This work was funded in part by the BBSRC (BB/1006478/1). PRR is the recipient of a Royal Society University Research Fellowship.

1. Strategic Considerations
<ol>
	<li>The choice of seed-crystals used for microseeding experiments will vary depending on the objective of the experiment. At the beginning of a project it is helpful to find several crystallization hits that can provide alternative starting points for crystal optimization. rMMS greatly reduces the need for crystal optimization because good-quality crystals are more likely to grow in the metastable zone of the phase diagram, i.e. in the region where following disturbance or nucleation the system returns to equilibrium. We therefore suggest using rMMS routinely as soon as the first crystals are obtained (or, more accurately, as soon as the first crystals stop growing).</li>
	<li>For the initial round of rMMS, a seed stock should be made with as much crystalline material as possible. If only one well is available that contains crystals, or if the crystals are small, it may be helpful to set up multiple repetitions of the original hit (without seeding) to increase the supply of crystals. If, however, several different hits are obtained, seed crystals can be harvested from several conditions and mixed together.</li>
	<li>To avoid phase separation, crystals grown in high-salt conditions should be harvested separately from crystals grown in high-PEG conditions. If crystals from several wells are mixed, a reservoir solution that is less likely to give salt crystals should be selected for seed suspension. For example, high concentrations of phosphate, sulfate, calcium and magnesium should be avoided.</li>
	<li>Later in a project it may be important to look for crystals with different unit cells in order to improve diffraction, avoid twinned crystals, and obtain crystals that are suitable for binding ligands. At this stage only the most suitable crystals (e.g. those that diffract best) should be used to make the seed stock. Sometimes repeated rounds of microseeding are required, where only the "best" crystals are used to make the seed stock for the subsequent round. If possible, a "neutral" precipitant such as PEG 3000 should be used to suspend the seed crystals in to encourage novel crystal contacts and to crystallize complexes that may be unstable in high-salt solutions 12.</li>
	<li>Classical microseeding experiments, where a single crystallization condition is used, are often helpful following the identification of a promising condition and during subsequent optimization. It is often necessary to dilute the seed stock in order to get the desired number of crystals per drop. A "combinatorial" microseeding experiment (where a series of seed stocks of increasing dilution are added to a crystallization condition) is a quick method for finding the optimal dilution of the seed stock. This approach is described below.</li>
</ol>
2. Preparation of the Seed Stock
<ol>
	<li>Make a rounded glass probe from a glass Pasteur pipette using a Bunsen flame.
	<ol>
		<li>Heat the pipette near the middle until it becomes soft, then quickly remove it from the flame and draw it out by pulling apart the ends. Aim to pull the glass down to a diameter below 0.25 mm.</li>
		<li>Break the glass at the point where it is around 0.25 mm and briefly plunge the broken end into the flame. Repeat this until a hemisphere of glass with a diameter of ~0.75 mm is formed on the end. This probe is useful for crushing crystalline material as it is appropriately sized for striking solid objects on the 0.01-1.0 mm scale and it does not damage the plastic surface of a crystallization tray sub-well or coverslip. Larger probes (~1.0 mm) may crush small crystals more effectively because the crystals are trapped between the glass and plastic.</li>
	</ol>
	</li>
	<li>Place a 1.5 ml microcentrifuge tube containing a Seed Bead on ice.</li>
	<li>Inspect a pre-established crystallization tray using a binocular microscope or crystal imaging system and select one or more appropriate wells from the tray from which to harvest crystalline material for seed stock generation. Any material can be used including fine needles, spherulites, microcrystals and irregular, poorly formed crystals. The seed stock should be made as soon as possible after the crystals stop growing, as older material is more likely to be partially cross-linked, thus reducing its suitability for use in seeding experiments. If any doubt exists as to whether the crystalline material to be harvested is salt or protein the well may be visualized using a UV fluorescence microscope or imaging system prior to opening, or the crystalline material may be subjected to in-situ X-ray diffraction analysis in the tray. When using the former approach protein crystals will fluoresce under UV irradiation, salt crystals will appear colorless.</li>
	<li>Open the selected crystallization tray well. For 96-well sitting drop trays cut through the upper plastic sealing sheet around the selected well using a scalpel blade. For 24-well hanging drop trays remove the coverslip using a pair of tweezers. Invert the coverslip and place it on a stable clean surface. Remove 50 &mu;l of the reservoir solution and transfer this liquid to the microcentrifuge tube containing the Seed Bead ensuring that the tube remains on ice throughout.</li>
