Multi‐modal adaptor‐clathrin contacts drive coated vesicle assembly
2021; Springer Nature; Volume: 40; Issue: 19 Linguagem: Inglês
10.15252/embj.2021108795
ISSN1460-2075
AutoresSarah M. Smith, Gabrielle Larocque, Katherine M. Wood, Kyle L. Morris, Alan M. Roseman, Richard B. Sessions, Stephen Royle, Corinne J. Smith,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoArticle6 September 2021Open Access Transparent process Multi-modal adaptor-clathrin contacts drive coated vesicle assembly Sarah M Smith Sarah M Smith orcid.org/0000-0002-5349-1902 School of Life Sciences, University of Warwick, Coventry, UK Search for more papers by this author Gabrielle Larocque Gabrielle Larocque orcid.org/0000-0001-8295-9378 Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK Search for more papers by this author Katherine M Wood Katherine M Wood orcid.org/0000-0003-3723-097X School of Life Sciences, University of Warwick, Coventry, UK Search for more papers by this author Kyle L Morris Kyle L Morris orcid.org/0000-0002-1717-8134 School of Life Sciences, University of Warwick, Coventry, UK Search for more papers by this author Alan M Roseman Alan M Roseman orcid.org/0000-0002-4783-2619 Division of Molecular and Cellular Function, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Search for more papers by this author Richard B Sessions Richard B Sessions orcid.org/0000-0003-0320-0895 School of Biochemistry, University of Bristol, Bristol, UK Search for more papers by this author Stephen J Royle Corresponding Author Stephen J Royle [email protected] orcid.org/0000-0001-8927-6967 Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK Search for more papers by this author Corinne J Smith Corresponding Author Corinne J Smith [email protected] orcid.org/0000-0003-3364-7946 School of Life Sciences, University of Warwick, Coventry, UK Search for more papers by this author Sarah M Smith Sarah M Smith orcid.org/0000-0002-5349-1902 School of Life Sciences, University of Warwick, Coventry, UK Search for more papers by this author Gabrielle Larocque Gabrielle Larocque orcid.org/0000-0001-8295-9378 Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK Search for more papers by this author Katherine M Wood Katherine M Wood orcid.org/0000-0003-3723-097X School of Life Sciences, University of Warwick, Coventry, UK Search for more papers by this author Kyle L Morris Kyle L Morris orcid.org/0000-0002-1717-8134 School of Life Sciences, University of Warwick, Coventry, UK Search for more papers by this author Alan M Roseman Alan M Roseman orcid.org/0000-0002-4783-2619 Division of Molecular and Cellular Function, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Search for more papers by this author Richard B Sessions Richard B Sessions orcid.org/0000-0003-0320-0895 School of Biochemistry, University of Bristol, Bristol, UK Search for more papers by this author Stephen J Royle Corresponding Author Stephen J Royle [email protected] orcid.org/0000-0001-8927-6967 Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK Search for more papers by this author Corinne J Smith Corresponding Author Corinne J Smith [email protected] orcid.org/0000-0003-3364-7946 School of Life Sciences, University of Warwick, Coventry, UK Search for more papers by this author Author Information Sarah M Smith1, Gabrielle Larocque2,5, Katherine M Wood1, Kyle L Morris1,6, Alan M Roseman3, Richard B Sessions4, Stephen J Royle *,2 and Corinne J Smith *,1 1School of Life Sciences, University of Warwick, Coventry, UK 2Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK 3Division of Molecular and Cellular Function, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK 4School of Biochemistry, University of Bristol, Bristol, UK 5Present address: Cellular Signalling and Cytoskeletal Function Laboratory, The Francis Crick Institute, London, UK 6Present address: Diamond Light Source Ltd, Harwell Science & Innovation Campus, Didcot, UK *Corresponding author. Tel: +44 24 7615 1931; E-mail: [email protected] *Corresponding author. Tel: +44 24 7652 2461; E-mail: [email protected] The EMBO Journal (2021)40:e108795https://doi.org/10.15252/embj.2021108795 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Clathrin-coated pits are formed by the recognition of membrane and cargo by the AP2 complex and the subsequent recruitment of clathrin triskelia. A role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the β2 subunit of AP2, and several binding sites have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of β2 hinge-appendage (β2HA). We find that the β2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, β2-appendage cross-links two adjacent terminal domains from different triskelia. Functional analysis of β2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the "sandwich site" on the appendage. We propose that β2-appendage binding to more than one triskelion is a key feature of the system and likely explains why assembly is driven by AP2. SYNOPSIS The AP2 complex recruits clathrin triskelia to the plasma membrane to build clathrin-coated pits, yet other roles for AP2, such as in cage assembly, are unexplored. Here, single particle cryo-EM analysis reveals how AP2 promotes clathrin assembly during endocytosis by crosslinking clathrin triskelia at different locations in the cage. β2-appendage of AP2 binds in at least two positions in the clathrin cage. Functional analysis of β2-clathrin interactions shows that endocytosis requires two clathrin interaction sites. β2-appendage