Exploiting Protein Engineering and Crystal Polymorphism for

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Exploiting Protein Engineering and Crystal Polymorphism for Successful X-ray Structure Determination Published as part of the Crystal Growth & Design virtual special issue on the 13th International Conference on the Crystallization of Biological Macromolecules (ICCBM13). Luc Bonnefond,†,# Pascale Schellenberger,‡,§,£ Jerôme Basquin,|| Gerard Demangeat,‡ Christophe Ritzenthaler,§ Robert Chênevert,^ Christian Balg,^ Magali Frugier,† Jo€elle Rudinger-Thirion,† Richard Giege,† Bernard Lorber,† and Claude Sauter*,† †

Architecture et Reactivite de l’ARN, Universite de Strasbourg et CNRS, IBMC, 15 rue Rene Descartes, 67084 Strasbourg, France Institut National de la Recherche Agronomique, INRA/UDS UMR 1131, 28 rue de Herrlisheim, 68021 Colmar, France § Institut de Biologie Moleculaire des Plantes, CNRS, Universite de Strasbourg, 12 rue du General Zimmer, 67084 Strasbourg, France Max Planck Institute of Biochemistry, Structural Cell Biology Department, Am Klopferspitz 18, 82152 Martinsried, Germany ^ Departement de chimie, PROTEO, Faculte des Sciences et de Genie, Universite Laval, Quebec G1 V 0A6, Canada

)

‡

bS Supporting Information ABSTRACT: The preparation of high quality crystals is a central issue in the process of determining 3D structures of biomolecules by X-ray crystallography. The success of this key step frequently depends upon the right choice and the rigorous characterization of the target. Further, the identification and refinement of the growth conditions of a supplementary crystalline polymorph may be profitable. Four representative examples illustrate how the critical parameters can be handled. These case studies include chemically and structurally different biological entities: bacterial RNA chaperone Hfq, human mitochondrial enzyme tyrosyl-tRNA synthetase, yeast exosome subcomplex, and icosahedral virus causing grapevine fanleaf disease. The winding paths which led to the determination of each structure at atomic resolution are described together with related crystallogenesis strategies.

’ INTRODUCTION Crystallography is a powerful method to determine the threedimensional structure of biological macromolecules and macromolecular assemblies. In six decades it has contributed a large body of data.1,2 However, every crystallographic project is a new challenge because it relies on the experimenter’s ability to prepare crystals diffracting X-rays at a sufficiently high resolution. As numerous physical, chemical, and biological parameters influence the formation of regular 3D lattices,35 the task can be complex. In particular, one of the most critical variables of biological crystal growth is the target itself. Indeed, many biomolecules are difficult to produce in large quantity as soluble entities with a suitable degree of purity and homogeneity, and they can be unstable upon handling.69 Fragile or polydisperse proteins, difficulty in finding a hit in crystallization screens, and poorly diffracting crystals are some of the many obstacles to the continuation of a crystallographic study. However, rescue strategies can be applied to projects stopped at any step in the preparationcrystallization crystallographic analysis flow chart (Figure 1). In particular, gene cloning and protein engineering technologies have opened up the possibility to design compact and r 2011 American Chemical Society

stable biological constructs that are more amenable to crystallization than the original targets.10,11 Given one or more constructs of the target, the next step is the search for and definition of precise crystallization conditions. This is usually carried out by screening hundreds to thousands of chemical mixtures covering a variety of crystallants, buffers, and pH values, as well as additives. Frequently, crystal polymorphism is observed; that is, a same molecule occurs in crystals differing by their habit or packing. With biological molecules or assemblies, polymorphism can originate from alternative molecular arrangements or conformations. For obvious reasons, large single crystals with well-developed morphologies are analyzed first whereas less attractive crystals—tiny needles, crystal clusters, or microcrystalline precipitates—are neglected. Our experience suggests that keeping track of all screening experiments is advisible. Nowadays, this is greatly facilitated by automated imaging systems and associated databases.12,13 Secondary crystal forms may be extremely useful when the Received: October 15, 2010 Revised: June 21, 2011 Published: August 18, 2011 4334

dx.doi.org/10.1021/cg101468p | Cryst. Growth Des. 2011, 11, 4334–4343

Crystal Growth & Design

Figure 1. Major steps of a crystallographic study.

