Towards an integrative structural biology approach: combining Cryo-TEM, X-ray crystallography, and NMR

Abstract

Cryo-transmission electron microscopy (Cryo-TEM) and particularly single particle analysis is rapidly becoming the premier method for determining the three-dimensional structure of protein complexes, and viruses. In the last several years there have been dramatic technological improvements in Cryo-TEM, such as advancements in automation and use of improved detectors, as well as improved image processing techniques. While Cryo-TEM was once thought of as a low resolution structural technique, the method is currently capable of generating nearly atomic resolution structures on a routine basis. Moreover, the combination of Cryo-TEM and other methods such as X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular dynamics modeling are allowing researchers to address scientific questions previously thought intractable. Future technological developments are widely believed to further enhance the method and it is not inconceivable that Cryo-TEM could become as routine as X-ray crystallography for protein structure determination.

Fig. 1

a 3.3 Å structure of a non-enveloped icosahedral virus using Cryo-TEM. b Image of a selected region were individual side chains are clearly visible in the Cryo-TEM reconstruction validating the high resolution of the reconstruction. Images kindly provided by Z. Hong Zhou Electron Imaging Center for Nanomachines (EICN) CNSI and Department of Microbiology, Immun & Mol. Genetics, UCLA, and adapted from [9]. EMDB Accession code: EMD-5160; Fitted PDB ID: 3IYL

Fig. 2

9 Å three-dimensional reconstruction of soluble gp140 HIV envelope glycoprotein trimers bound to three copies of the Fab fragment from 17b, a neutralizing antibody whose binding mimics that of the co-receptor. The structure revealed the presence of a previously unknown “activated” intermediate state, where three buried helices become exposed and potentially accessible to binding by entry inhibitors. a Top view of the trimer and b Side view of the trimer. Images kindly provided by Sriram Subramaniam, Lab of Cell Biology, National Cancer Institute, National Institutes of Health. Adapted from [16]. EMDB Accession code: EMD-5462

Fig. 3

Use of time-resolved Cryo-TEM to visualize ribosome dynamics and tRNA movement. Furthermore, using the ratio of particles residing in each state, the entire thermodynamic dynamic landscape of the transition between states was determined. This is the first example of how using Cryo-TEM structural information to calculate thermodynamic parameters. Image kindly provided by Niels Fischer and Holger Stark, MPI Gottingen. Adapted from [19]. EMDB Accession codes: EMD-1716, EMD-1717, EMD-1718, EMD-1719, EMD-1720, EMD-1721, EMD-1722, EMD-1723, EMD-1724, EMD-1725, EMD-1726, EMD-1727; Fitted PDB IDs: 3J4V, 3J52, 3J4Z, 3J50, 3J4Y, 3J51, 3J53, 3J54, 3J57, 3J58, 3J59, 3J5A, 3J5B, 3J5C, 3J5H, 3J5J, 3J5J, 3J5K

Fig. 4

Cryo-TEM reveals various conformational states of areolysin pore formation where a prepore state transitions to a functional toxin pore inserted in the target membrane (a). Combining X-ray crystallography, Cryo-TEM, and molecular dynamics modeling revealed a novel swirling membrane insertion mechanism to form the pore, allowing for an atomic resolution interpretation of the transition from monomer to prepore to the functional pore state (b). Images kindly provided by Matteo Dal Peraro, Biomolecular Modeling—LBM Institute of Bioengineering, School of Life Sciences École Polytechnique Fédérale de Lausanne—EPFL & Swiss Institute of Bioinformatics—SIB Adapted from [20]

Fig. 5

Using cryo-tomographic analysis of individual, native “fullerene” cone HIV-1 capsid, combined with the high resolution Cryo-TEM hexamer structures an all-atom molecular dynamics HIV-1 capsid model was created. This model highlights the threefold capsid protein C-terminal domain as an attractive therapeutic target. a Slice through cryo-tomographic volume of an individual mature capsid. Red arrows highlight CA pentamers. b The tomographic density matches the shape and size of the capsid, shown by the overlay of densities from the segmented capsid and the fullerene model (yellow). c Final molecular dynamics equilibrated all-atom capsid model. Images kindly provided by Peijun Zhang Univ. of Pittsburgh. Adapted from [22]. EMDB Accession codes: EMD-5582, EMD-5639; Fitted PDB IDs: 3J34, 3J4F, 3J3Y