Hampton Research

RAMC 1997 Poster Abstracts

Organized by Bob Cudney and Allan D'Arcy
Location: Le Bischenberg, France

P1 - Comparative Assessment of Microgravity- and Earth-Grown Crystals of Thaumatin and Aspartyl-tRNA Synthetase

Joseph D. Ng, Bernard Lorber and Richard Giege

UPR 9002, Structure des Macromolecules Biologiques et Mecanismes de Reconnaissance, Institut de Biologie Moleculaire et Cellulaire du CNRS, 15 rue Rene Descartes, F-67084 Strasbourg Cedex, France

The plant sweetening protein, thaumatin, from Thaumatococcus daniellii (molecular weight of 25 kD) and the thermophilic aspartyl-tRNA synthetase (ttDRS) from Thermus thermophilus (molecular weight of 120 kD) were studied here as model proteins for crystallization under microgravity aboard the U.S. Space Shuttle mission LMS. Following retrieval of the samples, post flight microscopy and X-ray analyses were carried out on the crystals grown in microgravity and on ground controls prepared in parallel. We have evaluated and compared the quality of space- and earth-grown thaumatin and ttDRS crystals for X-ray diffraction studies characterized by size, relative plots of I/sigma versus resolution and mosaicity. Both types of protein crystals grown in microgravity were generally larger in volume with fewer total crystals, diffract at higher intensities and have less mosaicity than their earth grown controls. The data presented here lend further support to the notion that protein crystals of improved quality can be obtained in a microgravity environment.

P2 - Flipper Devices And Other Tools For Freezing And Storing Of Protein Crystals

Christian Oefner, Albert Hoffmann and Allan D'Arcy

Department of Pharmaceutical Research F. Hoffmann-La Roche Ltd., CH 4070 Basel, Switzerland

In recent years freezing of protein crystals for data collection has become a well accepted and extremely important method. A number strategies and devices for rapid freezing of protein crystals at temperatures around 100K have been developed and some are now commercially available. The storage and transport of the frozen crystals has also become an important factor with the ever increasing use of high intensity synchrotron radiation sources. Ideally the experimenter would like to pre-select and characterize frozen crystals using in-house X-ray facilities to optimize the often limited and expensive time on synchrotron beamlines. This however requires a mounting and storage device which can be used independent of the goniostat geometry. The sample should be easily re-mountable in a defined orientation and its transfer to and from the storage container to the goniometer head should be facilitated by a sufficient We have developed a device based on a previously described flipper system which allows flash freezing in liquid nitrogen, mounting of the crystals in a pre-defined orientation and the possibility of transporting or long term storage of crystals. The system has been tested with the Oxford Cryostream using a MAR imaging plate, Hi-Star multi wire detector and on a 4 circle goniometer at the European Synchrotron Radiation Facility . The equipment and associated tools will be described and demonstrated.

P3 - Improved Quantitive Analysis Of Dynamic Light Scattering Experiments

Philippe BENAS(*), Bernard LORBER, Philippe DUMAS

UPR9002 IBMC-CNRS 15, rue RenÈ Descartes F67084 Strasbourg, France

Dynamic Light Scattering (DLS) is a powerful tool to obtain information on molecular size distribution in solution. It is used in many area and, in crystallization studies it can be of great interest to determine a sample polydispersity, a characteristics known to adversely affect the obtention of good quality crystals. Quantification of a DLS experiment amounts essentially to fit with a (discrete or continuous) distribution of exponential functions an experimental record representing the decay with time of the autocorrelation function of the intensity of light scattered by the sample. In its discrete version one seeks to determine the value of the baseline b and a set of amplitudes A_i and characteristic times Tau_i so that the experimental record C(t) of the autocorrelation function is given by: C(t) = Sum[A_i exp(-t/Tau_i)] + b {i=1,N} N being the number of molecular species. This corresponds to a well-known ill-conditioned problem that has no unique solution. The currently used software try to deconvolute the function C(t) with N=1 or 2 species without any additional information. We also perform the same kind of analysis but with two decisive modifications. First, as it is often applicable in practice, we allow the user to fix the (approximate) value of one Tau, say Tau_1, since the knowledge of the MW of the pure macromolecule to be crystallized is more the rule than the exception. Second, we have devised an algorithm based on Fourier Transform (a tool that should not intimidate crystallographers...) that allows to COMPLETELY dissociate determination of the baseline from the other parameters. Furthermore, the use of Fourier Transform allows to impose to the set of residuals, not merely to be minimal, but more satisfactorily to be statistically sound. The results are quite impressive since in a mixture of two proteins sufficiently different in MW (e.g. lysozyme 18kD and BSA 66kD), with only one molecular weight considered as known, we are able to satisfactorily retrieve the MW of the second species and the composition of the mixture to within 5%. Such results allow to envisage the possibility of studying the influence of various factors (salt, temperature, pH) on as simple association as dimerization.

