Difference between revisions of "2012Zhang Cryo-PS"
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Latest revision as of 14:33, 30 June 2020
Zhang, L., Yan, F., Zhang, S. et al. Structural basis of transfer between lipoproteins by cholesteryl ester transfer protein. Nat Chem Biol 8, 342–349 (2012). DOI: 10.1038/nchembio.796
"The cryo-PS-EM was modified from Adrian’s cryo-negative-stain (cryo-NS) protocol by combining our optimized NS and conventional cryo-EM protocols. Instead of air-drying the sample in the last step of the NS protocol, the sample was flash-frozen in liquid nitrogen temperature. Since the cryo-EM image of particle has reversed contrast to that from the Adrian’s cryo-NS protocol, but has consistent contrast to that from conventional cryo-EM image, we call this the cryo-PS-EM protocol.
We compared the particle shape and size of images of 53kDa CETP with the CETP crystal structure [PDB entry 2OBD 10]. Wide-field optimized NS-EM micrographs and selected particle views reveal the expected banana-shaped CETP with dimensions of ~125 x 30 Å (Supplementary Figs. 1A and B). When the CETP crystal structure is overlaid onto a reference-free class average of NS-EM images, a near perfect match in shape and size is found (Supplementary Figs. 1C and D), and interestingly, even the concave surface, C-terminal end (more globular) and N-terminal end (more tapered) of CETP are readily distinguished (Supplementary Fig. 1D). These studies validate direct NS-EM as a way to visualize the structure of CETP in other settings where it associates with various lipoproteins.
Wide-field cryo-PS-EM micrographs (Fig. 1A) and selected particle views (Fig. 1B) also display the banana-shaped CETP with a shape and dimensions similar to those observed from the optimized NS-EM protocol described above (Supplementary Fig. 1). Interestingly, some raw particle images show significant structural detail (Fig. 2, left column). For example, at specific viewing orientations, a manual noise-decreasing procedure performed on the raw particle images near the particle boundary (Fig. 2, middle column), revealed that the particle outer features are remarkably similar to the crystal structure (Fig. 2, right column). Thus, the particle internal features show parallel fringes that are well matched to the β-sheet strands within the C-terminal β-barrel domain of the crystal structure as well as end loops and holes (Figs. 2 A-C). Imaging of internal structural features was achieved by using EM imaging conditions of near Scherzer focus and higher doses (~60-70 e-/Å2) rather than conventional cryo-EM conditions–high-defocus (~2-3um) and low doses (~ 20 e-/Å2). These high-resolution images show that CETP can be visualized directly by this EM protocol, confirming our findings above using NS-EM. Moreover, the images show that the NS reagent, uranyl formate (UF), penetrates the molecular surface, challenging the conventional view that NS could only visualize the outer surface structure.
Images of the cryo-PS-EM images of CETP, which display a rich 2D structural detail, were used to reconstruct a 3D density-map of CETP using the single-particle reconstruction method. Again, the class averages and reconstructed 3D density map (at ~13 Å resolution, supplementary Fig. 2) show a banana-shaped density (~125 x 30 Å) with a shape and size similar to that of the crystal structure (Figs. 1C-F); once again, we observed a near perfect fit of the crystal structure into the envelope of the 3D density map, suggesting that the density map can be used to determine the CETP concave orientation (Fig. 1G), with the C-terminal (more globular end) and N-terminal (more tapered end) domains at ends of the long CETP axis. Some hydrophobic loops were observed outside the EM CETP envelope at the distal portions of the N- and C-terminal β-barrel domains, suggesting that these regions have structural flexibility (Fig. 1G arrows). However, the 3D reconstructions of CETP from our modified cryoNS-EM protocol did not show as much detail as observed in the 2D raw images and provide no additional insights beyond those of our optimized NS-EM protocol."
cryo-positive staining, cryo-PS, high-resolution image of a single small protein