This work was supported by a Burroughs Wellcome Investigator Award to YM and by a Brown-Coxe Postdoctoral Fellowship from Yale University to MD.

Materials:
<ul>
<li>Protein sample - Lysozyme (50 mg/ mL)</li>
<li>Hanging drop 24-well tray</li>
<li>Sitting drop 24-well tray</li>
<li>Microbatch crystallization 96 well tray</li>
<li>Crystallization solutions (either commercial available or home made)</li>
<li>Silicon grease</li>
<li>5 mL syringe without Luer-lock</li>
<li>Siliconized cover slides</li>
<li>Optical sealing tape</li>
<li>Paraffin</li>
<li>0.1-2 &mu;L micropipette with low-retention tips</li>
<li>Tweezers</li>
<li>Professional wipes</li>
</ul>
1. Hanging/sitting Drop Procedure:
<ol>
<li>For an initial screen one can use commercially available screens that exploit the sparse matrix incomplete factorial method of trial conditions. The sparse matrix incomplete factorial method is biased by and selected from known crystallization conditions for macromolecules (Carter and Carter, 1979; Cudney et al., 1994; Jancarik and Kim, 1991; Jancarik et al., 1991). If the crystallization condition is already known, a narrow screen can be assembled, varying the precipitant concentration and pH around the original hit. The most commonly successful precipitant is polyethylene glycol (PEG), followed by ammonium sulfate. Together, these two precipitants account for approximately 60% of all recorded macromolecular precipitants used for crystallization (Gilliland et al., 1994; Kimber et al., 2003; Page et al., 2003).</li>
<li>Prepare your crystallization solutions. Usually these conditions are combined from a precipitating agent, salt of some kind and a buffer to set the pH. In some cases additives may be added specifically for the protein of interest. All stock solution should be filtered using 0.22 &mu;m filter. Some of the stock solution might be very viscous (50% PEGs, glycerol, PEG-MME, etc ).
<ul>
<li>Prepare 1.5 mL of 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 M NaCl each in 0.1 M NaOAc pH 4.0, 4.3, 4.6 and 4.9 (total of 24 solutions)</li>
</ul></li>
<li>Thaw your protein sample and keep on ice. Protein stocks should be stored at -80&deg;C unless the protein of interest is sensitive to freeze/thaw cycles (if so, store at 4&deg;C). Typical protein concentrations are in the range of 5-50 mg/ mL, with higher concentrations usually giving better results.</li>
<li>Spin the sample 15min/18,000xg/4&deg;C - some proteins tend to partially aggregate and precipitate after a thaw step. These aggregates can promote amorphous precipitation of the rest of the protein molecules therefore thorough spin down will ensure you have only soluble molecules in your sample. Keep protein on ice until setting up the tray.</li>
<li>Determine the protein concentration from its absorbance at 280 nm using a UV spectrophotometer. The protein concentration can be calculated from the absorbance reading and the protein's extinction coefficient. The extinction coefficient can be calculated with the ProtParam tool on the Expasy website: <a href="http://ca.expasy.org/tools/protparam.html">http://ca.expasy.org/tools/protparam.html</a></li>
<li>Fill wells of the 24-well hanging/sitting drop tray with 500 &mu;L of precipitant solution according to the tray setup scheme (Figure 2B). Create a silicone grease ring around the edge of the well. The ring should have a small gap to prevent air pressure buildup as the well is sealed. In sitting drops, there is no need for grease since the well is sealed with tape (Figure 2A).</li>
<li>Take a cover slide and clean it with condensed air spray, or a professional wipe - avoiding dust will ease the interpretation of the crystallization drop and eliminate contaminations. For sitting drops, cover slide are not used. Instead, the well contains a shelf on which the protein and precipitant are mixed.</li>
<li>Put equal volumes of the protein sample and reservoir solution on the cover slide. Different volumes of protein and precipitant may be tried as part of optimization process. Using more protein than precipitant results in a net concentration of the protein in the drop after equilibration, which can be desirable for dilute protein samples, and to slow crystal nucleation or growth. Any other nonvolatile substance in the drop solution will also be concentrated by the same factor. When pipetting the protein and reservoir solutions onto the cover slide, be extremely careful to avoid bubble formation. This often occurs when air is blown out of the pipette.
<ul>
<li>Hanging drop: Load 2 &mu;L of 50 mg/ mL lysozyme in 0.02 M NaOAc pH 4.9 to the center of the cover slide and add to it 2 &mu;L of the reservoir solution.</li>
<li>Sitting drop: Load 2 &mu;L of 50 mg/ mL lysozyme solution to the center of the shelf and add to it 2 &mu;L of reservoir solution.</li>
</ul></li>
<li>Flip the cover slide gently and lay it down on the grease ring on top of the well. Press down gently so that air can escape from the well through the notch in the grease to prevent air pressure buildup in the well and keep the well sealed. Air pressure buildup in the well can raise the cover slide, compromising the well seal. In the sitting drop method, optically clear transparent tape is used to cover the wells. Hence, in this method sealing will take place every row/column.</li>
<li>Move on to the next well until tray is complete.</li>
<li>Check the tray upon setup - eliminating dust particles and other contaminations can reduce the false positive identification of protein crystals.</li>
<li>Use a scoring sheet to document your experiment.</li>
<li>Place the tray at the desired incubation temperature, generally between 4&deg;C and room temperature. 20&deg;C is most commonly successful. Take care to handle the tray gently and avoid any shaking. Be careful while opening and closing the incubator door. Vibrations or temperature changes during transfer or incubation can prevent crystal growth or negatively affect crystal quality. A shock absorbing material (e.g. packing foam) can be used as padding under the crystal trays.</li>
<li>Check the tray for crystals the following day, and then every few days, always handling the trays with care. Document any findings and drop morphologies in a tray specific scoring sheet. Crystals typically take 2-5 days to appear, although crystals occasionally appear much sooner (almost immediately) or later (up to several months!). Once crystallization conditions have been identified and the growth rate of the crystals is known, it is best to leave the trays undisturbed until crystals have appeared and grown to at least half of their final size. Leaving the trays undisturbed can reduce the number of crystal nucleation events, hence producing smaller numbers of larger crystals.</li>
</ol>
2. Microbatch Procedure:
<ol>
<li>Protein sample and precipitant preparation are as described above.</li>
<li>Air spray a new microbatch tray to avoid dust and other large particles.</li>
<li>In the microbatch tray, fill paraffin oil up to 3 mm high (just enough to cover the wells). Remove access of oil.</li>
<li>Load 1 &mu;L of protein solution (50 mg/ mL lysozyme) into the oil pre-filled well - make sure to pipette the solution directly to the bottom of the well (Figure 2A).</li>
<li>Load 1 &mu;L of precipitant solution into the well according to the tray setup scheme (Figure 2B) - make sure the drop sinks to the bottom of the well and fuses with the protein droplet.</li>
<li>Move to the next well until tray is complete.</li>
<li>Follow steps 11-14 of the hanging/sitting drop procedure.</li>
</ol>