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Crystallization Experiments. Screening for crystallization conditions was carried out in 96 well microplates (CrystalQuick, Greiner Bio-One) using commercial screens. Assays were set up using automated pipetting stations (Mosquito from TTP Labtech and Phoenix from Art Robbins). The Optisalt suite of 96 conditions from Qiagen was used as an additive screen for human mt-TyrRS. For gelled crystallization trials, a 2% (m/v) agarose stock solution was liquefied at 90 C in a heating block and kept at 35 C. The appropriate volume of warm agarose solution was added to the mixture of target and crystallant to reach a final polysaccharide concentration of 0.2% (m/v). Crystals were obtained by vapor diffusion (in hanging or sitting drops) at 20 C, and the optimized growth conditions of the described polymorphs were the following: Hfq yielded tetragonal crystals (space group I4) in 2 M NH4 sulfate and 0.1 M Tris-HCl pH 7.0, monoclinic crystals (P1) in 30% (m/v) PEG-3350 and 0.1 M Na citrate pH 5.4, hexagonal form I (P6) in 1.6 M NH4 sulfate and 0.1 M Tris-HCl pH 8.0, and hexagonal form II (P61) crystals in 25% (m/v) PEG-4000, 0.2 M Na acetate pH 4.6, and 0.2 M NH4 acetate, using 10 mg/mL protein solutions; mt-TyrRS tetragonal polymorph (P43212) was obtained in 30% (m/v) PEG-4000, 0.1 M Na acetate pH 4.6, and 0.2 M NH4 acetate with a 20 mg/ mL protein solution, whereas orthorhombic forms (P212121) were generated in the presence of YSA and TYA analogues by adding 100 mM Tris-HCl pH 7.5 and 1.2 M Na malonate, respectively; crystals of the exosome subcomplex (concentration 10.5 mg/mL) grew in 7.5% (m/v) PEG-20000, 0.1 M Tris-HCl pH 8.0, 10% (v/v) glycerol; GFLVTD solutions (6 mg/mL) led to cubic crystals (P213) in 7% (m/v) PEG-3350, 0.1 M Hepes Na pH 7.5; and the native GFLV-F13 at 1.5 2.2 mg/mL produced triclinic (P1) and monoclinic (P21) polymorphs in 3.75% (m/v) PEG-3350, 0.1 M Hepes Na pH 7.5, and 0.2% (m/v) agarose gel. Crystallographic Analyses. All crystals described below were mounted in nylon cryoloops (Hampton Research) or polymer litholoops (Molecular Dimension, Ltd.) and characterized using synchrotron radiation under cryogenic conditions. Crystallographic statistics are given in Table S1 of the Supporting Information. Crystal structures of Hfq in P1 (PDBid: 2YHT) and P6 (2Y90), mt-TyrRS with the TYA analogue in P212121 (3ZXI), and GFLV-F13 in P21 (2Y2T, 2Y2U, 2Y2 V) have been deposited with the Protein Database.

best-looking crystals turn out to be disappointing in terms of X-ray diffraction and useless for structure determination. Indeed, the optimization of several crystal polymorphs in parallel may accelerate the crystallographer’s work, which is ultimately to learning about the biological implication of the three-dimensional structure. Here we present an overview of four structural studies that provided challenges from the viewpoints of biochemistry, molecular biology, crystal growth, and crystallography. The examples chosen illustrate the production of highest diffraction quality crystals of the bacterial RNA chaperone Hfq, of the human mitochondrial enzyme tyrosyl-tRNA synthetase (mt-TyrRS), of a subcomplex of the yeast exosome composed of proteins Rrp44Rrp41-Rrp45, and of the icosahedral Grapevine fanleaf virus (GFLV). In the frame of this work, four new crystal structures were solved. The winding paths that eventually led to the determination of each structure at atomic resolution are described together with the related crystallogenesis strategy. Tips and tricks which are often absent from structural reports are described to guide beginners in the selection and refinement of the optimal biological target(s) and crystal polymorph(s).

’ RESULTS AND DISCUSSION

’ EXPERIMENTAL SECTION

RNA Chaperone Hfq: A Soluble Bacterial Protein with Floppy Tails. Hfq was first identified in E. coli as a host factor

Chemicals. All solutions used in the crystallization assays were prepared with sterile three-times-distilled water and chemicals of highest purity. The low-gelling temperature agarose (Tg = 28 C) used during the optimization of GFLV crystals was a gift from So.bi.gel. (Hendaye, France). Two stable analogues of tyrosyl-adenylate were employed as additives for the crystallization of TyrRS. Tyrosinol-AMP (TYA) was synthesized according to ref 14, and tyrosyl-sulfamoyl-AMP (YSA) was purchased from Intergrated DNA Technologies (Belgium). Preparation and Analysis of Biological Targets. Protein targets were expressed in Escherichia coli with a 6-histidine tag to facilitate their separation on a nickel substituted affinity column. GFLV particles were propagated in the systemic host plant Chenopodium quinoa and purified by a series of ultracentrifugation steps.15 The UV-absorbance of proteins and viruses was measured with a Nanodrop ND-100 spectrophotometer. Protein batches were analyzed by electrophoresis on polyacrylamide gels (SDS-PAGE) after denaturation at 95 C in the presence of sodium dodecyl sulfate and mercaptoethanol. Mean particle diameter, polydispersity, and size of aggregates were estimated using either a DynaPro DP801 (Protein Solutions, Inc.) or a Zetasizer NanoS (Malvern, U.K.) dynamic light scattering (DLS) instrument.1618

