Olek, M. | bioRxiv Science

bioRxiv2020

CryoET structures of immature HIV Gag reveal a complete six-helix bundle and stabilizing small molecules distinct from IP6

Gag is the major HIV-1 structural polyprotein precursor. The Gag SP1 domain with the last residues of CA have been hypothesized to form a six-helix bundle necessary for particle assembly, but this bundle has not been fully resolved. Here, we determined the structures of complete CA-SP1 six-helix bundle connecting to the NC domain, from both in vitro Gag assemblies and viral-like particles (VLPs) carrying a T8I mutation in SP1, to near-atomic resolutions using cryoET and subtomogram averaging. The structures revealed novel densities, however distinct from IP6, inside the six-helix bundle of Gag assemblies, stabilizing the immature lattice. Interestingly, the T8I mutation impaired proteolytic cleavage of Gag at both SP1 boundaries. Our findings signify the involvement of small molecules in immature Gag assembly and provide the structural basis for development of small molecule inhibitors that stabilize SP1 helix, thus interfere with PR-mediated virus maturation.

microbiology↗

bioRxiv2020

Outcomes of the 2019 EMDataResource model challenge: validation of cryo-EM models at near-atomic resolution

This paper describes outcomes of the 2019 Cryo-EM Map-based Model Metrics Challenge sponsored by EMDataResource (www.emdataresource.org). The goals of this challenge were (1) to assess the quality of models that can be produced using current modeling software, (2) to check the reproducibility of modeling results from different software developers and users, and (3) compare the performance of current metrics used for evaluation of models. The focus was on near-atomic resolution maps with an innovative twist: three of four target maps formed a resolution series (1.8 to 3.1 [A]) from the same specimen and imaging experiment. Tools developed in previous challenges were expanded for managing, visualizing and analyzing the 63 submitted coordinate models, and several novel metrics were introduced. The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual laboratory experiments and holdings of structure data archives such as the Protein Data Bank. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived from these benchmark maps by 13 participating teams, representing both widely used and novel modeling approaches. We also evaluate the pros and cons of the commonly used metrics to assess model quality and recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed density in the cryo-EM map.

biochemistry↗