Virani, S. | Search bioRxiv

bioRxiv2025

End-to-end automation of repeat-target cryo-EM structure determination in CryoSPARC

Single particle cryo-EM is a valuable and growing technique for life science and drug discovery. Currently, obtaining state-of-the-art results from cryo-EM data analysis requires a human in the loop to analyze intermediate results and make image processing decisions. This bottleneck limits the achievable throughput of structure determination, especially in high-throughput settings such as structure-based drug design. In this work, we develop an end-to-end automation strategy for repeat-target structure determination using new tools in CryoSPARC. We demonstrate completely hands-off processing of 21 challenging G protein-coupled receptor (GPCR) datasets. In 17 of 21 cases, automated processing meets or exceeds published resolution and map quality and, in several cases, provides significant improvement in receptor and ligand density that allows improved model building. Our results on both active and inactive state GPCRs show that our automation strategy generalizes easily to new target classes, and that complete automation of data processing is straightforward to achieve in CryoSPARC. We provide downloadable CryoSPARC Workflow files so that users can import, replicate, adapt and extend our automated workflow for their own targets, enabling cryo-EM to be applied at larger scales and to answer larger biological questions.

biophysics↗

bioRxiv2022

Variability of Phenylalanine side chain conformations facilitates promiscuity of Fatty acid binding in Cockroach milk proteins.

The pacific beetle cockroach, Diploptera punctata, is a viviparous cockroach that produces a milk-like substance to support the growing embryo with a brood sac. The structure of the in vivo grown crystals present in the gut of the embryo showed that the milk-derived crystals are heterogenous and are made of three proteins (called Lili-Mips). Multiple fatty acids could be modeled into the active site, and we hypothesized that each of the three isoforms of the protein bound to a different fatty acid. We previously reported that the recombinantly expressed Lili-Mip2 has a structure similar to the structure of the protein determined from in vivo crystals, and this single isoform also binds to several fatty acids. In this study, we aimed to probe the specificity and affinity of fatty acid binding and test the stability of different isoforms. We show that all the isoforms can bind to different fatty acids with very similar affinities, and the local abundance of a fatty acid determined bound fatty acid ratios. Lili-Mips thermostability is pH dependent, where stability is highest at acidic pH and declines as the pH increases to physiological levels near 7.0. The measurement of the pH in the gut lumen and the gut cells suggests that the pH in the gut is acidic and the pH inside the gut cells is closer to neutral pH. We propose that the protein has evolved to be highly stable in the acidic gut lumen and, when absorbed inside the gut cells, becomes less stable to enable the breakdown of the glycosylated lipo-protein complex to provide essential metabolites for survival and development of the embryo. The different orientations of Phe-98 and Phe-100 control the binding pocket volume and allow the binding of different chain-length fatty acids to bind with similar affinities.

biochemistry↗