Disentangling conformational states of macromolecules in 3D-EM through likelihood optimization

Although three-dimensional electron microscopy (3D-EM) permits structural characterization of macromolecular assemblies in distinct functional states, the inability to classify projections from structurally heterogeneous samples has severely limited its application. We present a maximum likelihood-based classification method that does not depend on prior knowledge about the structural variability, and demonstrate its effectiveness for two macromolecular assemblies with different types of conformational variability: the Escherichia coli ribosome and Simian virus 40 (SV40) large T-antigen.     

Simplified method for the collection, storage, and comet assay analysis of DNA damage in whole blood

Single-cell gel electrophoresis (comet assay) is one of the most common methods used to measure oxidatively damaged DNA in peripheral blood mononuclear cells (PBMC), as a biomarker of oxidative stress in vivo. However, storage, extraction, and assay workup of blood samples are associated with a risk of artifactual formation of damage. Previous reports using this approach to study DNA damage in PBMC have, for the most part, required the isolation of PBMC before immediate analysis or freezing in cryopreservative. This is very time-consuming and a significant drain on human resources. Here, we report the successful storage of whole blood in ~ 250 μl volumes, at − 80 °C, without cryopreservative, for up to 1 month without artifactual formation of DNA damage. Furthermore, this blood is amenable for direct use in both the alkaline and the enzyme-modified comet assay, without the need for prior isolation of PBMC. In contrast, storage of larger volumes (e.g., 5 ml) of whole blood leads to an increase in damage with longer term storage even at − 80 °C, unless a cryopreservative is present. Our “small volume” approach may be suitable for archived blood samples, facilitating analysis of biobanks when prior isolation of PBMC has not been performed.     

A Bayesian view on cryo-EM structure determination

Three-dimensional (3D) structure determination by single-particle analysis of cryo-electron microscopy (cryo-EM) images requires many parameters to be determined from extremely noisy data. This makes the method prone to overfitting, that is, when structures describe noise rather than signal, in particular near their resolution limit where noise levels are highest. Cryo-EM structures are typically filtered using ad hoc procedures to prevent overfitting, but the tuning of arbitrary parameters may lead to subjectivity in the results. I describe a Bayesian interpretation of cryo-EM structure determination, where smoothness in the reconstructed density is imposed through a Gaussian prior in the Fourier domain. The statistical framework dictates how data and prior knowledge should be combined, so that the optimal 3D linear filter is obtained without the need for arbitrariness and objective resolution estimates may be obtained. Application to experimental data indicates that the statistical approach yields more reliable structures than existing methods and is capable of detecting smaller classes in data sets that contain multiple different structures.     

Electron microscopy of Xrcc4 and the DNA ligase IV–Xrcc4 DNA repair complex

The DNA ligase IV–Xrcc4 complex is responsible for the ligation of broken DNA ends in the non-homologous end-joining (NHEJ) pathway of DNA double strand break repair in mammals. Mutations in DNA ligase IV (Lig4) lead to immunodeficiency and radiosensitivity in humans. Only partial structural information for Lig4 and Xrcc4 is available, while the structure of the full-length proteins and their arrangement within the Lig4–Xrcc4 complex is unknown. The C-terminal domain of Xrcc4, whose structure has not been solved, contains phosphorylation sites for DNA-PKcs and is phylogenetically conserved, indicative of a regulatory role in NHEJ. Here, we have purified full length Xrcc4 and the Lig4–Xrcc4 complex, and analysed their structure by single-particle electron microscopy. The three-dimensional structure of Xrcc4 at a resolution of ～37 Å reveals that the C-terminus of Xrcc4 forms a dimeric globular domain connected to the N-terminus by a coiled-coil. The N- and C-terminal domains of Xrcc4 locate at opposite ends of an elongated molecule. The electron microscopy images of the Lig4–Xrcc4 complex were examined by two-dimensional image processing and a double-labelling strategy, identifying the site of the C-terminus of Xrcc4 and the catalytic core of Lig4 within the complex. The catalytic domains of Lig4 were found to be in the vicinity of the N-terminus of Xrcc4. We provide a first sight of the structural organization of the Lig4–Xrcc4 complex, which suggests that the BRCT domains could provide the link of the ligase to Xrcc4 while permitting some movements of the catalytic domains of Lig4. This arrangement may facilitate the ligation of diverse configurations of damaged DNA.     