P4 - A 3D Microscope for Protein Crystal Growth

Carlo Ciatto, Thomas Gleichmann, Manfred S. Weiss, Dietmar Schwertner & Rolf Hilgenfeld

Department of Structural Biology & Crystallography, Institute of Molecular Biotechnology, Beutenbergstr. 11, D-07745 Jena, Germany

A 3D microscope (magnification up to 1200-fold) being developed in our department is used to visually follow protein crystal growth in both time and volume. Various growth techniques and robotics are employed to screen a large number of crystallization conditions. The ultimate goal of these experiments is to improve the crystal quality and to maximize the size of single mosaic domains in the crystals necessary for time-resolved Laue diffraction studies. Organic dyes such as methylene blue or mercurochrome can easily be diffused into protein crystals, as we demonstrated for blood coagulation factor XIII1 and concanavalin A2. When monitored through the microscope, this process provides data for the optimization of soaking techniques with heavy atom compounds or cryoprotectants3. It can also be used to detect cracks in the crystals as the dye will accumulate there. Therefore, these techniques provide a quick test for crystal perfection using the novel microscope.

1R. Hilgenfeld, A. Liesum, R. Storm, H.J. Metzner & H.E. Karges: FEBS Lett. 265, 110-112 (1990).
2A. Deacon, T. Gleichmann, S. Harrop, A.J. Gilboa, H. Price, J. Raftery, G. Bradbrook, J. Yariv & J.R. Helliwell: submitted.
3E. Stura & T. Gleichmann: Chapter 13 in A. Ducruix & R. Giege (eds.): Crystallization of Nucleic Acids and Proteins, in preparation.

P5 - Using ligand crystal contacts to produce protein-inhibitor complex crystals

Hans E. Parge, Richard Showalter, Laura Pelletier, Peter Dragovich and Susumu Katoh

Agouron Pharmaceuticals, Inc., Research Laboratories 11099 North Torrey Pines Road, La Jolla, CA 92037 USA

The macrocycle FK506 (MW=822) binds to the FK506-binding protein (FKBP) with low nano-molar affinity. Because FKBP is a small protein in the crystal structure of FKBP-FK506 complex (Van Duyne, et al., 1993) the ligand is involved in extensive crystal packing interactions. Likewise ligands, synthesized as part of our structure-based drug design efforts on FKBP, are found to mediate crystal packing. We will describe how an analysis of these ligand mediated crystal contacts identified a moiety that could be attached to a variety of ligands to produce crystals of FKBP-ligand complexes on demand. Furthermore, the packing environment directly around ligated active site could usually be used to suggest which on the many crystal forms produced should be used to obtain crystals of new FKBP-ligand complexes. Van Duyne, G.D., Standaert, R.F., Karplus, P.A., Schrieber, S.L. & Clardy, J. (1993) Atomic structures of the human immunophilin FKBP-12 complexes with the FK506 and rapamycin. J.Mol. Biol. 229 105-124.

P6 - APCF, Advanced Protein Crystallization Facility: Results of Recent Missions - Actual Facility Features

J. Stapelman, R. Bosch, L. Potthast, K. Fuhmann*, J. Helliwell**

DASA-DORNIER, * European Space Agency, ** University of Manchester

The APCF, a multi-user facility for protein crystal growth under reduced gravity, has been flown as a payload on four shuttle missions up to now (SPACEHAB 1, IML-2, USML-2, LMS). Scientific results of recent missions shall be presented together with detailed information on the current performance characteristics of the facility. In particular information of the Mach-Zehnder-Interferometer MZI, flown in APCF on the LMS mission in June 1996 for the first time, and of interferometric ground experiments shall be provided.

P7 - Crystallization of fumarate reductase of Shewanella putrefaciens MR-1

David Leys, Vincent Vileret, Alexandre Tsapin, Terrance Meyer, Jozef Van Beeumen

Universiteit Gent, Lab Of Protein Biochemistry & Protein Engineering, Ledeganckstraat 35, B-9000 Gent, Belgium

Fumarate reductase, a tetraheme flavocytochrome, was purified from the bacterium Shewanella putrefaciens. Contamination with two small peptides, as detected by mass spectrometry, initially made the crystallization quite difficult. Four totally different conditions gave crystals but only one condition allowed to prepare large crystals. We obtained a native data set up to 3.2 Å and are oon the way of increasing the resolution by improving crystal conditions and purity. A heavy atom derivative search is in preparation.

