Electron Cryomicroscopy and Three‐dimensional Computer Reconstruction of Biological Molecules

Abstract

Electron cryomicroscopy (cryo‐EM) single‐particle analysis is a technique for imaging individual molecules or macromolecules under native‐like conditions. When combined with complex computer algorithms, the resulting images can produce a three‐dimensional (3D) structure at resolutions now beyond 4 Å. At these resolutions, the protein backbone can be traced, and in many cases sufficient side chains identified to build a full atomistic model akin to that produced by X‐ray crystallography. Cryo‐EM can also approach problems, which are difficult or impossible for X‐ray diffraction or NMR. For example, cryo‐EM can be used to characterise the structural flexibility/dynamics of large macromolecules, which is not possible via any other current method. Cryo‐EM is also often the method of choice for studying large membrane proteins, which are generally difficult to crystallise. Additional specialised methods exist for 2D crystals or extended structures with helical symmetry.

Key Concepts:

  • Owing to the short wavelength of high voltage electrons, it is possible to resolve features at resolutions ∼1000× higher than light microscopes.

  • Liquid specimens cannot be used under the high vacuum of the microscope and must be either frozen in vitreous ice or chemically fixed before imaging.

  • Tens of thousands of randomly oriented 2D images are combined to produce a high‐resolution 3D volumetric structure.

Keywords: electron microscopy; 3D reconstruction; single particle; macromolecule; crystal

Figure 1.

Flow diagram of a typical single‐particle reconstruction for a particle of low symmetry. Beginning at the lower left, particles are located within the overall micrograph. A preliminary model is then generated from the raw particles. Projections of this model are generated. Raw particles are classified by comparing each particle to each projection. Particles in similar orientations are then aligned to one another and averaged. The averages, combined with the orientations, are then used to generate a new three‐dimensional (3D) model. This model is then used for another cycle of refinement. The procedure continues until convergence is achieved.

Figure 2.

Concept of projection theorem and common lines. A three‐dimensional model of an icosahedral particle is shown in two different orientations. Projections are generated in the same orientations, and fast Fourier transforms are calculated. These two Fourier transforms intersect as shown on the right. The amplitudes and phases are identical along the line of intersection, meaning they can be located in the Fourier images. The addition of a third projection would produce three common lines, which can be used to determine the orientation of the particles.

Figure 3.

(a) Two views of a 4.2 Å resolution reconstruction of the chaperonin mm‐cpn as determined by cryo‐EM single‐particle analysis. (b) Close‐up of several portions of the reconstructed density with individual atom positions shown. This demonstrates the level at which protein side chains can be resolved in cryo‐EM reconstructions at this resolution. Resolution is evaluated somewhat differently in cryo‐EM than X‐ray crystallography, so values are similar, but not directly comparable.

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Further Reading

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Ludtke, Steven J, and Chiu, Wah(Jun 2011) Electron Cryomicroscopy and Three‐dimensional Computer Reconstruction of Biological Molecules. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002987.pub2]