Single Particle EM


Single particle electron microscopy (EM) plays an important role in structural biology because it allows derivation of biologically relevant information about proteins and macromolecular complexes. A large amount of randomly oriented images of the specimen under study (so‐called particles) are collected from micrographs taken with an electron microscope. These particles are then computationally aligned and combined to yield the 3D structure, which is subsequently subjected to visualisation and interpretation. In most cases, the resolution attained with this technique precludes tracing of the polypeptide chain or the clear visualisation of the secondary structure elements. Nevertheless, the integrative combination of the information provided by the different structural techniques (X‐ray crystallography, EM, etc.) at different resolution levels has allowed a comprehensive interpretation of the structure. The recent advancements in instrumentation and computational procedures are now making it possible to obtain maps at sub‐nanometre, and even near‐atomic, resolution.

Key Concepts

  • Single particle electron microscopy (EM) allows the structural determination of proteins and macromolecular complexes.
  • The combination of the structure obtained by single particle EM and the high‐resolution information obtained by other structural techniques allows the derivation of biologically relevant information.
  • Structure determination at sub‐nanometre, and even near‐atomic, resolution is also being possible in a growing number of cases.
  • The biological material has to be specially prepared prior to be imaged in the electron microscope.
  • The 3D structure of the specimen is determined by collecting, aligning and combining a large number of randomly oriented particles of the specimen that are extracted from the microscope images.

Keywords: single particle electron microscopy; electron cryomicroscopy; structural biology; image processing; structure determination

Figure 1. Sample preparation. Sketches of the preparation of the biological material by negative staining and cryomicroscopy are shown in (a–d) and (e), respectively. Middle panel (a–e, right) shows sketches of the structure that would be projected in the acquired images from the preparations shown in (a–e, left). In negative staining, the sample is embedded in a stain medium (a) that produces images of the specimen with good contrast (a, right). However, this technique is prone to artefacts (b–d, right) coming from partial (b) or uneven (c) staining, or due to the specimen distortion (d). In cryomicroscopy, the sample is embedded in a layer of vitreous ice (e), ensuring preservation of the structure in near‐physiological conditions while imaged (e, right). Micrographs of a sample of bacteriophage T4 prepared with negative staining and cryomicroscopy are shown in (f) and (g), respectively. Note the better and inverted (hence negative) contrast in (f) with respect to (g). Also note that the viruses in (f) present artefacts and deformations compared to the good preservation in cryomicroscopy (g).
Figure 2. Electron microscope. A photograph and a schematic representation (courtesy of Dr. C. San Martín) are shown.
Figure 3. Structure determination. Particles of the specimen under study are randomly dispersed and oriented in the micrographs taken with the microscope (top left). First, particles are selected and extracted (top right). After alignment and classification, averages that represent the different 2D views of the specimen under study are obtained (bottom left). The relative 3D orientations of the average views are determined by angular assignment, after which the tomographic reconstruction can be carried out to yield the 3D structure (bottom right). This structure can be refined afterwards by iteratively combining angular assignment and 3D reconstruction and using the original particles instead of average views. (See main text for a more detailed explanation of the whole process.) (Material for this figure is by courtesy of Dr. E. Arias‐Palomo.)
Figure 4. Structure interpretation. (a) Rigid‐body fitting allowed interpretation of the mechanism of the eukaryotic chaperonin containing T‐complex polypeptide 1 (CCT) to assist the folding of actin by fitting its atomic structure into specific regions of the map (Llorca et al., ). (b) Flexible fitting allowed comparison of the single particle EM map of an archaeal prefoldin (a complex that stabilises and delivers unfolded proteins to a chaperonin for facilitated folding) with the atomic structure of a homologue (Martín‐Benito et al., ). (c, left) Rigid‐body fitting of the atomic structure of a trimer of infectious bursal disease virus T = 1 subviral particle into a map solved by single particle EM allowed assessment of the latter (Luque et al., ). At this resolution (close to 7Å), secondary structure prediction yielded good results (c, right) in the detection of α helices (green rods) and β sheets (blue planes), as compared with the atomic structure of the monomer (Fernandez et al., ). Material for this panel is by courtesy of Dr. D. Luque. (d, left) High‐resolution EM structure of the TRPV1 ion channel at 3.4 Å (Liao et al., ) allowed identification of many side‐chain densities and (d, right) enabled de novo atomic model building for most residues. (d) Reproduced from (Liao et al., ) © Nature Publishing Group.
Figure 5. Identification and compensation for the beam‐induced specimen movement. (a) The fast readout rate of modern DDD cameras allows recording of images as frames of a movie. Estimation of the translational motion in individual frames, or rather averages of the frames, can be carried out with computational techniques based, for instance, on cross‐correlation. For this panel, 60 frames from the same field of double‐layered rotavirus particles were taken. By comparing consecutive 10‐frame averages, the shifts undergone by the particles were estimated. The first 10‐frame average and three other representative ones are shown here. The white lines represent the shifts with regard to the previous 10‐frame average, scaled by a factor of 70× for illustrative purposes. (a) Reproduced from Brilot et al. () © Elsevier. (b) Motion correction. On the left, the average of the 60 frames was computed in a straightforward way, which results in a substantially blurred image. On the right, the 60 frames were first aligned to compensate for the shifts describing the particle movements, and their average shows substantial reduction of the blurring and improved contrast. Adapted from Grigorieff, © Creative Commons Licence.
Figure 6. Illustrative structures determined by single particle EM. Structures solved at near‐atomic resolution (a–c), at sub‐nanometre resolution (d–f) and at medium resolution (g) by single particle cryo‐EM are included as representative examples. Two representative structures of small proteins where the use of negative staining is of paramount importance are also shown (h–i). For all these structures, the actual resolution achieved is included. As an indication of their sizes, the molecular mass is indicated too. The structures in (a–h) are accessible through the Electron Microscopy Data Bank under the following accession codes: emd‐5001, emd‐1461, emd‐1508, emd‐1231, emd‐1466, emd‐1217, emd‐1102 and emd‐1190, respectively. The structure in (i) is from a previous work of ours. Bar, 5nm.
Figure 7. Recent major breakthroughs by high‐resolution single particle cryo‐EM. The use of direct detection device cameras and compensation for beam‐induced specimen motion has been of paramount importance. As in Figure , the actual resolution achieved and the molecular mass are indicated for all structures. Maps in (a–d) are accessible through the Electron Microscopy Data Bank under the following accession codes: emd‐5623, emd‐5778, emd‐2566 and emd‐2677, respectively. Bar, 5nm.


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Further Reading

Fernandez JJ, Sorzano COS, Marabini R and Carazo JM (2006) Image processing and 3‐D reconstruction in electron microscopy. IEEE Signal Processing Magazine 23 (3): 84–94.

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Fernández, José‐Jesús, and Valpuesta, José‐María(Mar 2015) Single Particle EM. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0021846.pub2]