P8 - Hardware for Cryocrystallography

Elspeth Garman and Mike Pickford

Laboratory of Molecular Biophysics, Oxford University, United Kingdom

Many laboratories have developed their own hardware and `gizmos' to facilitate cryocrystallography data collection, as well as for crystal transfer, storage and retrieval. The equipment which has been developed at the Laboratory of Molecular Biophysics over the last few years will be displayed together with a poster on their use and design features. This hardware includes: an alignment tip for a cryostat nozzle, pins for loops, `top-hats' for loops, magnetic goniometer adaptors, a loop making jig, a removable arc for a goniometer and various tools which we have found useful.

P9 - A novel capillary-free mounting system for protein crystals: Fine Adjustment of the crystal environment with the possibility of manipulating the crystal quality

Reiner Kiefersauer*, Rick Engh*, Takemasa Kawashima**, Robert Huber*

*Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany, am Klopferspitz 18a
**European Molecular Biology Laboratory Grenoble Outstation,
c/o Institut Laue Langevin B.P. 156, 38042 Grenoble Cedex France

For diffraction-data collection, protein crystals are usually maintained under constant humidity by mounting in a sealed glass capillary. The capillary interferes with X-ray diffraction intensity measurements by generating additional anisotropic absorption effects and interferes with optical measurements on the crystal. Capillary-free mounting and free access to the crystal would avoid these shortcomings and have additional advantages, such as the possibility of simple mechanical manipulations. In addition, controlled changes of humidity offers the possibility to investigate humidity-dependent shrinkage stages. A controlled stream of air allows an accurate adjustment of the humidity surrounded the crystal. The crystal is sucked on the pin of a micropipette. With a video system (CCD-camera) the two-dimensional shadow projections of crystals are recorded and by the method of back projection the crystal shape is reconstructed. The whole system is mounted on a X-ray camera for analyzing the crystal lattice. The combination of controlling the environment of the protein crystal (humidity, additional volatile substances) with accurate optical and X-ray measurements on the crystal system offers enormous possibilities for fine-tuned crystal engineering. Changes of the arrangement of the protein molecules in the unit cell can be optically detected in the time scale of seconds forced by the crystal environment. The pH of the crystal can be adjusted by adding e.g. ammonia to the air stream. Furthermore the water in the crystal can be easily replaced by alcohols or other volatile substances. The free access to the crystal allows new applications like micro-injections of the crystals, studying the influence of electric or magnetic field on the crystal system.

P10 - Crystallization of glucosamine 6-phosphate synthase from E. coli

G. Tepliakova (Obmolova)

EMBL, Hamburg Outstation, DESY, Notkestr. 85, D-22603 Hamburg, Germany

The initial crystallization screening of glucosamine 6-phosphate synthase from E. coli using the HR kits I and II gave well-shaped crystals under a number of conditions. The most promising of them contained high concentrations of salt (30% ammonium sulphate, 30% Na formate, or 1.4 M Li sulphate). Improvement of those conditions resulted in large (up to 1 mm) hexagonal bipyramidal crystals which diffracted to better than 2.2 A. The space group was P61 with a=b=63.5, c=334.3 A with two protein molecules in the asymmetric part. The problem was that the packing of the molecules was not uniform along the 6-fold axis as reflected by the rotation function and high merging R-factors. All attempts to improve crystal order by varying the current conditions failed. In order to obtain another crystal form, the protein was 'refreshed' by applying hydrophobic chromatography (though no impurities were detected), and a new search for crystallization conditions was performed. The rhombohedral (R32) crystals were obtained from 25-35% MPD and 50 mM salt (ammonium sulphate or NaCl). Using them, the structure was solved at 1.6 A resolution. It appeared that only a combination of different optimized parameters such as the freshly prepared protein, high protein concentration, presence of a substrate, and a proper choice of precipitating agents (MPD+salt) resulted in a new crystal form.

Obmolova, G., Badet-Denisot, M.-A., Badet, B., Teplyakov, A. (1994). Crystallization and preliminary X-ray analysis of the two domains of glucosamine 6-phosphate synthase from Escherichia coli. J. Mol. Biol. 242, 703-705.

P11 - A new method to slow vapor diffusion rate in macromolecule crystallization

Breed, J. and Zeth, K.

