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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).
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Figure 2. Electron microscope. A photograph and a schematic representation (courtesy of Dr. C. San Martín) are shown.
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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.)
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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 O, Martín-Benito J, Grantham J et al. (2001) The ‘sequential allosteric ring’ mechanism in the eukaryotic chaperonin-assisted folding of actin and tubulin. EMBO Journal 20: 4065–4075.). (b) Flexible fitting allowed comparison of the single particle EM map of an archaeal prefoldin (a complex that stabilizes and delivers unfolded proteins to a chaperonin for facilitated folding) with the atomic structure of a homologue (Martín-Benito J, Gómez-Reino J, Stirling PC et al. (2007) Divergent substrate-binding mechanisms reveal an evolutionary specialization of eukaryotic prefoldin compared to its archaeal counterpart. Structure 15: 101–110.). (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 D, Saugar I, Rodriguez JF et al. (2007) Infectious bursal disease virus capsid assembly and maturation by structural rearrangements of a transient molecular switch. Journal of Virology 81: 6869–6878.). At this resolution (close to 7 Å), secondary structure prediction yielded good results (c, right) in the detection of helices (green rods) and sheets (blue planes), as compared with the atomic structure of the monomer (Fernández JJ, Luque D, Castón JR et al. (2008) Sharpening high resolution information in single particle electron cryomicroscopy. Journal of Structural Biology 164: 170–175.). (d, left) Rigid-body fitting allowed comparison of the EM structure of the bacteriophage T7 connector to the atomic structure of a homologue (bacteriophage 29 connector). Secondary structure prediction on the 8 Å map allowed identification of putative helices in the stalk domain (Agirrezabala X, Martín-Benito J, Valle M et al. (2005) Structure of the connector of bacteriophage T7 at 8 Å resolution: structural homologies of a basic component of a DNA translocating machinery. Journal of Molecular Biology 347: 895–902.).
(Some material for this figure is by courtesy of Dr. D. Luque.)
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Figure 5. 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 cryoEM 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 at the European Bioinformatics Institute under the following accession codes: emd-5001 (Ludtke SJ, Baker ML, Chen DH et al. (2008) De novo backbone trace of GroEL from single particle electron cryomicroscopy. Structure 16: 441–448), emd-1461 (Zhang X, Settembre E, Xu C et al. (2008) Near-atomic resolution using electron cryomicroscopy and single-particle reconstruction. Proceedings of the National Academy of Sciences of the United States of America 105: 1867–1872), emd-1508 (Yu X, Jin L and Zhou ZH (2008) 3.88 Å structure of cytoplasmic polyhedrosis virus by cryo-electron microscopy. Nature 453: 415–419), emd-1231 (Agirrezabala X, Martín-Benito J, Valle M et al. (2005) Structure of the connector of bacteriophage T7 at 8 Å resolution: structural homologies of a basic component of a DNA translocating machinery. Journal of Molecular Biology 347: 895–902), emd-1466 (Kaufmann B, Chipman PR, Kostyuchenko VA et al. (2008) Visualization of the externalized VP2 N termini of infectious human parvovirus B19. Journal of Virology 82: 7306–7312), emd-1217 (Halic M, Gartmann M, Schlenker O et al. (2006) Signal recognition particle receptor exposes the ribosomal translocon binding site. Science 312: 745–747), emd-1102 (Rivera-Calzada A, Maman JD, Spagnolo L et al. (2005) Three-dimensional structure and regulation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Structure 13: 243–255), emd-1190 (Okorokov AL, Orlova EV, Kingsbury SR et al. (2004) Molecular structure of human geminin. Nature Structural and Molecular Biology 11: 1021–1022), respectively. The structure in (i) is from a previous work of ours (Martín-Benito J, Gómez-Reino J, Stirling PC et al. (2007) Divergent substrate-binding mechanisms reveal an evolutionary specialization of eukaryotic prefoldin compared to its archaeal counterpart. Structure 15: 101–110). Bar, 5 nm.
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