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Review
. 2001 Jan 8;152(1):F1-10.
doi: 10.1083/jcb.152.1.f1.

Molecular Machines: putting the pieces together

E Nogales  1 , N Grigorieff
Affiliations
Review

Molecular Machines: putting the pieces together

E Nogales et al. J Cell Biol. .
No abstract available

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Figures

Figure 5
Figure 5
Reconstruction of CCT bound to α-actin. The partially folded actin molecule binds at the top of the apical domains of two of the subunits in the top cavity. (a) Unbound CCT; (b and c) orthogonal views of the actin-bound CCT; and (d) section through the actin-bound CCT (Llorca et al. 1999a) (J.M. Valpuesta, Centro Nacional de Biotechnologia, Madrid, Spain). (e) Reconstruction of human TFIID (green) bound to TFIIA (yellow) and TFIIB (blue). The position of TBP within TFIID was localized using a TBP-specific antibody (magenta) (Andel et al. 1999).
Figure 1
Figure 1
Processing of images of single protein particles and 2-D crystals. (a) After selecting particles from images scanned into a computer, the particles are aligned and classified according to shape and orientation. The classification yields averages with improved signal to noise ratio that can be used as new references to realign the particles. After a few iterations of alignment and classification, the orientations corresponding to the views represented by the class averages are determined. The data is merged with data from other images after determining the imaging parameters (image defocus and magnification). Assuming the data set contains a sufficient number of different orientations, a 3-D structure is calculated. (b) The image of a 2-D crystal is scanned into a computer, it is Fourier-transformed, the noise between diffraction spots is masked out, and the filtered image is back-transformed. To correct for distortions in the crystal (unbending), a reference area containing between 10 and 100 unit cells is selected from the filtered image. An improved reference area is derived from the unbent image, and the original image is unbent a second time. Spot amplitudes and phases are then extracted from the unbent image and merged with data extracted from other images after determining the sample parameters (phase origin and crystal tilt) and imaging parameters (defocus, beam tilt, and amplitude scaling). A 3-D structure is then calculated using a simple Fourier transformation. If the crystals are sufficiently large (∼10 μm), amplitudes can also be obtained from electron diffraction patterns. After determining the crystal tilt, the diffraction amplitudes are merged with image data where they replace the image amplitudes (diffraction amplitudes are more reliable than amplitudes derived from images).
Figure 2
Figure 2
3-D reconstructions of (a) complex I (Grigorieff 1998), (b) clathrin cage (Smith et al. 1998) (C. Smith, Birbeck College, London, UK), and (c) yeast and vertebrate nuclear pore (Yang et al. 1998) (C. Akey, Boston University, Boston, MA).
Figure 3
Figure 3
Localization of subunits and active sites within large protein complexes. (a) 26S proteosome (core region in orange, lids in blue) (Walz et al. 1998): the use of gold-conjugated substrate was used to locate the position of the deubiquitinylating enzyme p37A (bottom) (Hölzl et al. 2000) (W. Baumeister, Max-Planck-Institute, Martinsried, Germany). (b) Insulin receptor: the location of the insulin-binding site was obtained using gold-labeled insulin (Luo et al. 1999) (F.P. Ottensmeyer, University of Toronto, Ontario, Canada). (c) TFIIH: four of its subunits were located using subunit-specific antibodies (Schultz et al. 2000) (P. Schultz, Centre National de la Recherche Scientifique, Illkirck, France).
Figure 4
Figure 4
Structure of F-actin and actin-binding cytoskeletal polymers and interacting proteins. (a) Undecorated F-actin, (b) F-actin decorated with cofilin (the helical screw is significantly altered) (McGough et al. 1997), (c) F-actin decorated with gelsolin, and (d) F-actin decorated with α-actinin (McGough et al. 1998) (A. McGough, Purdue University, West Lafayette, IN). (e) Helical reconstruction of a microtubule decorated with the motor domain of monomeric kinesin KIF1A (Kikkawa et al. 2000). One motor domain is shown in blue (M. Kikkawa, University of Tokyo, Tokyo, Japan).
Figure 6
Figure 6
CryoEM studies of the ribosome and the translocation cycle. (a) Location of tRNAs (green, magenta, and yellow) and elongation factors (orange and purple) during the translation cycle (Frank et al. 1999) (J. Frank, Wadsworth Center, Albany, NY). (b) Conformations of EF-G (Stark et al. 2000). Orthogonal views of difference densities fitted with EF-G. The blue mesh shows the density attributed to EF-G from difference mapping. The structure of EF-G was fitted using the crystal structure of EF-G–GDP, but allowing for movements of domains 3, 4, and 5 (dark green, light green, and orange) relative to domains 1 and 2 (magenta and blue). Areas of contact with the 30S or 50S ribosomal subunits are indicated. Density due to the ribosome is marked by an asterisk (Stark et al. 2000) (W. Wintermeyer, Universität Witten, Witten, Germany).
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