<ol>
	<li>Bergfors, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protein crystallization : techniques, strategies, and tips : a laboratory manual. , (1999).</li><li>Carter, C. W., Carter, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+crystallization+using+incomplete+factorial+experiments.">Protein crystallization using incomplete factorial experiments.</a> J Biol Chem. 254, 12219-12223 (1979).</li><li>Cudney, R., Patel, S., Weisgraber, K., Newhouse, Y., McPherson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+and+optimization+strategies+for+macromolecular+crystal+growth.">Screening and optimization strategies for macromolecular crystal growth.</a> Acta Crystallogr D Biol Crystallogr. 50, 414-423 (1994).</li><li>Dessau, M., Chamovitz, D. A., Hirsch, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression,+purification+and+crystallization+of+a+PCI+domain+from+the+COP9+signalosome+subunit+7+(CSN7).">Expression, purification and crystallization of a PCI domain from the COP9 signalosome subunit 7 (CSN7).</a> Acta Crystallogr Sect F Struct Biol Cryst Commun. 62, 1138-1140 (2006).</li><li>Gilliland, G. L., Tung, M., Blakeslee, D. M., Ladner, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+Macromolecule+Crystallization+Database,+Version+3.0:+new+features,+data+and+the+NASA+archive+for+protein+crystal+growth+data.">Biological Macromolecule Crystallization Database, Version 3.0: new features, data and the NASA archive for protein crystal growth data.</a> Acta Crystallogr D Biol Crystallogr. 50, 408-413 (1994).</li><li>Jancarik, J., Kim, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sparse+matrix+sampling:+a+screening+method+for+crystallization+of+proteins.">Sparse matrix sampling: a screening method for crystallization of proteins.</a> J Appl Cryst. 23, 409-411 (1991).</li><li>Jancarik, J., Scott, W. G., Milligan, D. L., Koshland, D. E., Kim, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+and+preliminary+X-ray+diffraction+study+of+the+ligand-binding+domain+of+the+bacterial+chemotaxis-mediating+aspartate+receptor+of+Salmonella+typhimurium.">Crystallization and preliminary X-ray diffraction study of the ligand-binding domain of the bacterial chemotaxis-mediating aspartate receptor of Salmonella typhimurium.</a> J Mol Biol. 221, 31-34 (1991).</li><li>Kimber, M. S., Vallee, F., Houston, S., Necakov, A., Skarina, T., Evdokimova, E., Beasley, S., Christendat, D., Savchenko, A., Arrowsmith, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+mining+crystallization+databases:+knowledge-based+approaches+to+optimize+protein+crystal+screens.">Data mining crystallization databases: knowledge-based approaches to optimize protein crystal screens.</a> Proteins. 51, 562-568 (2003).</li><li>Lorber, B., Sauter, C., Theobald-Dietrich, A., Moreno, A., Schellenberger, P., Robert, M. C., Capelle, B., Sanglier, S., Potier, N., Giege, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+growth+of+proteins,+nucleic+acids,+and+viruses+in+gels.">Crystal growth of proteins, nucleic acids, and viruses in gels.</a> Prog Biophys Mol Biol. 101, 13-25 (2009).</li><li>McPherson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Crystallization of biological macromolecules. , (1999).</li><li>McPherson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+protein+crystallization.">Introduction to protein crystallization.</a> Methods. 34, 254-265 (2004).</li><li>Page, R., Grzechnik, S. K., Canaves, J. M., Spraggon, G., Kreusch, A., Kuhn, P., Stevens, R. C., Lesley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shotgun+crystallization+strategy+for+structural+genomics:+an+optimized+two-tiered+crystallization+screen+against+the+Thermotoga+maritima+proteome.">Shotgun crystallization strategy for structural genomics: an optimized two-tiered crystallization screen against the Thermotoga maritima proteome.</a> Acta Crystallogr D Biol Crystallogr. 59, 1028-1037 (2003).</li></ol>