Fakultat fur Biologie, Postfach 5560, Universitat Konstanz, 78434 Konstanz, Germany

Slowing the rate of vapor diffusion within a hanging drop crystallization set up may lead to an improvement in eventual crystal size and/or quality. Such a slowing may be achieved by a number of means and we present an additional method here. A plastic insert separates the reservoir and the drop. A small hole, precision drilled to one of a range of specific diameters, allows vapor exchange between drop and reservoir solutions. The design my also be slightly altered for use with sitting drop set ups. We present results from a number of test proteins indicating a small but discernible improvement in protein crystal quality from the use of such inserts.

P12 - X-ray Structural analysis of Klebsiella Pneumoniae Nitrogenase Component 1 (Kp1)

David M. Lawson, Suzy M. Mayer, S. Mark Roe*, Carol A. Gormal & Barry E. Smith

Nitrogen Fixation Laboratory, John Innes Centre, Norwich NR4 7UH, United Kingdom
* Present address: Department of Biochemistry, University College London, London WC1E 6BT, United Kingdom

SBiological nitrogen fixation contributes about 90 M tonnes of nitrogen to agriculture every year. In contrast, about 60 M tonnes of nitrogen in the form of nitrogenous fertilizer is applied to agricultural soils, but up to 50% of this can be lost through denitrification and leaching. Thus the biological process, which is mediated by the enzyme nitrogenase, is of major importance to world food supplies. Nitrogenase is comprised of two component proteins: component 2 is the electron donor to component 1, and it is the latter which contains the substrate binding and reduction site. Previous structural studies on component 1 have indicated that the protein contains two unique metal-sulfur clusters: the iron-molybdenum cofactor, and the iron-sulfur P clusters1,2. Nevertheless, there is still some debate as to the precise details of these two clusters. We have performed extensive structural studies on component 1 from Klebsiella pneumoniae (Kp1) in order to resolve these ambiguities, and hopefully shed more light on the molecular details of thebiological nitrogen fixation process. Kp1 is oxygen sensitive and is therefore handled and crystallized in a temperature controlled glove box with oxygen levels kept below 1ppm. Crystals are grown by the liquid-liquid diffusion technique from a mixture of 14% PEG 6000 and 0.4M MgCl2 in 50mM tris-HCl at pH8.0. When flash-frozen to 100K in 25% ethylene glycol, these crystals remain air stable for the duration of the X-ray experiment 3.

1J.S. Kim & D.C. Rees (1992) Nature, 360, 553-560.

2J.T. Bolin et al. (1993) In: Molybdenum Enzymes, Cofactors, and Model Systems, (Eds: Stiefel, Coucouvanis & Newton), Chapter 12, pp.186-195, ACS.

3S.M. Roe, C.A. Gormal, B.E. Smith et al. (1997) Acta Cryst. D, 53, 227-228.

P13 - Crystallization and X-ray Structure of Azotobacter vinelandii molybdate-binding protein at 1.2E resolution

Clare E.M. Williams, David M. Lawson, Richard N. Pau & Lesley A. Mitchenall

Nitrogen Fixation Laboratory, John Innes Centre, Norwich NR4 7UH, United Kingdom

Molybdenum is required for the cofactors of redox enzymes (eg. nitrogenase) and is normally available in trace amounts as the soluble oxyanion molybdate (MoO42-). Azotobacter vinelandii has a specific, high affinity transport system for this oxyanion, which is an ATP-binding Cassette transporter1. The first component in this system is the periplasmic molybdate-binding protein (ModA) which binds both molybdate and tungstate, but not sulfate or phosphate. We have used high resolution X-ray crystallography to determine the molecular basis for this exquisite specificity. ModA was crystallized at 18degC by the hanging drop vapour diffusion technique using a mixture of 11-14%(w/v) PEG 4000 and 0.4M ammonium sulfate in 0.1M acetate buffer at pH 4.0. Large single crystals grew over a period of 1-2 days having dimensions up to 0.8 x 0.4 x 0.3mm. The structure was solved using SIRAS phases derived from X-ray data collected on the molybdate- and tungstate-bound forms of the protein which crystallize isomorphously. Subsequent analysis of the structure indicates that acetate buffer is crucial to the crystallization.

1.N.J. Mouncey, L.A. Mitchenall & R.N. Pau (1995). J. Bacteriol., 177, 5294-5302.

