Aller, P. | Search bioRxiv

bioRxiv2022

Redox-controlled structural reorganization and flavin strain within the ribonucleotide reductase R2b-NrdI complex monitored by serial femtosecond crystallography

Redox reactions are central to biochemistry and are both controlled by and induce protein structural changes. Here we describe structural rearrangements and crosstalk within the Bacillus cereus ribonucleotide reductase R2b-NrdI complex, a di-metal carboxylate- flavoprotein system, as part of the mechanism generating the essential catalytic free radical of the enzyme. Femtosecond crystallography at an X-ray free-electron laser was utilized to obtain structures at room temperature in defined redox states without suffering photoreduction. We show that the flavin in the hydroquinone state is under steric strain in the R2b-NrdI protein complex, presumably tuning its redox potential to promote superoxide generation. Moreover, a binding site in close vicinity to the expected flavin O2-interacton site is observed to be controlled by the redox state of the flavin and linked to the channel proposed to funnel the produced superoxide species from NrdI to the di-manganese site in protein R2b. These specific features are coupled to further structural changes around the R2b- NrdI interaction surface. The mechanistic implications for the control of reactive oxygen species and radical generation in protein R2b are discussed.

biochemistry↗

bioRxiv2022

xia2.multiplex: a multi-crystal data analysis pipeline

In macromolecular crystallography radiation damage limits the amount of data that can be collected from a single crystal. It is often necessary to merge data sets from multiple crystals, for example small-wedge data collections on micro-crystals, in situ room-temperature data collections, and collection from membrane proteins in lipidic mesophase. Whilst indexing and integration of individual data sets may be relatively straightforward with existing software, merging multiple data sets from small wedges presents new challenges. Identification of a consensus symmetry can be problematic, particularly in the presence of a potential indexing ambiguity. Furthermore, the presence of non-isomorphous or poor-quality data sets may reduce the overall quality of the final merged data set. To facilitate and help optimise the scaling and merging of multiple data sets, we developed a new program, xia2.multiplex, which takes data sets individually integrated with DIALS and performs symmetry analysis, scaling and merging of multicrystal data sets. xia2.multiplex also performs analysis of various pathologies that typically affect multi-crystal data sets, including non-isomorphism, radiation damage and preferential orientation. After describing a number of use cases, we demonstrate the benefit of xia2.multiplex within a wider autoprocessing framework in facilitating a multi-crystal experiment collected as part of in situ room-temperature fragment screening experiments on the SARS-CoV-2 main protease.

molecular biology↗