for the replication of bacteriophage Qβ. In the late 90s, it was discovered that this highly abundant bacterial protein is a riboregulator sharing sequence homology with Sm proteins of the eukaryotic spliceosome core. Hfq displays a strong affinity for A/U-rich single strand RNA sequences and acts as an RNA chaperone that facilitates the interaction of small regulatory RNAs (sRNAs) with their mRNA targets.19 Structural studies initiated on short (Staphylococcus aureus type) and long (E. coli type) Hfq forms (see Figure 2A), to better understand their role in the modulation of bacterial mRNA translation, revealed a general organization as a homohexamer.20,21 The protein from E. coli is very stable, is easily overexpressed, and constituted at first glance a good target for structural investigation. However, it possesses a floppy C-terminal tail that complicated crystallization and structure determination. The full-length sequence of E. coli Hfq encompasses 102 amino acids and possesses a cleavable N-terminal 6-His-tag. Crystals with cubic symmetry (Figure 2B) were observed in drops after 4 months. X-ray data collected on these crystals could not be phased by molecular replacement (MR) using homology models 4335

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Figure 2. From target selection to structure of E. coli Hfq. (A) Alignment of bacterial sequences on the left showing two types of Hfq monomers with a characteristic Sm-like domain composed of an α-helix and a five stranded β-sheet, followed by either a long (ECOLI for E. coli) or a short (STAAU for S. aureus) C-terminal extension. The latter was removed in the Hfq-Δ form at the position indicated by the scissors. On the right, the missing extensions are modeled on top of the green Hfq ring, which looks like an octopus with six tentacles. (B) Four crystal forms of Hfq, three of them showing related packing and geometry. The molecular content of their asymmetric unit is shown in green, and the rest of the unit cell content (symmetry related subunits), in blue. The twinned P6 polymorph led to a MR structure solution and to a model of the complete Hfq hexamer. Its refinement was subsequently performed with the P1 and P61 polymorphs. (C) Synoptic view of the Hfq project with the monthly chronology of major steps (m = month).

derived from Sm/Sm-like structures, and the crystals could not be reproduced. Later, a hexagonal polymorph was obtained in a second screen after another long incubation time. The selfrotation function (see Figure S1 of the Supporting Information) and data statistics derived from this crystal form indicated the occurrence of twinning (twinning ratio 28%). The size and the geometry of the unit cell were compatible with a fully symmetrical hexameric molecule. However, the presence of a single monomer of 11 kDa in the asymmetric unit led to an extremely low solvent content value (12%, VM = 1.4 Å3/Da). This observation

together with the long induction time of crystal growth and the issues with reproducibility suggested that the species crystallized was a fragment of the full-length protein. Sequence comparison highlighted the existence of two groups of Hfq monomers possessing either a short or a long C-terminal tail appended to the Sm-like domain. Accordingly, a shortened E. coli sequence was prepared in order to obtain a more compact monomer similar to the one found in S. aureus (Figure 2A). This shorter construct (named Hfq-Δ) readily produced two new triclinic and hexagonal crystal forms with cell parameters 4336



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Figure 3. From unstable protein to the first structure of human mt-TyrRS. (A) TyrRSs sharing an overall conserved monomer organization with some divergence at their C-termini. To improve the structural homogeneity of human mt-TyrRS, the C-terminal S4-like domain was removed without altering enzymatic activity. Abbreviations used: (N) N-terminal motif; (N-core) and (C-core) respectively N- and C-parts of the catalytic domain; (CP1) connective peptide; (α-ACB) α-helical anticodon-binding domain; (CW/Y) C-terminal domain homologous to tryptophanyl-tRNA synthetases; (S4-like) S4-like domain; (extra-Ct) additional C-terminal domain. The SDS-PAGE analysis of the ΔS4 construct on the right revealed its tendency to form intramolecular disulfide bridges (line). This behavior was not observed, and the enzymatic activity was preserved in the presence of a reducing agent (+line). The interpretation of the additional polypeptide chains is given on the right of the gel. Numbers correspond to the positions of the cysteine residues. (B) First tetragonal crystal polymorph (left) optimized by adding the substrate analogues (TYA and YSA) and testing a Se-methionine substituted protein (center). In both cases, the C-terminal domain remained ill-defined in the electron density maps. An orthorhombic polymorph (right) with improved diffraction properties was found by using an additive screen. Hence, the complete protein structure as well as that of the inhibitor could be built in the electron density. (C) Monthly chronology of major steps of the TyrRS project.