	<li>Thoroughly crush the crystals in the subwell (96-well tray) or coverslip (24-well tray) drops, using the glass probe prepared in section 2.1, viewing the results under a microscope. Small crystals may take several minutes to crush thoroughly. This step allows the experimenter to check that the crystals are easy to crush and are therefore not cross-linked, and also that they are not salt crystals. Salt crystals produce a distinctive click that can be heard and felt when they are crushed. If the crystals are tough and difficult to crush try to obtain and use fresher material.</li>
	<li>Remove 5 &mu;l of the reservoir solution from the Seed Bead tube and transfer to the equivalent sub-well (sitting drop experiment), or coverslip (hanging drop experiment) containing the crystalline material to be harvested. Pipette this liquid up and down 5-6 times to resuspend as much of the sub-well or coverslip contents as possible. Return the suspension to the Seed Bead tube and repeat this step twice more, ensuring that as much crystalline material is harvested as possible.</li>
	<li>Vortex the Seed Bead for two minutes, stopping every 30 sec to cool the tube on ice.</li>
	<li>Make a dilution series, diluting the seed stock in reservoir solution by a factor of 4 to 10 at each stage. Typically, too many crystals will be obtained when using an undiluted seed stock, so use the diluted seed stocks to control the number of crystals per well. In the first round of rMMS, it is important not to dilute the seed stock solution, since the greater the concentration of seeds the more crystallization hits will be obtained.</li>
	<li>If it is not to be used immediately, transfer the seed stock suspension and the diluted seed stock to a -20 &deg;C, or -80 &deg;C freezer for storage. To prevent repeated freeze-thaw cycles, which may reduce the efficacy of the seed stock, store this material in small volume aliquots, i.e. 10-20 &mu;l. Once frozen, seed stocks may be kept indefinitely until needed.</li>
</ol>
3. Establishment of Crystallization Trays
<ol>
	<li>Depending on the availability of crystallization facilities and the preferences of the experimenter, rMMS crystallization screening can be performed using either 24-well trays employing the hanging drop vapor diffusion method, or 96-well trays using the sitting drop vapor diffusion method. Crystallization trays for rMMS can be established either by hand, using a liquid handling robotic dispenser, or a combination of the two.</li>
	<li>A typical rMMS experiment set up by hand will comprise, 1.0 &mu;l protein solution, 1.0 &mu;l crystallization condition, and 0.5 &mu;l seed stock. A typical experiment set up using a crystallization robot will comprise, 0.3 &mu;l protein solution, 0.2 &mu;l crystallization condition, and 0.1 &mu;l seed stock.</li>
	<li>To perform rMMS screening in 24-well hanging drop trays, firstly, using a manual pipette, transfer 300 &mu;l of each condition from a 96-well condition crystallization screen in 10 ml single tube format into each well of 4 x 24-well pregreased crystallization trays.
	<ol>
		<li>Transfer 1 &mu;l of each crystallization condition from each 300 &mu;l reservoir onto the surface of a corresponding plastic coverslip from which both front and rear protective backing strips have been removed.</li>
		<li>To the 1 &mu;l of each crystallization condition add 1 &mu;l of protein solution followed by 0.5 &mu;l of crystal seed stock. Invert each coverslip such that the drop of liquid is downward facing and position above the appropriate well.</li>
		<li>Press downwards on the coverslip, compressing the sealing grease and forming a secure seal. Once all 96 drops are established, the tray should be transferred to an incubator or constant temperature room, usually in the range 4-18 &deg;C for storage.</li>
	</ol>
	</li>
	<li>To perform rMMS screening in 96-well sitting drop trays firstly transfer 20-50 &mu;l of each condition from a 96-well condition crystallization screen in deep well block format into each corresponding well of the crystallization tray. This transfer step may be performed using either a crystallization robot, or by manual transfer using an 8-channel pipette.
	<ol>
		<li>Transfer reservoir, protein and seed stock solutions to the drops by hand or with a robot using the volumes detailed in 3.1. Some commercially available crystallization robots do not contact dispense, and performing seed stock transfer using such a system may result in blockage of dispensing tips or associated tubing. The order of dispensing is variable: some robots dispense all solutions simultaneously. If this is not possible, dispense protein first, then reservoir, then seed stock.</li>
		<li>If a contact dispensing robot is not available transfer the seeds using a low volume glass syringe fitted with a blunt needle. Rinse the needle by passing it through the reservoir solution then dispense the seed stock into each drop.</li>
		<li>Seal the tray using a transparent sealing sheet and transfer to an incubator or constant temperature room for storage.</li>
	</ol>
	</li>
</ol>
4. Inspection of Crystallization Trays
<ol>
	<li>Visualize all crystallization experiments using either a binocular microscope or a crystal imaging system. The former generally affords the user greater control over view depth and the degree of magnification than the latter, but is more time consuming.</li>
	<li>Inspect each sub-well/coverslip in sequence and record any evidence of crystal formation. Experiments should be inspected once every 24 hr for 5 days following establishment and then subsequently once every 7 days for up to 4 weeks.</li>
	<li>Multiple cycles of rMMS are often required before optimal diffraction quality crystals are produced.</li>
</ol>

