In Situ Cryo-Electron Tomography: A Post-Reductionist Approach to Structural Biology

Cryo-electron tomography is a powerful technique that can faithfully image the native cellular environment at nanometer resolution. Unlike many other imaging approaches, cryo-electron tomography provides a label-free method of detecting biological structures, relying on the intrinsic contrast of frozen cellular material for direct identification of macromolecules. Recent advances in sample preparation, detector technology, and phase plate imaging have enabled the structural characterization of protein complexes within intact cells. Here, we review these technical developments and outline a detailed computational workflow for in situ structural analysis. Two recent studies are described to illustrate how this workflow can be adapted to examine both known and unknown cellular complexes. The stage is now set to realize the promise of visual proteomics a complete structural description of the cell's native molecular landscape. (C) 2015 Elsevier Ltd. All rights reserved

The promise and the challenges of cryo-electron tomography

Structural biologists have traditionally approached cellular complexity in a reductionist manner in which the cellular molecular components are fractionated and purified before being studied individually. This 'divide and conquer' approach has been highly successful. However, awareness has grown in recent years that biological functions can rarely be attributed to individual macromolecules. Most cellular functions arise from their concerted action, and there is thus a need for methods enabling structural studies performed in situ, ideally in unperturbed cellular environments. Cryo-electron tomography (Cryo-ET) combines the power of 3D molecular-level imaging with the best structural preservation that is physically possible to achieve. Thus, it has a unique potential to reveal the supramolecular architecture or 'molecular sociology' of cells and to discover the unexpected. Here, we review state-of-the-art Cryo-ET workflows, provide examples of biological applications, and discuss what is needed to realize the full potential of Cryo-ET

Whole Cell Cryo-Electron Tomography Reveals Distinct Disassembly Intermediates of Vaccinia Virus

At each round of infection, viruses fall apart to release their genome for replication, and then reassemble into stable particles within the same host cell. For most viruses, the structural details that underlie these disassembly and assembly reactions are poorly understood. Cryo-electron tomography (cryo-ET), a unique method to investigate large and asymmetric structures at the near molecular resolution, was previously used to study the complex structure of vaccinia virus (VV). Here we study the disassembly of VV by cryo-ET on intact, rapidly frozen, mammalian cells, infected for up to 60 minutes. Binding to the cell surface induced distinct structural rearrangements of the core, such as a shape change, the rearrangement of its surface spikes and de-condensation of the viral DNA. We propose that the cell surface induced changes, in particular the decondensation of the viral genome, are a prerequisite for the subsequent release of the vaccinia DNA into the cytoplasm, which is followed by its cytoplasmic replication. Generally, this is the first study that employs whole cell cryo-ET to address structural details of pathogen-host cell interaction

Structure of a type IV pilus machinery in the open and closed state.

This is the final version of the article. Available from eLife Sciences Publications via the DOI in this record.Proteins of the secretin family form large macromolecular complexes, which assemble in the outer membrane of Gram-negative bacteria. Secretins are major components of type II and III secretion systems and are linked to extrusion of type IV pili (T4P) and to DNA uptake. By electron cryo-tomography of whole Thermus thermophilus cells, we determined the in situ structure of a T4P molecular machine in the open and the closed state. Comparison reveals a major conformational change whereby the N-terminal domains of the central secretin PilQ shift by ~30 Å, and two periplasmic gates open to make way for pilus extrusion. Furthermore, we determine the structure of the assembled pilus.This work was supported by the Max Planck Society and the Deutsche Forschungsgemeinschaft (AV 9/6-1)