and geometry close to those of the initial hexagonal Hfq-FL crystals (Figure 2B). The self-rotation functions derived from diffraction data unambiguously confirmed the hexameric organization of Hfq (Figure S1 of the Supporting Information). Both polymorphs had similar solvent contents (3033%, VM = 1.71.8 Å3/Da), indicating a dense but reasonable packing for Hfq-Δ. A posteriori, the comparison of the triclinic and hexagonal packings indicated that twinning is very likely to occur when Hfq molecules

assemble (Figure 2B). Indeed, a P1-like arrangement with a headto-head stacking of Hfq donuts is easily obtained in a P6 environment by flipping the hexamers located in the next layer. A MR solution was eventually found for the first hexagonal polymorph which belonged to space group P6 and contained a single monomer in the unit cell. Structure refinement was difficult due to twinning, but the MR solution was used to generate a model of Hfq hexamer consistent with the data and helped solve the structures in the 4337



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Figure 4. From loose protein to structure of compact yeast exosome subcomplex. (A) On the left, schematic domain organization of eukaryotic Rrp44 (Dis3) compared to that of bacterial RNase II. Numbering refers to S. cerevisiae and E. coli sequences, respectively. The two exoribonucleases feature a similar modular domain arrangement with two cold shock domains (CSD1 and CSD2), a catalytic domain (RNB), and an S1 domain. In addition, Rrp44 has an N-terminal portion containing a predicted PIN domain that is not present in either RNase II or R. On the right, overall view of the crystallized complex between the exosome core Rrp41Rrp45 (blue and green, respectively) and Rrp44. Domains composing Rrp44 are displayed in different colors: the three OB-folds in yellow (CSD1), in orange (CSD2), and in salmon (C-terminal S1), the RNB nuclease in light red, and the endonucleolytic PIN in dark red. (B) On the left, SDS-PAGE analysis of a preparative chymotrypsin experiment. Improvement of crystal quality illustrated by the pictures taken at different time intervals of the optimization procedure. SEC profile of washed crystals. (C) Synoptic view of the Rrp44Rrp45Rrp41 complex study with monthly chronology of major steps.

P1 and P61 space groups.21 Altogether, the combination of sequence analysis and polymorph characterization led to the design of a truncated construct that proved to be more structurally compact and crystallized more reproducibly (Figure 2C). Indeed, the packing of Hfq molecules observed in the final structures could never take place in the presence of flexible C-terminal extensions (Figure 2A).

Human mt-TyrRS: An Unstable Eukaryotic Protein Produced in E. Coli. TyrRS belongs to the ubiquitous family of

aminoacyl-tRNA synthetases (aaRSs). AaRSs catalyze the specific covalent binding of amino acids on their cognate tRNAs and are responsible for the fidelity of the process of translating genetic information into proteins. TyrRSs have been extensively characterized biochemically and biophysically with over 40 reported 4338



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Figure 5. From monodisperse GFLV samples to virus structures. (A) From left to right: Purification protocol yielding virions pure according to their absorption properties in UV (A260nm/A280nm ratio = 1.80 to 1.97). No copurified protein was detected by SDS-PAGE. Lanes 1 and 2 correspond to 5 and 10 µg of virus, respectively. Electron micrograph of virions after negative staining (scale bar 100 nm). Signal intensity vs particle size distribution from dynamic light scattering analysis of virus in 0.15 M NaCl. Mean particle hydrodynamic diameter is 31.5 nm and polydispersity ∼5%. Variants GFLV-F13 and GFLV-TD gave comparable results. (B) Phase diagrams of the two variants superimposed to facilitate their comparison. Each diagram is a compilation of multiple independent grid screens carried out successively on a dozen virus batches. All crystallization assays were done in 1 µL sitting drops equilibrated at 20 C. Symbols stand for the major crystal form observed with a binocular microscope at a 50-fold magnification. Green symbols represent data from GFLV-F13 and open symbols those of GFLV-TD. Arrows indicate the paths followed during the optimization of the crystal growth of each virus, from initial crystals (labeled I and I 0) to best crystals (labeled C and C 0). Hatched curves correspond to the hypothetical solubility lines separating the regions above which the viruses do crystallize from those below, where they do not. Experimental solubilities (plotted as open and closed circles) are 1.3 mg/mL and 3.3 mg/mL in the presence of 7% (m/v) PEG-3350 for GFLV-F13 and GFLV-TD, respectively. (C) Comparison of the diffraction quality of GFLV-F13 crystals grown in solution (top, 5.5 Å resolution) or in gel (bottom, 3 Å resolution). Scale bars is 100 m. (D) Synoptic view of GFLV project with major steps.