binding to more than one clathrin triskelion is a key feature of clathrin-AP2 interaction, explaining why clathrin assembly is driven by AP2. Introduction Clathrin-mediated endocytosis (CME) is the major route of entry for receptors and their ligands into cells (Mettlen et al, 2018). A clathrin-coated pit is formed at the plasma membrane that selects cargo for uptake into the cell via a clathrin-coated vesicle. Clathrin cannot recognize membrane or cargo itself and so an adaptor protein binds the membrane, selects the cargo, and associates with clathrin leading to pit formation (Fig 1A). Several adaptor proteins have clathrin binding sites and colocalize with clathrin structures in cells but the assembly polypeptide-2 (AP2) complex (α, β2, µ2 and σ2 subunits) is thought to primarily initiate clathrin recruitment. Figure 1. Structural view of clathrin assembly during endocytosis Schematic diagram of clathrin-mediated endocytosis. The AP2 complex opens when it engages cargo and PI(4,5)P2, the β2 hinge and appendage (β2HA) become available for clathrin binding, initiating pit formation. Structure of β2HA (PDB code: 2G30). The appendage is divided into platform and sandwich subdomain, each with a tyrosine residue previously identified to be important for clathrin binding. The unstructured hinge region contains a clathrin-box motif (CBM, LLNLD) which binds the N-terminal domain (NTD) of clathrin heavy chain. Structure of a clathrin triskelion (PDB code: 3IYV). Three clathrin heavy chains (CHC) each with an associated light chain (CLC) are trimerized at their C-termini forming a tripod. Each leg is divided into proximal and distal segments, an ankle region and NTD. Clathrin assemblies. (i) An indigo triskelion is shown engaged with six other triskelia in a hexagonal barrel, coating a vesicle. Each edge is made from four leg segments for four different triskelia: two antiparallel proximal (P) regions on the outer surface and two antiparallel distal (D) regions below. Three edges (a–c) are shown schematically. (ii) The tripod of this triskelion (T0) is at a vertex, and below that, three NTDs (NTD1-3) are arranged, contributed by triskelia (purple) whose tripods are two or three edges away (T1-3). Right panels show the view from the vesicle towards the vertex. The positions of triskelia were mapped by downsampling the carbon backbones in 3IYV by 5 residues and smoothing their position in 3D space using a 25 residue window in IgorPDB. CLCs have been removed for clarity. Download figure Download PowerPoint The recruitment of clathrin by the β2 subunit is an essential step in CME. AP2 and clathrin arrive jointly at the membrane in a ratio of two AP2 complexes per triskelion (Cocucci et al, 2012). As the pit matures, the ratio decreases as clathrin polymerizes (Bucher et al, 2018). It is assumed that this polymerization—which is an innate property of clathrin triskelia—completes vesicle formation. However, AP2 is named after its ability to promote clathrin cage assembly in vitro (Zaremba & Keen, 1983; Pearse & Robinson, 1984), and a fragment of the β2 subunit of AP2, containing the hinge and appendage domains (β2HA), has been shown to promote the polymerization of clathrin (Gallusser & Kirchhausen, 1993; Shih et al, 1995; Owen et al, 2000). How these in vitro observations relate to endocytosis in cells is unclear. One intriguing but often overlooked idea is that AP2, via β2HA, serves a dual role in CME: initially recruiting clathrin to the plasma membrane and then driving coated vesicle assembly. There are two clathrin-binding locations on β2HA (Fig 1B). The first is a linear peptide motif within the hinge region (Owen et al, 2000; Lundmark & Carlsson, 2002), LLNLD, called the clathrin-box motif (CBM). The second clathrin-binding location is within the β2-appendage domain, however, its precise nature is debated (Chen & Schmid, 2020). The appendage domain has two sites that interact distinctly with different binding partners (Owen et al, 2000; Edeling et al, 2006; Schmid et al, 2006). The first, termed the sandwich (or side) domain, which surrounds Tyr 815, binds AP180, amphiphysin and eps15. A second site, termed the platform (or top) domain, surrounds residues Y888 and W841 (Fig 1B). This binds the adaptor proteins epsin, β-arrestin and autosomal recessive hypercholesterolemia (ARH) protein and functions independently from the sandwich domain. The roles of these sites in clathrin binding remain to be clarified. In vitro pull-down experiments highlight the potential importance of both Y888 and Y815 for clathrin binding but reports differ on their relative contribution (Owen et al, 2000; Edeling et al, 2006; Schmid et al, 2006). Our structural understanding of how clathrin engages with AP2 is incomplete. The N-terminal domain (NTD, Fig 1C) of clathrin heavy chain is a seven-bladed β-propeller with four adaptor protein binding sites (Willox & Royle, 2012). Atomic structures have revealed that CBMs bind promiscuously to these sites, with the AP2 CBM binding to the "CBM site" between blades 1 and 2 and also to the "arrestin site" between blades 4 and 5 (Muenzner et al, 2017). The location where β2-appendage binds clathrin is uncertain. Knuehl et al (2006) used biochemical approaches and yeast-2-hybrid studies to identify residues C682 and G710 on the heavy chain ankle region as a potential location for β2-appendage. Another potential location is where transforming acidic coiled-coil 3 (TACC3) binds clathrin (residues 