No conflicts of interest declared.

In this article we describe and demonstrate general current protocols for protein crystallization. Since it a multi-step procedure there are few considerations one needs to be aware of. When working with very small volumes (0.5-2 &mu;L), drying of the drop due to evaporation is a major concern. Hence, it is recommended to work in a well-controlled environment (with low air flow, high humidity and tight temperature control) and to adopt a technique that minimizes exposure of the drop to the open air. For this reason, it is essential to operate in a well-organized work environment. All reagents should be prepared prior to setting up the tray and lab equipment should be within easy reach (pipetors, tips, tubes, wipes, etc ).
As a general rule, take your protein out of storage as close as possible to the time you will be setting up the tray. That way, there are fewer environmental influences on your sample. It is very important to hold the protein tube by the neck of the tube and not on the bottom where the protein is, so the sample will not get heated from your body temperature. With the above precautions, the experimental margins of error can be minimized, thus increasing the reproducibility of the results.
There are many possible modifications to the protocols described here. One can alter protein concentration, protein/precipitant ratio, temperature (4&deg;C - 37&deg;C), pH and various additives can be added. Another relatively commonly used method for protein crystallization is dialysis. In this method, the protein solution is dialyzed against a precipitant solution through a semi-permeable membrane. Dialysis trials are more cumbersome to set up, but one advantage of dialysis is that precipitations conditions of low ionic strength can easily be tested. This is desirable if the protein is poorly soluble at low salt concentrations, since the protein may then crystallize as the salt is dialyzed away from the protein buffer. A solid phase (gel) can be used to control the rate of dialysis. For example, an agarose gel inside a capillary can be used as the diffusion interface between the protein and precipitant solutions (Figure 1C).
Increasing the concentration of a precipitating agent to a point just below supersaturation and then adjusting the pH or temperature to reduce the solubility of the protein can be used to crystallize samples with low protein concentration. Modification of pH can be accomplished very well with the vapor diffusion technique, when volatile acids and bases such as acetic acid, ammonium hydroxide or ammonium acetate are used (McPherson, 2004). 
 In the micro-batch technique a whole array of oil mixtures can be tested to cover the wells. Different oils have different evaporation permeabilities, and hence produce different rates of concentration changes of the protein/precipitant mixture under the oil.
Crystallization is a requisite step for obtaining structural information of macromolecules at near-atomic resolution. The methods described here are therefore fundamental to studying enzyme mechanisms and protein-protein or protein-nucleic acids interactions, and are ultimately one of the most important tools in structure-based drug design and development.

