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| Crystals of human hemoglobin grown at Hampton Research |
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Analysis of polycrystallinity in hen egg-white lysozyme using a pnCCD. S. Send, A. Abboud, W. Leitenberger, M. S. Weiss, R. Hartmann, L. Strueder and U. Pietsch. J. Appl. Cryst. (2012). 45, 517-522 [ doi:10.1107/S0021889812015038 ] Synopsis: An analysis of polycrystallinity in tetragonal hen egg-white lysozyme by means of energy-dispersive Laue diffraction techniques using a pnCCD detector is presented.
Protonation-state determination in proteins using high-resolution X-ray crystallography: effects of resolution and completeness. S. J. Fisher, M. P. Blakeley, M. Cianci, S. McSweeney and J. R. Helliwell. Acta Cryst. (2012). D68, 800-809 [ doi:10.1107/S0907444912012589 ]. Synopsis: A bond-distance analysis to determine the protonation states of ionizable amino acids has been made for trypsin at 1.2 Å resolution, subtilisin at 1.26 Å resolution and lysozyme at 0.65 Å resolution. This was effective for Asp and Glu but not for His.
Outrunning free radicals in room-temperature macromolecular crystallography. R. L. Owen, D. Axford, J. E. Nettleship, R. J. Owens, J. I. Robinson, A. W. Morgan, A. S. Doré, G. Lebon, C. G. Tate, E. E. Fry, J. Ren, D. I. Stuart and G. Evans. Acta Cryst. (2012). D68, 810-818 [ doi:10.1107/S0907444912012553 ]. Synopsis: A systematic increase in lifetime is observed in room-temperature protein and virus crystals through the use of reduced exposure times and a fast detector.
Crystallization and preliminary X-ray diffraction studies of the GhKCH2 motor domain: alteration of pH significantly improved the quality of the crystals. X. Qin, Z. Chen, T. Xu, P. Li and G. Liu. Acta Cryst. (2012). F68, 798-801 [ doi:10.1107/S1744309112016351 ]. Synopsis: The GhKCH2 motor domain was crystallized and the pH of the crystallization buffer was shown to have a significant effect on the crystal morphology and diffraction quality.
Crystallization of SHARPIN using an automated two-dimensional grid screen for optimization. B. Stieglitz, K. Rittinger and L. F. Haire. Acta Cryst. (2012). F68, 816-819 [ doi:10.1107/S1744309112022208 ]. Synopsis: The expression, purification and crystallization of an N-terminal fragment of SHARPIN are reported. Diffraction-quality crystals were obtained using a two-dimensional grid-screen seeding technique.
Combining in situ proteolysis and mass spectrometry to crystallize Escherichia coli PgaB. D. J. Little, J. C. Whitney, H. Robinson, P. Yip, M. Nitz and P. L. Howell. Acta Cryst. (2012). F68, 842-845 doi:10.1107/S1744309112022075. Synopsis: Construct engineering and crystallization of E. coli PgaB using in situ proteolysis and mass spectrometry is reported.
Rehydratable gel for rapid loading of nanoliter solution and its application in protein crystallization. Yuefang Li , Dameng Guo and Bo Zheng. RSC Adv., 2012, 2, 4857-4863 DOI: 10.1039/C2RA20511D. Synopsis: In the work presented, rehydratable polyacrylamide gel is introduced as a medium to uptake and store nanoliter protein solutions in microwells for multiplex bioanalysis. The polyacrylamide gel, produced and stored in the microwells, shrank by 97% upon dehydration and could be reversibly rehydrated to 95% of the initial volume by absorbing aqueous solution. We employed the rehydratable gel to load aqueous solutions of different proteins with molecular weights in the range of 14.7–250 kDa. The protein loading occurred simultaneously with the gel rehydration and reached saturated state in 5 min. The relative protein concentrations in the gel ranged from 92% to 53%, depending on the molecular weight of the proteins. Particularly, the rehydratable gel had a much higher protein loading efficiency than the fresh gel. We applied the protein-carrying gel to the crystallization of four model proteins in the microwells and produced diffraction-quality protein crystals. The rehydratable gel is simple to fabricate, efficient to load with protein, and has good capacity for storing the protein solutions in microwells with minimal dilution effects on the protein solution. The rehydratable gel incorporated microwell chip should be useful in multiplex analysis that requires small sample consumption and high throughput.
