Fig. 5: CLEM finds sites of arrested clathrin-mediated endocytosis (CME).

a Select portion of grid shown as a low magnification cryoEM image registered with cryo-fluorescence images of Dyn1(K44A)-GFP (green) and 500 nm fiducial markers (red). b The grid square highlighted in (a) (white square) is shown in a higher resolution map. Orange dots indicate the outline of the isolated plasma membrane. c The fluorescence overlay is shown. d The black box from (c) is shown enlarged. Black boxes indicate the location of tilt series acquisition for the tomograms shown in (e). e Examples of tomograms in XY (left) and XZ (right). f Examples of arrested CME sites with Dynamin 1 (K44A) tubules. In (e, f), orange and black arrows point to putative clathrin and dynamin densities, respectively. g Examples of arrested CME sites with large clathrin-decorated clusters. XY tomogram images in (e–g) are minimum intensity projections (mIPs) over 21 Gaussian-smoothed XY slices while XZ images are mIPs of 101 XZ slices. h A subtomogram average of clathrin (EMD-46973) is shown with two representative thresholds (left and middle columns, 0.14; right column, 0.08). Labels indicate putative proteins and domains contributing to observed densities. A rigid body fit of PDB ID:6YAI is shown (bottom row) within the subtomogram average showing the clathrin heavy chain domains proximal to the central vertex (blue), clathrin light chain (yellow), clathrin heavy chain N-terminal domain with distal leg (pink), and the β2 appendage of the adapter AP2 (orange). Magnified views of the heavy chain N-terminal domain (bottom-middle) and the β2 appendage are shown (bottom-right). i The clathrin vertex reconstruction is superimposed onto refined subtomogram positions reconstructing a clathrin coated pit (green) representative of those found in the 20 tomograms used in this analysis. Several lattice arrangements are visible within the single structure. The image is an average of 10 XY slices from a denoised tomogram. This condition is represented in grids 5-6 (Table 1). All clathrin data are from grid 6.