skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Enhanced X-ray diffraction of in vivo-grown μNS crystals by viscous jets at XFELs

Abstract

μNS is a 70 kDa major nonstructural protein of avian reoviruses, which cause significant economic losses in the poultry industry. They replicate inside viral factories in host cells, and the μNS protein has been suggested to be the minimal viral factor required for factory formation. Thus, determining the structure of μNS is of great importance for understanding its role in viral infection. In the study presented here, a fragment consisting of residues 448–605 of μNS was expressed as an EGFP fusion protein in Sf9 insect cells. EGFP-μNS(448–605) crystallization in Sf9 cells was monitored and verified by several imaging techniques. Cells infected with the EGFP-μNS(448–605) baculovirus formed rod-shaped microcrystals (5–15 µm in length) which were reconstituted in high-viscosity media (LCP and agarose) and investigated by serial femtosecond X-ray diffraction using viscous jets at an X-ray free-electron laser (XFEL). The crystals diffracted to 4.5 Å resolution. A total of 4227 diffraction snapshots were successfully indexed into a hexagonal lattice with unit-cell parameters a = 109.29, b = 110.29, c = 324.97 Å. The final data set was merged and refined to 7.0 Å resolution. Preliminary electron-density maps were obtained. While more diffraction data are required to solve the structure of μNS(448–605), themore » current experimental strategy, which couples high-viscosity crystal delivery at an XFEL with in cellulo crystallization, paves the way towards structure determination of the μNS protein.« less

Authors:
 [1];  [1];  [1];  [1]; ORCiD logo [1];  [1];  [1];  [2];  [1];  [1]; ORCiD logo [3];  [1];  [1];  [1];  [1];  [1]
  1. Arizona State Univ., Tempe, AZ (United States)
  2. SLAC National Accelerator Lab., Menlo Park, CA (United States). Linac Coherent Light Source (LCLS)
  3. Arizona State Univ., Tempe, AZ (United States); La Trobe Univ., Melbourne, VIC (Australia)
Publication Date:
Research Org.:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1637950
Grant/Contract Number:  
AC02-76SF00515; 1991; 1231306
Resource Type:
Accepted Manuscript
Journal Name:
Acta Crystallographica. Section F, Structural Biology Communications
Additional Journal Information:
Journal Volume: 76; Journal Issue: 6; Journal ID: ISSN 2053-230X
Publisher:
International Union of Crystallography
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; μNS; avian reovirus; in vivo crystallization; high-viscosity jets; serial crystallography; X-ray free-electron lasers

