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Title: High-throughput in situ X-ray screening of and data collection from protein crystals at room temperature and under cryogenic conditions

Protein crystallography has significantly advanced in recent years, with in situ data collection, in which crystals are placed in the X-ray beam within their growth medium, being a major point of focus. In situ methods eliminate the need to harvest crystals, a previously unavoidable drawback, particularly for often small membrane-protein crystals. Here, we present a protocol for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature (20-25 degrees C) and under cryogenic conditions. The Mylar in situ method uses Mylar-based film sandwich plates that are inexpensive, easy to make, and compatible with automated imaging, and that show very low background scattering. They support crystallization in microbatch and vapor-diffusion modes, as well as in lipidic cubic phases (LCPs). A set of 3D-printed holders for differently sized patches of Mylar sandwich films makes the method robust and versatile, allows for storage and shipping of crystals, and enables automated mounting at synchrotrons, as well as goniometer-based screening and data collection. The protocol covers preparation of in situ plates and setup of crystallization trials; 3D printing and assembly of holders; opening of plates, isolation of film patches containing crystals, and loading them onto holders; basicmore » screening and data-collection guidelines; and unloading of holders, as well as reuse and recycling of them. In situ plates are prepared and assembled in 1 h; holders are 3D-printed and assembled in <= 90 min; and an in situ plate is opened, and a film patch containing crystals is isolated and loaded onto a holder in 5 min.« less
Authors:
ORCiD logo [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [2] ;  [3] ;  [3] ;  [2] ;  [2] ; ORCiD logo [4] ;  [2] ; ORCiD logo [1]
  1. Univ. of Toronto, ON (Canada)
  2. Argonne National Lab. (ANL), Lemont, IL (United States)
  3. Univ. of Southern California, Los Angeles, CA (United States)
  4. Argonne National Lab. (ANL), Lemont, IL (United States); Univ. of Southern California, Los Angeles, CA (United States)
Publication Date:
Grant/Contract Number:
AC02-06CH11357
Type:
Accepted Manuscript
Journal Name:
Nature Protocols
Additional Journal Information:
Journal Volume: 13; Journal Issue: 2; Journal ID: ISSN 1754-2189
Publisher:
Nature Publishing Group
Research Org:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org:
National Institutes of Health (NIH) - National Institute of General Medical Sciences; National Institutes of Health (NIH) - National Cancer Institute
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE
OSTI Identifier:
1473603

Broecker, Jana, Morizumi, Takefumi, Ou, Wei-Lin, Klingel, Viviane, Kuo, Anling, Kissick, David J., Ishchenko, Andrii, Lee, Ming-Yue, Xu, Shenglan, Makarov, Oleg, Cherezov, Vadim, Ogata, Craig M., and Ernst, Oliver P.. High-throughput in situ X-ray screening of and data collection from protein crystals at room temperature and under cryogenic conditions. United States: N. p., Web. doi:10.1038/nprot.2017.135.
Broecker, Jana, Morizumi, Takefumi, Ou, Wei-Lin, Klingel, Viviane, Kuo, Anling, Kissick, David J., Ishchenko, Andrii, Lee, Ming-Yue, Xu, Shenglan, Makarov, Oleg, Cherezov, Vadim, Ogata, Craig M., & Ernst, Oliver P.. High-throughput in situ X-ray screening of and data collection from protein crystals at room temperature and under cryogenic conditions. United States. doi:10.1038/nprot.2017.135.
Broecker, Jana, Morizumi, Takefumi, Ou, Wei-Lin, Klingel, Viviane, Kuo, Anling, Kissick, David J., Ishchenko, Andrii, Lee, Ming-Yue, Xu, Shenglan, Makarov, Oleg, Cherezov, Vadim, Ogata, Craig M., and Ernst, Oliver P.. 2018. "High-throughput in situ X-ray screening of and data collection from protein crystals at room temperature and under cryogenic conditions". United States. doi:10.1038/nprot.2017.135.
@article{osti_1473603,
title = {High-throughput in situ X-ray screening of and data collection from protein crystals at room temperature and under cryogenic conditions},
author = {Broecker, Jana and Morizumi, Takefumi and Ou, Wei-Lin and Klingel, Viviane and Kuo, Anling and Kissick, David J. and Ishchenko, Andrii and Lee, Ming-Yue and Xu, Shenglan and Makarov, Oleg and Cherezov, Vadim and Ogata, Craig M. and Ernst, Oliver P.},
abstractNote = {Protein crystallography has significantly advanced in recent years, with in situ data collection, in which crystals are placed in the X-ray beam within their growth medium, being a major point of focus. In situ methods eliminate the need to harvest crystals, a previously unavoidable drawback, particularly for often small membrane-protein crystals. Here, we present a protocol for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature (20-25 degrees C) and under cryogenic conditions. The Mylar in situ method uses Mylar-based film sandwich plates that are inexpensive, easy to make, and compatible with automated imaging, and that show very low background scattering. They support crystallization in microbatch and vapor-diffusion modes, as well as in lipidic cubic phases (LCPs). A set of 3D-printed holders for differently sized patches of Mylar sandwich films makes the method robust and versatile, allows for storage and shipping of crystals, and enables automated mounting at synchrotrons, as well as goniometer-based screening and data collection. The protocol covers preparation of in situ plates and setup of crystallization trials; 3D printing and assembly of holders; opening of plates, isolation of film patches containing crystals, and loading them onto holders; basic screening and data-collection guidelines; and unloading of holders, as well as reuse and recycling of them. In situ plates are prepared and assembled in 1 h; holders are 3D-printed and assembled in <= 90 min; and an in situ plate is opened, and a film patch containing crystals is isolated and loaded onto a holder in 5 min.},
doi = {10.1038/nprot.2017.135},
journal = {Nature Protocols},
number = 2,
volume = 13,
place = {United States},
year = {2018},
month = {1}
}

Works referenced in this record:

Fixed target combined with spectral mapping: approaching 100% hit rates for serial crystallography
journal, July 2016
  • Oghbaey, Saeed; Sarracini, Antoine; Ginn, Helen M.
  • Acta Crystallographica Section D Structural Biology, Vol. 72, Issue 8, p. 944-955
  • DOI: 10.1107/S2059798316010834

A comprehensive review of the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins and complexes
journal, January 2015
  • Caffrey, Martin
  • Acta Crystallographica Section F Structural Biology Communications, Vol. 71, Issue 1, p. 3-18
  • DOI: 10.1107/S2053230X14026843

PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010
  • Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221
  • DOI: 10.1107/S0907444909052925