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 nuc...

<ol>
	<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. Crystallogr. 61 (Pt 6), 772-775 (2005).</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=Screening+and+optimization+methods+for+nonautomated+crystallization+laboratories.">Screening and optimization methods for nonautomated crystallization laboratories.</a> Methods Mol. Biol. 363, 131-151 (2007).</li><li>Xu, L., Butcher, S. 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=Crystallization+and+preliminary+X-ray+analysis+of+receptor-binding+protein+P2+of+bacteriophage+PRD1.">Crystallization and preliminary X-ray analysis of receptor-binding protein P2 of bacteriophage PRD1.</a> J. Struct. Biol. 131 (2), 159-1563 (2000).</li><li>Rangarajan, E. S., Izard, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+diffraction+of+full-length+human+selenomethionyl+metavinculin+crystals+by+streak-seeding.">Improving the diffraction of full-length human selenomethionyl metavinculin crystals by streak-seeding.</a> Acta. Crystallogr. Sect. F. Struct. Biol. Cryst. Commun. 66 (Pt 12), 1617-1620 (2010).</li><li>Kadirvelraj, R., Harris, P., 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=A+stepwise+optimization+of+crystals+of+rhamnogalacturonan+lyase+from+Aspergillus+aculeatus.">A stepwise optimization of crystals of rhamnogalacturonan lyase from Aspergillus aculeatus.</a> Acta. Crystallogr. D. Biol . Crystallogr. 58 (Pt 8), 1346-1349 (2002).</li><li>D'Arcy, A., Villard, F., 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=An+automated+microseed+matrix-screening+method+for+protein+crystallization.">An automated microseed matrix-screening method for protein crystallization.</a> Acta. Crystallogr. D. Biol. Crystallogr. 63 (Pt 4), 550-554 (2007).</li><li>Shaw Stewart, P. D., Kolek, S. 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. C., 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=Thirty+years+of+heme+catalases+structural+biology.">Thirty years of heme catalases structural biology.</a> Arch. Biochem. 525 (2), 102-110 (2012).</li></ol>

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 border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<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>&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 Phoenix 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 &mu;l pipette</td>
			<td>Starlab</td>
			<td>S7100-0125</td>
			<td></td>
		</tr>
		<tr>
			<td>ErgoOne 2-20 &mu;l pipette</td>
			<td>Starlab</td>
			<td>S7100-0221</td>
			<td></td>
		</tr>
		<tr>
			<td>ErgoOne 100-1000 &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 ...

improving-success-rate-protein-crystallization-random-microseed

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Biology

17.7K ビュー.  University of Bristol. ここでは、ランダムmicroseedマトリックススクリーニングのための一般的方法を記載する。この技術は、有意に、タンパク質結晶化スクリーニング実験の成功率を高める最適化の必要性を低減し、データ収集およびリガンド浸漬実験の結晶の安定供給を提供することが示されている。 