National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM108753
United States
Citation
Journal: Sci Adv / Year: 2020 Title: The structures of natively assembled clathrin-coated vesicles. Authors: Mohammadreza Paraan / Joshua Mendez / Savanna Sharum / Danielle Kurtin / Huan He / Scott M Stagg / Abstract: Clathrin-coated vesicles mediate trafficking of proteins and nutrients in the cell and between organelles. Proteins included in the clathrin-coated vesicles (CCVs) category include clathrin heavy ...Clathrin-coated vesicles mediate trafficking of proteins and nutrients in the cell and between organelles. Proteins included in the clathrin-coated vesicles (CCVs) category include clathrin heavy chain (CHC), clathrin light chain (CLC), and a variety of adaptor protein complexes. Much is known about the structures of the individual CCV components, but data are lacking about the structures of the fully assembled complexes together with membrane and in complex with cargo. Here, we determined the structures of natively assembled CCVs in a variety of geometries. We show that the adaptor β2 appendages crosslink adjacent CHC β-propellers and that the appendage densities are enriched in CCV hexagonal faces. We resolve how adaptor protein 2 and other associated factors in hexagonal faces form an assembly hub with an extensive web of interactions between neighboring β-propellers and propose a structural model that explains how adaptor binding can direct the formation of pentagonal and hexagonal faces.
History
Deposition
Mar 30, 2020
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Header (metadata) release
Apr 8, 2020
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Map release
Aug 19, 2020
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Update
Mar 6, 2024
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Current status
Mar 6, 2024
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Number selected: 360000 Details: These are subparticle images that were extracted from particle images. Each minicoat particle has 24 asymmetric vertices. From a total of 15000 minicoat particles, 360000 vertex subparticles ...Details: These are subparticle images that were extracted from particle images. Each minicoat particle has 24 asymmetric vertices. From a total of 15000 minicoat particles, 360000 vertex subparticles were extracted. The position of subparticles was calculated by the localized reconstruction script in scipion, and the subparticle images were extracted with RELION extraction tools.
Startup model
Type of model: OTHER Details: An asymmetric vertex was cut out of the minicoat cage map from the same dataset.
Final reconstruction
Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1) Details: After the final refinement in cisTEM, half-maps were generated in cisTEM and the FSC was calculated with EMAN FSC tools. Number images used: 305413
Initial angle assignment
Type: OTHER / Software - Name: cryoSPARC (ver. 2) Details: Using the Euler angles of the mini-coat cage particles and the position of the pentagonal faces in the map, initial hexagonal face Eulers were calculated. Using 2D classification and ...Details: Using the Euler angles of the mini-coat cage particles and the position of the pentagonal faces in the map, initial hexagonal face Eulers were calculated. Using 2D classification and homogenous refinement tools of cryoSPARC shifts, Euler angles were refined for binned-by-2 subparticle images.
Final angle assignment
Type: PROJECTION MATCHING / Software: (Name: cisTEM (ver. 1), RELION (ver. 3)) Details: The output of cryoSPARC was imported into cisTEM for unbinned refinement. Then, the initial output of cisTEM was used for RELION's per particle defocus estimation and beam tilt estimation. ...Details: The output of cryoSPARC was imported into cisTEM for unbinned refinement. Then, the initial output of cisTEM was used for RELION's per particle defocus estimation and beam tilt estimation. This was done step-by-step and after each estimation the map was refined in RELION. After defocus and beam tilt estimation with RELION converged, the vertex map was refined in cisTEM one last time.
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