 minutes, others require overnight. The methodology has been discussed in talks, but other than the paper by Lusty, no other manuscript was known to exist.

Treating the crystals for 30 minutes at 4 degrees Celsius was suggested by one researcher. Another person suggested adding 5 microliters of a 25% solution of glutaraldehyde to a Micro-Bridge in a 24 well plate. Let the crystal cross- link through the vapor phase. Periodically remove crystals to test diffraction and see if there is any evidence that the crystal is cross-linked by physical manipulation.

What is the distribution of screening hits for protein crystallization experiments?

The bar graph shows the distribution of crystallization hits obtained at the Hauptman- Woodward Medical Research Institute for 167 NESG and SGPP targets. Each target was set up with 1536 different crystallization cocktails and produced 1 or more hits during microbatch-under-oil crystallization screening.

Do people mount crystals directly from microplates?

Loop access seems to be the big issue facing people when trying to mount directly from a plate. Some researchers suggested using an acupuncture needle to move the crystal to one side of the drop so that it would be easier to loop.

One group did a study of plates to see which ones were the best for retrieving crystals. The MRC plate, and the Intelli-Plate were both deemed best.

Any tips for adding ligands to protein under high salt conditions?

Increase the ratio of ligand to protein by 3 to 5x. Adding ligand to the cryo-preservative can help. It is difficult when you have a low binging affinity; however success was reported even with micromolar inhibitors.

In cases where the sample is crystallized from conditions such as 3.5M sodium chloride, you can sometimes transfer the crystals to a solution of PEG added to the original cocktail to improve soaking.

Soak for longer periods of time. In some cases no evidence of the ligand was observed in the structure after a 24 hour soak, but after a one week soak, the ligand was clearly observed in the electron density.

Try to transfer the crystals to a solution with a different pH before adding the ligand.

Try to cross-link the crystals before soaking the ligand.

Solubility of the ligands can be a problem. DMSO can be used to increase the solubility of the ligand, and works as a cryoprotectant at concentrations of 20-30%.

Why is PEG 3350 so successful and popular?

Part of the success is due to over sampling. A lot of people use PEG 3350 and so a lot of crystallization conditions come from it.

PEG breaks down into aldehydes and peroxides. These compounds change the solution pH.

PEG is used for many things other than crystallization.

FLUKA now assays PEG that is purchased by Hampton Research for crystallization reagents. A study was done at Hampton Research to look at the stability of PEG solutions. Hundreds of bottles of PEG solutions were prepared and separately bottled for 2 years. Among other measurements pH, and conductivity were recorded. A fresh bottle was sacrificed for each measurement. Conductivity increases as the PEG breaks down. PEG solutions will degrade over time. Exposure to oxygen and light increases the rate of degradation.

PEG 3350 is an ingredient in food products and medications. Hence, it is regulated by the FDA. This means that it is well-characterized and does not have batch to batch variation. PEG 3350 is also the 'most stable' PEG.

To prepare PEG and keep it stable for a longer period of time, you need to get oxygen out of the bottle, keep it in the dark, and keep it cold (4 degrees Celsius or freezer storage is recommended after preparing the solution).

Regardless of how the solution is stored, it will break down over time. Solid PEG will break down over time as well.

If you can obtain crystals only from an aged cocktail, dope the drop with the aged cocktail (cut the protein solution with the aged cocktail) and use fresh and easier to obtain cocktail as the reservoir solution. You can get a lot of crystallization experiments from a few milliliters of vintage PEG containing cocktail solution if you only use it in the experiment drop.

Is microdialysis a popular method of crystallizing proteins?

A very large and vocal majority cried out, "no"!

Attendees felt that dialysis is a good approach, but requires too much sample, and is just too difficult to set up using traditional methods. People would use the method if there was a better way to set up the experiments.

A metal frame that holds the membrane flat and eases set up was described by one researcher. This frame eliminates the bubbles that can form during microdialysis experiments and block the liquid diffusion across the membrane.

Another person described a method using Eppendorf microcentrifuge tubes to set up dialysis experiments using 10microL of sample.

How commonly used is dynamic light scattering?

DLS is widely used, however it seems to be the general opinion that it works best in conjunction with other methods. If you use: 1) SDS-PAGE, 2) size-exclusion chromatography, and 3) dynamic light scattering to check your sample's purity you are much better off. Any one of the methods can mislead you, but all three provide solid evidence that your sample is likely to crystallize. 70% of samples will crystallize if all three methods produce "good" data on a sample.

What methods are used to determine the quality of protein samples prior to crystallization?

Many of the attendees routinely use mass spec (MALDI-TOF) to identify post- translational modifications. The size range of suitable samples was reported to be 2,000 to 90,000 Daltons. You can readily see contaminants. It was reported to be used with transmembrane proteins by one attendee. It only requires 15 minutes to run the experiment and "get the answer".

Other approaches included SDS-PAGE and size-exclusion chromatography, and dynamic light scattering.

Crystallization drop volume: small, medium, or large?

There seems to be a growing trend away from very small drop volumes. The general feeling was that too small a drop would decrease the likelihood of nucleation. MCSG now uses 300 nanoliter or protein plus 300 nanoliter of cocktail solution for their screening drops. Approximately one half of the crystals for MCSG come right from those screening drops.

A range of drop volumes are used, from 100 nanoliter plus 100 nanoliter up to 1 microliter. Many people seemed to agree that 600 or 800 nanoliter drops would lead to the formation of more crystal hits than smaller nanoliter drops.

Adding an extra volume of protein solution to the drop was deemed an effective strategy by one of the attendees. They felt that there was a significant amount of protein lost on the surface of the small volume drops.

How can you distinguish salt crystals from protein crystals?

Using a deep UV light source and a couple of filters you can usually distinguish protein from salt crystals. It doesn't work very well when a protein precipitate covers a salt crystal.

Newport makes a 260-290 band pass filter. The camera is from PLS Design and is the most expensive component of the system. The cost of an appropriate UV light source is approximately 9000 Euro.

What are some options for reformulation of your protein sample buffer?

A case was discussed where the protein sample is formulated with 500mM ammonium sulfate which was not considered ideal for crystallization trials. Options were presented for reformulation including, neutralized organic acids, TACSIMATE, these may be more amenable to cryopreservation of the crystals.

Phosphate buffer has gone out of fashion as a protein buffer because of the false leads (salt crystals) that can form during crystallization screening trials in the presence of calcium and magnesium. It was pointed out that it is a great buffer and works well for some proteins.

Also, the following paper by Jancarik et al was discussed as offering options for formulating the protein for crystallization trials. Acta Cryst. (2004). D60, 1670-1673 [ doi:10.1107/S0907444904010972 ] Optimum solubility (OS) screening: an efficient method to optimize buffer conditions for homogeneity and crystallization of proteins J. Jancarik, R. Pufan, C. Hong, S.-H. Kim and R. Kim.

Another discussion took place that mentioned the stargazer (real-time PCR) approach to look at chemicals that stabilize proteins for crystallization trials.

PNAS October 24, 2006 vol. 103 no. 43 15835-15840 Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination Masoud Vedadi, Frank H. Niesen, Abdellah Allali-Hassani, Oleg Y. Fedorov, Patrick J. Finerty, Jr., Gregory A. Wasney, Ron Yeung, Cheryl Arrowsmith, Linda J. Ball, Helena Berglund, Raymond Hui, Brian D. Marsden, Par Nordlund, Michael Sundstrom, Johan Weigelt, and Aled M. Edwards. 

When surface entropy reduction is used to enhance crystallization can you change the serine to something other than alanine? What about Tyrosine?

It was reported that for PSI2 they tried the surface entropy reduction method on about 20 proteins, of those there was only 1 successful application of the method.

The online server that identifies which residues to modify for surface entropy reduction is considered to be fast and effective (http://nihserver.mbi.ucla.edu/SER/). It was reported that a modeler required an entire year of effort to come to the same conclusions about which residues to modify, verifying the effectiveness and time-savings that this software offers.

Controlled proteolysis works as another approach to improving crystallizability of proteins. Test 8 to 10 proteolytic enzymes and run an SDS-PAGE gel to check the sample purity post-treatment. Concentrations used range from 0.01 to 0.001% of the protein concentration. pH is important to control the enzymes functions. In some cases, it may be worth trying to add a proteolytic enzyme directly to the crystallization drop.

What do crystals that have been crushed for seeding experiments look like?

If you look under a microscope at about 40x magnification the suspension of crushed crystals will not have any discernable single crystals. There should be an opalescence, or sheen to the mixture with particles that are too small to clearly distinguish.

Very small crystals are difficult to crush. Larger crystals are easier to crush. Allan D'Arcy uses the reservoir solution to suspend the seeds. He recommends first using micro tools to crush the crystals, followed by the seed bead. If the crystals are cross- linked and gummy when you try to crush them with a needle or probe, they will not produce a good seed stock. They should crumble like parmesan cheese when pressure is applied with the probe.

Fairly concentrated seed stock suspensions should be used when trying to screen for initial crystallization conditions. Dilute suspensions are used when micro-seeding to try to produce a few larger crystals.

Should you crush crystals grown from different cocktails together or separately?

If the cocktails are chemically close, you can pool the crystals and crush them together, otherwise, crush them separately.

Can you seed using the horse hair crushed using liquid nitrogen using the robotic delivery system?

No, it isn't very effective. The crushed hair seed stock settles out from solution rather quickly.

What methods do people use to measure the concentration of their protein samples?

