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

https://doi.org/10.1016/j.jmb.2015.09.030Get rights and content

Highlights

  • Cryo-electron tomography visualizes native frozen cells with molecular resolution.

  • Protein complexes are identified and characterized within the cellular environment.

  • Sample preparation and computational approaches are described in depth.

  • Two recent studies provide examples for characterizing known and unknown complexes.

  • The in situ structural analysis of every macromolecule in the cell is within reach.

Abstract

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.

Introduction

Understanding complex networks requires detailed knowledge about the properties and interactions of each of the network's components. This is especially true in molecular cell biology, where individual proteins are often involved in multiple cellular processes, frequently swapping interaction partners and changing states of assembly [1], [2]. Structural biologists have traditionally approached this cellular complexity in a reductionist manner by characterizing purified molecular components [3]. This “divide and conquer” strategy has been highly successful, as evidenced by the impressive number of entries in the Protein Data Bank [4]. However, the reductionist approach has several intrinsic limitations. Labile and membrane-embedded complexes can be easily damaged or distorted from their functional conformations during purification. Moreover, complexes frequently lose their biologically relevant associations once they are removed from the crowded cellular environment. It is well known in protein crystallography that missing cofactors can weaken the integrity of structures, while packing forces can stabilize non-physiological conformations [5].

Cryo-electron tomography (cryo-ET) avoids these pitfalls by imaging unperturbed cells, preserving the full spectrum of each molecule's conformations and interactions [6], [7], [8]. With several recent technological advances increasing both the versatility and the image quality of cryo-ET, it is now possible to identify and characterize macromolecules within a wide variety of cell types [9], [10], [11], [12], [13], [14], [15], [16]. Computational tools including template matching, subtomogram averaging, and classification enable the generation of molecular-resolution structures that accurately represent the physiological conformations of complexes within their functional environments [17], [18], [19]. In this review, we present a practical workflow for in situ structural analysis, with an eye toward the future of visual proteomics [20], [21].

Section snippets

Preparation of Thin Vitreous Samples and Cryo-ET Data Acquisition

Sample preparation for cellular cryo-ET follows a relatively straightforward workflow that is compatible with most cell types (Fig. 1). Cells that grow in solution, including bacteria, archaea, yeast, and many protists, are cultured in their standard medium and applied to carbon-coated copper electron microscopy (EM) grids just prior to freezing (Fig. 1a and b) [10], [22], [23], [24], [25]. Alternatively, adherent cells, such as Dictyostelium, mammalian cell lines, and neurons, are grown on top

Detection of Macromolecules Inside Cells

Several consecutive image-processing steps are necessary for the detection of macromolecules within cellular tomograms (Fig. 2). Tilt-series that were recorded with defocus can be subjected to contrast transfer function correction to improve their resolution [57], [83], [84]. This step is unnecessary for in-focus images acquired with the Volta phase plate. Denoising procedures (Fig. 2a), such as bandpass frequency filtering, non-local means filtering, and iterative reconstruction schemes [85],

In Situ Characterization of Known Complexes: 26S Proteasomes in Neurons

A recent study characterizing 26S proteasomes in neurons showcases the strength of in situ subtomogram averaging and classification [9]. In this section, we outline this study's computational workflow, which can be adapted to the investigation of other complexes with known structures (Fig. 3). The 26S proteasome is a macromolecular complex responsible for the regulated degradation of proteins that are misfolded, damaged, or no longer needed [119], [120]. These protein substrates are marked for

De Novo Analysis of Unknown Structures: Golgi Intracisternal Protein Arrays

In situ subtomogram averaging is also capable of characterizing previously unknown structures. In a recent cryo-ET study, intracisternal protein arrays were observed within the Golgi apparatus of FIB-milled Chlamydomonas reinhardtii cells (Fig. 4a) [16]. These structures were both abundant and highly regular, enabling de novo structure generation.

The initial template was a single low-pass-filtered subvolume containing two cisterna membranes connected by a protein array (Fig. 4b, top). After the

New Frontiers for Visual Proteomics

The functions of cellular complexes did not evolve in isolation. Thus, we believe that the holy grail of structural biology should be to directly observe molecular structures within their functional environments. Thanks to advances in sample preparation and cryo-ET, this vision is starting to become a reality. Although current studies have only examined a few types of macromolecules, extrapolating the in situ approach to its natural conclusion brings us to the concept of visual proteomics: the

Acknowledgements

This work was supported by an Alexander von Humboldt Foundation post-doctoral fellowship (to B.D.E.), the European Commission's grant agreement ERC-2012-SyG_318987-ToPAG, and the Deutsche Forschungsgemeinschaft Excellence Clusters CIPSM (Center for Integrated Protein Science Munich) and SFB 1035.

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