Citation Formats

Nagaratnam, Nirupa, Tang, Yanyang, Botha, Sabine, Saul, Justin, Li, Chufeng, Hu, Hao, Zaare, Sahba, Hunter, Mark, Lowry, David, Weierstall, Uwe, Zatsepin, Nadia, Spence, John C. H., Qiu, Ji, LaBaer, Joshua, Fromme, Petra, and Martin-Garcia, Jose M. Enhanced X-ray diffraction of in vivo-grown μNS crystals by viscous jets at XFELs. United States: N. p., 2020. Web. https://doi.org/10.1107/s2053230x20006172.
Nagaratnam, Nirupa, Tang, Yanyang, Botha, Sabine, Saul, Justin, Li, Chufeng, Hu, Hao, Zaare, Sahba, Hunter, Mark, Lowry, David, Weierstall, Uwe, Zatsepin, Nadia, Spence, John C. H., Qiu, Ji, LaBaer, Joshua, Fromme, Petra, & Martin-Garcia, Jose M. Enhanced X-ray diffraction of in vivo-grown μNS crystals by viscous jets at XFELs. United States. https://doi.org/10.1107/s2053230x20006172
Nagaratnam, Nirupa, Tang, Yanyang, Botha, Sabine, Saul, Justin, Li, Chufeng, Hu, Hao, Zaare, Sahba, Hunter, Mark, Lowry, David, Weierstall, Uwe, Zatsepin, Nadia, Spence, John C. H., Qiu, Ji, LaBaer, Joshua, Fromme, Petra, and Martin-Garcia, Jose M. Fri . "Enhanced X-ray diffraction of in vivo-grown μNS crystals by viscous jets at XFELs". United States. https://doi.org/10.1107/s2053230x20006172. https://www.osti.gov/servlets/purl/1637950.
@article{osti_1637950,
title = {Enhanced X-ray diffraction of in vivo-grown μNS crystals by viscous jets at XFELs},
author = {Nagaratnam, Nirupa and Tang, Yanyang and Botha, Sabine and Saul, Justin and Li, Chufeng and Hu, Hao and Zaare, Sahba and Hunter, Mark and Lowry, David and Weierstall, Uwe and Zatsepin, Nadia and Spence, John C. H. and Qiu, Ji and LaBaer, Joshua and Fromme, Petra and Martin-Garcia, Jose M.},
abstractNote = {μNS is a 70 kDa major nonstructural protein of avian reoviruses, which cause significant economic losses in the poultry industry. They replicate inside viral factories in host cells, and the μNS protein has been suggested to be the minimal viral factor required for factory formation. Thus, determining the structure of μNS is of great importance for understanding its role in viral infection. In the study presented here, a fragment consisting of residues 448–605 of μNS was expressed as an EGFP fusion protein in Sf9 insect cells. EGFP-μNS(448–605) crystallization in Sf9 cells was monitored and verified by several imaging techniques. Cells infected with the EGFP-μNS(448–605) baculovirus formed rod-shaped microcrystals (5–15 µm in length) which were reconstituted in high-viscosity media (LCP and agarose) and investigated by serial femtosecond X-ray diffraction using viscous jets at an X-ray free-electron laser (XFEL). The crystals diffracted to 4.5 Å resolution. A total of 4227 diffraction snapshots were successfully indexed into a hexagonal lattice with unit-cell parameters a = 109.29, b = 110.29, c = 324.97 Å. The final data set was merged and refined to 7.0 Å resolution. Preliminary electron-density maps were obtained. While more diffraction data are required to solve the structure of μNS(448–605), the current experimental strategy, which couples high-viscosity crystal delivery at an XFEL with in cellulo crystallization, paves the way towards structure determination of the μNS protein.},
doi = {10.1107/s2053230x20006172},
journal = {Acta Crystallographica. Section F, Structural Biology Communications},
number = 6,
volume = 76,
place = {United States},
year = {2020},
month = {5}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 1 work
Citation information provided by
Web of Science

Figures / Tables:

Table 1 Table 1: EGFP- μNS(448–605) SFX data-collection statistics.

Save / Share:

Works referenced in this record:

Cheetah : software for high-throughput reduction and analysis of serial femtosecond X-ray diffraction data
journal, May 2014

  • Barty, Anton; Kirian, Richard A.; Maia, Filipe R. N. C.
  • Journal of Applied Crystallography, Vol. 47, Issue 3
  • DOI: 10.1107/S1600576714007626

CrystFEL : a software suite for snapshot serial crystallography
journal, March 2012

  • White, Thomas A.; Kirian, Richard A.; Martin, Andrew V.
  • Journal of Applied Crystallography, Vol. 45, Issue 2
  • DOI: 10.1107/S0021889812002312

X-ray free-electron lasers
journal, November 2010


Solving structures of protein complexes by molecular replacement with Phaser
journal, December 2006

  • McCoy, Airlie J.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 63, Issue 1
  • DOI: 10.1107/S0907444906045975

Serial crystallography on in vivo grown microcrystals using synchrotron radiation
journal, February 2014


Overview of the CCP 4 suite and current developments
journal, March 2011

  • Winn, Martyn D.; Ballard, Charles C.; Cowtan, Kevin D.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4
  • DOI: 10.1107/S0907444910045749

Virus factories: associations of cell organelles for viral replication and morphogenesis
journal, February 2005

  • Novoa, Reyes R.; Calderita, Gloria; Arranz, Rocío
  • Biology of the Cell, Vol. 97, Issue 2
  • DOI: 10.1042/BC20040058

Introduction to protein crystallization
journal, December 2013

  • McPherson, Alexander; Gavira, Jose A.
  • Acta Crystallographica Section F Structural Biology Communications, Vol. 70, Issue 1
  • DOI: 10.1107/S2053230X13033141

A simple mechanical mixer for small viscous lipid-containing samples
journal, September 1998


Serial femtosecond crystallography: A revolution in structural biology
journal, July 2016

  • Martin-Garcia, Jose M.; Conrad, Chelsie E.; Coe, Jesse
  • Archives of Biochemistry and Biophysics, Vol. 602
  • DOI: 10.1016/j.abb.2016.03.036

Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation
journal, May 2017

  • Martin-Garcia, Jose M.; Conrad, Chelsie E.; Nelson, Garrett
  • IUCrJ, Vol. 4, Issue 4, p. 439-454
  • DOI: 10.1107/S205225251700570X

A pipeline for structure determination of in vivo -grown crystals using in cellulo diffraction
journal, March 2016

  • Boudes, Marion; Garriga, Damià; Fryga, Andrew
  • Acta Crystallographica Section D Structural Biology, Vol. 72, Issue 4
  • DOI: 10.1107/S2059798316002369

The history of X-ray free-electron lasers
journal, June 2012


Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix
journal, October 2019

  • Liebschner, Dorothee; Afonine, Pavel V.; Baker, Matthew L.
  • Acta Crystallographica Section D Structural Biology, Vol. 75, Issue 10
  • DOI: 10.1107/S2059798319011471

Mammalian Reovirus M3 Gene Sequences and Conservation of Coiled-Coil Motifs near the Carboxyl Terminus of the μNS Protein
journal, November 1999

  • McCutcheon, Aimee M.; Broering, Teresa J.; Nibert, Max L.
  • Virology, Vol. 264, Issue 1
  • DOI: 10.1006/viro.1999.9990

The Coherent X-ray Imaging instrument at the Linac Coherent Light Source
journal, April 2015

  • Liang, Mengning; Williams, Garth J.; Messerschmidt, Marc
  • Journal of Synchrotron Radiation, Vol. 22, Issue 3
  • DOI: 10.1107/S160057751500449X

A novel inert crystal delivery medium for serial femtosecond crystallography
journal, June 2015


Crystallizing membrane proteins using lipidic mesophases
journal, April 2009


Scaling and assessment of data quality
journal, December 2005

  • Evans, Philip
  • Acta Crystallographica Section D Biological Crystallography, Vol. 62, Issue 1, p. 72-82
  • DOI: 10.1107/S0907444905036693

XDS
journal, January 2010

  • Kabsch, Wolfgang
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2
  • DOI: 10.1107/S0907444909047337

Autoindexing diffraction images with iMosflm
journal, June 2013

  • Powell, Harold R.; Johnson, Owen; Leslie, Andrew G. W.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 69, Issue 7
  • DOI: 10.1107/S0907444912048524

Indexing in single-crystal diffractometry with an obstinate list of reflections
journal, April 1992


Real-time investigation of dynamic protein crystallization in living cells
journal, July 2015

  • Schönherr, R.; Klinge, M.; Rudolph, J. M.
  • Structural Dynamics, Vol. 2, Issue 4
  • DOI: 10.1063/1.4921591

In vivo protein crystallization in combination with highly brilliant radiation sources offers novel opportunities for the structural analysis of post-translationally modified eukaryotic proteins
journal, July 2015

  • Duszenko, Michael; Redecke, Lars; Mudogo, Celestin Nzanzu
  • Acta Crystallographica Section F Structural Biology Communications, Vol. 71, Issue 8
  • DOI: 10.1107/S2053230X15011450

Recent developments in CrystFEL
journal, March 2016

  • White, Thomas A.; Mariani, Valerio; Brehm, Wolfgang
  • Journal of Applied Crystallography, Vol. 49, Issue 2
  • DOI: 10.1107/S1600576716004751

Protein crystallization in living cells
journal, June 2018

  • Schönherr, Robert; Rudolph, Janine Mia; Redecke, Lars
  • Biological Chemistry, Vol. 399, Issue 7
  • DOI: 10.1515/hsz-2018-0158

The Avian Reovirus Genome Segment S1 Is a Functionally Tricistronic Gene That Expresses One Structural and Two Nonstructural Proteins in Infected Cells
journal, November 2001

  • Bodelón, Gustavo; Labrada, Lucı́a; Martı́nez-Costas, José
  • Virology, Vol. 290, Issue 2
  • DOI: 10.1006/viro.2001.1159

First lasing and operation of an ångstrom-wavelength free-electron laser
journal, August 2010


Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography
journal, February 2014

  • Weierstall, Uwe; James, Daniel; Wang, Chong
  • Nature Communications, Vol. 5, Issue 1
  • DOI: 10.1038/ncomms4309

