High-resolution Electron Microscopy of Helical Specimens: A Fresh Look at Tobacco Mosaic Virus

Abstract

The treatment of helical objects as a string of single particles has become an established technique to resolve their three-dimensional (3D) structure using electron cryo-microscopy. It can be applied to a wide range of helical particles such as viruses, microtubules and helical filaments. We have made improvements to this approach using Tobacco Mosaic Virus (TMV) as a test specimen and obtained a map from 210,000 asymmetric units at a resolution better than 5 A. This was made possible by performing a full correction of the contrast transfer function of the microscope. Alignment of helical segments was helped by constraints derived from the helical symmetry of the virus. Furthermore, symmetrization was implemented by multiple inclusions of symmetry-related views in the 3D reconstruction. We used the density map to build an atomic model of TMV. The model was refined using a real-space refinement strategy that accommodates multiple conformers. The atomic model shows significant deviations from the deposited model for the helical form of TMV at the lower-radius region (residues 88 to 109). This region appears more ordered with well-defined secondary structure, compared with the earlier helical structure. The RNA phosphate backbone is sandwiched between two arginine side-chains, stabilizing the interaction between RNA and coat protein. A cluster of two or three carboxylates is buried in a hydrophobic environment isolating it from neighboring subunits. These carboxylates may represent the so-called Caspar carboxylates that form a metastable switch for viral disassembly. Overall, the observed differences suggest that the new model represents a different, more stable state of the virus, compared with the earlier published model.

Hydrogen-bonding networks and RNA bases revealed by cryo electron microscopy suggest a triggering mechanism for calcium switches

“…1E). These loops are well-resolved in our cryoEM structure, unlike the previous lower-resolution cryoEM structure (9).…”

Self-organization of the vesicular stomatitis virus nucleocapsid into a bullet shape

“…Selection of B1,000 trunks of N-RNA resulted in B65,000 overlapping 612 Â 612 Å segments extracted every 10.2 Å along the trunk axis. The original in-plane angle and filament assignment of the images were recorded for further alignment validation 28 . Individual images were corrected for CTF by phase-flipping, lowpass filtered to 15 Å, normalized (mean ¼ 0; sigma ¼ 1) and masked by an accordingly rotated smooth-edged rectangular mask of 200 Â 560 Å.…”

“…For the same purpose, the same number of images per on-axis view was selected for reconstructions after each PM cycle. Cross-correlation-based selection and other standard single particle-based selection procedures that impose alignment restrains were applied 28 . After B20 cycles, individual subunits were clearly distinguishable ( Supplementary Fig.…”

Cryo-electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for immature virus assembly

“…The helical parameters of each tube were extracted using cryo-ET and subtomogram averaging (10,11,41). Real-space helical reconstruction was conducted by using extracted helical parameters (42). The pseudohexameric asymmetric units from all reconstructed tubes were averaged to obtain a final reconstruction of the HIV-1 CANC Y169S/L tubes at 9.4 Å (SI Materials and Methods).…”