Three-dimensional (3D) structure determination by single particle electron cryomicroscopy (cryoEM) involves the calculation of an initial 3D model, followed by extensive iterative improvement of the orientation determination of the individual particle images and the resulting 3D map. Because there is much more noise than signal at high resolution in the images, this creates the possibility of noise reinforcement in the 3D map, which can give a false impression of the resolution attained. The balance between signal and noise in the final map at its limiting resolution depends on the image processing procedure and is not easily predicted. There is a growing awareness in the cryoEM community of how to avoid such over-fitting and over-estimation of resolution. Equally, there has been a reluctance to use the two principal methods of avoidance because they give lower resolution estimates, which some people believe are too pessimistic. Here we describe a simple test that is compatible with any image processing protocol. The test allows measurement of the amount of signal and the amount of noise from overfitting that is present in the final 3D map. We have applied the method to two different sets of cryoEM images of the enzyme beta-galactosidase using several image processing packages. Our procedure involves substituting the Fourier components of the initial particle image stack beyond a chosen resolution by either the Fourier components from an adjacent area of background, or by simple randomisation of the phases of the particle structure factors. This substituted noise thus has the same spectral power distribution as the original data. Comparison of the Fourier Shell Correlation (FSC) plots from the 3D map obtained using the experimental data with that from the same data with high-resolution noise (HR-noise) substituted allows an unambiguous measurement of the amount of overfitting and an accompanying resolution assessment. A simple formula can be used to calculate an unbiased FSC from the two curves, even when a substantial amount of overfitting is present. The approach is software independent. The user is therefore completely free to use any established method or novel combination of methods, provided the HR-noise test is carried out in parallel. Applying this procedure to cryoEM images of beta-galactosidase shows how overfitting varies greatly depending on the procedure, but in the best case shows no overfitting and a resolution of ~6 Å. (382 words)

The ESCRT machinery mediates sorting of ubiquitinated transmembrane proteins to lysosomes via multivesicular bodies (MVBs) and also has roles in cytokinesis and viral budding. The ESCRT-III subunits are metastable monomers that transiently assemble on membranes. However, the nature of these assemblies is unknown. Among the core yeast ESCRT-III subunits, Snf7 and Vps24 spontaneously form ordered polymers in vitro. Single-particle EM reconstruction of helical Vps24 filaments shows both parallel and head-to-head subunit arrangements. Mutations of regions involved in intermolecular assembly in vitro result in cargo-sorting defects in vivo, suggesting that these homopolymers mimic interactions formed by ESCRT-III heteropolymers during MVB biogenesis. The C terminus of Vps24 is at the surface of the filaments and is not required for filament assembly. When this region is replaced by the MIT-interacting motif from the Vps2 subunit of ESCRT-III, the AAA-ATPase Vps4 can both bundle and disassemble the chimeric filaments in a nucleotide-dependent fashion.

SummaryThe 18 kDa TSPO protein is a polytopic mitochondrial outer membrane protein involved in a wide range of physiological functions and pathologies, including neurodegeneration and cancer. The pharmacology of TSPO has been extensively studied, but little is known about its biochemistry, oligomeric state, and structure. We have expressed, purified, and characterized a homologous protein, TspO from Rhodobacter sphaeroides, and reconstituted it as helical crystals. Using electron cryomicroscopy and single-particle helical reconstruction, we have determined a three-dimensional structure of TspO at 10 Å resolution. The structure suggests that monomeric TspO comprises five transmembrane α helices that form a homodimer, which is consistent with the dimeric state observed in detergent solution. Furthermore, the arrangement of transmembrane domains of individual TspO subunits indicates a possibility of two substrate translocation pathways per dimer. The structure provides the first insight into the molecular architecture of TSPO/PBR protein family that will serve as a framework for future studies.

Homologous DNA recombination (HR) by the RAD51 recombinase enables error-free DNA break repair. To execute HR, RAD51 first forms a presynaptic filament on single-stranded (ss) DNA, which catalyses pairing with homologous double-stranded (ds) DNA. Here, we report a structure for the presynaptic human RAD51 filament at 3.5–5.0Å resolution using electron cryo-microscopy. RAD51 encases ssDNA in a helical filament of 103Å pitch, comprising 6.4 protomers per turn, with a rise of 16.1Å and a twist of 56.2°. Inter-protomer distance correlates with rotation of an α-helical region in the core catalytic domain that is juxtaposed to ssDNA, suggesting how the RAD51–DNA interaction modulates protomer spacing and filament pitch. We map Fanconi anaemia-like disease-associated RAD51 mutations, clarifying potential phenotypes. We predict binding sites on the presynaptic filament for two modules present in each BRC repeat of the BRCA2 tumour suppressor, a critical HR mediator. Structural modelling suggests that changes in filament pitch mask or expose one binding site with filament-inhibitory potential, rationalizing the paradoxical ability of the BRC repeats to either stabilize or inhibit filament formation at different steps during HR. Collectively, our findings provide fresh insight into the structural mechanism of HR and its dysregulation in human disease.