Charge-controlled metastable liquid–liquid phase separation in protein solutions as a universal pathway towards crystallization. Fajun Zhang , Roland Roth , Marcell Wolf , Felix Roosen-Runge , Maximilian W. A. Skoda , Robert M. J. Jacobs , Michael Stzucki and Frank Schreiber. Soft Matter, 2012, 8, 1313-1316, DOI: 10.1039/C2SM07008A. Synopsis: We demonstrate that a metastable liquid–liquid phase separation (LLPS) in protein aqueous solutions can be induced by multivalent metal ions at room temperature. We determine the salt and protein partitioning in the two coexisting phases. The structure factor obtained by small angle X-ray scattering provides direct evidence for a short-ranged attraction, which leads to the metastability of the LLPS. An extended phase diagram with three control parameters (temperature, protein and salt concentration) provides a conclusive physical picture consistent with a criterion for the second virial coefficient. The presented isothermal control mechanism of the phase behavior opens new perspectives for the understanding of controlled phase behavior in nature. Furthermore, we discuss the application of this framework in predicting and optimizing conditions for protein crystallization.
Hofmeister effects of ionic liquids in protein crystallization: Direct and water-mediated interactions. Magdalena Kowacz , Abhik Mukhopadhyay , Ana Luísa Carvalho , José M. S. S. Esperança , Maria J. Romão and Luís Paulo N. Rebelo. CrystEngComm, 2012, Advance Article DOI: 10.1039/C2CE25129A. Synopsis: We have performed experiments on the crystallization of two low molecular weight, positively charged proteins, lysozyme and ribonuclease A, using ionic liquids as either crystallization additives or, in particular cases, as precipitating agents. The ionic liquids (ILs) have been ordered according to their salting-in/out ability and the relative position of these ionic liquids in this ranking has been rationalized by considering their hydration properties (positive–negative, hydrophobic–hydrophilic). The ability to screen the effective charge of cationic proteins and aid protein nucleation (salting-out) has been shown to be superior for large polarizable anions with low charge density, negatively hydrated-Cl-, Br-, [SCN]-, methane-[C1SO3]- and ethanesulfonates [C2SO3]-, than for anions with a relatively stable hydration shell, positively hydrated-lactate [Lac]-, butylsulfonate [C4SO3]- and acetate [Ac]-. Upon increasing the background salt concentration, where electrostatic interactions are already effectively screened, the ability of the IL ions to stabilize proteins in solution (salting-in) has been shown to increase as the ions are likely to migrate to the non-polar protein surface and lower protein–water interfacial tension. This tendency is enhanced as the focus moves from those ions with positively hydrated hydrophilic compartments (e.g. [Ac]-) to those with negatively hydrated groups (e.g. [C1SO3]-) and the prevailing hydrophobic hydration (e.g. [C4SO3]-). The observed inversion in the relative effect of ILs on protein crystallization with increasing ionic strength of the aqueous media has been interpreted as the differing effects of ion adsorption: charge screening and interfacial tension modification. Moreover, this work can further help in our understanding of the influence of ionic liquids on conformational changes of biomacromolecules in solution. Identification of the specific incorporation sites for choline and acetate ions, localized in two lysozyme crystals grown in pure IL solutions without any buffer or inorganic precipitant, can give us some insight into the role of the ionic liquid ions in protein structure development.
