Methods
Clathrin coat preparation
Clathrin triskelions containing heavy and light chains and AP complex proteins were obtained by Tris extraction from calf-brain coated vesicles according to protocols described in1. AP-containing fractions were subjected to further purification by hydroxyapatite chromatography on an Econo-Pac CHT-II column (BioRad). Fractions containing exclusively AP-2 proteins were pooled and dialyzed against “AP buffer” (100 mM MES, pH 7.0, 150 mM NaCl, 1 mM EDTA, 0.02% NaN3, 0.5 mM DTT). To obtain light chain-free clathrin coats, heavy chain triskelions were separated from the bound light chains by dialysis against a buffer containing 1.35 M KSCN, 50 mM TAE, pH 8.0, 2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, 0.02% NaN3, followed by gel filtration on a Superose 12 column (Amersham Biosciences) equilibrated with the same buffer. SDS-PAGE analysis (Supplementary Fig. 1a) showed better than 90% removal of the light chain.
Coats were assembled by mixing concentrated clathrin triskelions or light chain-free clathrin triskelions (1.3 mg/ml) and the purified AP-2 proteins (1 mg/ml) at a ratio of 3:1 (v/v). The mixture was dialyzed against “assembly buffer” (50mM Mes Na, pH 6.5, 2mM EDTA, 100mM NaCl, 2mM DTT) overnight at 4ºC. The relatively high NaCl concentration in the assembly buffer was used to facilitate formation of D6 barrels. Aggregated clathrin was removed by centrifugation in an Eppendorf centrifuge at 15,000 rpm at 4ºC for 10 min. Assembled coats were separated from unassembled triskelions by high-speed centrifugation at 60,000 rpm in a TLA-100.4 rotor (Beckman) at 4ºC for 12 min and then gently resuspended in “storage buffer” (25mM MES Na, pH 6.5, 2 mM DTT) to a final concentration of 1 mg/ml. The presence or absence of light chains had no effect on the yield of coat assembly. Samples of clathrin triskelions, light chain-free clathrin triskelions, AP-2 complex proteins as well as assembled coats were analyzed by SDS-PAGE (Supplementary Fig. 1a).
Electron cryomicroscopy data collection
Only fresh clathrin coat samples (within 1-3 days of the preparation) were used for electron microscopy to ensure the integrity of the specimen. The sample was diluted to 0.05 mg/ml coat concentration with assembly buffer just before freezing, applied to a Quantifoil® holey carbon grid (Quantifoil Micro Tools GmbH, Germany), blotted with filter paper and flash-frozen in liquid ethane at –180°C using a Reichert/Leica KF80 rapid-freezing unit. A series of freezing experiments was done in order to obtain ice of approximately 1000Å thickness. After optimal freezing conditions were found, a batch of 20 frozen grids was prepared and stored in a liquid nitrogen tank.
Grids with vitrified clathrin coats were imaged with an FEI Tecnai F20 microscope equipped with a field-emission gun and operated at 200 kV voltage using low-dose procedures (15 electron/Å2). Approximately 350 micrographs were recorded for each specimen at a nominal magnification of 50,000x and defocus levels ranging from -2 µm to -5 µm.
Image processing and initial model reconstruction
Electron micrographs were visually inspected on an optical diffractometer and only those showing clearly visible Thon rings2 and absence of drift and significant astigmatism were selected for digitization. The micrographs were digitized on a Zeiss SCAI scanner using a step size of 7 µm corresponding to 1.4 Å/pixel.
For initial processing the selected digitized micrographs were binned over 6x6 pixels corresponding to 8.4 Å/pixel. A set of single clathrin coat particles was selected interactively using the Ximdisp visualization softwar3. On average, there were 30 clathrin particles present on each micrograph. Although “hexagonal barrel” particles were in abundance, smaller “mini-coat” and larger “icosahedral” particles were also present (see Supplementary Fig. 1b). These particles were also selected and put into separate sets.