The Nanodrop ND-1000 system (http://www.nanodrop.com/nd-1000-overview.html) was used by about one-third of the audience. According to attendees you can place about 2 microliters of your sample on the detector, lower an arm to read the concentration of the sample (from fairly dilute to about 80mg/mL) all in a few seconds. You can retrieve some of the solution after the read. There is a single and 8 probe version of the device available.

Bradford, Biorad colorimetric assays were the other popular choice.

What methods do people use to concentrate protein samples for crystallization trials?

It was noted that centrifuge-based ultrafiltration cells can form a concentrated layer of glycerol on the surface of the ultrafiltration membrane.

People are using the Centricon, Centriprep, Minicon and Zeta-spin centrifuge-based ultrafiltration cells. It was reported that people using these systems would often stop the centrifuge every five minutes to mix the solution and then begin the centrifugation process again to eliminate the formation of a gradient.

A number of researchers brought up the use of an ultrafiltration cell that is stirred, using pressurized nitrogen. An example of this is the Amicon. The stirred cell eliminates the concentrated layer of protein, glycerol, etc... that often forms in a centrifuge-based ultrafiltration cell. The smallest volume of these cells currently available is 3 mL and using this you can concentrate a solution down to ~ 100microL volume.

How many people use ammonium sulfate to concentrate their samples? Only one or two people raised their hands. It was pointed out that Allan D'Arcy does this quick process and that it worked well for a number of samples. How many people place a dialysis tube filled with dilute protein solution over a bed of high molecular weight PEG to concentrate their samples?

Only a few people raised their hands.

Can we use data mining methods with the currently published crystallization conditions to come up with new crystallization screens or methods?

(Howard Einspahr) It’s a wonderful idea but a nightmare to implement. Information in the literature is not well described. There are reported crystallization conditions with a missing key ingredient. The BMCD is not longer current.

(Onkar Singh) With the variation in protein batches, different conditions and ways to measure protein concentration being used in all the different laboratories minimal information may be adequate.

(Martin Caffrey) Journals have page limits for articles, limiting the amount of detail you can use to describe your crystallization experiments.

(Howard Einspahr) Not any more with Acta cryst F.

Could we develop a CCP4 style database for crystallization?

(Shane Atwell) We would need a database with clean interfaces. It would have to be made available to the public. Someone would have to decide what data should be entered.

(Patrick Shaw Stewart) There isn’t much progress. The suites sold by companies are not modular. You buy it and have to use the entire suite, not just the sections you may like.

How do we communicate ideas between crystallization groups and to the general biological community who are just beginning to learn crystallization methods?

(Bob Cudney) There will be a newsgroup/bulletin board on the new Hampton web site that will provide a forum for communication amongst crystallizers.

(Larry Miercke) Why is the concentration of the buffer 0.1M in crystallization screens?

(Bob Cudney) The original Jancarik and Kim Crystal Screen was the result of a survey of the literature for successful crystallization conditions.

The low ionic strength screens use lower buffer concentrations to keep the ionic strength down.

You need a high enough buffer concentration in your crystallization screens to affect the pH of the final experiment drop. If the protein solution has an initial buffer concentration of 25-50mM you need to have a higher buffer concentration to effectively shift the pH of the crystallization experiment.

Should we vary the concentration and chemical composition of the buffer we use in our crystallization trials?

(Onkar Singh) Yes, vary the concentration of the buffer.

What is the best concentration of protein to use for an initial screen of crystallization conditions?

The majority of people polled work in the 5 to 10 mg/ml range of protein concentration.

(Bob Cudney) A continually updated survey of the literature shows an average protein concentration of 14.5 mg/ml.

(Patrick Shaw Stewart) This is going to be technique dependent. Batch experiments will require a higher protein concentration than vapor diffusion experiments. Looking at 800 experiments in the literature 4 were set up at < 2mg/ml and 4 were set up at >300mg/ml. The majority are set up in between that wide range of concentrations.
Add a larger volume of protein than crystallization cocktail to your experiment drop to increase the amount of protein in the experiment.

(Shane Atwell) Commercial screens seem to be targeted to a protein concentration of 10 mg/ml. Wizard screens seem to work best at a slightly higher protein concentration than the other screens.
The Hampton pre-crystallization test (PCT) was reported to work well.

What confidence level do people have when using crystals for drug design?

(Shane Atwell) Surface mutations are okay, but avoid active site mutations.

(Allan D’Arcy) Be sure to check the biological activity of the mutants.

How do we seal the end of the hollow tubes that hold the cryoloops to prevent liquid from being wicked into the tube through capillary action?

(Allan D’Arcy) Use superglue.

(Shane Atwell) The pins oxidize when using superglue to seal them. Use the thicker glue-like superglue.

(Howard Einspahr) Old school use sealing wax, it won’t oxidize the pins and repels liquid.
Mounted CryoLoops produced by Hampton Research are now sealed.

(Shirley Roberts) How does the wax behave in liquid nitrogen? No one knew the answer to this question.

What has been people’s experience with the litholoops?Mixed results.

(Onkar Singh) No luck with them.

(Shane Atwell) The litholoops tended to pull more liquid away from the sample than traditional loops.

Are good crystals dependant on the protein, the method, or both?


Should you mix your protein and cocktail drops when you set up screening experiments to crystallize your protein?

The general consensus seemed to be that you were better off not mixing the drops. The higher concentrations of protein and crystallization cocktail at the boundary between the two unmixed drops would set up a highly supersaturated boundary zone. This high level of supersaturation is more likely to initiate nucleation of the crystals.

Do you prefer round or square wells in your crystallization plates?

(Allan D’Arcy) Round wells provide better optics.

(Shirley Roberts) Image processing algorithms do not work in her group with round wells.

Flat bottom plates do not help localize the drop.

A protein sample looks promising. It is monodisperse by DLS, migrates as expected on SDS-PAGE yet you can’t produce crystals, what should you do?

The sample may be contaminated with something that you can’t resolve by the methods of analysis that you are using.

(Allan D’Arcy) Roughly 30% of proteins are soluble to a high concentration but do not produce crystals.

(Zygmunt Derewenda) Disorder. Mutation does nothing to improve crystallization. Using NMR analysis the structure was shown to be disordered. Look from a number of angles including secondary structure, tertiary structure structure prediction.

(Patrick Shaw Stewart) Weitzman Institute web service program to predict crystallization of a sample. Prilusky J., Felder C.E., Zeev-Ben-Mordehai T., Rydberg E., Man O., Beckmann J.S., Silman I. and Sussman J.L. (2005) FoldIndex© predicts whether a given protein is intrinsically disordered. Bioinformatics (in press).
See also http://www.disprot.org/predictors.php

Joel Sussman emphasized that the choice of predictor may depend on the question that you are asking. The Wiesmann interface is the best for identifying unfolded proteins and large unfolded regions. Some of the other predictors are better at identifying smaller floppy regions.

(Aengus MacSweeney) Heard of two people who used it and had good results.

(Howard Einspahr) Ligands can help. Purification protocols sometimes carry along with the protein natural ligands that can help the protein fold correctly.

(Aengus MacSweeney) If you have no ligand try 50mM substrate, anything can help.

(Shane Atwell) Ligands are as important as the protein. If the concentration of the ligand is high enough, anything from a related family will bind and may help to stabilize the protein.

Nanodrops. What is considered small these days? Is it difficult to scale up after initial nanodrop leads?

(Howard Einspahr) Less than 100 nanoliters is small. Going from 20 nanoliter drops containing crystals to larger drops is difficult. The literature contains all we need to bridge that gap. We should run experiments to figure this out.

(Shirley Roberts) Our group likes to use smaller drops. 100 – 150 nL drops do produce more salt crystals than larger drops. It can be difficult to retrieve crystals with so little mother liquor.

(Heather Ringrose) Use vapor diffusion vary the size of the protein portion of the experiment drop 200nL protein + 100nL precipitating agent.

(Beat Blattmann) Make it part of your routine with a robot to check the delivery on a daily basis. Make sure the drops are delivered to the correct location and are of the correct volume.

(Howard Einspahr) Are robotic experiments more reproducible than those set up by Humans?

(Shane Atwell) Side by side robot/technician experiments did not show a significant difference in reproducibility between humans and robots. Smaller drop volumes seem to be better. Smaller drops have a higher pressure than larger drops. The parameters of the drop change with drop volume.

Are robots really necessary?

Janet Newman) If the robot is not suitable for the process you end up with an expensive dust collector.

You need to make regular use of robotics. It’s not just the initial cost of the robot(s) but also the cost of technician time (for use, repair, and periodic maintenance), extended warrantees and disposables.

Nanodrops achieved with robotics were considered a breakthrough for one group. It lowered the cost per experiment and the sample requirements.

Robots can reduce the amount of sample required for screening.

Some industry participants report they are backing off on their use of robots and primarily use them for screening, doing optimization manually.

His-Tags Yes or No for crystallization. This topic comes up at every meeting and we will take a poll once again for the most recent public opinion on the subject.

If you have a choice of two proteins, one with a His-Tag and one without a His-Tag look at the level of expression before making a decision. If expression is the same for both then use the one without the Tag. It is best to cleave the Tag before concentrating the sample. An example presented described a protein that would not refold with the Tag but would refold without it.

(Janet Newman) Standardized reporting of crystallization experiments, how can we make it happen?

