Single-particle electron cryo-microscopy (cryo-EM) has become a popular method for high-resolution study of the structural and functional properties of proteins. However, sufficient expression and purification of membrane proteins holds many challenges. We describe methods to overcome these obstacles using ClC-rm1, a prokaryotic chloride channel (ClC) family protein from Ralstonia metallidurans, overexpressed in Escherichia coli (E. coli) BL21(DE3) strain. Mass spectrometry and electron microscopy analyses of purified samples revealed multiple contaminants that can obfuscate results of subsequent high-resolution structural analysis. Here we describe the systematic optimization of sample preparation procedures, including expression systems, solubilization techniques, purification protocols, and contamination detection. We found that expressing ClC-rm1 in E. coli BL21(DE3) and using n-dodecyl-β-D-maltopyranoside as a detergent for solubilization and purification steps resulted in the highest quality samples of those we tested. However, although protein yield, sample stability, and the resolution of structural detail were improved following these changes, we still detected contaminants including Acriflavine resistant protein AcrB. AcrB was particularly difficult to remove as it co-purified with ClC-rm1 due to four intrinsic histidine residues at its C-terminus that bind to affinity resins. We were able to obtain properly folded pure ClC-rm1 by adding eGFP to the C-terminus and overexpressing the protein in the ΔacrB variant of the JW0451-2 E. coli strain.

Frealign is a software tool designed to process electron microscope images of single molecules and complexes to obtain reconstructions at the highest possible resolution. It provides a number of refinement parameters and options that allow users to tune their refinement to achieve specific goals, such as masking to classify selected regions within a particle, control over the refinement of specific alignment parameters to accommodate various data collection schemes, refinement of pseudosymmetric particles, and generation of initial maps. This chapter provides a general overview of Frealign functions and a more detailed guide to using Frealign in typical scenarios.

The structures of many helical protein filaments can be derived from electron micrographs of their suspensions in thin films of vitrified aqueous solutions. The most successful and generally-applicable approach treats short segments of these filaments as independent "single particles", yielding near-atomic resolution for rigid and well-ordered filaments. The single-particle approach can also accommodate filament deformations, yielding sub-nanometer resolution for more flexible filaments. However, in the case of thin and flexible filaments, such as some amyloid-β (Aβ) fibrils, the single-particle approach may fail because helical segments can be curved or otherwise distorted and their alignment can be inaccurate due to low contrast in the micrographs. We developed new software called Frealix that allows the use of arbitrarily short filament segments during alignment to approximate even high curvatures. All segments in a filament are aligned simultaneously with constraints that ensure that they connect to each other in space to form a continuous helical structure. In this paper, we describe the algorithm and benchmark it against datasets of Aβ(1-40) fibrils and tobacco mosaic virus (TMV), both analyzed in earlier work. In the case of TMV, our algorithm achieves similar results to single-particle analysis. In the case of Aβ(1-40) fibrils, we match the previously-obtained resolution but we are also able to obtain reliable alignments and \~{}8-Å reconstructions from curved filaments. Our algorithm also offers a detailed characterization of filament deformations in three dimensions and enables a critical evaluation of the worm-like chain model for biological filaments.

Among all the factors that determine the resolution of a 3D reconstruction by single particle electron cryo-microscopy (cryoEM), the number of particle images used in the dataset plays a major role. More images generally yield better resolution, assuming the imaged protein complex is conformationally and compositionally homogeneous. To facilitate processing of very large datasets, we modified the computer program, FREALIGN, to execute the computationally most intensive procedures on Graphics Processing Units (GPUs). Using the modified program, the execution speed increased between 10 and 240-fold depending on the task performed by FREALIGN. Here we report the steps necessary to parallelize critical FREALIGN subroutines and evaluate its performance on computers with multiple GPUs.

Noroviruses are a leading cause of foodborne illnesses worldwide. Although GII.4 strains have been responsible for most norovirus outbreaks, the assembled virus shell structures have been available in detail for only a single strain (GI.1). We present high-resolution (2.6- to 4.1-Å) cryoelectron microscopy (cryo-EM) structures of GII.4, GII.2, GI.7, and GI.1 human norovirus outbreak strain virus-like particles (VLPs). Although norovirus VLPs have been thought to exist in a single-sized assembly, our structures reveal polymorphism between and within genogroups, with small, medium, and large particle sizes observed. Using asymmetric reconstruction, we were able to resolve a Zn2+ metal ion adjacent to the coreceptor binding site, which affected the structural stability of the shell. Our structures serve as valuable templates for facilitating vaccine formulations.