Subunit Positioning and Stator Filament Stiffness in Regulation and Power Transmission in the V1 Motor of the Manduca sexta V-ATPase

The vacuolar H⁺-ATPase (V-ATPase) is an ATP-driven proton pump essential to the function of eukaryotic cells. Its cytoplasmic V₁ domain is an ATPase, normally coupled to membrane-bound proton pump V_o via a rotary mechanism. How these asymmetric motors are coupled remains poorly understood. Low energy status can trigger release of V₁ from the membrane and curtail ATP hydrolysis. To investigate the molecular basis for these processes, we have carried out cryo-electron microscopy three-dimensional reconstruction of deactivated V₁ from Manduca sexta. In the resulting model, three peripheral stalks that are parts of the mechanical stator of the V-ATPase are clearly resolved as unsupported filaments in the same conformations as in the holoenzyme. They are likely therefore to have inherent stiffness consistent with a role as flexible rods in buffering elastic power transmission between the domains of the V-ATPase. Inactivated V₁ adopted a homogeneous resting state with one open active site adjacent to the stator filament normally linked to the H subunit. Although present at 1:1 stoichiometry with V₁, both recombinant subunit C reconstituted with V₁ and its endogenous subunit H were poorly resolved in three-dimensional reconstructions, suggesting structural heterogeneity in the region at the base of V₁ that could indicate positional variability. If the position of H can vary, existing mechanistic models of deactivation in which it binds to and locks the axle of the V-ATPase rotary motor would need to be re-evaluated.     

Classification of single-projection reconstructions for cryo-electron microscopy data of icosahedral viruses

We present a novel strategy for classification of heterogeneous electron microscopy data of icosahedral virus particles. The effectiveness of the procedure, which is based on classification of single-projection reconstructions (SPRs), is first investigated using simulated data. Of several reconstruction approaches examined, best results were obtained with algebraic reconstruction techniques (ART) when providing prior information about the reconstruction in the form of a starting volume. The results presented indicate that SPR-classification is sufficiently sensitive to classify assemblies with differences of only a few percent of the total mass. The usefulness of this procedure is illustrated by application to a heterogeneous cryo-electron microscopy dataset of adenovirus mutant dl313, lacking minor coat protein IX. These data were successfully divided into two distinct classes, in agreement with gel analysis and immuno-electron microscopy results. The classes yielded a wildtype-like reconstruction and a reconstruction representing the polypeptide IX-deficient dl313 virion. As the largest difference between these volumes is found at the location previously assigned to the external portion of minor coat protein polypeptide IIIa, questions arise concerning the current adenovirus model.     

SPI-EM: towards a tool for predicting CATH superfamilies in 3D-EM maps

In this paper the theoretical framework used to build a superfamily probability in electron microscopy (SPI-EM) is presented. SPI-EM is a new tool for determining the homologous superfamily to which a protein domain belongs looking at its three-dimensional electron microscopy map. The homologous superfamily is assigned according to the domain-architecture database CATH. Our method follows a probabilistic approach applied to the results of fitting protein domains into maps of proteins and the computation of local cross-correlation coefficient measures. The method has been tested and its usefulness proven with isolated domains at a resolution of 8 A and 12 A. Results obtained with simulated and experimental data at 10 A suggest that it is also feasible to detect the correct superfamily of the domains when dealing with electron microscopy maps containing multi-domain proteins. The inherent difficulties and limitations that multi-domain proteins impose are discussed. Our procedure is complementary to other techniques existing in the field to detect structural elements in electron microscopy maps like alpha-helices and beta-sheets. Based on the proposed methodology, a database of relevant distributions is being built to serve the community.