A widely studied achiral porphyrin, which is highly soluble in aqueous solutions (TPPS4), is shown to self‐assemble into helical nanotubes. These were imaged by electron cryo‐microscopy and a state‐of‐the‐art image analysis allows building a map at ∼5 Å resolution, one of the highest obtained so far for molecular materials. The authors were able to trace the apparent symmetry breaking to existing nuclei in the “as received samples”, while carefully purified samples show that both handnesses occur in equal amounts.

The breast and ovarian cancer suppressor BRCA2 controls the enzyme RAD51 during homologous DNA recombination (HDR) to preserve genome stability. BRCA2 binds to RAD51 through 8 conserved BRC repeat motifs dispersed in an 1127-residue region (BRCA2 targets RAD51 to ssDNA while inhibiting dsDNA binding and that these contrasting activities together bolster one another to stimulate HDR. Our work provides fresh insight into the mechanism of HDR in humans, and its regulation by the BRCA2 tumor suppressor.DNA recombination ͉ electron microscopy ͉ single-molecule fluorescence spectroscopy ͉ tumor suppressor H omologous DNA recombination (HDR) is essential in somatic cells not only for the error-free repair of DNA double-strand breaks (DSBs), but also for the restoration of DNA replication forks that stall at lesions in the template strand (1). The central event in HDR is the synapsis of a single-stranded (ss)DNA molecule with homologous duplex DNA, which initiates the strand exchange that leads to recombination. Recombinase molecules, conserved from RecA in prokarya to RAD51 in eukaryal cells, mediate strand exchange via distinct reactions grouped into the presynaptic, synaptic, and postsynaptic phases (2).The breast cancer suppressor protein, BRCA2, an ϳ384-kDa molecule of 3418 residues, is essential in vivo for RAD51-mediated HDR (3). Several regions of BRCA2 may contribute to this role (4). Human BRCA2 interacts with RAD51 through 8 copies of the BRC repeat motifs of Ϸ40 residues each, embedded in a 1127-residue fragment (BRCA2 ) within exon 11 (5), as well as through an unrelated carboxyl-terminal motif in exon 27 (6). The BRC repeats appear to be the primary locus for the BRCA2-RAD51 interaction; unlike the C-terminal motif, their sequence is evolutionarily conserved in simple eukaryotes like fungi, as well as in plants (7). In addition, exon 17 of human BRCA2 encodes an Ϸ800 residue DNA-binding domain (BRCA2 DBD ) containing 3 oligonucleotide-binding (OB) folds, ssDNA-binding modules also found in the abundant ssDNA-binding protein, replication protein A (RPA) (8). In a fungal BRCA2 homolog, the OB-foldcontaining, ssDNA-binding domain displaces RPA from DNA substrates in vitro to enable nucleation of the RAD51 filament by the single BRC repeat found in this organism (9). Moreover, fragments containing 2 to 4 of the 8 human BRC repeats fused to the ssDNA-binding BRCA2 [DBD] domain can partially complement HDR in BRCA2-deficient cells in vivo (10) and promote RAD51-dependent strand exchange in vitro (11). Together, these observations suggest that the BRCA2 [DBD] cooperates with the BRC repeats to target RAD51 to DNA substrates, acting as a recombination mediator.The precise role in HDR played by the multi-BRC repeat region of human BRCA2 is not yet clear. Indeed, the BRC repeats are reported to have activities that could potentially inhibit (12-14) as well as stimulate (11, 15, 16) RAD51 function, under different experimental conditions. We have previously purified the exon 11 encoded 1,127-residue domain (BRCA2...

The MRC binary file format is widely used in the three-dimensional electron microscopy field for storing image and volume data. Files contain a header which describes the kind of data held, together with other important metadata. In response to advances in electron microscopy techniques, a number of variants to the file format have emerged which contain useful additional data, but which limit interoperability between different software packages. Following extensive discussions, the authors, who represent leading software packages in the field, propose a set of extensions to the MRC format standard designed to accommodate these variants, while restoring interoperability. The MRC format is equivalent to the map format used in the CCP4 suite for macromolecular crystallography, and the proposal also maintains interoperability with crystallography software. This Technical Note describes the proposed extensions, and serves as a reference for the standard.

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