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	<li>Bergfors, T. <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.">Protein Crystallization.</a> IUL Biotechnology Series. , (2009).</li><li>Rupp, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomolecular Crystallography: Priciples, Practice and Application to Structural Biology. , (2010).</li><li>Babnigg, G., Joachimiak, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+protein+crystallization+propensity+from+protein+sequence.">Predicting protein crystallization propensity from protein sequence.</a> J. Struct. Funct. Genomics. 11 (1), 71-80 (2010).</li><li>Bergfors, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeds+to+crystals.">Seeds to crystals.</a> J. Struct. Biol. 142 (1), 66-76 (2003).</li><li>Ireton, G. C., Stoddard, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microseed+matrix+screening+to+improve+crystals+of+yeast+cytosine+deaminase.">Microseed matrix screening to improve crystals of yeast cytosine deaminase.</a> Acta. Crystallogr. D. Biol. Crystallogr. 60, 601-605 (2004).</li><li>Zhu, D. Y., Zhu, Y. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+protein+crystal+growth+through+dynamic+seeding.">Optimizing protein crystal growth through dynamic seeding.</a> Acta. Crystallogr. D. Biol. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Random+Microseeding:+A+Theoretical+and+Practical+Exploration+of+Seed+Stability+and+Seeding+Techniques+for+Successful+Protein+Crystallization.">Random Microseeding: A Theoretical and Practical Exploration of Seed Stability and Seeding Techniques for Successful Protein Crystallization.</a> Crystal Growth &amp; Design. 11 (8), 3432-3441 (2011).</li><li>Obmolova, G., Malia, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promoting+crystallization+of+antibody-antigen+complexes+via+microseed+matrix+screening.">Promoting crystallization of antibody-antigen complexes via microseed matrix screening.</a> Acta. Crystallogr. D. Biol. Crystallogr. 66, 927-933 (2010).</li><li>Villasenor, A. G., Wong, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustic+matrix+microseeding:+improving+protein+crystal+growth+with+minimal+chemical+bias.">Acoustic matrix microseeding: improving protein crystal growth with minimal chemical bias.</a> Acta Crystallogr D Biol Crystallogr. 66 (Pt 5), 568-5676 (2010).</li><li>Strynadka, N. C., James, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysozyme:+a+model+enzyme+in+protein+crystallography.">Lysozyme: a model enzyme in protein crystallography.</a> EXS. 75, 185-222 (1996).</li><li>Diaz, A., Loewen, P. 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We have nothing to disclose.