(Janet Newman) We should try to develop a standard, constant method to report crystallization information. Publish it in a standard way using the same method developed by the PDB for structures. Inconsistencies in the literature include the reporting of ammonium sulfate concentrations as both percent of saturation and as molarity. Molarity was agreed to be the correct way to report this. Other issues discussed included the reporting of PEG 3350 as PEG 4000 in the literature.

(Shane Atwell) The initial hits are never reported in the literature. You only see the final ‘optimized’ crystallization conditions that produced the crystal(s) used in the diffraction experiment. (Martin Caffrey) Data mining needs all of the conditions, those that fail as well as those that succeed in producing crystals.

(Howard Einspahr) Commercial crystallization screens are standardized, they are fixed in time. If you report conditions that gave you hits in these screens, then by default you also are reporting those that did not produce hits. Acta F will publish this in the supplementary information.

(Bob Cudney) Some vendors sell PEG in large batches using paper bags as a container. The PEG is not as stable when it is stored in paper bags. PEG 3350 is pharmaceutical grade, monodisperse, and has batch to batch consistency. PEG 4000 will have batch to batch variation.

(Allan D’Arcy) The Index screen was developed by Allan D’Arcy and Bob Cudney. Each of them surveyed the literature and came up with 48 solutions that they separately decided would be the best for an initial screen of crystallization conditions. Allan D’Arcy’s 48 were called ‘Hammer’. Bob Cudney’s 48 were called USA3. The genomics centers did not screen a number of different crystallization variables and may have been settled in a local minima for conditions.

(Patrick Shaw Stewart) Acta cryst has a macro that is available for formatting papers. Would it be possible for Acta F to do something similar?

(Howard Einspahr) Yes, great idea. A database like the PDB is only a receiver of information. It can only say, “Stay out”. There is no enforcement power. The PDB could not exclude deposition of coordinates until journals began to say, “No”. This was an expression of the will of the community. We as researchers have the power to help the databases get the data. “Make your voices heard”!

When should we screen temperature during crystallization trials and what range of temperature should we use?

A poll of the meeting participants showed that ~ 1/3 routinely screen temperature as a crystallization variable.

(Neil Grodsky) Use DLS to determine the effect of temperature on macromolecular samples before beginning crystallization trials. Place your sample in the DLS and make readings at 4, 13 and 21oC. Look at the results of these measurements to determine the ‘best’ temperature to use for crystallization trials. You should screen at least two temperatures. Based on experience in the laboratory he feels that for ~50% of the samples they work with temperature is an important variable.

(Bob Cudney) Routinely use microbatch under oil for screening experiments. They are well suited for screening temperature without the condensation issues normally associated with using vapor equilibration techniques. Experiments are set up at room temperature and scored after one day, the samples are moved to an incubator set at a temperature of 16 oC stored for one day and scored. The process is continued at temperatures of 4 and 30 oC.

(Alexander McPherson) Temperature is very important when working with detergents. Protein solubility is not nearly as affected by temperature as detergent solubility. If your protein is hydrophobic and tends to aggregate there is good reason to try to work with it at 37 oC.

(Martin Caffrey) We have designed lipids that will form a lipidic cubic phase in temperatures ranging from 4 to 60 oC. The primary lipid, mono olein forms a solid at 17 oC and so cannot be used for crystallization trials at 4 oC.

(Aengus MacSweeney) You can get condensation on the oil that could harm your crystallization experiments when changing temperatures.

(Bob Cudney) This condensation is climate dependent. It doesn’t occur in southern California.

(Lesley Haire) Temperature can be used to separate nucleation from growth phases. Set up the experiments at 4 oC where they are more highly supersaturated and nucleate (note this is case specific) and then transfer the experiments to 18 oC for the growth phase of the crystallization trials to prevent over-nucleation. Use the effect of temperature on protein solubility to control the level of supersaturation in your experiments.

(Allan D’Arcy) Be sure to test crystals for X-ray diffraction at room temperature. Loop mount your crystal and push it into an X-ray capillary sealing the ends of the capillary with clay (plasticine). If the crystal diffracts then, take the same crystal and use it in a cryogenic diffraction experiment. Make sure the crystal diffracts X-rays at room temperature before spending time trying to optimize cryo conditions. Someone brought up the fact that it is not uncommon to see significant differences in the quality of X-ray diffraction from ‘identical’ crystals taken from the same experiment plate or even the same drop.

(Howard Einspahr) Flash freezing is a big insult to the crystals. There is an increase in mosaic spread after flash freezing. Robert Thorne has shown that even when annealing works to lower mosaic spread it does not occur throughout the crystal. There are still regions of high mosaicity within the crystal. Freezing hurts the crystal mosaicity but pays off in the time you have to collect data before radiation damage becomes a problem. Elspeth Garman’s tip ; “Be the crystal”

Cloning and Expression - How Fast Can We Go?
Optimum speed of crystal growth?

Joe Luft reported his studies had shown there was an optimal speed of growth for a crystal but it varied from protein to protein.

Generally, no one reported having seen a correlation between speed of growth and the diffraction quality of the crystal.

Margaret O'Gara, Pfizer reported having successfully used silica hydrogels for crystal growth. Most people had been unsuccessful and didn't like using it.

Mass spectrometry and heavy metals

Examples were reported where reaction of protein solutions with heavy metals (Mercury compounds in particular) and then analysis with mass spectrometry had revealed binding of mercury to the protein. This was then confirmed when the protein was crystallised and the structure solved.

Can polydisperse be converted to mono-disperse?

Yes and no!

Detergents may help. Additives may help.

A change of buffer can often make the protein "happier".

A high speed spin (100,000 x g in an airfuge) can remove aggregates and leave a monodisperse supernatant.

One suggestion was to run the screen and look for positive leads and then add those components back into the protein and then rerun the light scattering experiment. No examples could be given of this having worked!

The preferred buffer for the protein was generally agreed to be one containing low salt, low buffer concentration and not phosphate. Glycerol is OK at low concentrations.

Good or bad precipitate?

Observing the drops at higher magnification than normal (100 to 1,000x) can sometimes be useful in deciding when a precipitate is amorphous or has some structure.

One comment was made that sometimes more buffer can be added directly to the drop and if its crystalline material then it would probably dissolve. If it is precipitate it will most likely not dissolve.

Also, adding a chromophore to the protein prior to crystallisation may help to make the distinction.

Crystallization and Molecular Biology

It was felt that other disciplines now appreciated the need for a molecular biology effort to work closely with protein crystallisation.

Industrial groups felt there was a definite need to have a molecular biologist within their structural group and the reason where this was not the case was mainly due to organisational problems.

A show of hands among the audience revealed 20 (out of 100) people were working in groups which had a dedicated molecular biology effort.

Spreading of drops containing detergent

Hampton Research have microbridges made from polypropylene which can be used for sitting drop experiments and give a nice drop even when they contain detergent.

Cross-linking in crystals

Several people apparently have unwanted cross-linking occurring in their crystals.

Anil Mistry reported this happening in on of his proteins and he believed it to be a problem associated with the cysteine residues in the protein. One solution would be to keep the crystals in a solution containing 200mM DTT which was kept fresh.

A recommendation was made for TCEP (sold by Pierce) as a reducing agent. It doesnt polymerise like DTT does and can be used successfully at 1/10th the concentration of DTT.

Treat the protein with a moderate concentration (30 mM) of DTT for a certain period of time (30 minutes) before setting the crystallization experiment.

Proteolytic digestion

People would like a standard set of proteases to be readily available.

Immobilised proteases (beads or plates) would be preferred.

New standard proteins?

People suggested there be readily available standard proteins for crystallization.

When to stop screening?

Some people were in favour of a fairly limited number of screens before reassessing the protein they were using.

Others reported using screens customised for an individual protein.

Screen for a while then use molecular biology to change the protein solubility and repeat screens.

A comment was made that statistically, a total of 400 solutions was a reasonable number.

Throwing away plates?

No one liked to throw away plates and people held onto them as long as possible.

What to do with a cloudy protein solution?

One comment was "give it back".

Other suggestions were:

Place at 4°C , add 15-20% glycerol, alter the buffer conditions. It was felt that if the cloudiness was reversible then this may well be a good sign.

One suggestion was to put the protein on a cover slip over a well solution of acetic acid or ammonium hydroxide and see if the cloudiness disappears due to pH change.

Data management

It was noted that several of the posters showed the application of Microsoft Access software to the handling of data generated from crystallisation trials.

The key problem here was generally felt to be getting people to input the data to keep the information up to date. One factor here would be to have the PC right next to the microscope. Ease of use would be the determining factor for a successful program. Everyone would like to have such a program and the consensus was that a figure of $1000 would be a reasonable sum to expect to pay for such a program. Again Margaret OGara pointed out that Leica sell a digital camera which comes with useful software which is aimed at general biology use but could be adapted for crystallisation applications.

Digital cameras

There was a high degree of interest in which digital cameras were suitable for collecting images of the set-ups. One recommended was the Pixera system sold by Leica. (Details from Margaret O'Gara at Pfizer) See web site for additional information: http://www.pixera.com/

Tag/No Tag?

Most people try to crystallise their protein with and without the tag.

A minority were quite happy to pursue crystallisation without looking at removing the tag and about 1/3 would remove the tag before attempting crystallization.

It was reported that the presence of the his tag can lead to protein insolubility at a pH of around 6.57.0.

The comment was made that with modern chromatographic techniques it should not be necessary to introduce tags purely as a purification tool.

Thrombin was the most popular cleavage site enzyme.

How to improve diffraction in DNA binding proteins.

