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

Abstract

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...

High-Resolution Cryo-EM Maps and Models: A Crystallographer's Perspective

“…We would like to postulate that it might be better to leave B factors undefined than to deposit their values that cannot represent any physical reality. The treatment of B factors may improve in the future if reciprocal-space refinement of the cryo-EM models becomes a standard procedure (Hryc et al, 2017). …”

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

“…S2). To perform this assessment, we generated a weighted map, derived solely from an atomic model that accounted for both ADP of all atoms and weak/negative density of all charged oxygen atoms, and compared it with the experimental map (41). The weighted map provides a better approximation of the experimental map by simulating map variability as opposed to treating all atoms equally.…”

Flexible Connectors between Capsomer Subunits that Regulate Capsid Assembly

“…E-loop-like structural elements occur in many capsids which range in size from T=1 to T=16 and include the well-studied bacteriophages P22 [28–30], T4 [31], T5 [32], T7 [33], as well as epsilon15 [34], BPP-1 [35], Sf6 [36], three bacterial encapsulins [37–39], Herpesvirus nucleocapsids [40] and have been inferred in many others, including ϕ29[41] and P2[42]. While the E-loop was first revealed as a structural element involved in forming the covalent bonds that stabilize the HK97 capsid [16], none of the examples above use such covalent crosslinks, so crosslinking is probably only a minor, late-acquired role for the E-loop.…”