<table><tbody><tr><td>Lysozyme</td><td></td><td>Sigma-aldrich</td><td>L6876-1G</td></tr><tr><td>24 well VDX Plate</td><td></td><td>Hampton research</td><td>HR3-142</td></tr><tr><td>24 well Cryschem Plate</td><td></td><td>Hampton research</td><td>HR3-158</td></tr><tr><td>Dow Corning Vacuum Grease</td><td></td><td>Hampton research</td><td>HR3-510</td></tr><tr><td>Siliconized glass circle coverslides</td><td></td><td>Hampton research</td><td>HR3-231</td></tr><tr><td>100% paraffin oil</td><td></td><td>Hampton research</td><td>HR3-411</td></tr><tr><td>1.88 inch wide Crystal Clear Sealing Tape</td><td></td><td>Hampton research</td><td>HR3-511</td></tr><tr><td>96 Well Imp@ct Plate (Microbatch plate)</td><td></td><td>Hampton research</td><td>HR3-098</td></tr></tbody></table>

protein crystallization for x-ray crystallography

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.

Crystallization is usually referred to as the bottleneck of X-ray crystallography. A sparse matrix incomplete factorial screen of precipitating conditions typically produces many different types of protein aggregation and precipitation, among them large single crystals. If the protein or precipitant concentrations are too high one can see brown matter with no distinct shape and size (amorphous precipitation). When the solution is undersaturated, the drop will often be completely clear and devoid of any kind of precipitation. Figure 3 shows several examples for precipitation phenomena and crystals (Dessau et al., 2006). More precipitation phenomena with more detailed interpretation can be found at <a href="http://xray.bmc.uu.se/terese/tutorials.html">http://xray.bmc.uu.se/terese/tutorials.html</a>.
	<img fo:content-width="5in" src="/files/ftp_upload/2285/2285fig1.jpg" fo:src="/files/ftp_upload/2285/2285fig1highres.jpg" alt="Protein crystallization phase diagram, nucleation process, experimental setup, precipitation analysis." data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. The principle of protein crystallization. 
The principle of protein crystallization. In a vapor diffusion experiment (A) equal volumes of precipitant and protein are present in the drop. Water will diffuse out and both the precipitant and protein concentration will be doubled until equilibrium is achieved between the drop and the reservoir solution. In batch crystallization (B) the precipitant and protein concentration do not change during the experiment. Point A - Protein stays undersaturated no crystals can be formed, Point B - Protein nucleation occur, crystals began to form and the concentration of protein in solution drops to saturation. Point C - Protein precipitates, but crystals may still grow. (C) Dialysis crystallization using a gel-plugged capillary. Crystals appear as salt diffuses out of the protein sample (and/or precipitant diffuses into the protein sample) across the gel plug.
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/2285/2285fig1highres.jpg" target="_blank">Click here to view larger image</a>.
	<img fo:content-width="5in" src="/files/ftp_upload/2285/2285fig2.jpg" fo:src="/files/ftp_upload/2285/2285fig2highres.jpg" alt="Protein crystallization process diagram showing vapor diffusion and microbatch methods." data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Outline of typical protein crystallization experiments. 
(A) Procedures for hanging drop vapor diffusion, sitting drop vapor diffusion, and microbatch protein crystallization. In each case, a small volume of concentrated protein sample is mixed with an equal or smaller volume of precipitant solution and allowed to equilibrate. (B) For the crystallization of chicken lysozyme, a 24-well tray is set up with various concentrations of sodium chloride (the precipitant) and with 0.1 M sodium acetate as the buffer at various pHs (4.0 - 4.9).
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/2285/2285fig2highres.jpg" target="_blank">Click here to view larger image</a>.
	<img fo:content-width="5in" src="/files/ftp_upload/2285/2285fig3.jpg" fo:src="/files/ftp_upload/2285/2285fig3highres.jpg" alt="Protein crystallization process, microscope images, crystal formation analysis, structural biology." data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Typical outcomes of a protein crystallization experiment. 
(A) Amorphous precipitation. When protein or precipitant (or both) are in high concentration. (B) Phase separation. Protein or detergent may separate to a different phase when mixed with certain precipitants at high concentration. (C) Rod shaped protein crystals of AtCSN7 obtained in polyethylene glycol 8000 and magnesium acetate (Dessau et al., 2006). (D) Lysozyme crystals obtained in 1.0 M NaCl and sodium acetate pH 4.9. (E) Under saturated drops usually remain clear. Photography by Moshe Dessau.
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/2285/2285fig3highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about Protein Crystallization for X-ray Crystallography at JoVE.com

