Cryo-electron microscopy (cryo-EM) of single-particle specimens is used to determine the structure of proteins and macromolecular complexes without the need for crystals. Recent advances in detector technology and software algorithms now allow images of unprecedented quality to be recorded and structures to be determined at near-atomic resolution. However, compared with X-ray crystallography, cryo-EM is a young technique with distinct challenges. This primer explains the different steps and considerations involved in structure determination by single-particle cryo-EM to provide an overview for scientists wishing to understand more about this technique and the interpretation of data obtained with it, as well as a starting guide for new practitioners.

Amyloid fibrils characterize a diverse group of human diseases that includes Alzheimer’s disease, Creutzfeldt-Jakob and type II diabetes. Alzheimer’s amyloid fibrils consist of amyloid-beta (Abeta) peptide and occur in a range of structurally different fibril morphologies. The structural characteristics of 12 single Abeta(1-40) amyloid fibrils, all formed under the same solution conditions, were determined by electron cryo-microscopy and three-dimensional reconstruction. The majority of analyzed fibrils form a range of morphologies that show almost continuously altering structural properties. The observed fibril polymorphism implies that amyloid formation can lead, for the same polypeptide sequence, to many different patterns of inter- or intra-residue interactions. This property differs significantly from native, monomeric protein folding reactions that produce, for one protein sequence, only one ordered conformation and only one set of inter-residue interactions.

The physiology of N-methyl-d-aspartate (NMDA) receptors is fundamental to brain development and function. NMDA receptors are ionotropic glutamate receptors that function as heterotetramers composed mainly of GluN1 and GluN2 subunits. Activation of NMDA receptors requires binding of neurotransmitter agonists to a ligand-binding domain (LBD) and structural rearrangement of an amino-terminal domain (ATD). Recent crystal structures of GluN1-GluN2B NMDA receptors bound to agonists and an allosteric inhibitor, ifenprodil, represent the allosterically inhibited state. However, how the ATD and LBD move to activate the NMDA receptor ion channel remains unclear. Here we applied X-ray crystallography, single-particle electron cryomicroscopy and electrophysiology to rat NMDA receptors to show that, in the absence of ifenprodil, the bi-lobed structure of GluN2 ATD adopts an open conformation accompanied by rearrangement of the GluN1-GluN2 ATD heterodimeric interface, altering subunit orientation in the ATD and LBD and forming an active receptor conformation that gates the ion channel.

The tumor suppressor protein adenomatous polyposis coli (APC) regulates cell protrusion and cell migration, processes that require the coordinated regulation of actin and microtubule dynamics. APC localizes in vivo to microtubule plus ends and actin-rich cortical protrusions, and has well-documented direct effects on microtubule dynamics. However, its potential effects on actin dynamics have remained elusive. Here, we show that the C-terminal "basic" domain of APC (APC-B) potently nucleates the formation of actin filaments in vitro and stimulates actin assembly in cells. Nucleation is achieved by a mechanism involving APC-B dimerization and recruitment of multiple actin monomers. Further, APC-B nucleation activity is synergistic with its in vivo binding partner, the formin mDia1. Together, APC-B and mDia1 overcome a dual cellular barrier to actin assembly imposed by profilin and capping protein. These observations define a new function for APC and support an emerging view of collaboration between distinct actin assembly-promoting factors with complementary activities.

The Wiener filter is a standard means of optimizing the signal in sums of aligned, noisy images obtained by electron cryo-microscopy (cryo-EM). However, estimation of the resolution-dependent ("spectral") signal-to-noise ratio (SSNR) from the input data has remained problematic, and error reduction due to specific application of the SSNR term within a Wiener filter has not been reported. Here we describe an adjustment to the Wiener filter for optimal summation of images of isolated particles surrounded by large regions of featureless background, as is typically the case in single-particle cryo-EM applications. We show that the density within the particle area can be optimized, in the least-squares sense, by scaling the SSNR term found in the conventional Wiener filter by a factor that reflects the fraction of the image field occupied by the particle. We also give related expressions that allow the SSNR to be computed for application in this new filter, by incorporating a masking step into a Fourier Ring Correlation (FRC), a standard resolution measure. Furthermore, we show that this masked FRC estimation scheme substantially improves on the accuracy of conventional SSNR estimation methods. We demonstrate the validity of our new approach in numeric tests with simulated data corresponding to realistic cryo-EM imaging conditions. This variation of the Wiener filter and accompanying derivation should prove useful for a variety of single-particle cryo-EM applications, including 3D reconstruction.

