New variants and in silico analyses in GRK1 associated Oguchi disease.

Biallelic mutations in G-Protein coupled receptor kinase 1 (GRK1) cause Oguchi disease, a rare subtype of congenital stationary night blindness (CSNB). The purpose of this study was to identify disease causing GRK1 variants and use in-depth bioinformatic analyses to evaluate how their impact on protein structure could lead to pathogenicity. Patients' genomic DNA was sequenced by whole genome, whole exome or focused exome sequencing. Disease associated variants, published and novel, were compared to nondisease associated missense variants. The impact of GRK1 missense variants at the protein level were then predicted using a series of computational tools. We identified twelve previously unpublished cases with biallelic disease associated GRK1 variants, including eight novel variants, and reviewed all GRK1 disease associated variants. Further structure-based scoring revealed a hotspot for missense variants in the kinase domain. In addition, to aid future clinical interpretation, we identified the bioinformatics tools best able to differentiate disease associated from nondisease associated variants. We identified GRK1 variants in Oguchi disease patients and investigated how disease-causing variants may impede protein function in-silico.

Need for Speed: Examining Protein Behavior during CryoEM Grid Preparation at Different Timescales.

A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (cryoEM) grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon, and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms (the fastest reported to date), to interrogate particle behavior at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin-film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimize. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic "silver bullet" for improving the quality of every cryoEM sample preparation.