Research ArticleSTRUCTURAL BIOLOGY

The structures of natively assembled clathrin-coated vesicles

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Science Advances  22 Jul 2020:
Vol. 6, no. 30, eaba8397
DOI: 10.1126/sciadv.aba8397
  • Fig. 1 Single-particle refinement of CCVs.

    (A) Refinement of CCVs into five different cage geometries. The tetrahedral mini-coat has the highest resolution among the geometries. The C2 basket is a previously uncharacterized geometry represented in two views in the dashed panel. The largest cages are the D6 barrel and the tennis ball. (B) Clathrin-focused subparticle refinement of the mini-coat asymmetric vertex further improves the resolution. Top: Asymmetric vertex refinement with colors representing different domains; resolution, 6.3 Å (FSC0.143). Middle: Side view of the vertex. Bottom: Side view with one CHC and CLC removed to show the trimerization helices.

  • Fig. 2 Atomic modeling of the CHC distal leg-tripod interactions.

    (A) The left panel shows the full vertex atomic model. The middle panel shows the same atomic model but highlights the unique interactions of the distal legs from two different CHC chains (green and cyan) with the trimerization domain of a third CHC (magenta). The top right panel is a side view of the model, and the bottom right panel shows only the three interacting CHCs. (B) The left panel shows the EM density map for the tripod interaction. The right panel shows the position of the QLML residues of the QLMLT motif extending down from the trimerization helix.

  • Fig. 3 Three-way interaction between β-propeller, ankle, and β2 appendage with their densities and crystal structures shown in dark blue, light blue, and orange, respectively.

    (A) Cross-linking of two β-propellers by a β2 appendage domain. Fitted crystal structures are PDB IDs 1BPO (clathrin) and 1E42 (β2 appendage). (B) All hexagonal faces of the mini-coat contain three copies of the β2 appendage density. The dashed boxes indicate magnified insets. The dashed orange arc in the bottom panel roughly outlines the interface between β-propellers and β2 appendage and is used as reference for (C) and (D). (C) Pentagonal faces of the mini-coat only have densities for clathrin. Bottom panel shows the movement of the right clathrin leg compared to its position in a hexagonal face as shown in (B). (D) Larger cages like the D2 baseball have β2 appendage densities in their pentagonal faces, unlike the mini-coat. However, the position of the β2 appendage in a pentagonal face is different than its position in a hexagonal face [compare (B) and (D)].

  • Fig. 4 Three different configurations of neighboring faces for a pentagonal face in all geometries and their relation to the population of adaptor appendages in the pentagonal face (colored by domain).

    Fractions show the number of pentagonal faces in each configuration out of the 12 total pentagonal faces. (A) Top: configuration 1. The neighboring faces are indicated with Pent and Hex. The CHC chains with β-propellers in the central pentagonal face are traced using the color code. Bottom: The same CHCs traced in the top panel are shown in a side view with no appendages present. Geometries with configuration 1 are indicated (T, D3, C2). The organization of the top and bottom panels are the same for (B) and (C). (B) Configuration 2. This configuration is unique to C2 basket. The bottom panel shows that some β-propellers are engaged with adaptor appendages. (C) Configuration 3. The four different geometries indicated in the bottom panel accommodate configuration 3, in which all β-propellers are engaged with adaptor appendages. The table shows a relationship between configuration and approximate fraction of β-propellers in the central pentagonal face of that configuration that are engaged with adaptor appendages.

  • Fig. 5 Adaptor-focused refinement revealing AP2 core density and a network of cross-linking densities (clathrin colored by domain).

    (A) β2 appendages of one hexagonal face and averaged-out density from the mini-coat cage refinement. Red box indicates the volume that was further resolved in subparticle alignment. (B) Subparticle alignment reveals the AP2 core density. The crystal structure of open AP2 conformation is fitted inside. (C) AP2 density at a slightly higher isosurface threshold than (B) to emphasize the two connecting densities. (D) Relative positions of clathrin hexagonal face (gray), clathrin β-propellers (blue), and AP2 core densities (orange) are shown. The clathrin hexagonal face density is removed to show the proximity of AP2 to 18 β-propellers. (E) Same views of (D) are shown here at a lower isosurface threshold to show the interaction web of β-propellers and the cluster of densities formed around AP2.

  • Fig. 6 Model for adaptor cluster–mediated organization of pentagonal and hexagonal faces (colored by domain).

    Auxilin is represented by green blobs sitting on top of β-propellers. Clathrin chains in red and magenta are not interacting with the adaptor cluster and hence can be removed. The orange adaptor cluster and the blue clathrin chains are interconnected with dashed lines. Dashed inset shows the schematic interaction network of β-propellers cross-linked by adaptors and the map on which it was based.

Supplementary Materials

  • Supplementary Materials

    The structures of natively assembled clathrin-coated vesicles

    Mohammadreza Paraan, Joshua Mendez, Savanna Sharum, Danielle Kurtin, Huan He, Scott M. Stagg

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