P14 - Screening for crystallization conditions of 10-formyltetra-hydrofolate dehydrogenase and its N-terminal domain

Sergey A. Krupenko and Conrad Wagner

Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232 USA

We screened the multifunctional, multi-domain enzyme, 10-formyltetrahydrofolate dehydrogenase for crystallization conditions. The enzyme is abundant in liver cytosol and is involved in folate metabolism converting 10-formyltetrahydrofolate to tetrahydrofolate. The enzyme is a tetramer of four identical 99 kDa subunits consisting of 902 amino acid residues each. Amino and carboxyl-termini of the protein are two functional non-related domains which are connected by a 100 amino acid residue intermediate domain. The enzyme is a unique example of a natural fusion protein where the amino-terminal domain bears the substrate-binding site (1) while the machinery of aldehyde dehydrogenase homologous carboxyl-terminal domain is applied as a catalytic center (2).

For the screening we used Crystal Screen and Crystal Screen II kits (Hampton Research) and the recombinant intact enzyme expressed in insect cells (3). Screening has been done by the method of sitting drop vapor diffusion in CrystalClear Strips (Hampton Research). Drop volume was 4 ul (2 ul of 10 mg/ml protein solution and 2 ul of reservoir solution) and 100 ul of the solution were added to the reservoir. Experiments have been done at 23 °C and 4 °C. Higher concentration of the protein (25 mg/ml) was applied for conditions where drop remained clear after two months. Totally about 250 conditions were screened by present and no crystal formation was observed.

Because failure to obtain crystals could be due to the fact that the entire protein is a very big molecule or that the intermediate domain might not be well ordered, we decided to crystallize the recombinant 310 amino acid residue amino-terminal domain of the enzyme. This has been shown to be a functional monomeric protein of 34kDa possessing hydrolase activity for the folate substrate (1). The same experimental conditions as for the full-length enzyme were applied. We found that the N-terminal domain at 4 °C formed crystals at low pH (4.6) and high ammonium sulfate concentration (2.0 M) or at high pH (8.5) and ammonium dihydrogen phosphate concentration (2.0 M). Further screening revealed that ammonium sulfate and concentration of protein sample 5-10 mg/ml. Further growing of the crystals and estimation of their parameters are in process.

1. Krupenko, S.A., Wagner, C., and Cook, R.J. (1997) J. Biol. Chem., 272, 10273
2. Krupenko, S.A., Wagner, C., and Cook, R.J. (1997) J. Biol. Chem., 272, 10266
3. Krupenko, S.A. et al. (1995) Protein Expr. & Purif. 6, 457

P15 - Crystallization of and Preliminary Diffraction Data for Retinol Dehydratase, a Vitamin-Metabolizing Sulfotranserase

John Gately Luz*, Marcia Newcomer#, Jochen Buck*

*Cornell University Medical College, Department of Pharmacology USA
#Vanderbilt University Medical College, Department of Biochemistry USA

Retinol dehydratase catalyzes the synthesis of the retro -retinoid anhydroretinol from retinol (vitamin A), using as a cofactor 3¹ -phosphoadenosine 5¹ -phosphosulfate (PAPS). The enzyme was previously cloned from the Sf-21 cell line, and its sequence is homologous to that of the sulfotransferases. To facilitate the biochemical characterization of the enzyme, retinol dehydratase was overexpressed in and purified from Trichoplusio ni High Five Cells. The purified recombinant enzyme was active and possessed a sulfotransferase activity, which had not been previously demonstrated experimentally. The purified enzyme was crystallized under a number of conditions and in a number of forms. Preliminary diffraction data demonstrate a resolution limit for crystals of the PAPS-boundholoenzyme of 2.1 Å and a space group of P21. This is the first reported crystallization of a sulfotransferase, and at least one of the crystal forms reported is suitable for structural analysis by x-ray crystallography, a study now underway.

P16 - More Crystallization Stories from the Protein Structure Group at York

Shirley Roberts (on behalf of the group)

University Of York, Chem Dept, Protein Structure Group, York Y01500 UNITED KINGDOM

The protein structure group, based at the University Chemistry Department in York, is large and multidisciplinary. Areas of genetic engineering, molecular biology, biochemistry, X-ray crystallography and molecular modelling are combined to investigate protein structure and function, protein-ligand interactions, oligonucleotide structures and drug design. There are several people within the group who are commited solely to growing protein crystals, whilst many have protein crystallisation as a key step ( or hold-up ) in their research project. At RAMC 1996, a poster was presented containing various tips and stories from within the group. This year a new poster will be presented alongside last years with some new and follow up stories. Topics covered include chemical modification of proteins as an aid to crystallisation, electrospray mass spectroscopy, various buffer stories and the use of additives and pH in obtaining crystals.