One comment advised using short strands of DNA in complex with the protein and ensuring that the DNA was not too long to "overhang" the folded protein.

Also the annealing processes mentioned in the poster and talk by Clare Williams may be of benefit.

Birefringence & diffraction

No correlation was noted between birefringence and diffraction.

Soaking or co-crystallization?

There was an approx. 50:50 split between those who go for soaking and those who prefer co-crystallisation. The consensus seemed to be that given the choice then co-crystals were preferred. Some groups go for both apo crystals and co-crystals to maximise the chances of success.

It was pointed out that often it is feasible to use apo crystals as seeds to obtain complex crystals.

An instance was noted when soaking in the crystallisation conditions using ammonium sulfate was not possible since the sulfate ion was found to block the active site of the protein. So, soaking experiments required the crystal to be transferred to PEG conditions prior to inhibitor soaking experiments.

The issue of conformational chance and crystal contacts may lead to problems during soaking. Tightly bound inhibitors can crack crystals during soaking in contacts are restricted. Consider the size of the inhibitor when soaking (i.e. might be better to co-crystallize than soak with a large inhibitor).

Dissolve the highest concentration of inhibitor into 100% DMSO and mix with protein. If anything precipitates out, spin and set supernatant in hopes that some bound inhibitor stayed in the supernatant.

Mix dilute protein with dilute inhibitor then concentrate the mixture. Or mix dilute protein with dilute inhibitor and run over a size exclusion column, then concentrate.

Soak crystal in solution with a solid inhibitor. Try this with heavy atoms too.


Most people would use a robot for initial screening trials.

A smaller number would want a robot for optimisation studies.

Most people said no, they would not be interested in a robot capable of screening in a 96-well format mainly due to the problems that would raise in results analysis. Those that would use it would only do so for screening.

A useful innovation would be if automation was capable of "screening" the trials and dismissing large numbers of useless drops and perhaps picking out "interesting" ones.

One problem with automated result analysis arises from the need to scan up and down through a drop to get the full "result" from that experiment.

One possible innovation would involve a joystick controlled microscope stage and a microscope connected to a PC-linked camera.

When doing set-ups manually it is often possible to make changes to the design of the experiment "on-the-fly"; this is not be possible with automated screening robotics.

One person suggested using color schemes to score crystals.

Temperature for optimal crystal nucleation/growth?

One person reported using a mini-incubator with the capacity to hold 6-12 plates. This will allow a wider variety of temperatures to be screened on a smaller scale. This person reported obtaining an increased number of initial screen hits by cycling the same screen across several temperatures using standard proteins. Also it was his experience with Ribonuclease A that cycling through a temperature gradient lead to a larger crystal. For example use temperature T1 to nucleate, increase temperature to T2 to etch crystal back, reduce temperature to T1 and continue to grow the crystal and repeat until the desired results are achieved.

About 2/3 of the audience routinely screen at two temperatures. About 2/3 screen at 40°C and most of the audience use 20°C. A few people screen at intermediate temperatures and some screen at temperatures greater than 20°C.

Subtle differences in temperature such as the difference between 4 and 8 degrees Celsius can be significant.

Alex McPherson does his initial screens at 20°C then moves the plates around after about 10 days to 37°C, 4°C etc. He also pointed out that he finds hydrophobic proteins (or proteins which oil-out) work better at 37°C and therefore temperatures above room temperature should not be ignored.

It was pointed out that when using an incubator to control temperature the vibration/mechanical movement of the motor within the incubator adds another factor to the crystallisation. One person reported removing the compressor unit from the incubator to remove the vibration source.

Different host cells

Try different host cells in a bauculovirus system to see different levels of glycosylation. Or use inhibitors to prevent glycosylation. Or remove the glycosylation for enzymes.


Note that 50% v/v glycerol is required as a cryoprotectant in 0.2 M magnesium formate.


Try beta-mercaptoethanol as a crystallization additive.

Best temperature

What is the best temperature for sample storage? Try an ammonium sulfate precipitation and store at 4°C. When ready to set up sample, spin and resuspend in the crystallization buffer. Others suggest flash freezing in liquid nitrogen and then freezing the sample at -80°C.

Cryo Tips

Experiencing and increase in mosaic spread? Try optimizing the cryoprotectant conditions, try optimizing the crystallization reagent, consider the speed of transfer, the speed of freezing, and the size of the crystal as variables.

Block the cryostream for a few seconds or even minutes to remove ice when you have poor cryo conditions.

Ice on crystal in cryostream? Poor liquid nitrogen onto crystal in cryostream.

Another crystallization variable

Try a variety of buffers effective at the same pH when optimizing crystallization conditions since sometimes the buffer will bind the protein or the buffer may prevent a desired ligand from binding.

His tags

His tags were discussed again. It appears there is no consensus on whether to leave the tail on or chop it off. Everyone seemed to agree to leave the His tag one, try a screen and if no crystals are obtained, chop off the His tag and repeat the screen.

Hydrophobic protein

When working with a hydrophobic protein add the detergent during the purification procedure. Also try adding sodium chloride along with the detergent during purification.


Try using agarase to dissolve agarose gels when growing crystals in agarose gels.


When using polyamines for RNA and DNA crystallization keep in mind that polyamines effect both sample solubility and crystal quality.

Looking for new additives to try?

Looking for new additives to try? Iodoacetic acid, iodoacetamide, and trifluoroethanol.

Filter the protein

People who prefer to filter the protein sample prior to crystallization experiments suggest trying one of the following: 300 kD, 0.02 micron, 0.1 micron, or 0.2 micron filters.

Production of glycoproteins with predefined glycosylation

During the RAMC 97 Meeting there was discussion about the production of glycoproteins with predefined glycosylation. Annie Hassell provided the following reference after the meeting: Davis, SJ, Puklavec, MJ, Ashford, DA, Harlos, K, Jones, EY, Stuart, DI, & Williams AF. Expression of soluble recombinant glycoproteins with predefined glycosylation: application to the crystallization of the T-cell glycoprotein CD-2. Protein Engineering 6: 229-232 (1993).

By: Terese Bergfors

Poisoning to abolish excess nucleation. Problem: You have lots of showers of crystals in lots of drops no matter what you do. Solution: Do an additive screen on one or more of your hits. Out of 96 additives, chances are good that at least one will abolish all nucleation, i.e. the drops stay clear. This is now your poison additive. Step 2: Seed into the poisoned drop. Now it is you that controls the amount of nucleation added. Step 3: Vary the seed concentration to titrate exactly the amount of nuclei needed in the poisoned drop. Note: There is a published example of this idea in Acta D where they seeded a clear drop, hence my idea for a poison additive screen (I have to look up the reference on my computer).


By: J.A. Marquez

Incubate your crystallization experiments at a temperature at least 25 degrees Celsius below the Tm of your protein.


By: Kirsten Kahler

Use protein seeds from an unrelated protein to seed a new protein that has not crystallized through standard screening and no crystals of the protein of interest are available.


By: Kirsten Kahler

Use grease on the end of a tool to pop bubbles in a drop.


By: Laura Pelletier

If a room temperature soaking system results in loss of diffraction, adapt to 4 degrees Celsius to decrease the diffusion rate.


By: Beat Blattmann

To enhance reproducibility for the scale-up alays use the same polymer type of your crystallization plates.


By: Paul Reichert

Boil for two minutes your low soluble protein binders (small molecules) before complexing with your proteins.


By: Helen Gingell

If you seal wells with grease and coverslips don't make a complete circle of grease as when you place the slide on top of the grease the pressure in the well - make a small gap with a pipette tip so that when you press the coverslip down it seals quickly and firmly - the grease is squishes in to fill the gap.


By: Nham Nguyen

For protein complex : Pt substrate for screen I got one hit after six days (small crystal). I used the same condition as screening but instead of set up I incubated Pt substrate at 4 degrees Celsius for 6 days and set up, I got crystal the next day, twenty times bigger.


By: Christian Oefner

Try different ways of mixing the crystallization drop when setting up. For example, protein plus precipitant or vice versa, mixing, etc. Might yield different amount or size of crystals.


By: Paris Ward

I noticed the evaporation drying rate of certain additives in the Hampton screen and also detergent screen. I started pre-plating with different volumes into regularly used plates them putting them into the freezer to preserve the deepwell blocks and future plates from evaporation.


By: Jean-Paul Renaud

If you want to crystallize your protein don't use precipitants. Use crystallants. And please use the term crystallant or crystallizing agent in your publications and lectures.


By: Jean-Paul Renaud

Don't rely exclusively on automated screening and don't limit yourself to commercial crystallization kits. Try to mix yourself protein solution with a small set of crystallant solutions and see what happens under binoculars. You will learn a lot about your protein's behavior. First clue: All drops remain clean, protein concentration probably too low. Precipitate everywhere, protein concentration probably too high. Mixed results, go ahead with screening.


Use acupuncture needles as a micro tool to manipulate crystals in a drop or to remove skin.

Use acupuncture needles as a micro tool to manipulate crystals in a drop or to remove skin.

By: Tobias Merz

First read McPherson's book or any other reasonable source of knowledge before you start your crystallization experiments.


By: Tobias Merz

Check the homogeneityof your protein sample with as many methods as available.


By: Tobias Merz

If you use PEGs for crystallization experiments be aware they easily oxidize and can change behavior (in case repeated trials do not work anymore).