The resolution of subtomogram averages calculated from cryo-electron tomograms (cryo-ET) of crowded cellular environments is often limited owing to signal loss in, and misalignment of, the subtomograms. By contrast, single-particle cryo-electron microscopy (SP-cryo-EM) routinely reaches near-atomic resolution of isolated complexes. We report a method called 'tomography-guided 3D reconstruction of subcellular structures' (TYGRESS) that is a hybrid of cryo-ET and SP-cryo-EM, and is able to achieve close-to-nanometer resolution of complexes inside crowded cellular environments. TYGRESS combines the advantages of SP-cryo-EM (images with good signal-to-noise ratio and contrast, as well as minimal radiation damage) and subtomogram averaging (three-dimensional alignment of macromolecules in a complex sample). Using TYGRESS, we determined the structure of the intact ciliary axoneme with up to resolution of 12 Å. These results reveal many structural details that were not visible by cryo-ET alone. TYGRESS is generally applicable to cellular complexes that are amenable to subtomogram averaging.

Rotaviruses, like other non-enveloped, double-strand RNA (dsRNA) viruses, package an RNA-dependent RNA polymerase (RdRp) with each duplex of their segmented genomes. Rotavirus cell entry results in loss of an outer protein layer and delivery into the cytosol of an intact, inner capsid particle (the “double-layer particle” or DLP). The RdRp, designated VP1, is active inside the DLP; each VP1 achieves many rounds of mRNA transcription from its associated genome segment. Previous work has shown that one VP1 molecule lies close to each fivefold axis of the icosahedrally symmetric DLP, just beneath the inner surface of its protein shell, embedded in tightly packed RNA. We have determined a high-resolution structure for the rotavirus VP1 RdRp in situ, by local reconstruction of density around individual fivefold positions. We have analyzed intact virions (“triple-layer particles” or TLPs), non-transcribing DLPs and transcribing DLPs. Outer layer dissociation enables the DLP to synthesize RNA, in vitro as well as in vivo, but appears not to induce any detectable structural change in the RdRp. Addition of NTPs, Mg2+, and S-adenosyl methionine, which allows active transcription, results in conformational rearrangements, in both VP1 and the DLP capsid shell protein, that allow a transcript to exit the polymerase and the particle. The position of VP1 (among the five symmetrically related alternatives) at one vertex does not correlate with its position at other vertices. This stochastic distribution of site occupancies limits long-range order in the 11-segment, dsRNA genome.

Many DNA transactions are crucial for maintaining genomic integrity and faithful transfer of genetic information but remain poorly understood. An example is the interplay between nucleotide excision repair (NER) and transcription, also known as transcription-coupled DNA repair (TCR). Discovered decades ago, the mechanisms for TCR have remained elusive, not in small part due to the scarcity of structural studies of key players. Here we summarize recent structural information on NER/TCR factors, focusing on bacterial systems, and integrate it with existing genetic, biochemical, and biophysical data to delineate the mechanisms at play. We also review emerging, alternative modalities for recruitment of NER proteins to DNA lesions.

We describe an implementation of maximum likelihood classification for single particle electron cryo-microscopy that is based on the FREALIGN software. Particle alignment parameters are determined by maximizing a joint likelihood that can include hierarchical priors, while classification is performed by expectation maximization of a marginal likelihood. We test the FREALIGN implementation using a simulated dataset containing computer-generated projection images of three different 70S ribosome structures, as well as a publicly available dataset of 70S ribosomes. The results show that the mixed strategy of the new FREALIGN algorithm yields performance on par with other maximum likelihood implementations, while remaining computationally efficient.

Double-stranded RNA (dsRNA) viruses transcribe and replicate RNA within an assembled, inner capsid particle; only plus-sense mRNA emerges into the intracellular milieu. During infectious entry of a rotavirus particle, the outer layer of its three-layer structure dissociates, delivering the inner double-layered particle (DLP) into the cytosol. DLP structures determined by X-ray crystallography and electron cryomicroscopy (cryoEM) show that the RNA coils uniformly into the particle interior, avoiding a "fivefold hub" of more structured density projecting inward from the VP2 shell of the DLP along each of the twelve 5-fold axes. Analysis of the X-ray crystallographic electron density map suggested that principal contributors to the hub are the N-terminal arms of VP2, but reexamination of the cryoEM map has shown that many features come from a molecule of VP1, randomly occupying five equivalent and partly overlapping positions. We confirm here that the electron density in the X-ray map leads to the same conclusion, and we describe the functional implications of the orientation and position of the polymerase. The exit channel for the nascent transcript directs the nascent transcript toward an opening along the 5-fold axis. The template strand enters from within the particle, and the dsRNA product of the initial replication step exits in a direction tangential to the inner surface of the VP2 shell, allowing it to coil optimally within the DLP. The polymerases of reoviruses appear to have similar positions and functional orientations.