crystal structures.22 Humans have both cytosolic and mitochondrial versions. Besides the classical aminoacylation function, the cytosolic form has cytokine activity, it is involved in angiogenesis after fragmentation,23 and mutations in the mitochondrial enzyme are correlated with diseases.24 Unlike phylogenetically related bacterial TyrRSs, human mt-TyrRS aminoacylates tRNATyr from

bacteria and other organisms, including tRNATyr from human cytosol.25 A crystallographic project was initiated to gain an insight into the structural basis of this peculiar function of human mt-TyrRS and the differences with cytosolic TyrRS, and to gain further understanding of human mitochondrial tRNA aminoacylation systems.26 4339



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Table 1. Main Prerequisites along the Five Steps of Crystallographic Studies of Soluble Biomolecules (See Figure 1) and Suggested Remedies step target preparation and quality control

prerequisite

remedies if prerequisite not fulfilled

target expressed and stable

change solvent composition, pH, ionic strength, add ligands or analogues, reducing agent, protease inhibitors try target varianta or expression in another host

soluble to >1 mg/mL

change solvent composition, pH, ionic strength add reducing agent, ligands, or detergents try target variant

target pure in PAGE, UV absorbance, EM, ...

add one or more steps to purification protocol try target variant

search for crystallization conditions

target homogeneous in SEC, IEF, DLS, MS, ...

try various additives

target crystallized

try target variant increase target concentration change crystallant, pH, ionic strength, temperature add small ligands, inhibitors, or macromolecular partners try other chemical or biochemical additives modify target (proteolysis, deglycosylation, methylation)a try target variant

optimization of crystallization conditions

crystals large enough and reproducible

vary temperature vary target and crystallant concentrations to cover a wide range of supersaturation, establish a phase diagram try seeding crystallize in diffusive media (agarose gel, ...)

crystal characterization

adequate diffraction properties:

change crystallization conditions and temperature

cryosolvent available,

change crystallization method (batch, counterdiffusion ...)

isotropic diffraction at high resolution,

crystallize in diffusive media (agarose gel, ...)

no twinning

add ligands, inhibitors, or other additives search for another crystal polymorph try target variant

structure determination

phasing and refinement successful

include heavy atoms or selenomethionines stabilize floppy regions with additives, selected mutations, or surface modifications search for another crystal polymorph try target variant

a

A target variant is a biomolecule that has been modified (e.g., by sequence or domain insertion/deletion, point mutations, chemical modifications) in order to alter its physicalchemical properties (solubility, homogeneity, stability). In the process of selecting a target and/or designing variants, predictive information from bioinformatic tools about folding and the potential presence of unstructured or mobile domains may be useful.

TyrRSs are homodimeric enzymes (Figure 3A). The subunits of bacterial and mitochondrial TyrRSs consist of a catalytic domain and an anticodon-binding domain followed by a S4-like domain. Initially, human mt-TyrRS was expressed as the mature full-length protein. Each subunit of the 100 kDa homodimer was 452 residues long and included a 6-His C-term tag. A one-step affinity purification protocol was not sufficient to produce a protein that was stable enough for crystallization. Samples were polydisperse in DLS, and additional chromatographic steps did not improve this. Moreover, the protein aggregated at concentrations above 2 mg/mL and, although it crystallized, yielded needleshaped crystals whose diffraction was limited using synchrotron radiation to 9 Å resolution under cryogenic conditions. Sequence comparison and previous crystallographic studies27 suggested that a shorter construct lacking the S4-like domain would be more structurally compact. This ΔS4 variant encompassing 350 residues was active in tRNA tyrosylation, monodisperse in DLS, and soluble at up to 20 mg/mL. However, it still produced multiple bands on SDS-PAGE and lost its activity over time. Additional

analyses suggested that thiol groups of cysteine in monomers could form multiple types of disulfide bonds (Figure 3A). Treatment with Ellman’s reagent (5,50 -dithiobis-2-nitrobenzoic acid or DTNB, an oxidative agent28) led to an inactive enzyme that yielded crystals of poor quality. By keeping the protein under reducing conditions (5 mM dithioerythritol systematically added to buffers), inappropriate SS bond formation was prevented and the enzymatic activity was preserved. Tetragonal bipyramids were obtained that diffracted X-rays to a resolution of 4 Å using a Classics screen (Nextal-Qiagen). By fine-tuning the initial drop composition (optimal volume ratio of protein/reservoir solutions 2:1), the kinetics of nucleation and crystal growth were altered and the diffraction limit was extended to 2.7 Å.29 These data could be used to build a model of TyrRS where the catalytic domain was well ordered whereas the anticodon binding domain was poorly resolved (Figure 3B). To select for a more stable conformer, the enzyme was cocrystallized with transition state analogues (TYA, YSA). These attempts improved neither the diffraction limit nor map readability, and the anomalous 4340


