Supersaturation-controlled microcrystallization and visualization analysis for serial femtosecond crystallography

Dan Bi Lee, Jong Min Kim, Jong Hyeon Seok, Ji Hye Lee, Jae Deok Jo, Ji Young Mun, Chelsie Conrad, Jesse Coe, Gerrett Nelson, Brenda Hogue, Thomas A. White, Nadia Zatsepin, Uwe Weierstall, Anton Barty, Henry Chapman, Petra Fromme, John Spence, Mi Sook Chung, Chang Hyun Oh, Kyung Hyun Kim

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Abstract

Time-resolved serial femtosecond crystallography with X-ray free electron laser (XFEL) holds the potential to view fast reactions occurring at near-physiological temperature. However, production and characterization of homogeneous micron-sized protein crystals at high density remain a bottleneck, due to the lack of the necessary equipments in ordinary laboratories. We describe here supersaturation-controlled microcrystallization and visualization and analysis tools that can be easily used in any laboratory. The microcrystallization conditions of the influenza virus hemagglutinin were initially obtained with low reproducibility, which was improved by employing a rapid evaporation of hanging drops. Supersaturation-controlled microcrystallization was then developed in a vapor diffusion mode, where supersaturation was induced by evaporation in hanging drops sequentially for durations ranging from 30 sec to 3 min, depending on the protein. It was applied successfully to the microcrystal formation of lysozyme, ferritin and hemagglutinin with high density. Moreover, visualization and analysis tools were developed to characterize the microcrystals observed by light microscopy. The size and density distributions of microcrystals analyzed by the tools were found to be consistent with the results of manual analysis, further validated by high-resolution microscopic analyses. Our supersaturation-controlled microcrystallization and visualization and analysis tools will provide universal access to successful XFEL studies.

Original languageEnglish
Article number2541
JournalScientific reports
Volume8
Issue number1
DOIs
Publication statusPublished - Dec 1 2018

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ASJC Scopus subject areas

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Cite this

Lee, D. B., Kim, J. M., Seok, J. H., Lee, J. H., Jo, J. D., Mun, J. Y., Conrad, C., Coe, J., Nelson, G., Hogue, B., White, T. A., Zatsepin, N., Weierstall, U., Barty, A., Chapman, H., Fromme, P., Spence, J., Chung, M. S., Oh, C. H., & Kim, K. H. (2018). Supersaturation-controlled microcrystallization and visualization analysis for serial femtosecond crystallography. Scientific reports, 8(1), [2541]. https://doi.org/10.1038/s41598-018-20899-9