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.
Highlights
Classical X-ray structure analysis requires the growth of large, well diffracting crystals, which has been a bottleneck in the process of obtaining three-dimensional structures of proteins, for membrane proteins and post-translationally modified proteins
This study reports the first SFX diffraction from EGFPNS(448–605) crystals, which led to initial electron-density maps that allowed the clear identification of two enhanced green fluorescent protein (EGFP) proteins as well as the identification of electron density for NS(448–605)
While the interpretation of the NS(448–605) density requires more data to be collected at a higher photon flux, the results clearly indicate that SFX at X-ray free-electron laser (XFEL) using viscous jets is the method of choice to solve the structure of EGFP-NS(448–605) and likely other crystals grown in vivo
Summary
Classical X-ray structure analysis requires the growth of large, well diffracting crystals, which has been a bottleneck in the process of obtaining three-dimensional structures of proteins, for membrane proteins and post-translationally modified proteins. A recent report suggests that in vivo protein crystallization could be feasible for recombinant proteins (for a review, see Schonherr et al, 2018). A typical standard in vitro crystallization pipeline involves protein expression, purification, crystallization optimization, and crystal harvesting and cryoprotection. In vivo crystallization enables crystal growth in the cells that express the protein, bypassing the protein purification and crystallization steps (Banerjee et al, 2018; Boudes et al, 2016). Cells are lysed and crystals are harvested and cryoprotected for data collection. The host cells are not lysed and the crystal-containing cells are delivered to the X-ray beam by standard sample-delivery methods with no need for crystal harvesting or cryoprotection (Boudes et al, 2016)
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