Further image processing was done using the IMAGIC software package4. Selected particles were windowed out into 128x128 pixel images and assembled into a stack. Particle images in the stack were normalized and band-pass filtered to remove spatial frequencies lower than 1/800 Å-1 and higher than 1/40 Å-1. No CTF correction was done at this stage. Normalized and filtered particles were translationally aligned against the rotationally averaged total stack sum. The aligned particles were then partitioned into classes corresponding to different orientations of the clathrin coats in the ice layer using an iterative procedure including multireference alignment (MRA), multivariate statistical analysis (MSA) and classification steps as described in5. After 5-10 iterations, the alignment and classification procedures produced stable class averages. Euler angles of the selected class averages were assigned using the angular reconstitution method5 followed by three-dimensional reconstruction and application of corresponding symmetry.
Figure S1. Coat assembly and image analysis. (a) SDS-polyacrylamide gel electrophoresis of the clathrin and AP-2 complexes used to make the assemblies described in this paper. Lane 1: AP-2. The s chain is not visible in the Figure. Lane 2: clathrin. Lane 3: light-chain free clathrin. Lane 4: Coats, assembled from clathrin and AP-2. Lane 5: Coats, assembled from light-chain free clathrin and AP-2. The bands corresponding to heavy-chain (HC), light-chains LCa and LCb (LCs), AP-2 large-chain (a/b) and AP-2 medium chain (m2) are labeled. (b) Unprocessed electron micrograph of a field of clathrin coats in vitreous ice. Black arrows: mini-coats; white arrow: soccer ball; the remaining coats are hexagonal barrels. (c) Gallery of class averaged images, as calculated using IMAGIC6. (d) Distribution of image orientations, plotted as a polar-angle diagram, viewed along the q = 0° axis. The orientations have all been determined within one asymmetric unit of the D6 symmetry. (e) Fourier shell correlation7 as a function of spatial frequency, for the ncs averaged map of the proximal segment (solid line) and for noise (dotted line). Using the FSC = 0.5 resolution criterion, the density map would have a resolution of about 12 Å, whereas the FSC curve intersects the 3s curve at a resolution of about 6 Å. The recently introduced FSC = 0.143 criterion8 suggests a resolution of 7.9 Å. A resolution of 7.9 Å is most consistent with the features seen in the density map (Figure S2) and has therefore been chosen as the resolution cutoff.
Figure S2. Resolution assessment. (a) Thon rings, calculated as a rotational average of the sum of power spectra of all particles in a single micrograph with estimated defocus of –3.5 mm. (b) Comparison of observed density with calculated density for two different resolution cutoffs. The upper and lower panels are orthogonal views. At the top of each panel is the observed density in the averaged image reconstruction (blue), with the model of Ybe et al9 fit as described. In the center of each panel is the density calculated from the model, with a cutoff at 7.9 Å resolution (red). At the bottom of each panel is the density calculated with a cutoff at 12 Å resolution (green). The upper two parts of each panel are the same as Fig. 3a,b.
Figure S3. Homology models for CHCR0-7. These were determined from sequence alignment 9 and from the X-ray structure of CHCR6, using MODELLER10,11. The shaded elements are those included in the crystal structures of the terminal-domain-linker12 and of a segment of proximal leg9. The CHCR6 repeat (residues 1280-1428) is completely included in the proximal-segment crystal structure, which covers residues 1210-1516. We built homology models for CHCRs 0-5 and 7, extracting directly from the structure the parts of CHCR5 and CHCR7 included within it. From the junctions between CHCR5 and 6 and CHCR6 and 7 in the crystal structure, which closely resemble each other, we modelled junctions between other successive repeat elements. The N-terminal part of CHCR0 is contained in the crystal structure of the N-terminal-domain-linker12. The homology model agrees well with all but the N-terminal hairpin (marked with an asterisk in Fig. 4b). In our final model for CHCR0, residues 395-483 derive from the crystal structure and 484-542 from homology with CHCR6. Colors are the same as in Fig. 4a.
Figure S4. Packing of heavy-chains in the clathrin coat. (a) View from outside the coat of an edge, showing antiparallel proximal segments (green and red) and the distal segments (blue and yellow) just beneath them. (b) Side view of the same edge. Colors as in (a). (c) Cross sectional view, showing that the orientation of helices in the distal segments is rotated by about 90° with respect to the orientation of helices in the proximal segment.
Figure S5. Aligned sequences of the last 85 residues (in the mammalian proteins) of the clathrin heavy chains: bovine (BT), human (HS), fruit fly (DM), yeast (SC). The region of the helical tripod is shown.
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