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 »
- Authors:
-
[1];
[3];
- Arizona State Univ., Tempe, AZ (United States)
- SLAC National Accelerator Lab., Menlo Park, CA (United States). Linac Coherent Light Source (LCLS)
- 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. doi: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}
}
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Figures / Tables:
Table 1: EGFP- μNS(448–605) SFX data-collection statistics.
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