Analysing the lattice transition of thin filaments in striated muscle

Thin filaments, through interaction with thick filaments, form the contractile apparatus of striated muscle. Therefore, the length and arrangement of the thin filaments are of key importance to the function of the muscle. The thin filaments from adjacent sarcomeres are anchored at the Z-disc. In 1968 Pringle predicted that thin filament are organised in the Z-disc in a rhomboid lattice rather than a square lattice. Previous experimental evidence has been insufficient to verify Pringle’s suggestion. In the A-band the thin filaments interdigitate with the thick filaments on a hexagonal lattice, hence from the Z-disc to the A-band, there is a transition of the lattice from square to hexagonal. In this project, I have firstly used Fourier analysis and electron tomography to investigate the thin filament lattice in the Z-disc. I have used electron tomography to determine how the lattice transition occurs between the Z-disc and the A-band. Electron tomography of these samples also allowed me to determine the lengths of thin filaments, showing unequivocally that they are of variable lengths in cardiac muscle

Structural basis of mitochondrial membrane bending by the I-II-III2-IV2 supercomplex

Mitochondrial energy conversion requires an intricate architecture of the inner mitochondrial membrane1. Here we show that a supercomplex containing all four respiratory chain components contributes to membrane curvature induction in ciliates. We report cryo-electron microscopy and cryo-tomography structures of the supercomplex that comprises 150 different proteins and 311 bound lipids, forming a stable 5.8-MDa assembly. Owing to subunit acquisition and extension, complex I associates with a complex IV dimer, generating a wedge-shaped gap that serves as a binding site for complex II. Together with a tilted complex III dimer association, it results in a curved membrane region. Using molecular dynamics simulations, we demonstrate that the divergent supercomplex actively contributes to the membrane curvature induction and tubulation of cristae. Our findings highlight how the evolution of protein subunits of respiratory complexes has led to the I-II-III2-IV2 supercomplex that contributes to the shaping of the bioenergetic membrane, thereby enabling its functional specialization

Cryo-electron microscopy reveals two distinct type IV pili assembled by the same bacterium

Type IV pili are flexible filaments on the surface of bacteria, consisting of a helical assembly of pilin proteins. They are involved in bacterial motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). Here, we use cryo-electron microscopy and mass spectrometry to show that the bacterium Thermus thermophilus produces two forms of type IV pilus ('wide' and 'narrow'), differing in structure and protein composition. Wide pili are composed of the major pilin PilA4, while narrow pili are composed of a so-far uncharacterized pilin which we name PilA5. Functional experiments indicate that PilA4 is required for natural transformation, while PilA5 is important for twitching motility. Type IV pili are flexible filaments on the surface of bacteria, consisting of a helical assembly of pilin proteins. Here, Neuhaus et al. show that the bacterium Thermus thermophilus produces two forms of type IV pilus, differing in structure, protein composition, and function.Peer reviewe

Development and Implementation of In-Focus Phase Contrast TEM for Materials and Life Sciences

Transmission electron microscopy (TEM) allows the imaging of objects from materials sciences and biology with a resolution of a few nm to a few Å. Biological systems can be reconstructed in 3D under physiological conditions using cryo TEM. However, the low signal-to-noise ratio (SNR) of individual images hampers resolution. Weak-phase objects such as native biological samples can be visualized only by phase contrast, which is generated in conventional TEM by defocusing. The defocus technique yields weak contrast and incomplete transfer of object information, which makes reconstruction difficult. Therefore, generating contrast by placing a phase plate in the back focal plane of the objective lens is desirable. This allows for artefact-free imaging of weak-phase objects with strong contrast. For TEM, phase plates have been realised only in the form of a thin carbon film which causes loss of signal and resolution. This work presents the first technical implementation of an electrostatic (Boersch) phase plate for TEM and gives the experimental proof-of-principle for this device. The Boersch phase plate generates maximum phase contrast while avoiding resolution loss. It consists of a miniaturised electrostatic einzel lens that shifts the phase of the unscattered wave. Obstruction effects are minimised by optically magnifying the focal plane. The advancement to an entirely obstruction-free phase plate is outlined which generates pseudo-topographic (Hilbert) contrast. The enhancement of electron-microscopic reconstruction by such a Hilbert phase plate is demonstrated for electron tomography of unstained cell sections