EMRinger: side chain–directed model and map validation for 3D cryo-electron microscopy

Barad, Benjamin A.; Echols, Nathaniel; Wang, Ray Yu-Ruei; Cheng, Yifan; DiMaio, Frank; Adams, Paul D.; Fraser, James S.

doi:10.1038/nmeth.3541

Cited by 887 publications

(663 citation statements)

References 30 publications

Supporting

Mentioning

642

Contrasting

Order By: Relevance

“…Inclusion of the surrounding subunits was critical to ensure accurate modeling without clashes at the molecular interfaces, both within and across asymmetric units. A final measure of model quality derived from the full image dataset was based on MolProbity (13), which indicated excellent stereochemistry of the model, the cross-correlation between model and map, and the EMRinger score (14), revealing good side-chain placement within the density (Table S1). Model validation was further performed by building independent versions of the seven models from the two independent maps that resulted from the gold-standard protocol (4) (Fig.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

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

Hryc

Chen

Afonine

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

113

118

View full text Add to dashboard Cite

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.ecently, cryo-EM maps with associated atomic coordinates have been reported at resolutions better than 4 Å (1, 2). However, few have been subjected to rigorous evaluation of the reliability of the observed features or of the correlation between the experimental map and its corresponding model at the residue level. Typically, such correlation is reported in terms of a curve known as the Fourier shell correlation (FSC), which is a function of spatial frequency (3, 4). Although informative, it does not assure the authenticity of local features, nor does it indicate which features in the model agree or disagree with observed density. Ideally, a molecular model can be used to generate a map that replicates the experimental map in most or all of its details, and thus constitutes a trustworthy and informative model for the specimen's structure at the reported resolution. This study examines the agreement and/or disagreement between the model and the experimental map density, determined at nearatomic resolution. These efforts establish the groundwork for a quantitative assessment of a cryo-EM structure. The methods described here were applied to the capsid of P22 bacteriophage, which infects Salmonella and has been extensively studied through biochemistry, genetics, and biophysics (5-8). ResultsCryo-EM Images and Reconstructions. To study the P22 structure, we used a 300-keV electron cryomicroscope (JEM-3200FSC; JEOL Ltd.) and a Direct Electron detector (DE-20; operated in integrating mode) to collect frozen, hydrated P22 bacteriophage images (Fig. 1A and Table S1). Signal was detectable out to 3-Å resolution ( Fig. S1 and Movie S1). A total exposure of 37.5 e − /Å 2 was fractionated into 24 frames during a 1.5-s exposure. All frames were dose-weighted (4) and used to refine particle orientation parameters; empirically, we found that using image frames 1 to 6 (a cumulative exposu...

show abstract

Section: Resultsmentioning

confidence: 99%

“…A final MolProbity check was completed to assess stereochemistry (13). To assess fit to density, cross-correlation was computed during phenix.real_space_refine (4) and an EMRinger was computed (14) (Table S1). …”

Section: Methodsmentioning

confidence: 99%

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

Hryc

Chen

Afonine

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

113

118

View full text Add to dashboard Cite

show abstract

“…The correlations for both the FSC and the per-residue assessment showed improvements when properly weighted, further demonstrating that our model provides a good approximation of the experimental data. Finally, we computed an EM-ringer score (42).…”

Section: Methodsmentioning

confidence: 99%

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

Roh

Hryc

Jeong

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

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-Å 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.nderstanding the function of a molecular machine typically requires determination of the structural components at atomic or near-atomic resolution. Although X-ray crystallography can provide atomic details, the crystal lattice forces may unnaturally constrain the structure. Moreover, solution methods, such as NMR spectroscopy, can reveal short-range dynamic behavior in the absence of information on the entire complex (1, 2). Electron cryo-microscopy (cryo-EM) has made substantial progress toward achieving atomic resolution for a broad range of molecular machines by averaging thousands to millions of single-particle images that are assumed to have identical conformations (3-6).Cryo-EM has also been used to determine multiple structures of a single specimen preparation containing conformational and compositional heterogeneity (7,8). The focused classification and refinement approaches (7, 8) have been used for two primary purposes: sorting out localized structural heterogeneity among particles (9, 10) and improving feature resolvability of flexible domains through local refinement (11, 12). These molecular machines frequently are composed of multiple protein subunits that can generate correlated and/or uncorrelated motions (13), and no experimental technique has yet captured the atomic structures of individual subunits within a single molecular machine in solution. GroEL, Escherichia coli chaperonin (13,14), are composed of 14 chemically identical protomers (13,14) and have been structurally characterized by both X-ray crystallography (15, 16) and cryo-EM (5, 17-19). However, these studies have not revealed any heterogeneity among the protomers in each oligomer. Here we used a 3D classification strategy for cryo-EM data (10) to investigate structural heterogeneity among chemically identical subunits and their conformational v...

show abstract

“…Binding to Raptor requires RagA/B to be GTP loaded, while RagC/D must be GDP loaded. Nutrients are thought to induce the RagA/B GTP -RagC/D GDP active state via a series of dedicated sensors that, in turn, control GTPase Activating Proteins (GAPs) and guanine nucleotide exchange factors (GEFs) specific for either Rag component (Bar-Peled et al, 2012;Barad et al, 2015;Chantranupong et al, 2016;Saxton et al, 2016;Wolfson et al, 2016;Zoncu et al, 2011). For example, the Gator1 complex has been shown to function as a GAP that promotes GTP hydrolysis by RagA/B, thus causing mTORC1 detachment from the lysosome when nutrient levels are low Panchaud et al, 2013).…”

Section: Introductionmentioning

confidence: 99%