Enhanced X-ray diffraction of in vivo-grown μNS crystals by viscous jets at XFELs
Category
Published on
Type
journal-article
Author
Nirupa Nagaratnam and Yanyang Tang and Sabine Botha and Justin Saul and Chufeng Li and Hao Hu and Sahba Zaare and Mark Hunter and David Lowry and Uwe Weierstall and Nadia Zatsepin and John C. H. Spence and Ji Qiu and Joshua LaBaer and Petra Fromme and Jose M. Martin-Garcia
Citation
Nagaratnam, N. et al., 2020. Enhanced X-ray diffraction of in vivo-grown μNS crystals by viscous jets at XFELs. Acta Crystallographica Section F Structural Biology Communications, 76(6), pp.278–289. Available at: http://dx.doi.org/10.1107/s2053230x20006172.
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), 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
Funding
NSF-STC Biology with X-ray Lasers (NSF-1231306)