Single-particle electron cryo-microscopy and computational image classification can be used to analyze structural variability in macromolecules and their assemblies. In some cases, a particle may contain different regions that each display a range of distinct conformations. We have developed strategies, implemented within the Frealign and cisTEM image processing packages, to focus classify on specific regions of a particle and detect potential covariance. The strategies are based on masking the region of interest using either a 2-D mask applied to reference projections and particle images, or a 3-D mask applied to the 3-D volume. We show that focused classification approaches can be used to study structural covariance, a concept that is likely to gain more importance as datasets grow in size, allowing the distinction of more structural states and smaller differences between states. Finally, we apply the approaches to an experimental dataset containing the HIV-1 Transactivation Response (TAR) element RNA fused into the large bacterial ribosomal subunit to deconvolve structural mobility within localized regions of interest, and to a dataset containing assembly intermediates of the large subunit to measure structural covariance.

Non-enveloped viruses of different types have evolved distinct mechanisms for penetrating a cellular membrane during infection. Rotavirus penetration appears to occur by a process resembling enveloped-virus fusion: membrane distortion linked to conformational changes in a viral protein. Evidence for such a mechanism comes from crystallographic analyses of fragments of VP4, the rotavirus-penetration protein, and infectivity analyses of structure-based VP4 mutants. We describe here the structure of an infectious rotavirus particle determined by electron cryomicroscopy (cryoEM) and single-particle analysis at about 4.3 Å resolution. The cryoEM image reconstruction permits a nearly complete trace of the VP4 polypeptide chain, including the positions of most side chains. It shows how the two subfragments of VP4 (VP8(*) and VP5(*)) retain their association after proteolytic cleavage, reveals multiple structural roles for the β-barrel domain of VP5(*), and specifies interactions of VP4 with other capsid proteins. The virion model allows us to integrate structural and functional information into a coherent mechanism for rotavirus entry.

The advent of direct electron detectors has enabled the routine use of single-particle cryo-electron microscopy (EM) approaches to determine structures of a variety of protein complexes at near-atomic resolution. Here, we report the development of methods to account for local variations in defocus and beam-induced drift, and the implementation of a data-driven dose compensation scheme that significantly improves the extraction of high-resolution information recorded during exposure of the specimen to the electron beam. These advances enable determination of a cryo-EM density map for β-galactosidase bound to the inhibitor phenylethyl β-D-thiogalactopyranoside where the ordered regions are resolved at a level of detail seen in X-ray maps at ∼ 1.5 Å resolution. Using this density map in conjunction with constrained molecular dynamics simulations provides a measure of the local flexibility of the non-covalently bound inhibitor and offers further opportunities for structure-guided inhibitor design.

We demonstrate a significant anisotropic magnification distortion, found on an FEI Titan Krios microscope and affecting magnifications commonly used for data acquisition on a Gatan K2 Summit detector. We describe a program (mag_distortion_estimate) to automatically estimate anisotropic magnification distortion from a set of images of a standard gold shadowed diffraction grating. We also describe a program (mag_distortion_correct) to correct for the estimated distortion in collected images. We demonstrate that the distortion present on the Titan Krios microscope limits the resolution of a set of rotavirus VP6 images to ∼7 Å, which increases to ∼3 Å following estimation and correction of the distortion. We also use a 70S ribosome sample to demonstrate that in addition to affecting resolution, magnification distortion can also interfere with the classification of heterogeneous data.

The contrast observed in images of frozen-hydrated biological specimens prepared for electron cryo-microscopy falls significantly short of theoretical predictions. In addition to limits imposed by the current instrumentation, it is widely acknowledged that motion of the specimen during its exposure to the electron beam leads to significant blurring in the recorded images. We have studied the amount and direction of motion of virus particles suspended in thin vitrified ice layers across holes in perforated carbon films using exposure series. Our data show that the particle motion is correlated within patches of 0.3-0.5 μm, indicating that the whole ice layer is moving in a drum-like motion, with accompanying particle rotations of up to a few degrees. Support films with smaller holes, as well as lower electron dose rates tend to reduce beam-induced specimen motion, consistent with a mechanical effect. Finally, analysis of movies showing changes in the specimen during beam exposure show that the specimen moves significantly more at the start of an exposure than towards its end. We show how alignment and averaging of movie frames can be used to restore high-resolution detail in images affected by beam-induced motion.