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Title: Towards in cellulo virus crystallography

Viruses are a significant threat to both human health and the economy, and there is an urgent need for novel anti-viral drugs and vaccines. High-resolution viral structures inform our understanding of the virosphere, and inspire novel therapies. Here we present a method of obtaining such structural information that avoids potentially disruptive handling, by collecting diffraction data from intact infected cells. We identify a suitable combination of cell type and virus to accumulate particles in the cells, establish a suitable time point where most cells contain virus condensates and use electron microscopy to demonstrate that these are ordered crystalline arrays of empty capsids. We then use an X-ray free electron laser to provide extremely bright illumination of sub-micron intracellular condensates of bacteriophage phiX174 inside living Escherichia coli at room temperature. We have been able to collect low resolution diffraction data. Despite the limited resolution and completeness of these initial data, due to a far from optimal experimental setup, we have used novel methodology to determine a putative space group, unit cell dimensions, particle packing and likely maturation state of the particles.
Authors:
 [1] ;  [1] ;  [2] ; ORCiD logo [3] ; ORCiD logo [4] ;  [1] ;  [2] ; ORCiD logo [3] ;  [5] ;  [4] ;  [4] ; ORCiD logo [6] ; ORCiD logo [1] ;  [5] ;  [2]
  1. Univ. of Oxford (United Kingdom); Science and Technology Facilities Council (STFC), Harwell Campus, Oxford (United Kingdom). Diamond Light Source, Ltd.
  2. Univ. of Helsinki (Finland)
  3. Science and Technology Facilities Council (STFC), Harwell Campus, Oxford (United Kingdom). Diamond Light Source, Ltd.
  4. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  5. Univ. of Oxford (United Kingdom)
  6. Univ. of Oxford (United Kingdom); Univ. of Helsinki (Finland)
Publication Date:
Grant/Contract Number:
AC02-05CH11231
Type:
Accepted Manuscript
Journal Name:
Scientific Reports
Additional Journal Information:
Journal Volume: 8; Journal Issue: 1; Journal ID: ISSN 2045-2322
Publisher:
Nature Publishing Group
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES
OSTI Identifier:
1465475

Duyvesteyn, Helen M. E., Ginn, Helen M., Pietilä, Maija K., Wagner, Armin, Hattne, Johan, Grimes, Jonathan M., Hirvonen, Elina, Evans, Gwyndaf, Parsy, Marie-Laure, Sauter, Nicholas K., Brewster, Aaron S., Huiskonen, Juha T., Stuart, David I., Sutton, Geoff, and Bamford, Dennis H.. Towards in cellulo virus crystallography. United States: N. p., Web. doi:10.1038/s41598-018-21693-3.
Duyvesteyn, Helen M. E., Ginn, Helen M., Pietilä, Maija K., Wagner, Armin, Hattne, Johan, Grimes, Jonathan M., Hirvonen, Elina, Evans, Gwyndaf, Parsy, Marie-Laure, Sauter, Nicholas K., Brewster, Aaron S., Huiskonen, Juha T., Stuart, David I., Sutton, Geoff, & Bamford, Dennis H.. Towards in cellulo virus crystallography. United States. doi:10.1038/s41598-018-21693-3.
Duyvesteyn, Helen M. E., Ginn, Helen M., Pietilä, Maija K., Wagner, Armin, Hattne, Johan, Grimes, Jonathan M., Hirvonen, Elina, Evans, Gwyndaf, Parsy, Marie-Laure, Sauter, Nicholas K., Brewster, Aaron S., Huiskonen, Juha T., Stuart, David I., Sutton, Geoff, and Bamford, Dennis H.. 2018. "Towards in cellulo virus crystallography". United States. doi:10.1038/s41598-018-21693-3. https://www.osti.gov/servlets/purl/1465475.
@article{osti_1465475,
title = {Towards in cellulo virus crystallography},
author = {Duyvesteyn, Helen M. E. and Ginn, Helen M. and Pietilä, Maija K. and Wagner, Armin and Hattne, Johan and Grimes, Jonathan M. and Hirvonen, Elina and Evans, Gwyndaf and Parsy, Marie-Laure and Sauter, Nicholas K. and Brewster, Aaron S. and Huiskonen, Juha T. and Stuart, David I. and Sutton, Geoff and Bamford, Dennis H.},
abstractNote = {Viruses are a significant threat to both human health and the economy, and there is an urgent need for novel anti-viral drugs and vaccines. High-resolution viral structures inform our understanding of the virosphere, and inspire novel therapies. Here we present a method of obtaining such structural information that avoids potentially disruptive handling, by collecting diffraction data from intact infected cells. We identify a suitable combination of cell type and virus to accumulate particles in the cells, establish a suitable time point where most cells contain virus condensates and use electron microscopy to demonstrate that these are ordered crystalline arrays of empty capsids. We then use an X-ray free electron laser to provide extremely bright illumination of sub-micron intracellular condensates of bacteriophage phiX174 inside living Escherichia coli at room temperature. We have been able to collect low resolution diffraction data. Despite the limited resolution and completeness of these initial data, due to a far from optimal experimental setup, we have used novel methodology to determine a putative space group, unit cell dimensions, particle packing and likely maturation state of the particles.},
doi = {10.1038/s41598-018-21693-3},
journal = {Scientific Reports},
number = 1,
volume = 8,
place = {United States},
year = {2018},
month = {2}
}

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TakeTwo: an indexing algorithm suited to still images with known crystal parameters
journal, July 2016
  • Ginn, Helen Mary; Roedig, Philip; Kuo, Anling
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Gas dynamic virtual nozzle for generation of microscopic droplet streams
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