In this paper we have described a general method for rMMS protein crystallization screening. We have demonstrated using two test proteins a significant enhancement in crystallization success rate using this method. Diffraction analysis using synchrotron radiation of a subset of crystals generated using rMMS and non-rMMS methods revealed little variation in diffraction quality between crystals grown using either method, although previous authors have reported that good quality crystals are more likely to grow in rMM...

From its initial application by Perutz, Kendrew and co-workers in determining the structures of hemoglobin and myoglobin, to the modern high throughput automated pipelines of the structural genomics consortia, macromolecular X-ray crystallography has afforded us an unprecedented structural glimpse into the protein world. This technique remains the most widely applicable experimental method that permits the direct visualization of protein structure at atomic, or near atomic resolution (i.e. in the 1-3 &Aring; range). A prerequisite for X-ray diffraction to be applied to a protein is that it must first be crystallized, and it is this stage of the process that remains the single greatest rate-limiting step in structure determination by diffraction methods 1, 2. Despite significant advances in our understanding of the process of protein crystallization, and major improvements in the quality and availability of crystallization screens, trays, and related technologies, it remains impossible to reliably predict the likelihood of crystallization success 3. Biochemical and biophysical methods can be applied to assess whether a protein of interest displays favorable characteristics for crystal nucleation and growth, i.e. is it well-folded, homogeneous, monodisperse, etc., however, these insights in no way provide a definitive predictor of crystallization propensity. 	
	Seeding has long been purported to be a viable method for improving the number, size, and quality of existing crystals or crystalline material 4-7. This approach is based on the premise that a condition that supports crystal nucleation may not be optimal for subsequent crystal growth and vice versa. By transferring nucleated material from one condition to another, one may attempt to effectively decouple these processes, thus giving access to new, as yet unexplored crystallization space, and as a result increasing the overall success rate of a screening experiment. Established methods have been documented for (i) macroseeding, the transfer of a single crystal in its entirety from one condition to another 8, (ii) streak seeding, the transfer of nucleated material, generally obtained by the application of directional pressure using for example a cat's whisker to the surface of an existing crystal, followed by subsequent passage of the whisker through a new crystallization drop 9, and (iii) "classical" microseeding, the transfer of crystal "seeds", generated by harvesting crushed crystals (or crystalline material), into conditions similar to those that yielded the seeds 10. Notably all three of these methods are time consuming and poorly scalable, certainly in comparison to what is achievable with modern liquid handling crystallization robotics. These factors have contributed, on some level at least, to the perception that seeding is a method to be visited only when other approaches have failed to bear fruit.	
	Random matrix microseeding (rMMS) is a recent methodological innovation that combines the benefits of traditional microseeding with those of high throughput screening and scalability 11-13. This approach relies on the generation of a seed stock, produced from nucleated crystalline material, which may be aliquoted into/onto each sub-well/coverslip within a standard 96-condition crystallization screen. This method is applicable to both sitting or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 24-well or 96-well tray format. rMMS has been demonstrated experimentally to significantly increase crystallization success rate, and produce crystals of greater diffraction quality and quantity 11, 13, 14, and represents an innovative tool in the crystallographers' arsenal of approaches in the ongoing effort towards crystallization success. Here we describe a general method for rMMS and provide sample data illustrating the effectiveness of this technique.