By: Zhanna Druzina

BME (2-mercaptoethanol) does it again. I will routinely add 1 microliter of 14 M BME (2-mercaptoethanol is typically made available as a 14.3 M solution) into the reservoir after drop set up. It worked in multiple cases to get biggers better diffracting crystals or even to get crystals of proteins that never crystallized before. If not hits appear in initial screening after months, seed the trays with seed stock, precpiptate, spherulites or even phase separation containing drops. Seed, seed and seed again. Works often and well. For controlled seeding (when seed stock concentration matters) add seed stock in the reservoir before mixing it with the protein in a tray.


By: Pierre Poussin-Courmontagne

There is a parameter which is rarely identified crystallization screening (vapor diffusion) it is the crystallization plate. Despite testing different crystallization kits, different technique, temperatures, protein concentration, ratios, etc., try at least different plates. With standard proteins in different plates we observed different results with one plate compared to another. In the lab we found one protein which gave crystals in one plate but not in others.


By: Martine Stihle

Too many small crystals or needles? If you seed with another form of crystals you may have at the end drops with nice big crystals from two forms.


By: Celine Ronin

Fill in the reservoir not with the condition of crystallization but with a high concentration of Sodium chloride, Ammonium sulfate (1M to 3M), PEG 3350, PEG 6000 (20 to 60%) (cf Newman Acta Cryst (2005) D61, 490-493). To optimize a hit, ask every people in the lab to make few drops. Just give them the materials (solution of crystallization, protein, coverslip, etc.) and no more information (not the size of the drop, not the order of adding the components, etc.) Let's see what happens!

No hits, what next
By: Anil Mistry, Pfizer

No hits from a screen, what next? You can always set up another screen! Or, you can make a list of all drops that have precipitate and use the precipitate as a potential seed stock. Streak seed from the precipitate into new drops that have been set up at 50% and 75% of the original screening solution. There may be nucleation sites or microscopic seeds in the precipitates that may grow at lower precipitate saturation. Better still, streak seed from the precipitate to a clear zone into the same drop to recycle the drop.


Reproducible seeding
By: Anna Aagaard, AstraZeneca

For reproducible micro-seeding by hand use a cryoloop to fish out your seed from the seed stock and transfer them to the drop. Use a 0.3-0.4 mm cryoloop.


Reduce to enlarge
By: Zhanna Druzina, SGX Pharma

For bigger crystals try to add 0.5-1 microliter of 14 M beta-mercaptoethanol to the reservoir after the protein drop was set up.


Difficult ligands
By: Annie Hassell, GlaxoSmithKline

Problem: Can grow crystal but no protein-ligand crystals. Tip: Take the conditions from the apo crystals and develop a focused optimization screen (24 well maximum). Screen complexes using cross seeding and the focused screen and three drop ratios (1:1, 2:1 and 2:3).


Time for a change
By: Annie Hassell, GlaxoSmithKline

Problem: Poor crystal quality. Tip: Change tray type or crystallization method. For example, initial screens done in sitting drop tray and crystal quality improved in 96 well hanging drop tray.


Avoid the rut
By: Brandon Collins, Boehringer-Ingelheim

Every project, every protein, every construct is unique. Be careful of knowing too much. Just because things did or did not work in the past does not mean things will work that way for the next project.


Get heavy to stabilize

Problem: Poor diffraction. Tip: Heavy atom soaks to stabilize floppy regions of the protein.

Lose focus
By: Joseph Luft, Hauptman-Woodward Institute

Don't focus all of your optimization efforts on a single crystallization condition. If you have several different crystallization conditions identified for a sample go after them. Crystals of the same protein produced from different chemical conditions and/or temperatures will have unique physical properties. These properties will determine how easy the crystal can be looped (physical stability), cryoprotected and ultimately how well the crystal diffracts X-rays. Avoid single points of failure, go after several hits.


Ligand seeding
By: Doug Marcotte, Biogen IDEC

If ligands can't be soaked into crystal or co-crystallization is ligand specific try seeding into drops that contain ligand of interest. When soaking crystals with insoluble ligands try adding the cryoprotectant to the soaking solution. This can help solubilize the ligand and also cryoprotect (so less handling). Doesn't work so well when salt is the cryoprotectant, but may well when it's glycerol, DMSO or ethylene glycol.


Mostly clear
By: Annie Hassell, GlaxoSmithKline

Problem: Drops are all/mostly clear. Tip: Remove stabilizing agents (salt, glycerol, etc) from the protein buffer. Then do crystallization screens.


Hydrate or die
By: Vaheh Oganesyan, MedImmune

At suboptimal protein concentrations the interface between protein solution and crystallization screen solution may exhibit excessive precipitation. To avoid this, before adding screen solution add 1 microliter of water. Downside of this is equilibration will take slightly longer. Upside of this is decreased osmotic shock for protein and less precipitation.


What failure looks like
By: Janet Newman, CSIRO

Put one conditions of 40% TCA into your standard screen. This should precipitate out all of your protein, so that you have an idea of what heavy precipitate should look like.


Think global act local
By: Edward Snell, Hauptman-Woodward Medical Research Institute

Don't consider a crystallization result in isolation. Look for neighbors in chemical space and use those results to provide chemical directions for optimization. If a cryocooled crystal does not diffract well. You cannot tell if it is the crystal, cryoprotectant or cooling that is causing the problem. Look at room temperature data before moving on.


X marks the spot
By: Mei Xu, Novartis

Consider the case of poor nucleation and seeding did not work. Tip: Mix protein and well solution then use pipette tip to cross the drop into branched shape. Crystal may grow in the branches of the drop.


Robotic seeding
By: Paris Ward, GlaxoSmithKline

To increase you choices of producing more optimal crystal condition or conditions using seeds, try the following. Program a small volume liquid handler to dispense your protein and seed solution directly into 96 well commercial screens and/or an additive screen.


Delete to succeed
By: Heidi Schubert, University of Utah

Recent success with loop deletions. Sequence alignments reveal either charged loops and/or loop insertions relative to homologues. I have removed 3 to 34 amino acids and retained high expression soluble protein and novel crystals.


PEG to the rescue
By: Annie Hassell, GlaxoSmithKline

Problem: Unstable protein. Tip: Add 1-5% low molecular weight (200 to 1,000) PEG directly to the protein and then screen.


By: Jim Pflugrath, Rigaku

Start with big crystals! Add at least 5% glycerol to everything.


Cryo stabilization
By: Laura Pelletier, SGX Pharmaceuticals

Stabilize crystals in cryo by adding protein buffer components into the cryo. For example, most commonly I add 100 to 150 mM NaCl plus reservoir components plus cryoprotectant(s).


Play with the ratio
By: Ayse Sinem Ozyurt, SGX Pharmaceuticals

Play with protein-mother liquor ratio, especially with low solubility proteins. Try different concentration of protein coupled with streak seeding.


Repeat after me
By: Nancy Bump, Millennium Pharmaceuticals

After looking at the results of initial screening or of additive screen, pick several of the best "hits" and screen in 96 well format, 6 or 8 identical drops of each favorite before scaling up. Helps to eliminate "one offs" and save time.


Complex it
By: Paul Reichert, Schering Plough Research Institute

Apo protein is monidisperse but won't crystallize. Complex it! Complex it! Complex it!


Matrix seeding
By: Armando Villasenor, Roche Palo Alto

If your crystal seeds withstand large serial dilutions, try matrix seeding via the reservoir by doing the following. 1) Create crystal seeds as described by Allan D'Arcy. 2) Dispense seed into reservoirs containing reservoir. 3) Aispirate/dispense to mix seed in reservoir. 4) Dispense mother liquor droplet containing seed onto protein drop.


Salt that sucker out
By: Neil Grodsky, Pfizer Global Research & Development, La Jolla Labs

If you do not see any crystal growth in several days after set-up (more than 1 to 2 weeks) and the drop are not all clear, add salt (such as ammonium sulfate) to 0.5 M to the drops. Even though ammonium sulfate salt crystals might form, you might actually get protein crystals. This worked for me recently.


Helpful urea
By: Gloria Borgstahl, Eppley Institute UNMC

Non denaturing (less than 3 M) concentrations of urea can be helpful to solubilize your protein.


Precise dosing
By: Simon Low, Pfizer, Inc.

Try stoichiometric levels of multivalents, cations as additives. They may be necessary for crystallization but sometimes the levels found in commercial screens are too high and toxic.


Cook to crystallize
By: Liping Wang, GlaxoSmithKline

Heat treatment of protein complex to obtain diffraction quality crystals. Original complex has no initial crystal hits. Heat treat protein complex (25 to 80 degrees Celsius) for various times (5 to 30 minutes). Centrifuge to get rid of aggregated protein and screen again.


Gap it
By: Paul Reinfelds, Illinois Institute of Technology

When using pre greased trays, take a toothpick and remove a bit of grease from each well. Now as you push down on your cover slip, you turn it a few degrees. This will allow the air to escape and the turn will form an airtight seal over each well.


By: Jackie Day, Pfizer St Louis

Thermostability. Monitor the effect of additives, buffers, ligands, etc. on melt temp of your protein. We have seen in multiple cases that the most thermostable construct, buffers, additive yields the best or only crystals. How? We use Bio-Rad's iQ5 iCycler (a PCR instrument) as it has 5 sets of filters for excitational emission and hydrophobic dyes that fluoresce upon binding (protein unfolding).


Sticky situation
By: Michael Wiener, University of Virgina

Problem: Crystals adhering to plastic of sitting drop plate, and mechanical dislodging (by cryoloop, tool, etc) does not work. Solution: Stan a fine gauge syringe needle into the plastic, near but not into the crystal. This often distorts/disrupts the plastic near the crystal and breaks the seal.