Nanolitre-scale crystallization using acoustic liquid-transfer technology. A. G. Villasenor, A. Wong, A. Shao, A. Garg, T. J. Donohue, A. Kuglstatter and S. F. Harris. Acta Cryst. (2012). D68, 893-900 doi:10.1107/S0907444912016617. Synopsis: Acoustic droplet ejection achieves precise, tipless, non-invasive transfer of diverse aqueous solutions, enabling nanolitre-scale crystallization trials. The rapid and scalable technique demonstrated successful crystal growth with diverse targets in drop volumes as small as 20 nl.
A universal indicator dye pH assay for crystallization solutions and other high-throughput applications. J. Newman, R. A. Sayle and V. J. Fazio. Acta Cryst. (2012). D68, 1003-1009 doi:10.1107/S0907444912018768. Synopsis: A rapid plate-based pH assay has been developed that takes advantage of the automation available in a protein-crystallization laboratory.
Proline: Mother Nature's cryoprotectant applied to protein crystallography. T. A. Pemberton, B. R. Still, E. M. Christensen, H. Singh, D. Srivastava and J. J. Tanner. Acta Cryst. (2012). D68, 1010-1018 doi:10.1107/S0907444912019580. Synopsis: The amino acid L-proline is shown to be a good cryoprotectant for protein crystals. Four examples are provided; the range of proline used for cryoprotection is 2.0-3.0 M.
Reduction of radiation damage and other benefits of short wavelengths for macromolecular crystallography data collection. R. Fourme, V. Honkimaeki, E. Girard, K. Medjoubi, A.-C. Dhaussy
and R. Kahn. J. Appl. Cryst. (2012). 45, 652-661 doi:10.1107/S0021889812019164. Synopsis: X-ray photons with energy higher than usual improve both the number and the quality of diffraction data from a given macromolecular crystal.
An anti-settling sample delivery instrument for serial femtosecond Crystallography. L. Lomb, J. Steinbrener, S. Bari, D. Beisel, D. Berndt, C. Kieser, M. Lukat, N. Neef and R. L. Shoeman. J. Appl. Cryst. (2012). 45, 674-678 doi:10.1107/S0021889812024557. Synopsis: A simple and robust instrument, which overcomes the crystal settling that impairs serial femtosecond crystallography experiments, is described.
A gradual desiccation method for improving the efficiency of protein crystallization screening. Q.-Q. Lu, X.-Z. Xie, R.-Q. Chen, Z.-Q. Wu, Q.-D. Cheng, P. Shang and D.-C. Yin. J. Appl. Cryst. (2012). 45, 758-765 doi:10.1107/S0021889812025757. Synopsis: A modification to the vapor diffusion protein crystallization method, named the gradual desiccation method, is reported. It was found that this method can significantly enhance crystallization of proteins.
Fine-needle capillary mounting for protein microcrystals. M. Makino, I. Wada, N. Mizuno, K. Hirata, N. Shimizu, T. Hikima, M. Yamamoto and T. Kumasaka. J. Appl. Cryst. (2012). 45, 785-788 doi:10.1107/S0021889812024545. Synopsis: It is demonstrated that a cryocrystallographic mounting method using a fine-needle capillary is suitable for protein microcrystals.
Crystallization of Pseudomonas aeruginosa AmrZ protein: development of a comprehensive method for obtaining and optimization of protein-DNA crystals. E. E. Pryor, D. J. Wozniak and T. Hollis. Acta Cryst. (2012). F68, 985-993 doi:10.1107/S1744309112025316. Synopsis: Crystallization of the complex of the transcription factor AmrZ with DNA was accomplished through the combination of established and newly developed methods. Here, a general method to obtain and optimize crystals of protein-DNA complexes consisting of these combined procedures is described.
Single-drop optimization of protein crystallization. A. Meyer, K. Dierks, D. Hilterhaus, T. Klupsch, P. Mühlig, J. Kleesiek, R. Schöpflin, H. Einspahr, R. Hilgenfeld and C. Betzel. Acta Cryst. (2012). F68, 994-998 doi:10.1107/S1744309112024074. Synopsis: A device has been developed to optimize crystal-growth conditions by experiments in a single drop.
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