Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation

Jose M. Martin-Garcia, Chelsie E. Conrad, Garrett Nelson, Natasha Stander, Nadia A. Zatsepin, James Zook, Lan Zhu, James Geiger, Eugene Chun, David Kissick, Mark C. Hilgart, Craig Ogata, Andrii Ishchenko, Nirupa Nagaratnam, Shatabdi Roy-Chowdhury, Jesse Coe, Ganesh Subramanian, Alexander Schaffer, Daniel James, Gihan Ketwala & 10 others Nagarajan Venugopalan, Shenglan Xu, Stephen Corcoran, Dale Ferguson, Uwe Weierstall, John C.H. Spence, Vadim Cherezov, Petra Fromme, Robert F. Fischetti, Wei Liu

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

Crystal structure determination of biological macromolecules using the novel technique of serial femtosecond crystallography (SFX) is severely limited by the scarcity of X-ray free-electron laser (XFEL) sources. However, recent and future upgrades render microfocus beamlines at synchrotron-radiation sources suitable for room-temperature serial crystallography data collection also. Owing to the longer exposure times that are needed at synchrotrons, serial data collection is termed serial millisecond crystallography (SMX). As a result, the number of SMX experiments is growing rapidly, with a dozen experiments reported so far. Here, the first high-viscosity injector-based SMX experiments carried out at a US synchrotron source, the Advanced Photon Source (APS), are reported. Microcrystals (5-20μm) of a wide variety of proteins, including lysozyme, thaumatin, phycocyanin, the human A 2A adenosine receptor (A 2A AR), the soluble fragment of the membrane lipoprotein Flpp3 and proteinase K, were screened. Crystals suspended in lipidic cubic phase (LCP) or a high-molecular-weight poly(ethylene oxide) (PEO; molecular weight 8000000) were delivered to the beam using a high-viscosity injector. In-house data-reduction (hit-finding) software developed at APS as well as the SFX data-reduction and analysis software suites Cheetah and CrystFEL enabled efficient on-site SMX data monitoring, reduction and processing. Complete data sets were collected for A 2A AR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of A 2A AR, phycocyanin, proteinase K and lysozyme were determined at 3.2, 3.1, 2.65 and 2.05Å resolution, respectively. The data demonstrate the feasibility of serial millisecond crystallography from 5-20μm crystals using a high-viscosity injector at APS. The resolution of the crystal structures obtained in this study was dictated by the current flux density and crystal size, but upcoming developments in beamline optics and the planned APS-U upgrade will increase the intensity by two orders of magnitude. These developments will enable structure determination from smaller and/or weakly diffracting microcrystals.

Original languageEnglish
Pages (from-to)439-454
Number of pages16
JournalIUCrJ
Volume4
DOIs
Publication statusPublished - Jan 1 2017

Fingerprint

Crystallography
Microcrystals
Synchrotrons
microcrystals
Phycocyanin
Adenosine A2A Receptors
Synchrotron radiation
Photons
Endopeptidase K
crystallography
synchrotron radiation
Membrane Proteins
Muramidase
Viscosity
Radiation
membranes
proteins
Proteins
Membranes
adenosines

Keywords

  • Advanced Photon Source
  • high-viscosity injector
  • serial millisecond crystallography
  • synchrotron radiation

ASJC Scopus subject areas

  • Chemistry(all)
  • Biochemistry
  • Materials Science(all)
  • Condensed Matter Physics

Cite this

Martin-Garcia, J. M., Conrad, C. E., Nelson, G., Stander, N., Zatsepin, N. A., Zook, J., ... Liu, W. (2017). Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation. IUCrJ, 4, 439-454. https://doi.org/10.1107/S205225251700570X

Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation. / Martin-Garcia, Jose M.; Conrad, Chelsie E.; Nelson, Garrett; Stander, Natasha; Zatsepin, Nadia A.; Zook, James; Zhu, Lan; Geiger, James; Chun, Eugene; Kissick, David; Hilgart, Mark C.; Ogata, Craig; Ishchenko, Andrii; Nagaratnam, Nirupa; Roy-Chowdhury, Shatabdi; Coe, Jesse; Subramanian, Ganesh; Schaffer, Alexander; James, Daniel; Ketwala, Gihan; Venugopalan, Nagarajan; Xu, Shenglan; Corcoran, Stephen; Ferguson, Dale; Weierstall, Uwe; Spence, John C.H.; Cherezov, Vadim; Fromme, Petra; Fischetti, Robert F.; Liu, Wei.

In: IUCrJ, Vol. 4, 01.01.2017, p. 439-454.

Research output: Contribution to journalArticle

Martin-Garcia, JM, Conrad, CE, Nelson, G, Stander, N, Zatsepin, NA, Zook, J, Zhu, L, Geiger, J, Chun, E, Kissick, D, Hilgart, MC, Ogata, C, Ishchenko, A, Nagaratnam, N, Roy-Chowdhury, S, Coe, J, Subramanian, G, Schaffer, A, James, D, Ketwala, G, Venugopalan, N, Xu, S, Corcoran, S, Ferguson, D, Weierstall, U, Spence, JCH, Cherezov, V, Fromme, P, Fischetti, RF & Liu, W 2017, 'Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation', IUCrJ, vol. 4, pp. 439-454. https://doi.org/10.1107/S205225251700570X
Martin-Garcia JM, Conrad CE, Nelson G, Stander N, Zatsepin NA, Zook J et al. Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation. IUCrJ. 2017 Jan 1;4:439-454. https://doi.org/10.1107/S205225251700570X
Martin-Garcia, Jose M. ; Conrad, Chelsie E. ; Nelson, Garrett ; Stander, Natasha ; Zatsepin, Nadia A. ; Zook, James ; Zhu, Lan ; Geiger, James ; Chun, Eugene ; Kissick, David ; Hilgart, Mark C. ; Ogata, Craig ; Ishchenko, Andrii ; Nagaratnam, Nirupa ; Roy-Chowdhury, Shatabdi ; Coe, Jesse ; Subramanian, Ganesh ; Schaffer, Alexander ; James, Daniel ; Ketwala, Gihan ; Venugopalan, Nagarajan ; Xu, Shenglan ; Corcoran, Stephen ; Ferguson, Dale ; Weierstall, Uwe ; Spence, John C.H. ; Cherezov, Vadim ; Fromme, Petra ; Fischetti, Robert F. ; Liu, Wei. / Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation. In: IUCrJ. 2017 ; Vol. 4. pp. 439-454.
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