Big low tech
By: Margarete Neu, GSK Stevenage, UK

Getting bigger crystal by low tech / low cost counter diffusion. If you are faced with either no nucleation or showers of crystals and the usual tricks including seeding do not work, try this: On a cover slide, set the protein and precipitant drop (example 1 microliter plus 1 microliter) separate, but very close to each other. Then, with a whisker or pipette tip streak through the drops to form a connecting bridge between the protein and precipitant solution. Invert cover slide and place over well. Crystals will form along the gradient and "self screen" for best conditions.


Insoluble compounds
By: Elizabeth Fry, Abbott Laboratories

When working with compounds for co-crystallization, if the compounds are highly insoluble in protein buffer (50 micromolar or less) we often employ low concentration complexing. We dilute the protein and then add in diluted compound, so tha the compound is added close, or at least closer to a concentration where it is soluble and the content of DMSO in the protein sample remains less than 2%. The protein-compound complex is then concentrated for crystallization trials. This has helped us with several projects with highly insoluble compounds.


Ionic liquids
By: Christopher Bonagura, Exelixis, Inc.

In experiments using model proteins we found that Ionic Liquids (IL's) specifically 1-Butyl-2-methyl imidazolium chloride gave increased numbers of crystallization outcomes compared to the IL controls. A large number of the crystals obtained had precipitated outcomes in the IL controls. In many other cases the IL and crystal had an improved morphology (needles to plates, plates to 3D crystals) over the IL controls. Tip: Using an IL such as 1-Butyl-2-methyl imidazolium chloride as an additive to improve chances of getting a crystal from conditions which otherwise would give precipitate. Marc Pusey, MI Research, Inc. Our favorite cryoprotectant. 1x UCP (Ultimate Cryo Protectant) 8% Glycerol, 8% Ethylene glycol, 9% Sucrose, 2% Glucose. We make a 2x solution. Generally add this 1:1 with reservoir. The ratio can be modified, for example 1.2 microliter 2x UCP : 0.8 microliter reservoir, or 1.4 microliter 2x UCP : 0.6 microliter reservoir, or 0.6 microliter 2x UCP : 1.4 microliter reservoir, etc. I believe this has been successful in cryoprotecting some 70% of all of our systems, resulting in more than 50 solutions of these targets regardless of previous cryogenic treatments. Author in unknown to me, but the credit is published in a singled Hencrickson paper. I was tipped off 6 years ago.


Se-met stuff
By: Shirley Robert, York Structural Biology Laboratory

If you can't get your Se-met protein to crystallize try leaving ou the DTT/TCEP during purification and crystallization.Sometimes there are disulfide bridges near crystallization contacts that need preserving for crystallization to take place. Collected the Se edge is still possible.


DMSO cryo
By: Rich Romero, SGX Pharmaceuticals

Try 20 to 30% DMSO as cryo. This has worked well in a number of cases for me and I've added this to a very short list of cryos that I personally use. Note: If your mother solution contains a high salt concentration the DMSO will cause it to precipitate out of solution. So beware!


Stack em up
By: Beat Blattmann, University of Zurich

4 degrees Celsius crystallization plates prepared at room temperature always have a condensation problem on the plate seal. To avoid condensation cover the finished plates with two lids and plate it in the cold room for 20 minutes or on top of a cold metal block, The will reduce the temperature of the reservoir solution while the 2 lids delay the temperature change from the top long enough to avoid condensation.


Anti skid
By: Barbra Pagarigan, Celgene

Ever been manually sealing a plate only have it slip out from under you? The result is usually death for hanging drop plates, and with sitting drop plates your best bet is hoping the drop is not splashed onto the seal above. To ensure the plate stays put when applying pressure, we use a "grip pad" in our lab. Simply place the grip pad onto the bench, set plate on top and seal as usual. The grip pad prevents the plate from slipping out from under the compression tool, usually a brayer, used for ensuring the please seal is applied correctly. The grip pad can be cut from the material commercially available for lining tool shop drawers. In addition, we created a fixed plate holder that encloses the entire plate to guarantee the brayer does not slip off the plate when sealing, a common occurrence when manually sealing many plates. Our plate holder is custom cut from a hard rubber to fit both thee 24 well and 96 well plates. The plates sit slightly above the platform to ensure both ends are sealed and for easy removal. The sides of the platform are rounded to ensure the brayer has a smooth path of travel.


By: Patrick Shaw Stewart

If your protein is not very concentrated, set up microbatch screening experiments with 0.1 microliter screening solution plus 0.5 microlier protein. Use Al’s oil to allow concentration in the drop. That way you are less likely to get salt crystals before you get protein crystals.

Patrick Shaw Stewart, Douglas Instruments

Fluidigm optimization
By: Neil Grodsky

When optimizing leads obtained from the Fluidigm chips in a vapor diffusion format, screen different ratios of protein to well volumes in drops.

Neil Grodsky, Pfizer Global R&D

He's not heavy, he's my crystal
By: Janet Newman

Wrap some lead tape (available from golf shops) around the lid of a cryo cap then the cap sits the appropriate way in a dewar of liquid nitrogen for easy storage of your frozen crystal.

Janet Newman, In Stilla Consulting

Be the crystal
By: Elspeth Garman

When manipulating crystals from their growing drop, and soaking them, try to imagine that you are the crystals, and how you would feel being poked with a loop or a needle, or what it would be like to have the pH temperature, osmotic pressure around you suddenly change without warning. It might help your procedures!

Elspeth Garman, Oxford University

Water replacement
By: Elspeth Garman

If cryo-cooling is not giving satisfactory results, check how you are making up the cryoprotectant solution. The water in the mother liquor should be replaced by the cryoprotectant agent, rather than diluting the mother liquor. This factor is the single most common factor causing trouble which is easily rectified.

Elspeth Garman, Oxford University

Glass seeds
By: Gaby, Novartis

Seeding with pieces of glass, broken cover slides. The protein crystals sometimes grow along the end of the glass.

Gaby, Novartis

Don't change
By: Shirley Roberts

If you are having problems repeating or transferring crystallizations from 96 well to 24 well plates, for optimization stick with the 96 well plates. The single well low profile Greiner plate case easily be set up by hand or robot. There is plenty of room for larger drops and crystals are accessible for fishing. There is even a ledge for a small drop of cryo.

Shirley Roberts, University of York

By: Jens-Christian Poulsen

Use seeding for screening when you have only precipitate in your first screens.

Jens-Christian Poulsen, University of Copenhagen

Silicon carbide
By: Christopher Browning

Seeding drops with silicon carbide. Silicon carbide whiskers used to deliver DNA into cell nucleus. Microscopic in size but contain suitable surface for protein molecules to aggregate and nucleate for crystal growth. Uniform size of whiskers will allow reproducible crystallization set ups. Thereby could be used in initial screening trials to induce nuclei for crystals to grow.

Christopher Browning, IGBMC

What about it
By: Christopher Browning

What about the production of protein crystals in transgenic yeast containing the genes of Bacillus thuringiensis. Bacteria too small. Yeaste cells of reasonable size. Bacillus thuringiensis produce protein crystals in vivo, thus utilize this technique to produce crystals in yeast with overexpression of target protein. Or oocytes.

Christopher Browning, IGBMC

Light em up
By: Christopher Browning

Use luminal in protein crystal identification. Luminol is used in forensics to stain blood. Uses UV to light up blood stains. Add luminal to drops with crystals. Luminol binds to protein crystals. Addition of UV light and protein crystal will fluoresce. Salt crystals should not fluoresce.

Christopher Browning, IGBMC

Smooth operator
By: Claude Sauter

Smooth soaking in agarose gel. Grow your crystals under the usual conditions with 0.1 to 0.3% agarose gel and let the soaking solution (ligands, heavy atoms) diffuse smoothly through the gel.

Claude Sauter, IBMC-CNRS-Strasbourg

Two to one
By: Heather Ringrose

In setting up nanoliter screens use 2:1 protein:reagent. Higher hit rate. Three examples so far.

Heather Ringrose, Pfizer Global Research & Development

By: Lesley Haire

Original suggestion from Steve Swerdon, NIMR. For crosslinking crystals, put glutaraldehyde into the moatt of a Douglas Instruments VaporBatch plate to crosslink crystals in droplets dispensed under Al's oil.

Lesley Haire, NIMR

Sit and spin
By: Dean Devonshire

When doing microbatch screening, briefly centrifuge the plate at low speed. That not only ensures protein and precipitants mix and form a single drop, but also separates particulate matter. Any immediate precipitate is pelleted softly allowing the majority of the drop to be easier to visualize under the microscope. This might also generate two concentration phases ofprotein. Crystals may form from the precipitate or the clear phase.

Dean Devonshire, Medivir

Oily drops
By: Joe Luft

Give yourself plenty of time to retrieve crystals from a hanging or sitting drop experiment. Add a drop of paraffin oil over the experiment drop. You will have plenty of time to mount crystals, test them with dye (remove a crystal to another drop of oil to dye so other crystals in the drop can still be used), crushed, etc. This works especially well when you find crystals that may be protein in an old dried out plate that cannot tolerate much additional evaporation.

Joe Luft, Hampton Woodward Institute

Greasing with Cyberlab Robot
By: Bryan Prince

Have you experienced grease blow out with your robot? Large globs of grease on your tray? Try this: Make a disc about 3/8" wide, the same diameter of the grease cartridge. In the middle 1/8", make a slot that will hold a 0-ring in place. Drill a small threaded hole in one end ~1/8" deep. This allows you to retrieve the disc when the cartridge is empty.

