Accurate model annotation of a near-atomic resolution cryo-EM map

Electron cryomicroscopy (cryo-EM) has been used to determine the atomic coordinates (models) from density maps of biological assemblies. These models can be assessed by their overall fit to the experimental data and stereochemical information. However, these models do not annotate the actual density values of the atoms nor their positional uncertainty. Here, we introduce a computational procedure to derive an atomic model from a cryo- EM map with annotated metadata. The accuracy of such a model is validated by a faithful replication of the experimental cryo-EM map computed using the coordinates and associated metadata. The functional interpretation of any structural features in the model and its utilization for future studies can be made in the context of its measure of uncertainty. We applied this protocol to the 3.3-Å map of the mature P22 bacteriophage capsid, a large and complex macromolecular assembly.With this protocol, we identify and annotate previously undescribed molecular interactions between capsid subunits that are crucial to maintain stability in the absence of cementing proteins or cross-linking, as occur in other bacteriophages.National Institutes of Health (U.S.) (Grant P41GM103832)National Institutes of Health (U.S.) (Grant R01GM079429)National Institutes of Health (U.S.) (Grant PN2EY016525)National Institutes of Health (U.S.) (Grant P01GM063210)Robert A. Welch Foundation (Grant Q1242

Structural Insights Into Cardiolipin Replacement by Phosphatidylglycerol in a Cardiolipin-lacking Yeast Respiratory Supercomplex

Cardiolipin is a hallmark phospholipid of mitochondrial membranes. Despite established significance of cardiolipin in supporting respiratory supercomplex organization, a mechanistic understanding of this lipid-protein interaction is still lacking. To address the essential role of cardiolipin in supercomplex organization, we report cryo-EM structures of a wild type supercomplex (I

4.4 Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virus

This study uses high-resolution cryo-electron microscopy to provide a complete structural model of the VEEV alphavirus, bridging the gap between incomplete crystal structures and lower resolution electron microscopy analyses

Beyond the Backbone: The Next Generation of Pathwalking Utilities for Model Building in CryoEM Density Maps

Single-particle electron cryomicroscopy (cryoEM) has become an indispensable tool for studying structure and function in macromolecular assemblies. As an integral part of the cryoEM structure determination process, computational tools have been developed to build atomic models directly from a density map without structural templates. Nearly a decade ago, we created Pathwalking, a tool for de novo modeling of protein structure in near-atomic resolution cryoEM density maps. Here, we present the latest developments in Pathwalking, including the addition of probabilistic models, as well as a companion tool for modeling waters and ligands. This software was evaluated on the 2021 CryoEM Ligand Challenge density maps, in addition to identifying ligands in three IP3R1 density maps at ~3 Å to 4.1 Å resolution. The results clearly demonstrate that the Pathwalking de novo modeling pipeline can construct accurate protein structures and reliably localize and identify ligand density directly from a near-atomic resolution map

Subunit conformational variation within individual GroEL oligomers resolved by Cryo-EM

Single-particle electron cryo-microscopy (cryo-EM) is an emerging tool for resolving structures of conformationally heterogeneous particles; however, each structure is derived from an average of many particles with presumed identical conformations. We used a 3.5-angstrom cryo-EM reconstruction with imposed D7 symmetry to further analyze structural heterogeneity among chemically identical subunits in each GroEL oligomer. Focused classification of the 14 subunits in each oligomer revealed three dominant classes of subunit conformations. Each class resembled a distinct GroEL crystal structure in the Protein Data Bank. The conformational differences stem from the orientations of the apical domain. We mapped each conformation class to its subunit locations within each GroEL oligomer in our dataset. The spatial distributions of each conformation class differed among oligomers, and most oligomers contained 10-12 subunits of the three dominant conformation classes. Adjacent subunits were found to more likely assume the same conformation class, suggesting correlation among subunits in the oligomer. This study demonstrates the utility of cryo-EM in revealing structure dynamics within a single protein oligomer.Y

Validated near-atomic resolution structure of bacteriophage epsilon15 derived from cryo-EM and modeling

High-resolution structures of viruses have made important contributions to modern structural biology. Bacteriophages, the most diverse and abundant organisms on earth, replicate and infect all bacteria and archaea, making them excellent potential alternatives to antibiotics and therapies for multidrug-resistant bacteria. Here, we improved upon our previous electron cryomicroscopy structure of Salmonella bacteriophage epsilon15, achieving a resolution sufficient to determine the tertiary structures of both gp7 and gp10 protein subunits that form the T = 7 icosahedral lattice. This study utilizes recently established best practice for near-atomic to high-resolution (3–5 Å) electron cryomicroscopy data evaluation. The resolution and reliability of the density map were cross-validated by multiple reconstructions from truly independent data sets, whereas the models of the individual protein subunits were validated adopting the best practices from X-ray crystallography. Some sidechain densities are clearly resolved and show the subunit–subunit interactions within and across the capsomeres that are required to stabilize the virus. The presence of the canonical phage and jellyroll viral protein folds, gp7 and gp10, respectively, in the same virus suggests that epsilon15 may have emerged more recently relative to other bacteriophagesNational Institutes of Health (U.S.) (Grant R01GM079429)National Institutes of Health (U.S.) (Grant P41GM103832)National Institutes of Health (U.S.) (Grant PN2EY016525)National Institutes of Health (U.S.) (Grant R56AI075208)Robert A. Welch Foundation (Q1242)National Institutes of Health (U.S.) (Grant GM063210)United States. Dept. of Energy (Contract DEAC0205CH11231

Orthopedic Hardware Type Impacts Case Complexity in Conversion Total Hip Arthroplasty Surgery

Background: Conversion total hip arthroplasty (THA) includes a variety of operations and prior implants. The implant present before conversion may influence the outcome and complexity of the procedure. The group hypothesized that conversion arthroplasty for patients with intramedullary nails (IMNs) is more complex from a surgical and resource utilization perspective than for those with screw fixation. Methods: THA conversion cases were reviewed retrospectively from 2012 to 2020 from 6 surgeons across 3 institutions. The included cohort had 106 patients with fixation in the proximal femur for prior traumatic events. Demographics, operative data, outcomes, and implant information were collected from the medical record. The conversion THA group was categorized by preoperative fixation type: closed reduction and percutaneous pinning/screw fixation (CRPP) or IMN. Results: No age or body mass index differences were observed between the cohorts. Prior to conversion THA, IMN patients had undergone more surgeries than CRPP (P < .05). Perioperatively, the IMN cohort sustained increased blood loss (P < .001), had longer surgeries (P < .0001), had longer length of hospital stays (P < .01), necessitated trochanteric plates more often (P < .05), were readmitted more (P < .05), and required additional follow-up surgery (P < .01) than the CRPP cohort. Conclusions: Conversion THA of a prior IMN implant is associated with worse perioperative outcomes than conversion of a CRPP construct. Surgeons, health systems, and payors should consider these differences when caring for these distinct groups of patients