Protein-to-structure pipeline for ambient-temperature in situ crystallography at VMXi

Mikolajek, Halina; Sanchez-Weatherby, Juan; Sandy, James; Gildea, Richard J.; Campeotto, Ivan; Cheruvara, Harish; Clarke, John D.; Foster, Toshana; Fujii, Sotaro; Paulsen, Ian T.; Shah, Bhumika S.; Hough, Michael A.

Abstract

The utility of X-ray crystal structures determined under ambient-temperature conditions is becoming increasingly recognized. Such experiments can allow protein dynamics to be characterized and are particularly well suited to challenging protein targets that may form fragile crystals that are difficult to cryo-cool. Room-temperature data collection also enables time-resolved experiments. In contrast to the high-throughput highly automated pipelines for determination of structures at cryogenic temperatures widely available at synchrotron beamlines, room-temperature methodology is less mature. Here, the current status of the fully automated ambient-temperature beamline VMXi at Diamond Light Source is described, and a highly efficient pipeline from protein sample to final multi-crystal data analysis and structure determination is shown. The capability of the pipeline is illustrated using a range of user case studies representing different challenges, and from high and lower symmetry space groups and varied crystal sizes. It is also demonstrated that very rapid structure determination from crystals in situ within crystallization plates is now routine with minimal user intervention.

Citation

Mikolajek, H., Sanchez-Weatherby, J., Sandy, J., Gildea, R. J., Campeotto, I., Cheruvara, H., Clarke, J. D., Foster, T., Fujii, S., Paulsen, I. T., Shah, B. S., & Hough, M. A. (2023). Protein-to-structure pipeline for ambient-temperature in situ crystallography at VMXi. IUCrJ, 10(4), 420-429. https://doi.org/10.1107/s2052252523003810

• Journal Article Type: Article
• Acceptance Date: Apr 27, 2023
• Online Publication Date: May 19, 2023
• Publication Date: 2023-07
• Deposit Date: Jun 26, 2023
• Publicly Available Date: Jun 30, 2023
• Journal: IUCrJ
• Electronic ISSN: 2052-2525
• Publisher: International Union of Crystallography
• Peer Reviewed: Peer Reviewed
• Volume: 10
• Issue: 4
• Pages: 420-429
• DOI: https://doi.org/10.1107/s2052252523003810
• Keywords: room temperature; in situ; multi-crystal; crystallization pipelines; automated data collection; structural biology; radiation damage; X-ray crystallography; VMXi
• Public URL: https://nottingham-repository.worktribe.com/output/20842006
• Publisher URL: https://journals.iucr.org/m/issues/2023/04/00/lz5063/index.html
• Additional Information: Received: 14 February 2023; Accepted: 27 April 2023; Published online: 19 May 2023; Supplementary materials: This article has supporting information; Copyright: © 2023 Halina Mikolajek et al.; Licence: Creative Commons Attribution (CC-BY)