Bryan Prince
Pharmacia & Upjohn Company: d.bryan.prince@am.pnu.com

Mounting Needles
By: Bryan Prince

Greasing Your Own Plates, the Easy Way

Make a 5 ml grease cartridge with a P200 tip. The size of the cartridge reduces hand strain & fatigue.


Bryan Prince
Pharmacia & Upjohn Company: d.bryan.prince@am.pnu.com

Improve Your Crystals in Size, Shape, and Quantity
By: Nham Nguyen

After you have crystals open the coverslip, remove the mother liquid in the droplet, dissolve the crystal with 3 ul of H20 or buffer and add 3 ul of well solution. Close the coverslip. The crystal will appear again. My crystals diffract ~ 4 A, sometimes I get twins that diffract ~2.5 A.

Nham Nguyen
University of British Columbia: nham@laue.biochem.ubc.ca

Pickled Crystals‹Unusual Additives! (A True Story)
By: Michael Hickey

Pickle juice was added as an additive to a mutant form of a crystallized native protein. The mutant could not be crystallized in near similar conditions of the native. By chance, the components of pickle juice were read and found to contain compounds used in crystallization (i.e. Glycerol, PEG 400, citric acid, acetic acid, alum and a few vitamins). This juice (Sweet & Snappy Vlassic brand) was filtered (0.45 µl and pH¹ed to neutrality. It was then added to various PEG¹s (that crystallized the native form) and set up with the mutant. Crystals formed after 1 week! Trying to "add back" single components of the pickle juice to determine which component was responsible gave no crystals, the pickle juice (~1%) was necessary. Hint/ Tip: Commercially available food/ detergent solutions ought not to not be discounted as additives for crystallization!

Michael Hickey
Agouron Pharmaceuticals: micheal.hickey@agouron.com

Using DLS to Test for Irreversible Aggregation
By: Anil Mistry

Generally, people concentrate a protein to 10 mg/ml or higher, then dilute 2-fold when setting up their hanging drops by doing a 1:1 minx. What if in concentrating to 10 mg/ml aggregation has been induced which is irreversible, such when pipetting your 1:1 mix hanging drop, it already contains aggregates‹a bad starting point.

(For monodisperse protein samples)
Using DLS test the limit of concentrated protein, i.e., the maximum concentration that can be achieved before a polydisperse signal is obtained. Then test samples, up to this limit, by concentrating up to this limit and test for irreversible aggregation by diluting a concentrated sample to a number of levels and test for monodispersity. With the sensitivity of current DLS equipment even samples at 10 mg/ml should be measurable. In this way and within the limits of your DLS machine it should be possible to find out whether you will have aggregates when you dilute your concentrated protein 2-fold when setting up a hanging drop.

Anil Mistry
Parke-Davis Pharmaceuticals: anil.mistry@wl.com

Drop Drying Technique (courtesy of Ron Rubin in our lab at Parke Davis)
By: Anil Mistry

When setting up drops with a protein which has low solubility a low starting concentration has to be used. Setup larger drops (5-10 ul) and leave them to stand "dry" for 3-5 minutes at room temperature /4°C prior to inverting over a well of a Linbro plate, this should allow some pre-concentration.

Anil Mistry
Parke-Davis Pharmaceuticals: anil.mistry@wl.com

Temperature Variation
By: Irene Weber

To grow crystals at different temperatures around room temperature search the lab for spots that are consistently at higher or lower temperatures. A difference of several degrees can be found. Temperature shifts can be easily made by moving crystals to a different place. (Check office shelves too!) [Discovered by Charles Reed in my lab]

Irene Weber
Thomas Jefferson University: weber@asterix.jci.tju.edu

TCEP as a Reducing Agent
By: Barbara Brandhuber

Use TCEP instead of DTT as a reducing agent in your protein solution. It isn¹t oxidized as quickly as DTT. Be sure to watch the pH of your solutions because it is very acidic.

Barbara Brandhuber
Array BioPharma: bbrandhuber@arraybiopharma.com

Raise the DMSO?
By: Melissa Harris

If higher DMSO does not damage your protein, try higher concentrations of DMSO (10-20%) for crystallization. It can help when dealing with insoluble compounds and is an excellent cryo-protectant. Freeze directly from drop!

Melissa Harris
Pharmacia & Upjohn: melissa.s.harris@am.pnu.com

Taking Picture of Crystal w/o Optical Microscope Instead of Dissect Scope
By: Ching Yuan

Ching Yuan
Baylor College of Medicine: ching@emily.bioch.bcm.tmc.edu

Advice for the Follically - Challenged Crystallographer
By: Jonathon Hadden

It has been long accepted that crystallizers with an abundance of facial hair have highly successful careers (Leeds University‹personal observation). This was thought to stem from the fact that matter found its way from the hair into the trials and acted as a nucleation centre-BUT SERIOUSLY; Addition of small grains of sand to a crystallization drop that is "close to producing" crystals can aid in nucleation and crystal growth.

Jonathon Hadden
University of Leeds: bmbjmh@leeds.ac.uk

Cross Seeding to Generate Crystals of a Related Protein or Protein/Inhibitor Complex
By: Margaret O'Gara

If your protein or protein complex fails to crystallize try seeding from crystals of the same protein (if it's a protein you want to crystallize) or a related protein for apo protein crystals. Serial micro seeding works best, make sure you look at the drops after seeding to identify any visible crystal seeds in there (i.e. not new crystals)

Margaret O'Gara
Pfizer Central Research: margaret_ogara@pfizer.sandwich.com

Floating Boat in the Sea - Right Side Up or Down?
By: Betty Yu

Single dehydrant/reservoir Hanging Drop/ Sitting Drop vapor diffusion using microbatch plate in suitable container.

Application for Hampton Screening:
- Low Ionic Strength Screen
- All Additive Screens
- Nucleic Acid Mini Screen
- All Detergent Screens

Betty Yu
NCI-FCRDC/SAIC: yub@mail.ncifcrf.gov

"No More 4°C"
By: Marie Anderson

Prepare trays for crystallisation, leave at 4°C, and fill polystyrene box or flat container with ice. Imbed a metal plate in the ice and set out cover slips. When you¹re ready to set out crystallisations place the trays in the bed of the ice and prepare drops, when finished transfer to 4°C. Simple but it works.

Marie Anderson
ASTRA: marieanderson@hassle.se.astra.com

Crystal Annealing
By: Clare Stevenson

As I said in my talk, give it a go. You might be surprised!!

Clare Stevenson
Nitrogen Fixation Laboratory: clare.williams@bbsrc.ac.uk

Soft Crystals
By: Allan D'Arcy

If you have crystals that are very sensitive to being touched (they break) or stick to the glass or dish, use a pointed strip of parafilm to move them. Otherwise, grow the crystals on parafilm and punch "wells" around the crystals to move it.

Allan D'Arcy
Hoffmann-La Roche: allan.darcy@roche.com

Buffer Screening
By: Anil Mistry

To find the best buffer system which will keep your protein happy, stable, and soluble for concentration prior to crystallisation, setup your protein (at 1-2 mg/ml) in hanging drops over 1 ml well solutions containing a series of buffers at various pH¹s, with various additives/stabilizers, etc. (but no precipitant!). Checking for drops which are clear will give an idea of which solutions keep the protein happy. In addition, as the system attains equilibrium some in situ concentration of protein will be induced due to the bulk difference between the drop and well volumes, hence an idea of how the protein behaves upon concentration in this solution will also be observed. Modifying a clear or slightly clear drop by adding a higher concentration of buffer to the well may even produce crystals. However, the main piece of information this method can produce is an idea of which buffer system and additives to put your protein into prior to concentration a crystallisation screening.

Anil Mistry
Parke-Davis Pharmaceuticals: anil.mistry@wl.com

Know when enough is enough!
By: Brent Segelke

As a graduate student, I spent countless hours and quantities of protein trying to get crystals of a single construct. We never got crystals. Another group got the structure of a homologous protein that was auto-digestive. Has we stopped after 400 trials and altered our construct, perhaps we would have faired betterŠ we couldn¹t have faired any worse. There is a reasonable statistical argument to demonstrate that 400 trials are a good limit.

Brent Segelke
Lawrence Livermore Laboratories: segelke1@llnl.gov

Crosslinking of Crystals
By: Clare Stevenson

When crystals are fragile or you want to transfer them to a different mother liquor e.g. for heavy atom soaking, why not try crosslinking your crystals with 0.1% glutaraldehyde in your mother liquor. This can be done by adding the glutaraldehyde directly to the drop or placing it next to the drop and allowing for vapour diffusion. This crosslinking enabled us to solve Mod A at 1.2 A resolution.

Clare Stevenson
Nitrogen Fixation Laboratory: clare.williams@bbsrc.ac.uk

Slow Cryosoaks for Improved Crystal Stability
By: Bryan Prince & Melissa Harris

In a sitting drop well, cryoprotectant (20% Glycerol in crystallization buffer) should be dribbled down the side of the well in the following manner: See drawing.

Bryan Prince & Melissa Harris
Pharmacia & Upjohn Company: d.bryan.prince@am.pnu.com

Cryoprotectant Additive
By: Laura Pelletier

I tried adding 1-10 mg/ml BSA in the cryoprotectant when soaking crystals that would crack. The one time it was used, the crystal did not crack and froze nicely. I don¹t know if the BSA was the reason for successful freezing. I was wondering if anyone else has tried this.

