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

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.

doi:10.1107/s2053230x20006172

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

Journal Article · 28 May 2020 · Acta Crystallographica. Section F, Structural Biology Communications

DOI: https://doi.org/10.1107/s2053230x20006172 · OSTI ID:1637950

Nagaratnam, Nirupa ^[1]; Tang, Yanyang ^[1]; Botha, Sabine ^[1]; Saul, Justin ^[1];

^[1]; Hu, Hao ^[1]; Zaare, Sahba ^[1]; Hunter, Mark ^[2]; Lowry, David ^[1]; Weierstall, Uwe ^[1];

^[3]; Spence, John C. H. ^[1]; Qiu, Ji ^[1]; LaBaer, Joshua ^[1]; Fromme, Petra ^[1]; Martin-Garcia, Jose M. ^[1]

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)

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.

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Research Organization:: SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)

Sponsoring Organization:: USDOE

Grant/Contract Number:: AC02-76SF00515

OSTI ID:: 1637950

Journal Information:: Acta Crystallographica. Section F, Structural Biology Communications, Journal Name: Acta Crystallographica. Section F, Structural Biology Communications Journal Issue: 6 Vol. 76; ISSN 2053-230X

Publisher:: International Union of CrystallographyCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
μNS
avian reovirus
in vivo crystallization
high-viscosity jets
serial crystallography
X-ray free-electron lasers

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

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