Crystal Growth & Design signal from Sel-Met substituted protein was too weak for de novo structure phasing. In spite of this, both analogues were clearly visible in the active site of the enzyme (Figure 3B) and a dozen data sets indicated that the resolution of 2.7 Å was the physical limit of this tetragonal polymorph. To generate new polymorphs, we tested a combination of 17 additives (OptiSalt suite) both on truncated TyrRS alone or in the presence of analogues. Two new conditions were found, one for each analogue. Orthorhombic crystals diffracting X-rays to 2.2 Å resolution were obtained in the presence of YSA and 100 mM Tris-HCl pH 7.5, which shifted the pH from 4.6 to 6.5. Data from these crystals could be used to unambiguously build and refine the entire polypeptide.30 The same packing geometry was observed with the second analogue (TYA) in a different solvent condition, but the resolution was limited to 2.8 Å (Table 1S of the Supporting Information). Like Hfq, success came from the combination of protein engineering and polymorph selection (Figure 3C). Yeast Exosome: The Quest for a Compact Subcomplex. Exosomes are conserved macromolecular complexes essential for RNA degradation. They are responsible for the main cellular 30 -50 nuclease activity and the partial shortening or complete breakdown of a wide variety of RNA transcripts.31,32 These particles are present in both the nucleus and the cytoplasm of eukaryotic cells, from yeast and protozoa to mammals. Their structural core composed of nine subunits is referred to as Exo-9. Six subunits assemble into a hexameric ring, and the other three are positioned on one side of the ring. The overall molecular architecture of Exo9 is conserved in bacterial and archaeal RNA-degrading complexes but with simpler subunit compositions and divergent catalytic mechanisms.33,34 In order to better understand the evolutionary-conserved interactions in the exosome, we have undertaken the crystallization of the ternary complex of yeast Rrp44 with the exosome core subunits Rrp41Rrp45. The full-length protein Rrp44 was crystallized first, but it only diffracted to 20 Å. As the structure of its catalytic domain was already determined (Figure 4A), we then focused on the Rrp44 N-terminal “pin domain” but were unsuccessful in producing crystals. Results from pull-down experiments suggested that this domain interacts with the proteins Rrp41 and Rrp45 of the exosome complex. Limited proteolysis of Rrp44 by chymotrypsin showed that removal of the first 25 residues did not interfere with the binding of partners. Finally, a stable complex was formed by adding an excess of Rrp41 and Rrp45 to the recombinant Rrp44Δ25, which was isolated by size exclusion chromatography (SEC). During screening, crystals of this complex were grown in the presence of PEG-20000 by vapor diffusion in sitting drops. However, they never diffracted to beyond 7.5 Å. Recrystallization was introduced as a supplementary purification step in the protocol of the complex production. Preparative bacth crystallization assays were performed in 1.5 mL microcentrifuge tubes by mixing 500 μL of crystallant solution and 500 μL of protein complex solution. Microcrystals were harvested by centrifugation and redissolved in SEC buffer. These samples yielded crystals by vapor diffusion, which diffracted X-ray to a resolution of 3 Å.35 The key to success with this project relied on the design of a construct following the removal of floppy extensions by proteolysis, on the formation of a compact complex after identification of interacting partners, and on preparative crystallization. Grapevine Fanleaf Virus: Two Viruses in One. The last case study deals with the isolation and the crystallization of a large native particle, the Grapevine fanleaf virus (GFLV). This major causal agent of grapevine degeneration disease occurs in vineyards

