Alignment of conduits for the nascent polypeptide chain in the ribosome- Sec61 complex

Science

Volumn 278, Issue 5346, 1997, Pages 2123-2126

  Beckmann, Roland ^a   Bubeck, Doryen ^b   Grassucci, Robert ^b   Penczek, Pawel ^c   Verschoor, Adriana ^b   Blobel, Günter ^a   Frank, Joachim ^c  

^a ROCKEFELLER UNIVERSITY   (United States)

^b WADSWORTH CENTER   (United States)

^c STATE UNIVERSITY OF NEW YORK   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

PROTEIN;

ARTICLE; CRYOELECTRON MICROSCOPY; ENDOPLASMIC RETICULUM; IMAGE RECONSTRUCTION; INTRACELLULAR TRANSPORT; NONHUMAN; PRIORITY JOURNAL; PROTEIN TRANSPORT; RIBOSOME SUBUNIT; TRANSPORT KINETICS;

BINDING SITES; BIOLOGICAL TRANSPORT; ENDOPLASMIC RETICULUM, ROUGH; FUNGAL PROTEINS; IMAGE PROCESSING, COMPUTER-ASSISTED; MACROMOLECULAR SUBSTANCES; MEMBRANE PROTEINS; MICROSCOPY, ELECTRON; RIBOSOMES; SACCHAROMYCES CEREVISIAE; SACCHAROMYCES CEREVISIAE PROTEINS;

EID: 0031473345     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.278.5346.2123     Document Type: Article

References (33)