<table><tbody><tr><td>MRC 96 well crystallization trays</td><td>Molecular Dimensions Ltd</td><td>MD11-00-100</td><td>Non-UV compatible, for screens established by robot</td></tr><tr><td>ClearView sealing sheets</td><td>Molecular Dimensions Ltd</td><td>MD6-01S</td><td></td></tr><tr><td>Hen egg white lyzozyme</td><td>Sigma-Aldrich</td><td>L6876</td><td>~95% purity</td></tr><tr><td>Bovine liver catylase</td><td>Sigma-Aldrich</td><td>C9322</td><td>&amp;gt;95% purity</td></tr><tr><td>Xylanase</td><td>Hampton Research</td><td>HR7-104</td><td></td></tr><tr><td>Thaumatin from Thaumatococcus daniellii</td><td>Sigma-Aldrich</td><td>T7630</td><td></td></tr><tr><td>Thermolysin from Bacillus thermoproteolyticus rokko</td><td>Sigma-Aldrich</td><td>P1512</td><td></td></tr><tr><td>JCSG-plus HT-96 screen</td><td>Molecular Dimensions Ltd</td><td>MD1-40</td><td>For screens established by robot</td></tr><tr><td>PACT premier HT-96 screen</td><td>Molecular Dimensions Ltd</td><td>MD1-36</td><td>For screens established by robot</td></tr><tr><td>Morpheus HT-96 screen</td><td>Molecular Dimensions Ltd</td><td>MD1-47</td><td>For screens established by robot</td></tr><tr><td>Crystal Ph&amp;#8211;nix liquid handling system</td><td>Art Robbins Instruments</td><td>602-0001-10</td><td></td></tr><tr><td>Seed bead kit</td><td>Hampton Research</td><td>HR2-320</td><td></td></tr><tr><td>Binocular stereo microscope</td><td>Leica</td><td>M165C</td><td></td></tr><tr><td>Scalpel blades</td><td>Sigma-Aldrich</td><td>S2646-100EA</td><td></td></tr><tr><td>ErgoOne 0.1-2.5 &amp;mu;l pipette</td><td>Starlab</td><td>S7100-0125</td><td></td></tr><tr><td>ErgoOne 2-20 &amp;mu;l pipette</td><td>Starlab</td><td>S7100-0221</td><td></td></tr><tr><td>ErgoOne 100-1000 &amp;mu;l pipette</td><td>Starlab</td><td>S7100-1000</td><td></td></tr><tr><td>JCSG-plus screen</td><td>Molecular Dimensions Ltd</td><td>MD1-37</td><td>For screens established by hand</td></tr><tr><td>PACT premier screen</td><td>Molecular Dimensions Ltd</td><td>MD1-29</td><td>For screens established by hand</td></tr><tr><td>Morpheus screen</td><td>Molecular Dimensions Ltd</td><td>MD1-46</td><td>For screens established by hand</td></tr><tr><td>Tweezers</td><td>Sigma-Aldrich</td><td>T5415-1EA</td><td></td></tr><tr><td>CrystalClene coverslips 18 mm</td><td>Molecular Dimensions Ltd</td><td>MD4-17</td><td></td></tr><tr><td>2 ml glass Pasteur pipettes</td><td>Sigma-Aldrich</td><td>Z722669</td><td></td></tr><tr><td>Vortex mixer</td><td>Fisher Scientific</td><td>02-215-360</td><td></td></tr><tr><td>24 well XRL crystallization tray</td><td>Molecular Dimensions Limited</td><td>MD3-11</td><td>For screens established by hand</td></tr><tr><td>30% (w/v) PEG 8000, 0.2 M ammonium sulfate, 0.1 M sodium cacodylate pH 6.5</td><td></td><td></td><td></td></tr><tr><td>20% (w/v) PEG 8000, 0.2 M magnesium acetate, 0.1 M sodium cacodylate pH 6.5</td><td></td><td></td><td></td></tr><tr><td>20% (w/v) PEG 6000, 100 mM citric acid pH 5.0</td><td></td><td></td><td></td></tr></tbody></table>

improving the success rate of protein crystallization by random microseed matrix screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

(A) Example of an rMMS experiment
To demonstrate the effectiveness of rMMS screening we applied this method to the crystallization of hen egg white lysozyme (HEWL) and bovine liver catalase (BLC). Both these enzymes are eminently crystallizable and are structurally well characterized targets 15, 16. As such both provide excellent test subjects with which to illustrate the enhanced crystallization success rate achievable with rMMS. Crystallization experiments were established in 96-well...

