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Cryo-EM of multiple cage architectures reveals a universal mode of clathrin self-assembly

Nature Structural & Molecular Biology volume 26, pages 890–898 (2019)Cite this article

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Abstract

Clathrin forms diverse lattice and cage structures that change size and shape rapidly in response to the needs of eukaryotic cells during clathrin-mediated endocytosis and intracellular trafficking. We present the cryo-EM structure and molecular model of assembled porcine clathrin, providing insights into interactions that stabilize key elements of the clathrin lattice, namely, between adjacent heavy chains, at the light chain–heavy chain interface and within the trimerization domain. Furthermore, we report cryo-EM maps for five different clathrin cage architectures. Fitting structural models to three of these maps shows that their assembly requires only a limited range of triskelion leg conformations, yet inherent flexibility is required to maintain contacts. Analysis of the protein–protein interfaces shows remarkable conservation of contact sites despite architectural variation. These data reveal a universal mode of clathrin assembly that allows variable cage architecture and adaptation of coated vesicle size and shape during clathrin-mediated vesicular trafficking or endocytosis.

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Fig. 1: Identification of multiple clathrin cage architectures.
Fig. 2: The consensus hub substructure from all and individual clathrin cage architectures.
Fig. 3: Clathrin cage stabilization.
Fig. 4: Structural features of the TxD, coiled-coil and light chain interactions.
Fig. 5: Contacts between triskelion legs for different cage architectures.

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Data availability

Structural data have been deposited into the Worldwide Protein Data Bank (wwPDB), the Electron Microscopy Data Bank (EMDB) and EMPIAR85. EM density maps were deposited in the EMDB with accession numbers EMD-0114, 0115, 0116, 0118 and 0120 for the 28 mini coat, 32 sweet potato, 36 D6 barrel, 36 tennis ball and 37 big apple, respectively. The corresponding hub substructure maps were deposited as EMD-0121, 0122, 0123, 0124 and 0125, respectively. The consensus hub substructure map was deposited with the accession number EMD-0126. The atomic coordinates for the consensus hub were deposited with the PDB accession code 6SCT. Particle stacks associated with EMD-0114–0120 were deposited to EMPIAR as 10294. Particle stacks associated with EMD-0114–0120 without phase flipping and suitable for subparticle extraction were deposited to EMPIAR as 10295. Particle stacks associated with EMD-0121–0125 and EMD-0126 were deposited to EMPIAR as 10296. Other data are available upon reasonable request.

Code availability

BUDE is available under a free academic license from the developer Richard Sessions (http://www.bris.ac.uk/biochemistry/research/bude). All utilities and scripts are available on github (https://github.com/kylelmorris).

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Acknowledgements

C.J.S. was funded by BBSRC grant nos. BB/K003461/1 and BB/N008391/1 and K.L.M. by BBSRC travel award no. BB/L018888/1. C.J.S. was a Royal Society Leverhulme Trust Senior Research Fellow. Y.C. was funded by National Institute of Health (NIH) award nos. R01GM098672 and P50GM082250. Y.C. is a Howard Hughes Medical Institute investigator. R.B.S. and A.A.I. thank the EPSRC for support (grant no. EP/N013573/1). J.R.J. was funded by the EPSRC via the MOAC Doctoral Training Centre. M.H. was supported by the Medical Research Council Doctoral Training Partnership (grant no. MR/J003964/1). M.B. was funded by BBSRC (MIBTP) grant no. BB/J014532/1. We acknowledge the use of EM facilities at the MRC-LMB (Cambridge, UK), NeCEN and UCSF Mission Bay for data collection and in particular V.K. Ragunath for microscope operational assistance at the MRC-LMB. We acknowledge Diamond for access and support of the Cryo-EM facilities at the UK national electron bio-imaging centre (eBIC), proposals EM13142 and EM13909, funded by the Wellcome Trust, MRC and BBSRC. Sample preparation and development was supported by I. Hands-Portman, Warwick Life Sciences Imaging Suite (now Advanced Bioimaging Research Technology Platform), using equipment funded by the Wellcome Trust (grant no. 055663/Z/98/Z). We thank S. Royle, T. Burnley (CCP-EM), J. Huiskonen and S. Scheres for helpful discussions.

Author information

Author notes

  1. Kyle L. Morris

    Present address: Medical Research Council London Institute of Medical Sciences, London, UK

  2. Shenping Wu

    Present address: Yale University, West Haven, CT, USA

  3. Michael Baker

    Present address: School of Biosciences, University of Birmingham, Birmingham, UK

  4. Jean-Paul Armache

    Present address: Department of Biochemistry and Molecular Biology, The Huck Institutes of the Life Sciences, Penn State University, University Park, PA, USA

Authors and Affiliations

  1. School of Life Sciences, University of Warwick, Coventry, UK

    Kyle L. Morris, Joseph R. Jones, Mary Halebian, Michael Baker, Alexander D. Cameron & Corinne J. Smith

  2. Howard Hughes Medical Institute, University of California, San Francisco, CA, USA

    Shenping Wu & Yifan Cheng

  3. Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA

    Jean-Paul Armache & Yifan Cheng

  4. School of Biochemistry, University of Bristol, Bristol, UK

    Amaurys Avila Ibarra & Richard B. Sessions

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Contributions

C.J.S. conceived the overall project. C.J.S and Y.C. supervised research. K.L.M., J.R.J., M.H., S.W., M.B., A.A.I., R.B.S., A.D.C. and C.J.S. performed research. K.L.M. designed and developed the experimental analysis strategy, performed EM and image analysis, collected data, prepared the samples and purified the proteins. J.R.J. constructed the cage library. K.L.M. and A.D.C. conducted modeling. R.B.S. and A.A.I. performed BUDE and Rosetta calculations. M.B assisted with protein preparation and M.H. with data acquisition. S.W. assisted with data acquisition. J.P.A. assisted with data analysis. C.J.S. and K.L.M. wrote the initial manuscript with assistance and editing by all authors equally.