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worldwide. It is exclusively transmitted from grapevine to grapevine by the ectoparasitic nematode Xiphinema index,36 and no efficient treatment is known. GFLV is an icosahedral virus of pseudo T = 3 symmetry, a with capsid composed of 60 identical subunits. Our aim was to solve the structure of GFLV in order to understand the molecular mechanism(s) governing the transmission of viruses by nematodes. This case illustrates two major obstacles of virus crystallography: the limited quantity that can be obtained (here 1 mg virus per kg plant leaves) and the difficulty to engineer the target. Crystallization assays were prepared on two variants of GFLV, strain F13 (GFLV-F13) derived from infectious transcripts,37 and a natural mutant differing by a single amino acid substitution in the capsid protein (GFLV-TD). To increase the reproducibility of the crystallization assays, a strict quality control using four analytical methods was systematically applied to each virus preparation. UV absorption, electrophoresis, DLS, and electron microscopy were used to verify the purity, the monodispersity, and the size of the particles. Indeed, virus batches that did not meet the quality level shown in Figure 5A yielded small ill-shaped crystals or no crystals at all. GFLV-TD crystals with a polyhedral habit were found in initial screening experiments (10 mg/mL GFLV solution in 10% m/v PEG-3350, 0.1 M Hepes pH 7.5) but did not exceed 30 μm in length and were accompanied by numerous dendrites and spherulites. This indicated that supersaturation was too high. For this reason, the concentrations of virus and crystallant were gradually decreased, as shown by the arrows in the phase diagram (Figure 4B). This led to a smaller number of crystals with a better defined habit (i.e., sharp edges and well-developed faces) and greater volume. After four rounds of adjustments, a single crystal was obtained in every 1 μL sitting drop prepared manually in Crystalquick microplates and equilibrated against 50 μL reservoirs. Final concentrations (45 mg/mL virus in 78% PEG3350) produced pyramids belonging to the cubic space group P213 and reaching 300 μm across. The diffraction limit of these GFLV-TD crystals grown in solution ranged from 3.5 to 4.5 Å on average, and one sample among 50 exhibited a well-resolved diffraction pattern with reflections beyond 3 Å. The conditions delimited for GFLV-TD gave numerous tiny crystals with GFLV-F13. The composition of the crystallization medium had to be shifted toward lower virus and crystallant concentrations using the same procedure as above (Figure 4B). Good GFLV-F13 crystals reaching 200 μm in length were grown at 1.82.2 mg/mL virus and 34% (m/v) PEG-3350, i.e. approximately at concentrations half of those required to produce the best GFLV-TD crystals. However, these crystals did not diffract X-rays to a resolution better than 68 Å. To enhance their diffraction power, crystals were grown in the presence of 0.2% (m/v) agarose gel as described.38 Finally, a triclinic (space group P1) and a monoclinic (space group P2) crystal form were obtained under the same conditions. Gel grown crystals were dissected from the gel, mounted in litholoops, and cryocooled. They both diffracted X-rays better than any crystal grown previously in solution under otherwise identical conditions (Figure 4C). Complete data sets were collected at 3 Å and at 3.7 Å resolution for the triclinic and the monoclinic forms, respectively (Table S1 of the Supporting Information). The structure of GFLV-TD was solved by MR starting from a cryoelectron microscopy 3D reconstruction using cubic data at 2.7 Å. In turn, the refined model served to determine the structure of GFLV-F13 in triclinic and monoclinic systems.39 4341


Crystal Growth & Design Although both viruses crystallized in the same crystallant, their habit and space group differed, but virions were packed with comparable density (Figure S2 of the Supporting Information). Altogether, this study led to three independent structures, two for the native virus and one for a transmission defective variant (Figure 4D). Thus, the initial cubic crystal form not only led to the structure of GFLV-TD variant but also provided a starting point to crystallize the native virus and to solve its structure.40 General Discussion and Outlook. On the basis of the four case studies, major prerequisites to the success of structural investigations are summarized in Table 1. As can be seen, the ultimate remedies are to play with the biological target or to search for an alternate polymorph. Importance of Target Choice and Characterization. The selection of a suitable construct and the preparation of pure, homogeneous samples were critical in all four above studies. This step can be automated using standard molecular biology methods.41 In the context of structural genomics initiatives, high throughput expression platforms have been set up that significantly increase the number of constructs tested in crystallization.4245 In lowthroughput approaches like the ones reported here, promising variants with compact domains or minimal complexes can be identified by sequence analysis or limited proteolysis in any biochemistry lab. Others, such as surface entropy modification46,47 and surface residue methylation,4851 may also be useful. Besides target design, the accurate characterization of every protein batch is crucial to ensure reproducible sample quality and thereby crystallizability. Analytical methods such as SEC, DLS, and mass spectrometry, are now common tools to characterize the target molecules or particles. Finally, biological activity assays should not be neglected when designing new molecules. Advantages of Crystal Polymorphism for Structural Biology. Crystal polymorphism is a well studied area of crystal growth of small molecules52 where much effort is made to master the production of selected polymorphs.53 In biocrystallography, polymorphism is sometimes considered a disadvantage because it multiplies the number of crystal forms that must be optimized and characterized. However, following up on different initial hits can be very worthwhile. As shown with GFLV, grid screening is possible with a limited amount of material and gives rapid access to partial solubility/crystallization phase diagrams. Also, polymorphs growing under various buffer conditions can be useful to capture different conformational states and so contribute data on the dynamics of the biomolecules.

