Cryo‐Electron Tomography for Structural Characterization of Macromolecular Complexes

Abstract

Cryo-electron tomography (cryo-ET) is an emerging 3-D reconstruction technology that combines the principles of tomographic 3-D reconstruction with the unmatched structural preservation of biological material embedded in vitreous ice. Cryo-ET is particularly suited to investigating cell-biological samples and large macromolecular structures that are too polymorphic to be reconstructed by classical averaging-based 3-D reconstruction procedures. This unit aims to make cryo-ET accessible to newcomers and discusses the specialized equipment required, as well as the relevant advantages and hurdles associated with sample preparation by vitrification and cryo-ET. Protocols describe specimen preparation, data recording and 3-D data reconstruction for cryo-ET, with a special focus on macromolecular complexes. A step-by-step procedure for specimen vitrification by plunge freezing is provided, followed by the general practicalities of tilt-series acquisition for cryo-ET, including advice on how to select an area appropriate for acquiring a tilt series. A brief introduction to the underlying computational reconstruction principles applied in tomography is described, along with instructions for reconstructing a tomogram from cryo-tilt series data. Finally, a method is detailed for extracting small subvolumes containing identical macromolecular structures from tomograms for alignment and averaging as a means to increase the signal-to-noise ratio and eliminate missing wedge effects inherent in tomographic reconstructions.

Higher-Order Architecture of Rhodopsin in Intact Photoreceptors and Its Implication for Phototransduction Kinetics

“…the phi angle) was searched during the alignment procedure. We performed the alignment and averaging iteratively using the av3 toolbox (Fö rster and Hegerl, 2007) or PEET (Cope et al, 2011); both programs gave similar results. For averaging with PEET, we followed a procedure implemented in the software: one rotation axis was defined as a vector between two detected points along the bilayer of the disk membrane.…”

Complexity and ultrastructure of infectious extracellular vesicles from cells infected by non-enveloped virus

“…Reconstructions of tomograms and 3-D surface model rendering were performed using the weighted back-projection method from the IMOD software package (Kremer et al, 1996). Volume averaging of virions inside vesicles was performed using PEET software (Cope et al, 2011;Nicastro et al, 2006) and the 3-D surface model of virions was rendered through averaged and low pass filtered subvolumes from smoothed tomograms. Nonlinear anisotropic diffusion (NAD) filtering was used to generate 3-D surface models of inner mat-like structures in the infectious microvesicles, to provide better density continuity and reduced noise.…”

“…Alignment and averaging were performed using the PEET software (which is part of the IMOD package). Classification analyses using principal component analysis (PCA) were performed on a set of aligned sub-volumes (Cope et al, 2011) to group structurally homogeneous particles using clusterPca in PEET. The majority of virions (820 out of 1,022) were identified in class 1, 160 virions in class 2, 32 virions in class 3, and 10 virions in class 4.…”

Nanoscale Imaging of Protein Molecules at the Postsynaptic Density

“…Then, after averaging large numbers of such projections, it is possible to compute the 3D reconstruction of the macromolecular assembly at a resolution in the subnanometer realm, which can reveal atomic-scale interactions between the constituent subunits. In TEM tomography of cells, the ability to average tomograms can give an improved resolution in the 2-3 nm range [ 38 ]. TEM tomography is beginning to be considered as a promising method for imaging large molecular complexes in neurons [ 39 -43 ], but there are still signifi cant challenges for imaging molecules at neuronal synapses, and especially in brain synapses.…”