Laura Pelletier
Agouron Pharmaceuticals: pell@agouron.com

To Determine the Optimal Concentration of Your Protein for Screen I and Screen II
By: Jaru Jancarik

Try (set up) drop no. 6 first (of Screen I). It should produce light precipitation‹if ppt appears too heavy, reduce the protein concentration by 1/2 and try again. If there is no precipitation in drop 6 try drop 4. If there is no precipitation in either of the drops concentrate the protein 2 fold and try again.

Jaru Jancarik
UC Berkeley/Chemistry: j_jancarik@lbl.gov

Mounting Needles
By: Frederick de Mare

Make a thin needle out of a glass capillary. Fish out crystal needles from drop with the capillary by holding the capillary parallel to the crystal. This will pick up less mother liquor (reduce background) and put less stress on the crystal.

Frederick de Mare
Pharmacia & Upjohn: fredrick.demare@eu.pnu.com

Check Both Liquid Nitrogen + Stream Flash Cooling
By: Hans Parge

If your crystal does not freeze well in the cold stream try liquid nitrogen or vice versa.

Hans Parge
Agouron Pharmaceuticals: hans.parge@agouron.com

90% Solutions
By: Anna Stevens

When optimizing or making solutions for a random scan, omit the buffer (@ final O.1 M) so that your stock is 90%. Prior to putting your stock in the well, add 100 uL of 1M buffer of your choice. Next, add 900 uL of 90% stock and mix. This reduces the number of tubes for crystallization (precipitant) stocks and allows flexibility in buffer identity and pH range. Be sure to make plenty of (~20mL) of precipitant, you¹ll need 900 uL per buffer.

Anna Stevens
Monsanto Company: anna.m.stevens@monsanto.com

Recycle Your Precipitate
By: Neali Armstrong

If your protein refolding reaction has a low yield and produces lots of precipitate try collecting the precipitate, resolubilize in GuHCl, and refold again. This material is sometimes more pure than the washed inclusion bodies.

Neali Armstrong
Columbia University: naa15@columbia.edu

Preserve Hampton Solutions
By: Cheryl Janson

To preserve Hampton solution when you or a co-worker gets a "hit" and suspect the Hampton solution may be "magic" or you cannot reproduce crystals with lab-made solution‹make the reservoir solution from lab ingredients and use your homemade solution for reservoirs. Use the Hampton "magic" solution only for the drops thus using a few ul per experiment rather than O.5 ml. Saves having a 48 well screening solution set with one tube empty and the rest at 8 ml and still allows the superstitions or in Alex's case, the contaminated solution to reproduce crystals.

Cheryl Janson
SmithKline Beecham: cheryl_a_janson@sbphrd.com

Concentration of Protein without Aggregation
By: Paul Reichert

Use centipreps (millipore) for concentrating protein >O.5 ml. Protein concentrating away from membrane. (No micro high concentration, ppt on membrane), works very nicely for a number of proteins.

Paul Reichert
Schering-Plough Research Institute: paul.reichert@spcorp.com

Don't Throw Away Without Looking Close
By: Kalevi Visuri

Look closely at your old test tubes when cleaning the place. Proteins do crystallize on the walls of the tube when stored in a cold room. I picked up my tubes from the wastebasket and an X-ray was made from an old supersaturated protein tube.

Kalevi Visuri
Macrocrystal Oy: management@macrocrystal.com

HPLC Profile
By: Glenn Dale

Keep an HPLC (Reverse phase) profile of your protein before crystallisation and after crystal formation. It can be used as a quality control and tells you if any modifications have occurred.

Glenn Dale
Hoffmann-La Roche: glenn.dale@roche.com

Rapid Preliminary Screening of Protein for Aggregation Using Protein Quantities
By: Tom Zarembinski

When only small amounts of protein are available, it is not feasible to screen many compounds which promote monodispersim using a dynamic light scattering machine. To detect aggregation, we use a pseudo-native gel approach: 1l of protein is mixed with 1 l additive from Hampton Additive/ or Detergent Screens. These samples are then incubated at room temperature for 20-30 minutes and then placed in 2x sample buffer containing no DTT, no SDS and are not boiled. These samples are run on a standard SDS-PAGE gel. We have screened many additives using this approach and it has given us leads for subsequent optimization of protein buffers.

Tom Zarembinski
Argonne National Laboratory: tomz@igg.anl.gov

Soaking Crystals to Improve Resolution
By: Irene Weber

Try soaking poorly diffracting crystals in higher concentration of precipitate‹ammonium sulfate or PEG. It may take several weeks so test after 1 or 2 months.

Irene Weber
Thomas Jefferson University: weber@asterix.jci.tju.edu

Iodoacetic Acid
By: Bernie Santarsiero

Add a small amount of (~ 1%) iodoacetic acid to buffer solutions. This helps prevent aggregation by carboxymethylation of cys. Also, iodoacetic acid seems to help form salt bridges and aid in crystallization.

Bernie Santarsiero
NIFG: bds@adrenaline.berkeley.edu

The Glycerol Effect
By: Anil Mistry

Glycerol has many benefits but also some drawbacks. We found it to be beneficial with one protein we were working with; this protein is a transpeptidase called Mur A. The protein is quite soluble and could concentrate to 20 mg/ml but it lost activity over time when stored at ­80°C. We therefore dialysed it into 50% v/v Glycerol to see if the activity could be retained for longer, this would allow us to make a large batch rather than regular smaller batches, for crystallisation. On dialysis we found a significant reduction in the volume of protein, so much so that the protein had concentrated from 5-10 mg/ml to almost 50 mg/ml. Activity was also found to be retained with no significant loss after 6 months at ­80°C. This gave us a method for storing large batches of Mur A at ­80°C, without losing activity and also resulted in a sample pre-concentrated for crystallisation and containing a cryo-protectant. Other sugars gave similar effects; sucrose, sorbitol, etc., but none were as effective as glycerol in achieving the 50 mg/ml final concentration.

Anil Mistry
Parke-Davis Pharmaceuticals: anil.mistry@wl.com

Extreme Soak
By: Jirundon Yuvaniyama

For soaking crystals with compounds with limited solubility I have tried two "extreme ways" (although not much- and more experiments should be tried):

- Leave some solid compound in the soaking solution.
- Dissolve some compound in n-octanol and layer the octanol solution on top of the soaking experiment.

These two provide (hopefully) more or less constant concentration of the compound in the soaking solution. The octanol layer may help reduce air oxidation by preventing direct contact of soaking solution to air.

I found that 200 µl of soaking solution in a well of the 24 well Linbro plate is a good volume to work with:
--Not too little that possibly causes concentration change due to evaporation (Sealed well) and not too much that the crystals get lost in the solution.

Jirundon Yuvaniyama
Mahidol University: scjyv@mahidol.ac.th

Don't Flip
By: Dennise Dombroski

When removing crystals from a hanging drop, I sometimes find that the biggest crystals fall against the coverslip and are impossible to resuspend without damage. I had our glass shop make a stand to transfer the coverslip to that enabled me to manipulate the crystals more easily.

Dennise Dombroski
University of British Columbia: dombroski@byron.biochem.ubc.ca

Shape of Drop
By: Ursula Kamlott

One of my proteins produced only zillions of tiny useless crystals. When I mixed the drops the conventional way-mixing well, overlaying, etc. the protein with precipitant solution. Large, gorgeous crystals were produced when I crossed the drop, creating a gradient within the drop. This worked best, setting up sitting drops with vapor diffusion.  Ursula Kamlott Hoffmann-La Roche: ursula.kammlott@roche.com

Mass spectrometry
By: David Leys

We found mass spectrometry like ESMS and MALDI highly efficient in determining impurities and/or microheterogeneities in our protein sample/batch. In most cases it is a simple, straight forward method which requires a minimum amount of sample. In some cases it has shown to detect impurities/microheterogeneities when other techniques did not.

By: Elspeth Garman

When making up cryoprotectant solutions containing glycerol, put a test tube of glycerol in a beaker of warm water. The viscosity falls and it is easier to pipette accurately.

Purest is not the best
By: Michal Harel

A protein which was purified and showed some faint bands of contaminants on a native gel was crystallized and solved successfully. The same protein, purified by HPLC and resulting in a single band native gel did not crystallize.

Low molecular weight PEGs
By: Lesley Haire

When screening with low molecular weight PEGs try microbatch. Crystals appear rapidly with PEG 400-2000. To convert to vapor diffusion use 0.2 M buffer in the well and a 1:1 drop ratio. Try using a positive displacement pipette such as the Anachem Microman 1 - 10 microliter. These are much more accurate.

By: Mike Sintchak

When working with crystals that grow fairly rapidly (one day) try the following. Pipette multiple protein drops (2 to 4 works best) onto the cover slide. Using a single pipette tip, get the reservoir solution to mix with the first drop. Now, go back to into the reservoir with the same tip to get the reservoir solution for the second drop. Continue for the remaining drops with the same tip. In certain cases, seeding starts very quickly, so by using the same tip one can introduce minute seeds to successive drops. Use the same cover slide with multiple drops to minimize evaporation.

Filtering the protein
By: Naomi Chayen

To reduce the number of crystals and increase their size, try filtering the protein solution prior to setting up the experiment. Try the following filter sizes: 0.22, 0.1 micron and 300 kD molecular weight cut-off. Try the Millipore centrifugal filters.