Abstract
COPII vesicles transport proteins from the endoplasmic reticulum to the Golgi apparatus. Previous COPII-cage cryo-EM structures lacked the resolution necessary to determine the residues of Sec13 and Sec31 that mediate assembly and flexibility of the COPII cage. Here we present a 12-Å structure of the human COPII cage, where the tertiary structure of Sec13 and Sec31 is clearly identifiable. We employ this structure and a homology model of the Sec13–Sec31 complex to create a reliable pseudoatomic model of the COPII cage. We combined this model with hydrogen/deuterium-exchange MS analysis to characterize four distinct contact regions at the vertices of the COPII cage. Furthermore, we found that the two-fold symmetry of the Sec31 dimeric region in Sec13–Sec31 is broken upon cage formation and that the resulting hinge is essential to form the proper edge geometry in COPII cages.
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Acknowledgements
This work was supported by the US National Institutes of Health through grant R01GM086892 (S.M.S.), the American Heart Association through grant 0835300N (S.M.S.), the US National Science Foundation Division of Materials Research through grant DMR-06-54118 (A.G.M.) and the State of Florida (A.G.M.). We thank W. Balch (Scripps Research Institute, La Jolla, California, USA) for providing the Sec13–31 expression construct. We thank C.L. Hendrickson and F. Xian for helpful discussions.
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J.O., H.H. and N.B. performed the specimen preparation and biochemistry. J.O. collected the cryo-EM data. S.M.S. performed the cryo-EM reconstructions. Q.Z. collected the MS data and analyzed it with the help of A.G.M. A.J.N. performed the modeling studies. A.J.N., Q.Z., A.G.M. and S.M.S. wrote the manuscript.
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Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–3, 5, Supplementary Table 1 and Supplementary Note (PDF 2222 kb)
Supplementary Data set
Supplementary Figure 4a (PDF 908 kb)
Supplementary Data set
Supplementary Figure 4b (PDF 516 kb)
Supplementary Movie 1
12 Å reconstruction of the Sec13-31 COPII cage, radially colored. The human Sec13-31 homology model, colored by chain, is then docked into the density map. An asymmetric unit of the density map is segmented out and MDFF is applied to the homology model. Octahedral symmetry is imposed when introducing 23 additional copies of the Sec13-31 atomic model. The 24 Sec13-31 atomic models are again subjected to MDFF. Multiple views of the resulting atomic model are shown at a vertex, colored as in Figure 6. At the end of the movie, an individual flexibly fitted Sec13- 31 unit is shown. The two Sec13 chains are aligned, superimposed, and colored as in Figure 2. The resulting hinge with a range of 17° in the plane normal to the 4-fold axis is shown. (MOV 30685 kb)
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Noble, A., Zhang, Q., O'Donnell, J. et al. A pseudoatomic model of the COPII cage obtained from cryo-electron microscopy and mass spectrometry. Nat Struct Mol Biol 20, 167–173 (2013). https://doi.org/10.1038/nsmb.2467
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DOI: https://doi.org/10.1038/nsmb.2467