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 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.

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 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.

Improved electron detectors and image-processing techniques will allow the structures of macromolecules to be determined from tens of thousands of single-particle cryo-EM images, rather than the hundreds of thousands needed previously.

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.

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.