Watch this Scientific Journal Video about Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening at JoVE.com

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

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17.8K Views.  University of Bristol. Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

Video: Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

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

When high quality crystals are obtained that diffract X-rays, the crystal structure may be solved at near atomic resolution. The conditions to crystallize proteins, DNAs, RNAs, and their complexes can however not be predicted. Employing a broad variety of conditions is a way to increase the yield of quality diffraction crystals. Two fully automated systems have been developed at the MRC Laboratory of Molecular Biology (Cambridge, England, MRC-LMB) that facilitate crystallization screening against 1,920 initial conditions by vapor diffusion in nanoliter droplets. Semi-automated protocols have also been developed to optimize conditions by changing the concentrations of reagents, the pH, or by introducing additives that potentially enhance properties of the resulting crystals. All the corresponding protocols will be described in detail and briefly discussed. Taken together, they enable convenient and highly efficient macromolecular crystallization in a multi-user facility, while giving the users control over key parameters of their experiments.

Automated systems and protocols for the routine preparation of a large number of screens and nanoliter crystallization droplets for vapor diffusion experiments are described and discussed.

When high quality crystals are obtained that diffract X-rays, the crystal structure may be solved at near atomic resolution. The conditions to crystallize ...

Biochemistry

From Constructs to Crystals &#8211; Towards Structure Determination of &#946;-barrel Outer Membrane Proteins

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, &#945;-helical and &#946;-barrel. While &#945;-helical membrane proteins can be found in nearly all cellular membranes, &#946;-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite &#946;-barrel outer membrane protein (OMP), general protocols are presented for the production of target &#946;-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.

&#946;-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of &#946;-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% ...

Chemistry

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction phase problem. Novel structures that lack a suitable homology model are often derivatized with heavy atoms to provide experimental phase information. The presented protocol efficiently generates derivatized protein crystals by combining random microseeding matrix screening with derivatization with a heavy atom molecule I3C (5-amino-2,4,6-triiodoisophthalic acid). By incorporating I3C into the crystal lattice, the diffraction phase problem can be efficiently solved using single wavelength anomalous dispersion (SAD) phasing. The equilateral triangle arrangement of iodine atoms in I3C allows for rapid validation of a correct anomalous substructure. This protocol will be useful to structural biologists who solve macromolecular structures using crystallography-based techniques with interest in experimental phasing.

This article presents a method to generate protein crystals derivatized with I3C (5-amino-2,4,6-triiodoisophthalic acid) using microseeding to generate new crystallization conditions in sparse matrix screens. The trays can be set up using liquid dispensing robots or by hand.

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

On-Chip Crystallization and Large-Scale Serial Diffraction at Room Temperature

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since cryogenic temperatures are non-physiological and may prevent, deter, or even alter protein structural dynamics, a robust method for routine X-ray diffraction experiments at room temperature is highly desirable. The crystal-on-crystal device and its accompanying hardware and software used in this protocol are designed to enable in situ X-ray diffraction at room temperature for protein crystals of different sizes without any sample manipulation. Here we present the protocols for the key steps from device assembly, on-chip crystallization, optical scanning, crystal recognition to X-ray shot planning and automated data collection. Since this platform requires no crystal harvesting nor any other sample manipulation, hundreds to thousands of protein crystals grown on chip can be introduced into an X-ray beam in a programmable and high-throughput manner.

This contribution describes how to set up protein crystallization on crystal-on-crystal devices and how to perform automated serial data collection at room temperature using the on-chip crystallization platform.

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 ...

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.

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 ...

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.

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

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

From Constructs to Crystals – Towards Structure Determination of β-barrel Outer Membrane Proteins

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

On-Chip Crystallization and Large-Scale Serial Diffraction at Room Temperature

High-Throughput Protein Crystallization via Microdialysis

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography

Protein Crystallization for X-ray Crystallography

Optimization of Crystal Growth for Neutron Macromolecular Crystallography