Protein Crystallization for X-ray Crystallography

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

protein-crystallization-for-x-ray-crystallography

Nanyang Technological University

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JoVE Journal

Biology

63.3K Views.  Yale University. The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.

Video: Protein Crystallization for X-ray Crystallography

Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology

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Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since ...

High-Throughput Protein Crystallization via Microdialysis

Understanding the structure-function relationships of macromolecules, such as proteins, at the molecular level is vital for biomedicine and modern drug discovery. To date, X-ray crystallography remains the most successful method for solving three-dimensional protein structures at atomic resolution. With recent advances in serial crystallography, either using X-ray free electron lasers (XFELs) or synchrotron light sources, protein crystallography has progressed to the next frontier, where the ability to acquire time-resolved data provides important mechanistic insights into the behavior of biological molecules at room temperature. This protocol describes a straightforward high-throughput (HTP) workflow for screening crystallization conditions through the use of a 96-well dialysis plate. These plates follow the Society for Biomolecular Screening (SBS) standard and can be easily set up using any standard crystallization laboratory. Once optimal conditions are identified, large quantities of crystals (hundreds of microcrystals) can be produced using the dialyzer. To validate the robustness and versatility of this approach, four different proteins were crystallized, including two membrane proteins.

High-Throughput Protein Crystallization via Microdialysis

The presented protocol describes a straightforward approach for screening protein crystallization conditions and crystal growth using a 96-well high-throughput dialysis plate. The use of dialyzer tubes for the large-scale growth of microcrystals is also demonstrated for serial crystallography and MicroED applications.

Understanding the structure-function relationships of macromolecules, such as proteins, at the molecular level is vital for biomedicine and modern drug ...

High-Throughput Screening for Protein Crystallization

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into an ordered lattice amenable to diffraction remains challenging. The crystallization of biomolecules is largely experimentally defined, and this process can be labor-intensive and prohibitive to researchers at resource-limited institutions. At the National High-Throughput Crystallization (HTX) Center, highly reproducible methods have been implemented to facilitate crystal growth, including an automated high-throughput 1,536-well microbatch-under-oil plate setup designed to sample a wide breadth of crystallization parameters. Plates are monitored using state-of-the-art imaging modalities over the course of 6 weeks to provide insight into crystal growth, as well as to accurately distinguish valuable crystal hits. Furthermore, the implementation of a trained artificial intelligence scoring algorithm for identifying crystal hits, coupled with an open-source, user-friendly interface for viewing experimental images, streamlines the process of analyzing crystal growth images. Here, the key procedures and instrumentation are described for the preparation of the cocktails and crystallization plates, imaging the plates, and identifying hits in a way that ensures reproducibility and increases the likelihood of successful crystallization.

Author Spotlight: High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography  

This protocol details high-throughput crystallization screening, ranging from the 1,536 microassay plate preparation to the end of a 6 week experimental time window. Details are included about the sample setup, the imaging obtained, and how users can perform analyses using an artificial intelligence-enabled graphical user interface to quickly and efficiently identify macromolecular crystallization conditions.

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into ...