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17. A. Verschoor, J. R. Warner, S. Srivastava, R. A. Grassucci, J. Frank, Nucleic Acids Res., in press.
18. 2]. The identity of the proteins was confirmed by specific antibodies to Sec61α (Sec61p) andSec61β (Sbh1p) (17).
19. 2]. The identity of the proteins was confirmed by specific antibodies to Sec61α (Sec61p) andSec61β (Sbh1p) (17).
20. S. Panzner, L. Dreier, E. Hartmann, S. Kostka, T. A. Rapoport, Cell 81, 561 (1995)
21. 2.
22. 2. After centrifugation for 60 min at 240,000g at 4°C, six fractions were collected manually. For saturation assays, the first two fractions were pooled as the unbound fraction and the following two as the bound fraction. Fractions were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and stained with SYPRO Red. The amount of protein was quantitated with the STORM system (red fluorescence) and NIH image. For immunoblotting, proteins were precipitated, separated on 10 to 20% SDS-PAGE gradient gels, transferred to a nitrocellulose membrane, incubated consecutively with anti-Sec61α or anti-Sec61β and horseradish peroxidase-conjugated donkey antibodies to rabbit, and detected by ECL as described (Amersham).
23. R. Beckmann et al., data not shown.
24. Incubation to form the ribosome-Sec61 complex was performed as described (16), and the mixture was diluted with 4 volumes of water immediately before it was applied to the grid. Grids for cryoelectron microscopy were prepared as described [T. Wagenknecht, R. Grassucci, J. Frank, J. Mol. Biol. 199, 137 (1988); J. Dubochet et al., Q. Rev. Biophys. 21, 129 (1988)]. Micrographs were recorded under low-dose conditions on a Philips EM 420, with 1.5-μm defocus and magnification of 52,200 ± 2% as checked by a tobacco mosaic virus standard.
25. Incubation to form the ribosome-Sec61 complex was performed as described (16), and the mixture was diluted with 4 volumes of water immediately before it was applied to the grid. Grids for cryoelectron microscopy were prepared as described [T. Wagenknecht, R. Grassucci, J. Frank, J. Mol. Biol. 199, 137 (1988); J. Dubochet et al., Q. Rev. Biophys. 21, 129 (1988)]. Micrographs were recorded under low-dose conditions on a Philips EM 420, with 1.5-μm defocus and magnification of 52,200 ± 2% as checked by a tobacco mosaic virus standard.
26. P. A. Penczek, R. Grassucci, J. Frank, Ultramicroscopy 53, 251 (1994).
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28. Micrographs were checked for drift, astigmatism, and presence of Thon rings by optical diffraction. Scanning was done with a step size of 25 μm corresponding to 4.78 Å on the object scale, on a Perkin-Elmer PDS 1010 A microdensitometer. Particles were selected by an automated selection procedure that differed from the one previously described [K. R. Lata, P. Penczek, J. Frank, Ultramicroscopy 58, 381 (1995)] in that the particle candidates were compared directly with the reference set of 87 quasi-evenly spaced projections (19) of an existing reconstruction of the ribosome from yeast (12). A total of 13, 178 particles were picked. The reconstruction was done with two independent approaches to obtain the orientations of the projections. In the first approach, an existing reconstruction of the ribosome from yeast (12) was used as a reference in the 3D projection alignment procedure (19). In the second approach, an initial reconstruction was obtained with the simultaneous minimization technique [P. Penczek, J. Zhu, J. Frank, Ultramicroscopy 63, 205 (1996)]. In both cases, four steps of the 3D projection alignment procedure (19) were applied with a 2° angular interval. In each step, the refined 3D structure was calculated with 70% of trie best matching particles (on the basis of the value of the cross-correlation coefficient). Both reconstructions proved to be indistinguishable within the measured resolution range. The final resolution, estimated with the Fourier shell correlation with a cutoff value at 0.5 [B. Böttcher, S. A. Wynne, R. A. Crowther, Nature 386, 88 (1997)], was 26 Å.
29. Micrographs were checked for drift, astigmatism, and presence of Thon rings by optical diffraction. Scanning was done with a step size of 25 μm corresponding to 4.78 Å on the object scale, on a Perkin-Elmer PDS 1010 A microdensitometer. Particles were selected by an automated selection procedure that differed from the one previously described [K. R. Lata, P. Penczek, J. Frank, Ultramicroscopy 58, 381 (1995)] in that the particle candidates were compared directly with the reference set of 87 quasi-evenly spaced projections (19) of an existing reconstruction of the ribosome from yeast (12). A total of 13, 178 particles were picked. The reconstruction was done with two independent approaches to obtain the orientations of the projections. In the first approach, an existing reconstruction of the ribosome from yeast (12) was used as a reference in the 3D projection alignment procedure (19). In the second approach, an initial reconstruction was obtained with the simultaneous minimization technique [P. Penczek, J. Zhu, J. Frank, Ultramicroscopy 63, 205 (1996)]. In both cases, four steps of the 3D projection alignment procedure (19) were applied with a 2° angular interval. In each step, the refined 3D structure was calculated with 70% of trie best matching particles (on the basis of the value of the cross-correlation coefficient). Both reconstructions proved to be indistinguishable within the measured resolution range. The final resolution, estimated with the Fourier shell correlation with a cutoff value at 0.5 [B. Böttcher, S. A. Wynne, R. A. Crowther, Nature 386, 88 (1997)], was 26 Å.
30. Micrographs were checked for drift, astigmatism, and presence of Thon rings by optical diffraction. Scanning was done with a step size of 25 μm corresponding to 4.78 Å on the object scale, on a Perkin-Elmer PDS 1010 A microdensitometer. Particles were selected by an automated selection procedure that differed from the one previously described [K. R. Lata, P. Penczek, J. Frank, Ultramicroscopy 58, 381 (1995)] in that the particle candidates were compared directly with the reference set of 87 quasi-evenly spaced projections (19) of an existing reconstruction of the ribosome from yeast (12). A total of 13, 178 particles were picked. The reconstruction was done with two independent approaches to obtain the orientations of the projections. In the first approach, an existing reconstruction of the ribosome from yeast (12) was used as a reference in the 3D projection alignment procedure (19). In the second approach, an initial reconstruction was obtained with the simultaneous minimization technique [P. Penczek, J. Zhu, J. Frank, Ultramicroscopy 63, 205 (1996)]. In both cases, four steps of the 3D projection alignment procedure (19) were applied with a 2° angular interval. In each step, the refined 3D structure was calculated with 70% of trie best matching particles (on the basis of the value of the cross-correlation coefficient). Both reconstructions proved to be indistinguishable within the measured resolution range. The final resolution, estimated with the Fourier shell correlation with a cutoff value at 0.5 [B. Böttcher, S. A. Wynne, R. A. Crowther, Nature 386, 88 (1997)], was 26 Å.
31. R. Gilmore and G. Blobel, Cell 42, 497 (1985).
32. In trying to gauge the correct threshold value, we were led by two criteria: (i) We observed the structure as the threshold was increased. There is normally a "plateau," a range of threshold values within which the appearance (or the volume encompassed) varies only slightly. (ii) Three-dimensional connectivity must not be violated, which means that, in this case, we could not choose a threshold, within the plateau defined above, that makes the connecting rod disappear. Thus, the plateau was further narrowed. These criteria were applied separately in the preparation of the 3D representations of the ribosome and the channel. Because of the residual uncertainty in the molecular boundaries, the measurements for pore size and the distance between channel and ribosome have an uncertainty of 25%.
33. We thank A. Fischer for the purification of the Sec61 complex; members of the Blobel lab and R. Agrawal for discussions; S. Darst and A. Malhotra for discussions, support with the electron microscopy, and assistance with the image processing; A. Heagle for help with the illustrations; and the National Center for Supercomputer Applications, University of Illinois at Urbana-Champaign, for computing support. Supported by grants from NIH (1R01 GM29169) and NSF (BIR 9219043) (to J.F.) and a fellowship of the Deutsche Foschungsgemeinschaft (to R.B.).