Corresponding author

Correspondence to Corinne J. Smith.

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The authors declare no competing interests.

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Peer review information Katarzyna Marcinkiewicz was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Integrated supplementary information

Supplementary Figure 1 Cryo-EM model validation.

(a) Example density for the final model and fully refined map of the consensus hub structure is shown. Examples are taken from heavy chain repeats, the trimerisation domain, and the light chain. The model has an emRinger score of 0.15 and map-vs-model FSC0.5 4.38 Å. (b) Per-residue B-factor and emRinger scores are shown. (c) BUDE energy scoring of the consensus hub structure for the heavy and light chains. Solid line values represent averages for all possible rotamers. Dotted line shows values for the rotamers adopted in the final model (corresponding to Fig. 3b). (d) Rosetta energy score profiles for the same structure as in (c). These are shown pre and post relaxation by dotted and solid lines respectively. The cross-correlation values between Rosetta and BUDE energy profiles are also given, coloured by domain as defined in (a). (e) Comparison of the proximal heavy chain crystal structure with the cryo-EM model with bulky side chains shown for sequence register reference.

Supplementary Figure 2 Cryo-EM model energy characterisation.

The consensus hub map and model are shown with colouring according to the Rosetta energy analysis. The schematic insets highlight in red where the model displayed sits within the map density. The disease-relevant mutations are highlighted as in Fig. 3c. Note that the back model view is equivalent to Fig. 3c (left) and the bottom model view equivalent to Fig. 3c (right).

Supplementary Figure 3 Residues at the trimerisation domain and yeast two-hybrid rescue mutations.

(a) The cysteine residues 1565, 1569 and 1573 previously implicated in hub assembly. (b) Cysteine residues shown in (a) displayed with corresponding cryo-EM density. (c-d) Rescue mutations associated with light chain binding found in yeast two-hybrid experiments published previously (Chen, C. Y. et al. Embo Journal 21, 6072-6082, 2002) shown on the molecular model presented in this study: (c) W105R rescued by K1326E and (d) W127R rescued by K1415E.

Supplementary Figure 4 Heavy chain leg angular variation and leg twisting.

(a) Representations of the angular changes between proximal, distal junction, distal and terminal segments for all the legs in each cage type. The arrows indicate the angular variation, or degree of swing, at the proximal to distal joint region, the distal to ankle region, and the distal joint and distal domain. (b) An example heavy chain leg of the 36 barrel is shown from the structure in this study (left) compared to the equivalent leg (right) of a previously published 36 barrel (Fotin, A. et al. Nature 432, 573–579, 2004). The arrows indicate the direction of the helix long axes in each leg structure and the proximal (1), distal junction (2), distal segments (3) and terminal segments (4) are shown. In (c) these segments are compared and viewed in cross section. For those marked by *, a rotation of the leg is found compared to the previous cryo-EM model of a clathrin cage from Fotin et al., cited above.

Supplementary Figure 5 Geometric environment, angular conformation and protein contacts.

(a) Characterisation of clathrin heavy chain conformation by local geometric environment. The heavy chain conformation adopted in a cage is defined by following the heavy chain position relative to hexagonal (H) or pentagonal (P) faces to arrive at a signature of the type: P-HP-HP-P, as shown in the diagram. Every geometric environment that a heavy chain is in, across all cage types, has been characterised in this manner. (b) Five domains of the heavy chain that maintain a constant structure in all heavy chain conformations are shown for one example conformation. Pivot points and the angles between these domains are marked 1 to 4. The angles between these pivot points characterise a particular heavy chain conformation. (c) Plots of the angles between the proximal/distal/distal junction/ankle of every heavy chain for the three architectures modelled at the whole cage level are shown. Individual points are coloured according to the geometric environment of the corresponding heavy chain. (d) Variation of heavy chain conformation for the six geometric environments common to all cage architectures, coloured by cage type. (e) All heavy chain leg angles plotted with individual points coloured by cage type.

Supplementary Figure 6 Protein contacts determined for each cage and geometric environment.

Contact density plots for the 28 mini coat, 36 barrel and 36 tennis ball cage architectures comparing all contacts (a, c, e), with contacts for legs that share the same geometric environment within these cages respectively (b, d, f).

Supplementary information

Supplementary Information

Supplementary Figures 1–6, Supplementary Tables 1–4 and Supplementary Notes 1–4.

Reporting Summary

Supplementary Video 1

Regions of flexibility and stability within the whole mini coat cage. Video showing that density occupancy varies across the cage indicating flexibility within the clathrin cage structure. The top panels show the whole cage and the density for the hubs. The middle panel (left) focuses on the TxD and (right) the proximal-proximal heavy chain contact. The bottom panel focuses on the proximal heavy chain in connection with another distal heavy chain and the light chain. The localized reconstruction volume is shown as mesh for reference.

Supplementary Video 2

Overall whole cage model fit for the 28 mini coat, 36 barrel and 36 tennis ball. The three cages are shown in sequence with their respective models.

Supplementary Video 3

The fit of triskelia legs to the 28 mini coat, 36 barrel and 36 tennis ball. The three cages are shown in sequence in the left panel and the fit of each triskelion leg shown in the right panel. The legs are shown in groups according to their geometric signature.

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Morris, K.L., Jones, J.R., Halebian, M. et al. Cryo-EM of multiple cage architectures reveals a universal mode of clathrin self-assembly. Nat Struct Mol Biol 26, 890–898 (2019). https://doi.org/10.1038/s41594-019-0292-0

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