Microseed matrix-screening (rMMS): introduction, theory, practice and a new technique for membrane protein crystallization in LCP

Stefan. A. Kolek, Patrick. D. Shaw Stewart, Bastian Brauning

Douglas Instruments Ltd, Research, Douglas House, East Gartson Hungerford, RG17 7HD, United Kingdom

 

Random Microseed Matrix-Screening (rMMS), where seed crystals are added automatically to random crystallization screens, is a significant recent breakthrough in protein crystallization [1].  During the eight years since the method was published, theoretical understanding of the method has increased [2 - 4], and several important practical variations of the basic method have emerged [5, 6].  We will briefly describe some of these variations, including cross-seeding, and introduce a novel method of making LCP seed stocks by scaling up LCP crystallization conditions.  We will also describe a method of generating seed gradients across a plate so that the number of crystals in each LCP bolus can be varied, with a practical example. 

 

[1] D'Arcy, A., Villard, F, and Marsh, M. "An automated microseed matrix-screening method for protein crystallization." Acta Crystallographica Section D: Biological Crystallography 63.4  (2007): 550-554. 

[2] Shaw Stewart, P. D., Kolek, S. A., Briggs, R. A., Chayen, N. E., & Baldock, P. F. (2011). Random microseeding: a theoretical and practical exploration of seed stability and seeding techniques for successful protein crystallization.Crystal Growth & Design, 11(8), 3432-3441. 

[3] D'Arcy, A., Bergfors, T., Cowan-Jacob, S. W., & Marsh, M. (2014). Microseed matrix screening for optimization in protein crystallization: what have we learned?. Acta Crystallographica Section F: Structural Biology Communications, 70(9), 1117-1126.

[4] Shaw Stewart, P. D, & Mueller-Dieckmann, J. (2014). Automation in biological crystallization.Acta Crystallographica Section F: Structural Biology Communications, 70(6), 686-696.

[5] Obmolova, G., Malia, T. J., Teplyakov, A., Sweet, R. W., & Gilliland, G. L. (2014). Protein crystallization with microseed matrix screening: application to human germline antibody Fabs. Structural Biology and Crystallization Communications, 70(8). 

[6] Abuhammad, A., Lowe, E. D., McDonough, M. A., Shaw Stewart, P. D., Kolek, S. A., Sim, E., & Garman, E. F. (2013). Structure of arylamine N-acetyltransferase from Mycobacterium tuberculosis determined by cross-seeding with the homologous protein from M. marinum: triumph over adversity. Acta Crystallographica Section D: Biological Crystallography, 69(8), 1433-1446.