[Show abstract][Hide abstract] ABSTRACT: The use of an alkane mixture that remains liquid at 77 K to freeze specimens has advantages over the use of a pure alkane that is solid at 77 K. It was found that a mixture of methane and ethane did not give a cooling rate adequate to produce vitreous ice, but a mixture of propane and ethane did result in vitreous ice. Furthermore, the latter mixture produced less damage to specimens mounted on a very thin, fragile holey carbon substrate.
Microscopy and Microanalysis 11/2008; 14(5):375-9. DOI:10.1017/S1431927608080781 · 1.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In an attempt to determine why cooling with liquid helium actually proved disadvantageous in our electron cryotomography experiments, further tests were performed to explore the differences in vitreous ice at approximately 82 and approximately 12 K. Electron diffraction patterns showed clearly that the vitreous ice of interest in biological electron cryomicroscopy (i.e., plunge-frozen, buffered protein solutions) does indeed collapse into a higher density phase when irradiated with as few as 2-3 e-/A2 at approximately 12 K. The high density phase spontaneously expanded back to a state resembling the original, low density phase over a period of hours at approximately 82 K. Movements of gold fiducials and changes in the lengths of tunnels drilled through the ice confirmed these phase changes, and also revealed gross changes in the concavity of the ice layer spanning circular holes in the carbon support. Brief warmup-cooldown cycles from approximately 12 to approximately 82 K and back, as would be required by the flip-flop cryorotation stage, did not induce a global phase change, but did allow certain local strains to relax. Several observations including the rates of tunnel collapse and the production of beam footprints suggested that the high density phase flows more readily in response to irradiation. Finally, the patterns of bubbling were different at the two temperatures. It is concluded that the collapse of vitreous ice at approximately 12 K around macromolecules is too rapid to account alone for the problematic loss of contrast seen, which must instead be due to secondary effects such as changes in the mobility of radiolytic fragments and water.
[Show abstract][Hide abstract] ABSTRACT: Electron cryotomography is the highest-resolution structural technique currently available that can be applied to unique objects such as flexible large protein complexes, irregular viruses, organelles and small cells. Specimens are preserved in a near-native, 'frozen-hydrated' state by vitrification. The thickness of the vitreous ice must be optimized for each specimen, and gold fiducials are typically added to facilitate image alignment. Here, we describe in detail our protocols for electron cryotomography sample preparation including (i) introduction of fiducial markers into the sample and (ii) sample vitrification. Because we almost exclusively use an automated, climate-controlled plunge-freezing device (the FEI Vitrobot) to vitrify our samples, we discuss its operation and parameters in detail. A session in which eight grids are prepared takes 1.5-2 h.
[Show abstract][Hide abstract] ABSTRACT: Electron cryotomography can be used to solve the three-dimensional structures of individual large macromolecules, assemblies, and even small intact cells to medium (approximately 4-8 nm) resolution in a near-native state, but restrictions in the range of accessible views are a major limitation. Here we report on the design, characterization, and demonstration of a new "flip-flop" rotation stage that allows facile and routine collection of two orthogonal tilt-series of cryosamples. Single- and dual-axis tomograms of a variety of samples are compared to illustrate qualitatively the improvement produced by inclusion of the second tilt-series. Exact quantitative expressions are derived for the volume of the remaining "missing pyramid" in reciprocal space. When orthogonal tilt-series are recorded to +/-65 degrees in each direction, as this new cryostage permits, only 11% of reciprocal space is left unmeasured. The tomograms suggest that further improvement could be realized, however, through better software to align and merge dual-axis tilt-series of cryosamples.
[Show abstract][Hide abstract] ABSTRACT: While the structures of nearly every HIV-1 protein are known in atomic detail from X-ray crystallography and NMR spectroscopy, many questions remain about how the individual proteins are arranged in the mature infectious viral particle. Here, we report the three-dimensional structures of individual HIV-1 virus-like particles (VLPs) as obtained by electron cryotomography. These reconstructions revealed that while the structures and positions of the conical cores within each VLP were unique, they exhibited several surprisingly consistent features, including similarities in the size and shape of the wide end of the capsid (the "base"), uniform positioning of the base and other regions of the capsid 11nm away from the envelope/MA layer, a cone angle that typically varied from 24 degrees to 18 degrees around the long axis of the cone, and an internal density (presumably part of the NC/RNA complex) cupped within the base. Multiple and nested capsids were observed. These results support the fullerene cone model for the viral capsid, indicate that viral maturation involves a free re-organization of the capsid shell rather than a continuous condensation, imply that capsid assembly is both concentration-driven and template-driven, suggest that specific interactions exist between the capsid and the adjacent envelope/MA and NC/RNA layers, and show that a particular capsid shape is favored strongly in-vivo.