High-resolution cryo-electron microscopy of clathrin coats

Clathrin-coated vesicles are the means by which selected integral membrane proteins are removed from the cell surface or trans-Golgi network and transported to the endosomal system. The clathrin cage-like structure that forms the outside of the vesicle has unusual molecular organization: clathrin triskelions (trimers with three legs radiating from a central hub) form a lattice of pentagons and hexagons that curves around to enclose the vesicle. In this work we applied high-resolution single-particle cryo-electron microscopy and homology-based modeling techniques to build pseudo-atomic structures of clathrin coats of different designs and performed structural analysis of clathrin lattice assembly and disassembly.; We computed 3D cryo-EM reconstructions of light chain-bound and light chain-free hexagonal barrel coats at 8.0 A resolution. Using available crystal structures of clathrin leg segments and sequence alignment of conserved heavy chain repeats, we modeled three-dimensional structures for most parts of the clathrin leg. We docked the modeled structures into the computed EM density map and built a complete pseudo-atomic model of an entire 20 MDa hexagonal barrel clathrin coat.; Binding sites of light chains on a clathrin coat were identified by computing the difference between the light chain-bound and light chain-free density maps. The light chains were shown to form extended helical interfaces with loops of the heavy chains. We obtained 3D cryo-EM reconstructions of tennis ball coats and performed comparative conformational analysis of triskelions extracted from hexagonal barrel and tennis ball coats. The analysis revealed the existence of a rigid hub structure invariant at every vertex of the lattice and showed that clathrin coats adapt to different packing requirements by altering hydrophobic contacts between clathrin legs forming an edge of a clathrin polyhedron.; 3D reconstruction of clathrin coats decorated with auxilin showed the mode of interaction between auxilin and clathrin. We propose a model of clathrin coat disassembly, in which auxilin targets Hsc70, a molecular chaperon, to the core of the rigid hub structure.