TY - JOUR
T1 - Enhanced X-ray diffraction of in vivo-grown μnS crystals by viscous jets at XFELs
AU - Nagaratnam, Nirupa
AU - Tang, Yanyang
AU - Botha, Sabine
AU - Saul, Justin
AU - Li, Chufeng
AU - Hu, Hao
AU - Zaare, Sahba
AU - Hunter, Mark
AU - Lowry, David
AU - Weierstall, Uwe
AU - Zatsepin, Nadia
AU - Spence, John C.H.
AU - Qiu, Ji
AU - LaBaer, Joshua
AU - Fromme, Petra
AU - Martin-Garcia, Jose M.
N1 - Publisher Copyright:
© 2020.
PY - 2020/5/29
Y1 - 2020/5/29
N2 - 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.
AB - 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.
KW - NS
KW - X-ray free-electron lasers
KW - avian reovirus
KW - high-viscosity jets
KW - in vivo crystallization
KW - serial crystallography
KW - μ
UR - http://www.scopus.com/inward/record.url?scp=85086135316&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85086135316&partnerID=8YFLogxK
U2 - 10.1107/S2053230X20006172
DO - 10.1107/S2053230X20006172
M3 - Article
C2 - 32510469
AN - SCOPUS:85086135316
SN - 1744-3091
VL - 76
SP - 278
EP - 289
JO - Acta Crystallographica Section F:Structural Biology Communications
JF - Acta Crystallographica Section F:Structural Biology Communications
ER -