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

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 A 2A adenosine receptor (A 2AAR), 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 collectedmore » for A 2AAR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of A 2AAR, 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. Furthermore, these developments will enable structure determination from smaller and/or weakly diffracting microcrystals.« less
Authors:
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  1. Arizona State Univ., Tempe, AZ (United States)
  2. Arizona State Univ., Tempe, AZ (United States); National Cancer Institute, Frederick, MD (United States)
  3. Argonne National Lab. (ANL), Lemont, IL (United States)
  4. Univ. of Southern California, Los Angeles, CA (United States)
  5. Paul Scherrer Inst. (PSI), Villigen (Switzerland)
Publication Date:
Grant/Contract Number:
AC02-06CH11357
Type:
Accepted Manuscript
Journal Name:
IUCrJ
Additional Journal Information:
Journal Volume: 4; Journal Issue: 4; Journal ID: ISSN 2052-2525
Publisher:
International Union of Crystallography
Research Org:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org:
National Science Foundation (NSF); National Institutes of Health (NIH), National Cancer Institute; National Institutes of Health (NIH); USDOE
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND TECHNOLOGY; serial millisecond crystallography; synchrotron radiation; Advanced Photon Source; high-viscosity injector
OSTI Identifier:
1374440