Automated Protein Crystallization and Real-Time Data Collection

Crystallization and In Situ Room Temperature Data Collection Using the Crystallization Facility at Harwell and Beamline VMXi, Diamond Light Source

Protocols for robotic protein crystallization using the Crystallization Facility at Harwell and in situ room temperature data collection from crystallization plates at Diamond Light Source beamline VMXi are described. This approach enables high-quality room-temperature crystal structures to be determined from multiple crystals in a straightforward manner and provides very rapid feedback on the results of crystallization trials as well as enabling serial crystallography. The value of room temperature structures in understanding protein structure, ligand binding, and dynamics is becoming increasingly recognized in the structural biology community. This pipeline is accessible to users from all over the world with several available modes of access. Crystallization experiments that are set up can be imaged and viewed remotely with crystals identified automatically using a machine learning tool. Data are measured in a queue-based system with up to 60&#176; rotation datasets from user-selected crystals in a plate. Data from all the crystals within a particular well or sample group are automatically merged using xia2.multiplex with the outputs straightforwardly accessed via a web browser interface.

Author Spotlight: Advancing Protein Structure Analysis for Drug Development 

We present a protocol for the crystallization of proteins using the crystallization facility in the Research Complex at Harwell and subsequent in situ X-ray crystallographic data collection from crystals within the plates at Diamond&#39;s Versatile Macromolecular Crystallography in situ (VMXi) beamline. We describe sample requirements, crystallization protocols, and data collection guidelines.

Protocols for robotic protein crystallization using the Crystallization Facility at Harwell and in situ room temperature data collection from ...

Optimization of Crystal Growth for Neutron Macromolecular Crystallography

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX beamlines having been built and increased availability of structure refinement software. However, the neutron sources currently available for NMX are significantly weaker than equivalent sources for X-ray crystallography. Despite advances in this field, significantly larger crystals will always be required for neutron diffraction studies, particularly with the tendency to study ever-larger macromolecules and complexes. Further improvements in methods and instrumentation suited to growing larger crystals are therefore necessary for the use of NMX to expand.
In this work, we introduce rational strategies and a crystal growth bench (OptiCrys) developed in our laboratory that combines real-time observation through a microscope-mounted video camera with precise automated control of crystallization solutions (e.g., precipitant concentration, pH, additive, temperature). We then demonstrate how this control of temperature and chemical composition facilitates the search for optimal crystallization conditions using model soluble proteins. Thorough knowledge of the crystallization phase diagram is crucial for selecting the starting position and the kinetic path for any crystallization experiment. We show how a rational approach can control the size and number of crystals generated based on knowledge of multidimensional phase diagrams.

Structural studies of biomacromolecules by crystallography require high-quality crystals. Here we demonstrate a protocol that can be used by OptiCrys (a fully automated instrument developed in our lab) and/or microdialysis buttons for growing large high-quality crystals based on knowledge of the crystallization phase diagram.

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX ...

Protein Crystallization

Protein crystallization, obtaining a solid lattice of biomolecules, elucidates protein structure and enables the study of protein function. Crystallization involves drying purified protein under a combination of many factors, including pH, temperature, ionic strength, and protein concentration. Once crystals are obtained, the protein structure can be elucidated by x-ray diffraction and computation of an electron density model.
This video introduces protein crystallization and shows a general procedure. Protein expression and purification, crystallization, and x-ray diffraction are covered in the procedure. Applications of protein crystallization include in silico drug design, binding site determination, and membrane protein structure analysis.
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Protein crystallization is the process of obtaining a latticed solid form of a protein. These crystals are especially valuable to structural biologists, assisting in the study of protein function. Other techniques, such as mass spec or SDS-PAGE, can only provide information on the one-dimensional structure of proteins. Protein crystallization is complemented by the techniques of recombinant protein expression and x-ray diffraction. This video will show the principles of protein crystallization, a general laboratory procedure, and several of its applications in the biochemical field.
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The first step required in the process is to obtain milligram quantities of very pure protein, typically using recombinant protein expression. The gene corresponding to the protein of interest is cloned into an expression vector, and the expressed protein is fused to an affinity tag, such as poly-histidine, to assist in the purification by affinity chromatography. To learn more, see this collection&#39;s video on affinity chromatography.
Formation of the purified protein into crystals is dependent on the proper combination of many factors, including pH, ionic strength, concentrations of precipitant and protein, temperature, and rate of equilibration. The most common method used is vapor diffusion, of which there are two categories: hanging drop and sitting drop. A droplet containing pure protein, buffer, and precipitant, which is an ionic solid that binds water molecules, reducing water availability for the protein and mimicking higher protein concentration, is in an enclosed microwell with a reservoir with a more highly concentrated mixture of the same buffer and precipitant. At the beginning, the concentrations of protein and precipitant are too low to cause crystallization. During the course of the experiment, water vaporizes from the droplet and collects in the reservoir; a decrease in the amount of water in the droplet causes the system to become supersaturated, and nucleation, followed by crystallization, can occur. The net transfer of water from the droplet is in equilibrium, and the system is maintained until the process is complete.
To visualize the 3D structure, x-ray diffraction is used. To obtain x-ray data from a crystal, it is placed in a monochromatic x-ray beam, where it is exposed to the beam at all angles. Each exposure provides an image, where each spot is a diffracted x-ray, which emerges from the crystal and is registered by a detector. The data are combined to produce a model of the arrangement of atoms within the crystal. The resulting crystal structure demonstrates the 3-dimensional placement of the atoms, with a typical resolution of 2 angstroms.

