RESUMO
Crystallization of recombinant proteins in living cells is an exciting new approach for structural biology that provides an alternative to the time-consuming optimization of protein purification and extensive crystal screening steps. Exploiting the potential of this approach requires a more detailed understanding of the cellular processes involved and versatile screening strategies for crystals in a cell culture. Particularly if the target protein forms crystalline structures of unknown morphology only in a small fraction of cells, their detection by applying standard visualization techniques can be time consuming and difficult owing to the environmental challenges imposed by the living cells. In this study, a high-brilliance and low-background bioSAXS beamline is employed for rapid and sensitive detection of protein microcrystals grown within insect cells. On the basis of the presence of Bragg peaks in the recorded small-angle X-ray scattering profiles, it is possible to assess within seconds whether a cell culture contains microcrystals, even in a small percentage of cells. Since such information cannot be obtained by other established detection methods in this time frame, this screening approach has the potential to overcome one of the bottlenecks of intracellular crystal detection. Moreover, the association of the Bragg peak positions in the scattering curves with the unit-cell composition of the protein crystals raises the possibility of investigating the impact of environmental conditions on the crystal structure of the intracellular protein crystals. This information provides valuable insights helping to further understand the in cellulo crystallization process.
RESUMO
Alphaviruses are (re-)emerging arboviruses of public health concern. The nsP3 gene product is one of the key players during viral replication. NsP3 comprises three domains: a macro domain, a zinc-binding domain and a hypervariable region. The macro domain is essential at both early and late stages of the replication cycle through ADP-ribose (ADPr) binding and de-ADP-ribosylation of host proteins. However, both its specific role and the precise molecular mechanism of de-ADP-ribosylation across specific viral families remains to be elucidated. Here we investigate by X-ray crystallography the mechanism of ADPr reactivity in the active site of Getah virus macro domain, which displays a peculiar substitution of one of the conserved residues in the catalytic loop. ADPr adopts distinct poses including a covalent bond between the C''1 of the ADPr and a conserved Togaviridae-specific cysteine. These different poses observed for ADPr may represent snapshots of the de-ADP-ribosylation mechanism, highlighting residues to be further characterised.
