Patrick D. Shaw Stewart, Stefan A. Kolek, Jack Stubbs, Peter Baldock
Douglas Instruments Ltd,
Douglas House, East Garston, Berkshire, RG17 7HD, UK
Serial data collection usually requires relatively small crystals that are well-ordered. Microseeding is an effective way to generate such samples. During the ten years since the random microseed matrix-screening (rMMS) method was published, understanding of the theoretical advantages of the method has increased [2 – 4], and several practical variations of the method have emerged. Moreover seeding can be carried out in a microbatch-under-oil setup, which is easy to scale up, volume-wise, and allows easy interpretation of phase diagrams. By combining these techniques, control can be increased and sample quality for both routine and advanced data collection improved.
Protein structure determination by cryoEM requires expensive equipment that has low throughput. It is therefore wasteful to examine samples that can be shown in advance to be aggregated, since such samples are unlikely to be suitable. We used a high-throughput screening approach with dynamic light scattering to explore 96 chemical conditions with as little as 10 µL of protein solution to identify conditions with reduced aggregation.
REFERENCES:
[1] D’Arcy, Allan, Frederic Villard, and May Marsh. “An automated microseed matrix-screening method for protein crystallization.” Acta Crystallographica Section D: Biological Crystallography 63.4 (2007): 550-554.
[2] Shaw Stewart, Patrick D., et al. “Random microseeding: a theoretical and practical exploration of seed stability and seeding techniques for successful protein crystallization.” Crystal Growth & Design 11.8 (2011): 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., & 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, Areej, et al. “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 (2013): 1433-1446.
[7] Kolek, S. A., Bräuning, B., & Shaw Stewart, P. D. (2016). A novel microseeding method for the crystallization of membrane proteins in lipidic cubic phase. Acta Crystallographica Section F: Structural Biology Communications, 72(4), 307-312.
[8] Falke, S., Dierks, K., Blanchet, C., Graewert, M., Cipriani, F., Meijers, R., Svergun, D. and Betzel, C., 2018. Multi-channel in situ dynamic light scattering instrumentation enhancing biological small-angle X-ray scattering experiments at the PETRA III beamline P12. Journal of synchrotron radiation, 25(2).