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Now that we have covered the principles of protein crystallization, let us look at a generalized protocol.
To begin the procedure an expression vector containing the gene of interest is transformed into cells. The cells are incubated and at mid-log phase, expression is initiated by adding an inducer, such as IPTG, which triggers transcription of the gene&#39;s mRNA. After protein expression, the crude material is suspended in lysis buffer, and then clarified by centrifugation.
The clarified lysate is then loaded onto a nickel column, and the polyhistidine-tagged protein binds to the column while all other biomolecules are washed away.
Once several milligrams of pure protein have been obtained, it is ready for crystallization by vapor diffusion. A 24-well hanging/sitting drop tray is filled with varying concentrations of sodium chloride and sodium acetate buffer solutions. For the sitting drop method, equal volumes of protein and reservoir solution are pipetted onto the shelf above each well, and then the tray is covered with transparent tape. The tray is then placed in an incubation chamber, and the wells are monitored for growth the following day, then every few days.
Once a proper crystal has been obtained it is ready for x-ray diffraction analysis. The crystal is mounted on a goniometer to position the crystal at selected orientations. The crystal is illuminated with a monochromatic beam of x-rays at all angles, producing a diffraction pattern. The software converts the two-dimensional images, taken at different orientations, to a three-dimensional model of the density of electrons within the crystal by determining the positions of the atoms in the crystal.
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Now that we have reviewed a procedure, let&#39;s review some useful applications of protein crystallization, and another crystallization technique.
Protein crystallization may be used for in silico drug design. The three-dimensional structure of Influenza virus&#39;s polymerase basic protein 2, which has been linked to viral infection in mammals, was determined by crystallization and x-ray diffraction. Potential binding sites in the protein are visualized, and with the use of a docking program, a three-dimensional molecule was designed that would insert into a cleft in the protein.
Co-crystallization of protein-DNA complexes is also a useful technique. DNA-binding proteins modulate a wide variety of biological functions such as transcription and DNA polymerization and DNA repair; and crystal structures of these complexes can provide insight into protein function, mechanism, and the nature of the specific interaction. The E. coli protein SeqA, a negative regulator of DNA replication, was co-crystallized with hemimethylated DNA.
Integral membrane proteins such as G-protein coupled receptors, or GCPRs, are difficult to crystallize due to their limited amount of polar surface area available for forming crystal lattice contacts, which has led to the development of fusion-protein-assisted protein crystallization. Genes encoding &#946;2 adrenergic receptor, a GCPR, and a lysozyme were inserted into an expression vector. The crystallization of the &#946;2AR- lysozyme fusion protein was achieved due to the increased extracellular hydrophilic surface over the naturally hydrophobic &#946;2AR, provided by the lysozyme, necessary for forming packing interactions in the crystal lattice.
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You&#39;ve just watched JoVE&#39;s video on protein crystallization. This video described its principles, a generalized protocol, and some its uses in the biomedical field. Thanks for watching!
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Protein Crystallization for X-ray Crystallography

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High-Throughput Protein Crystallization via Microdialysis

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography

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Optimization of Crystal Growth for Neutron Macromolecular Crystallography

Protein Crystallization