Enhanced X-ray diffraction of in vivo-grown ?NS crystals by viscous jets at XFELs.

ABSTRACT: ?NS 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 ?NS protein has been suggested to be the minimal viral factor required for factory formation. Thus, determining the structure of ?NS is of great importance for understanding its role in viral infection. In the study presented here, a fragment consisting of residues 448-605 of ?NS was expressed as an EGFP fusion protein in Sf9 insect cells. EGFP-?NS(448-605) crystallization in Sf9 cells was monitored and verified by several imaging techniques. Cells infected with the EGFP-?NS(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 ?NS(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 ?NS protein.

SUBMITTER: Nagaratnam N 

PROVIDER: S-EPMC7278499 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature