A maximum-likelihood approach to multi-reference image refinement is presented. In contrast to conventional cross-correlation refinement, the new approach includes a formal description of the noise, implying that it is especially suited to cases with low signal-to-noise ratios. Application of this approach to a cryo-electron microscopy dataset revealed two major classes for projections of simian virus 40 large T-antigen in complex with an asymmetric DNA-probe, containing the origin of simian virus 40 replication. Strongly bent projections of dodecamers showed density that may be attributed to the complexed double-stranded DNA, while almost straight projections revealed a twist in the relative orientation of the hexameric subunits. This new level of detail for large T-antigen projections was not detected using conventional techniques. For a negative stain dataset, maximum-likelihood refinement yielded results that were practically identical to those obtained using conventional multi-reference refinement. Results obtained using simulated data suggest that the efficiency of the maximum-likelihood approach may be further enhanced by explicitly incorporating the microscope contrast transfer function in the image formation model.

www.elsevier.com/locate/yjsbi A novel method for improvement of visualization of power spectra for sorting cryo-electron micrographs and their local areas

www.elsevier.com/locate/patcog Improved Bayesian image denoising based on wavelets with applications to electron microscopy

www.elsevier.com/locate/yjsbi Fast, robust, and accurate determination of transmission electron microscopy contrast transfer function

journal homepage: www.elsevier.com/locate/yjsbi Exploiting desktop supercomputing for three-dimensional electron microscopy

journal homepage: www.elsevier.com/locate/yjsbi Automatic particle selection from electron micrographs using machine

Three-dimensional structure of a wide range of biological specimens can be computed from images collected by transmission electron microscopy. This information integrated with structural data obtained with other techniques (e.g., X-ray crystallography) helps structural biologists to understand the function of macromolecular complexes and organelles within cells. In this paper, we compare two threedimensional transmission electron microscopy techniques that are becoming more and more related (at the image acquisition level as well as the image processing one): electron tomography and single-particle analysis. The first one is currently used to elucidate the three-dimensional structure of cellular components or smaller entire cells, whereas the second one has been traditionally applied to structural studies of macromolecules and macromolecular complexes. Also, we discuss possibilities for their integration with other structural biology techniques for an integrative study of living matter from proteins to whole cells.

Abstract not found

DNA replication is strictly regulated through a sequence of steps that involve many macromolecular protein complexes. One of them is the replicative helicase, which is required for initiation and elongation phases. A MCM helicase found as a prophage in the genome of Bacillus cereus is fused with a primase domain constituting an integrative arrangement of two essential activities for replication. We have isolated this helicase–primase complex (BcMCM) showing that it can bind DNA and displays not only helicase and primase but also DNA polymerase activity. Using single-particle electron microscopy and 3D reconstruction, we obtained structures of BcMCM using ATPcS or ADP in the absence and presence of DNA. The complex depicts the typical hexameric ring shape. The dissection of the unwinding mechanism using site-directed mutagenesis in the Walker A, Walker B, arginine finger and the helicase channels, suggests that the BcMCM complex unwinds DNA following the extrusion model similarly to the E1 helicase from papillomavirus.