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,
Nagarajan Venugopalan,
Shenglan Xu,
Stephen Corcoran,
Dale Ferguson,
Uwe Weierstall,
John C. H. Spence,
Vadim Cherezov,
Petra Fromme,
Robert F. Fischetti,
Wei Liu
Affiliations
Jose M. Martin-Garcia
School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA
Chelsie E. Conrad
School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA
Garrett Nelson
School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA
Natasha Stander
School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA
Nadia A. Zatsepin
School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA
James Zook
School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA
Lan Zhu
School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA
James Geiger
School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA
Eugene Chun
School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA
David Kissick
Advanced Photon Source, Argonne National Laboratory, 9700 South Cass Avenue, Lemont, IL 60439, USA
Mark C. Hilgart
Advanced Photon Source, Argonne National Laboratory, 9700 South Cass Avenue, Lemont, IL 60439, USA
Craig Ogata
Advanced Photon Source, Argonne National Laboratory, 9700 South Cass Avenue, Lemont, IL 60439, USA
Andrii Ishchenko
Department of Chemistry, Bridge Institute, University of Southern California, 3430 South Vermont Avenue, MC 3303, Los Angeles, CA 90089, USA
Nirupa Nagaratnam
School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA
Shatabdi Roy-Chowdhury
School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA
Jesse Coe
School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA
Ganesh Subramanian
School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA
Alexander Schaffer
School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA
Daniel James
Paul Scherrer Institute, 5232 Villigen, Switzerland
Gihan Ketwala
Department of Physics, Arizona State University, PO Box 871504, Tempe, AZ 85287, USA
Nagarajan Venugopalan
Advanced Photon Source, Argonne National Laboratory, 9700 South Cass Avenue, Lemont, IL 60439, USA
Shenglan Xu
Advanced Photon Source, Argonne National Laboratory, 9700 South Cass Avenue, Lemont, IL 60439, USA
Stephen Corcoran
Advanced Photon Source, Argonne National Laboratory, 9700 South Cass Avenue, Lemont, IL 60439, USA
Dale Ferguson
Advanced Photon Source, Argonne National Laboratory, 9700 South Cass Avenue, Lemont, IL 60439, USA
Uwe Weierstall
School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA
John C. H. Spence
Department of Physics, Arizona State University, PO Box 871504, Tempe, AZ 85287, USA
Vadim Cherezov
Department of Chemistry, Bridge Institute, University of Southern California, 3430 South Vermont Avenue, MC 3303, Los Angeles, CA 90089, USA
Petra Fromme
School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA
Robert F. Fischetti
Advanced Photon Source, Argonne National Laboratory, 9700 South Cass Avenue, Lemont, IL 60439, USA
Wei Liu
School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA
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 (5–20 µm) of a wide variety of proteins, including lysozyme, thaumatin, phycocyanin, the human A2A adenosine receptor (A2AAR), 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 8 000 000) 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 A2AAR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of A2AAR, phycocyanin, proteinase K and lysozyme were determined at 3.2, 3.1, 2.65 and 2.05 Å resolution, respectively. The data demonstrate the feasibility of serial millisecond crystallography from 5–20 µm 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.