’ ASSOCIATED CONTENT

bS

Supporting Information. Analysis of the crystal symmetry and packing of Hfq and GFLV, as well as crystallization conditions and crystallographic statistics for all polymorphs discussed in the manuscript. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Telephone: +33 388 417 102. Fax: +33 388 602 218. Present Addresses #

Department of Biophysics and Biochemistry, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

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Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, U.K.

’ ACKNOWLEDGMENT The authors thank E. Conti, C. Florentz, and D. Suck for fruitful discussion and constant support, as well as the European Synchrotron Radiation Facility (ESRF, Grenoble, France), the Swiss Light Source (SLS, Villigen, Switzerland), and the Elettra Synchrotron Light Source (Trieste, Italy) for beam-time allocation. This research was supported by the French Centre National de la Recherche Scientifique (CNRS), the French Ministry for Research (ACI-BCMS 042358), the University of Strasbourg, the Institut National de Recherche Agronomique (INRA), the European Molecular Biology Laboratory (EMBL), and the Max Planck Institute. L.B. was the recipient of a fellowship from the French Ministry for Research. P.S. was supported by a fellowship from Departement Sante des Plantes et Environnement (SPE-INRA) and the Conseil Interprofessionnel des Vins d’Alsace (CIVA). C.S. benefited from the European Marie Curie actions (IHP Program, Contract Numbers HPMF-2000-00434 and MERG-CT-2004-004898). ’ REFERENCES (1) Giege, R.; Sauter, C. HFSP J. 2010, 4, 109. (2) Dutta, S.; Zardecki, C.; Goodsell, D. S.; Berman, H. M. J. Appl. Crystallogr. 2010, 43, 1224–1229. (3) Ducruix, A., Giege, R., Ed. Crystallization of nucleic acids and proteins: a practical approach, 2nd ed.; Oxford University Press: Oxford, New York, 1999. (4) McPherson, A. Introduction to Macromolecular Crystallography, 2nd ed.; Wiley-Blackwell: Hoboken, NJ, 2009. (5) Sauter, C.; Lorber, B.; McPherson, A.; Giege, R. In International Tables of Crystallography; Crystallography of Biological Macromolecules, 2nd ed.; John Wiley and Sons: Chichester, U.K., 2011; Vol. F, in press. (6) Giege, R.; Dock, A. C.; Kern, D.; Lorber, B.; Thierry, J.-C.; Moras, D. J. Cryst. Growth 1986, 76, 554–561. (7) Dale, G. E.; Oefner, C.; D’Arcy, A. J. Struct. Biol. 2003, 142, 88–97. (8) Moreno, A.; Theobald-Dietrich, A.; Lorber, B.; Sauter, C.; Giege, R. Acta Crystallogr., D 2005, 61, 789–792. (9) Lee, J. E.; Fusco, M. L.; Abelson, D. M.; Hessell, A. J.; Burton, D. R.; Saphire, E. O. Acta Crystallogr., D 2009, 65, 1162–1180. (10) Psakis, G.; Polaczek, J.; Essen, L. O. J. Struct. Biol. 2009, 166, 107–111. (11) Derewenda, Z. S. Acta Crystallogr., D 2010, 66, 604–615. (12) Peat, T.; de La Fortelle, E.; Culpepper, J.; Newman, J. Acta Crystallogr., D 2002, 58, 1968–1970. (13) Walter, T. S.; Diprose, J. M.; Mayo, C. J.; Siebold, C.; Pickford, M. G.; Carter, L.; Sutton, G. C.; Berrow, N. S.; Brown, J.; Berry, I. M.; Stewart-Jones, G. B. E.; Grimes, J. M.; Stammers, D. K.; Esnouf, R. M.; Jones, E. Y.; Owens, R. J.; Stuart, D. I.; Harlos, K. Acta Crystallogr., D 2005, 61, 651–657. (14) Bernier, S.; Akochy, P.; Lapointe, J.; Chênevert, R. Bioorg. Med. Chem. 2005, 13, 69–75. (15) Schellenberger, P.; Andret-Link, P.; Schmitt-Keichinger, C.; Bergdoll, M.; Marmonier, A.; Vigne, E.; Lemaire, O.; Fuchs, M.; Demangeat, G.; Ritzenthaler, C. J. Virol. 2010, 84, 7924–7933. (16) Mikol, V.; Hirsch, E.; Giege, R. J. Mol. Biol. 1990, 213, 187–195. (17) Zulauf, M.; D’Arcy, A. J. Cryst. Growth 1992, 122, 102–106. (18) Ferre-D’Amare, A. R.; Burley, S. K. Methods Enzymol. 1997, 276, 157–166. (19) Brennan, R.; Link, T. Curr. Opin. Microbiol. 2007, 10, 125–133. 4342


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