Structure of the hDmc1-ssDNA filament reveals the principles of its architecture

In eukaryotes, meiotic recombination is a major source of genetic diversity, but its defects in humans lead to abnormalities such as Down's, Klinefelter's and other syndromes. Human Dmc1 (hDmc1), a RecA/Rad51 homologue, is a recombinase that plays a crucial role in faithful chromosome segregation during meiosis. The initial step of homologous recombination occurs when hDmc1 forms a filament on single-stranded (ss) DNA. However the structure of this presynaptic complex filament for hDmc1 remains unknown. To compare hDmc1-ssDNA complexes to those known for the RecA/Rad51 family we have obtained electron microscopy (EM) structures of hDmc1-ssDNA nucleoprotein filaments using single particle approach. The EM maps were analysed by docking crystal structures of Dmc1, Rad51, RadA, RecA and DNA. To fully characterise hDmc1-DNA complexes we have analysed their organisation in the presence of Ca2+, Mg2+, ATP, AMP-PNP, ssDNA and dsDNA. The 3D EM structures of the hDmc1-ssDNA filaments allowed us to elucidate the principles of their internal architecture. Similar to the RecA/Rad51 family, hDmc1 forms helical filaments on ssDNA in two states: extended (active) and compressed (inactive). However, in contrast to the RecA/Rad51 family, and the recently reported structure of hDmc1-double stranded (ds) DNA nucleoprotein filaments, the extended (active) state of the hDmc1 filament formed on ssDNA has nine protomers per helical turn, instead of the conventional six, resulting in one protomer covering two nucleotides instead of three. The control reconstruction of the hDmc1-dsDNA filament revealed 6.4 protein subunits per helical turn indicating that the filament organisation varies depending on the DNA templates. Our structural analysis has also revealed that the N-terminal domain of hDmc1 accomplishes its important role in complex formation through domain swapping between adjacent protomers, thus providing a mechanistic basis for coordinated action of hDmc1 protomers during meiotic recombination

Cryoelectron Tomography of HIV-1 Envelope Spikes: Further Evidence for Tripod-Like Legs

A detailed understanding of the morphology of the HIV-1 envelope (Env) spike is key to understanding viral pathogenesis and for informed vaccine design. We have previously presented a cryoelectron microscopic tomogram (cryoET) of the Env spikes on SIV virions. Several structural features were noted in the gp120 head and gp41 stalk regions. Perhaps most notable was the presence of three splayed legs projecting obliquely from the base of the spike head toward the viral membrane. Subsequently, a second 3D image of SIV spikes, also obtained by cryoET, was published by another group which featured a compact vertical stalk. We now report the cryoET analysis of HIV-1 virion-associated Env spikes using enhanced analytical cryoET procedures. More than 2,000 Env spike volumes were initially selected, aligned, and sorted into structural classes using algorithms that compensate for the “missing wedge” and do not impose any symmetry. The results show varying morphologies between structural classes: some classes showed trimers in the head domains; nearly all showed two or three legs, though unambiguous three-fold symmetry was not observed either in the heads or the legs. Subsequently, clearer evidence of trimeric head domains and three splayed legs emerged when head and leg volumes were independently aligned and classified. These data show that HIV-1, like SIV, also displays the tripod-like leg configuration, and, unexpectedly, shows considerable gp41 leg flexibility/heteromorphology. The tripod-like model for gp41 is consistent with, and helps explain, many of the unique biophysical and immunological features of this region

A new method for the determination of the wave aberration function for high resolution TEM 1. Measurement of the symmetric aberrations.

A new method for the accurate determination of the symmetric coefficients of the wave aberration function has been developed. The relative defoci and displacements of images in a focus series are determined from an analysis of the phase correlation function between pairs of images, allowing the restoration of an image wave even when focus and specimen drift are present. Subsequently, the absolute coefficients of both defocus and 2-fold astigmatism are determined with a phase contrast index function. Overall this method allows a very accurate automated aberration determination even for largely crystalline samples with little amorphous contamination. Using experimental images of the complex oxide Nb16W18O94 we have demonstrated the new method and critically compared it with existing diffractogram based aberration determinations. A series of protocols for practical implementation is also given together with a detailed analysis of the accuracy achieved. Finally a focal series restoration of Nb16W18O94 with symmetric aberrations determined automatically using this method is presented

A new method for the determination of the wave aberration function for high-resolution TEM.; 2. Measurement of the antisymmetric aberrations.

A new method is presented for the determination of the antisymmetric coefficients of the wave aberration function from a tableau of tilted illumination images. The approach is based on measurements of the apparent defocus and two-fold astigmatism using a phase correlation function and phase contrast index calculated from a short focus series acquired at each tilt. This method is shown to be suitable for a wide range of specimens and is sufficiently accurate for exit plane wave restoration at 0.1 nm resolution. Experimental examples of this approach are provided and the method is compared to results obtained from measurements of conventional power spectra

Comprehensive characterization of a FEGTEM

Several improvements have been made in the methodology of determining imaging aberrations - especially three-fold astigmatism coma and spherical aberration - from measurements of image shifts and diffractogram shapes, to characterise a microscope recently installed in Oxford. The combined use of shift and diffractogram measurements has been develop to address tilt calibration problems, and a new approach based on simpler measurements devised

Indirect super resolved microscopy in the TEM

An exit surface wavefunction restoration from a focal series of images of a complex oxide Nb16W18O94 recorded on a recently installed FEGTEM at Oxford is presented. The basic cation lattice is readily determined from a simple examination of the restored modulus whereas the restored phase provides additional information about the anion sublattice as confirmed by multislice image simulations

Super-resolution by aperture synthesis: tilt series reconstruction in CTEM

A substantial resolution improvement has been achieved - increasing the continuous transfer limit from 0.23 nm to better than 0.14 nm - in a reconstruction of a set of images made with differently tilted illumination. The transfer function envelopes mean that differently tilted images effectively cover different regions of Fourier space, allowing a synthesis over an area four times that covered by conventional axial imaging with similar illumination coherence. The image processing required no more than standard series restoration filters, but new techniques were needed to establish the imaging conditions with sufficient accuracy and to identify a common origin in each image. As a route to higher resolution, the approach has in most respects considerably less stringent requirements than that of focal series restoration. © 1995 Elsevier Science B.V. All rights reserved