Avian Reovirus μNS Protein Forms Homo-Oligomeric Inclusions in a Microtubule-Independent Fashion, Which Involves Specific Regions of Its C-Terminal Domain
journal, February 2010

  • Brandariz-Nuñez, Alberto; Menaya-Vargas, Rebeca; Benavente, Javier
  • Journal of Virology, Vol. 84, Issue 9
  • DOI: 10.1128/JVI.02534-09

Features and development of Coot
journal, March 2010

  • Emsley, P.; Lohkamp, B.; Scott, W. G.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
  • DOI: 10.1107/S0907444910007493

The New Era of Microcrystallography
journal, July 2018

  • Banerjee, Sanchari; Montaville, Pierre; Chavas, Leonard M. G.
  • Journal of the Indian Institute of Science, Vol. 98, Issue 3
  • DOI: 10.1007/s41745-018-0086-0

Structural Evidence for a Dehydrated Intermediate in Green Fluorescent Protein Chromophore Biosynthesis
journal, March 2010

  • Pletneva, Nadya V.; Pletnev, Vladimir Z.; Lukyanov, Konstantin A.
  • Journal of Biological Chemistry, Vol. 285, Issue 21
  • DOI: 10.1074/jbc.M109.092320

Avian reovirus nonstructural protein μNS forms viroplasm-like inclusions and recruits protein σNS to these structures
journal, February 2004


Current trends in protein crystallization
journal, July 2016


Protein crystallization by capillary counterdiffusion for applied crystallographic structure determination
journal, April 2003

  • Ng, Joseph D.; Gavira, José A.; Garcı́a-Ruı́z, Juan M.
  • Journal of Structural Biology, Vol. 142, Issue 1
  • DOI: 10.1016/S1047-8477(03)00052-2

Solvent content of protein crystals
journal, April 1968


Carboxyl-Proximal Regions of Reovirus Nonstructural Protein μNS Necessary and Sufficient for Forming Factory-Like Inclusions
journal, May 2005


On the treatment of negative intensity observations
journal, July 1978


Selective Detection of Protein Crystals by Second Harmonic Microscopy
journal, October 2008

  • Wampler, Ronald D.; Kissick, David J.; Dehen, Christopher J.
  • Journal of the American Chemical Society, Vol. 130, Issue 43
  • DOI: 10.1021/ja805983b

Protein coding assignment of avian reovirus strain S1133.
journal, January 1994


Protein crystallization in vivo
journal, April 2006


Matthews coefficient probabilities: Improved estimates for unit cell contents of proteins, DNA, and protein-nucleic acid complex crystals
journal, September 2003

  • Kantardjieff, Katherine A.; Rupp, Bernhard
  • Protein Science, Vol. 12, Issue 9
  • DOI: 10.1110/ps.0350503

X-ray free-electron lasers—principles, properties and applications
journal, March 2003

  • Pellegrini, Claudio; Stöhr, Joachim
  • Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment, Vol. 500, Issue 1-3
  • DOI: 10.1016/S0168-9002(03)00739-3

Avian Reovirus Morphogenesis Occurs Within Viral Factories and Begins with the Selective Recruitment of σNS and λA to μNS Inclusions
journal, August 2004

  • Tourı́s-Otero, Fernando; Cortez-San Martı́n, Marcelo; Martı́nez-Costas, José
  • Journal of Molecular Biology, Vol. 341, Issue 2
  • DOI: 10.1016/j.jmb.2004.06.026

XGANDALF – extended gradient descent algorithm for lattice finding
journal, August 2019

  • Gevorkov, Yaroslav; Yefanov, Oleksandr; Barty, Anton
  • Acta Crystallographica Section A Foundations and Advances, Vol. 75, Issue 5
  • DOI: 10.1107/S2053273319010593

In vivo protein crystallization opens new routes in structural biology
journal, January 2012

  • Koopmann, Rudolf; Cupelli, Karolina; Redecke, Lars
  • Nature Methods, Vol. 9, Issue 3
  • DOI: 10.1038/nmeth.1859

Avian reovirus infections: -EN- -FR- -ES-
journal, August 2000


Natively Inhibited Trypanosoma brucei Cathepsin B Structure Determined by Using an X-ray Laser
journal, November 2012


The History of Avian Reovirus
journal, July 2000

  • van der Heide, Louis
  • Avian Diseases, Vol. 44, Issue 3
  • DOI: 10.2307/1593104

    Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.