457–507; Burgess et al, 2018; Hood et al, 2013). However, a full picture of how the β2HA interacts with assembled clathrin, central to the mechanism of clathrin recruitment, remains elusive. Recently, two structural studies have visualized contradictory modes of binding for the β2-appendage in clathrin assemblies. Using cryo-electron tomography, Kovtun et al investigated the structure of assembled clathrin and a form of AP2 lacking the alpha appendage and hinge region on lipid membranes containing cargo peptides and PI(4,5)P2 (Kovtun et al, 2020). They observed density beneath the clathrin vertex enclosed by one terminal domain and the ankle regions of two triskelion legs (see Fig 1D for orientation). In contrast, Paraan et al isolated native coated vesicles from bovine brain and obtained a structure using single particle analysis. They observed density consistent with the β2-appendage, however it was in a different location, between two adjacent terminal domains (Paraan et al, 2020). In order to address the paradox, we have analysed the structure of purified clathrin bound to the β2HA using single particle cryo-EM approaches. We find that the β2-appendage binds in at least two positions on clathrin, within the same sample, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions and reconciling the differing observations in the literature. Our functional analysis of β2HA-clathrin interactions reveals that endocytosis requires hinge and appendage interaction sites, with the Tyr 815 sandwich site being more important for productive vesicle formation than the Tyr 888 platform site. In consolidating all available structural and functional information, we find that β2-appendage binding to more than one clathrin triskelion is a key feature of the system and likely explains how clathrin assembly is driven by AP2. Results The appendage of β2 is critical for coated vesicle formation We previously developed a strategy to trigger clathrin-coated vesicle formation in cells, termed "hot-wired endocytosis" (Wood et al, 2017). It works by inducibly attaching a clathrin-binding protein (clathrin "hook") to a plasma membrane "anchor" using an FKBP-rapamycin-FRB dimerization system; and this is sufficient to trigger endocytosis (Fig 2A). Using the hinge and appendage of the β2 subunit of the AP2 complex (FKBP-β2HA-GFP) as a clathrin hook allows us to examine endocytosis that is driven by the interaction of β2HA and clathrin, that is, independent of other clathrin-adaptor interactions. Hot-wired endocytosis can be detected in live cells by visualizing the formation of intracellular bright green puncta that also contain an antibody to the extracellular portion of the anchor. These puncta move inside the cell, away from the plasma membrane and we have shown previously that they are clathrin-coated vesicles that have pinched off from the surface and are competent for traffic inside the cell (Wood et al, 2017). Using FKBP-β2HA-GFP as a clathrin hook, the formation of numerous puncta was observed, while a control construct (FKBP-GFP) elicited no response (Fig 2B and C). Figure 2. The β2 appendage is crucial for hot-wired clathrin-mediated endocytosis Schematic diagram of hot-wired endocytosis. Under normal conditions the AP2 complex engages with cargo and PI(4,5)P2 at the plasma membrane (PM) and the β2 hinge and appendage become available for clathrin engagement. AP3 acts analogously at the early endosome (EE). In hot-wired endocytosis a clathrin hook, e.g. β2 hinge and appendage (FKBP-β2HA-GFP) is attached to a plasma membrane anchor CD8-mCherry-FRB inducibly by rapamycin application. Endocytosis is measured by uptake of a fluorescent antibody that binds the plasma membrane anchor. Representative confocal micrographs of cells expressing the plasma membrane anchor (CD8-mCherry-FRB) and the indicated hooks (green). Cells were incubated with anti-CD8-Alexa647 (red) and treated with rapamycin (200 nM, 15 min). Endocytic vesicles coinciding in both green and red channels (yellow in merge) were quantified in B. Scale bar, 10 µm. Scatter dot plot shows the number of intracellular GFP-positive vesicles that contained anti-CD8 Alexa647 per cell, bars indicate mean ± SD. Number of experiments = 3. P-values from Dunnett's post hoc test that were < 0.1 are shown above. Download figure Download PowerPoint An analogous construct from the AP3 complex, FKBP-β3HA-GFP, with the hinge and appendage of β3, was not competent for hot-wiring (Fig 2B and C). This is a surprising result for two reasons: first, the clathrin-box motif in the hinge of β3 binds clathrin in vitro (Dell'Angelica et al, 1998), and second, we had assumed that the role of the clathrin hook in the hot-wiring system was solely to recruit clathrin initially, with downstream polymerization being driven by clathrin alone. To investigate this result in more detail, we tested whether the hinges of β2 or β3 were competent for hot-wiring. Despite the presence of a clathrin-box motif in both hinges, with the appendage domains removed neither FKBP-β2H-GFP nor FKBP-β3H-GFP was able to induce endocytosis (Fig 2B and C). Next, we transplanted the appendage of β3 onto the β2 hinge, and the appendage of β2 onto the β3 hinge. We observed hot-wiring with FKBP-β3Hβ2A-GFP but not with FKBP-β2Hβ3A-GFP (Fig 2B and C). Thus, the β2 appendage was able to drive endocytosis with a β3 hinge but the β2 hinge alone or in the presence of the β3 appendage could not. These results indicate firstly that the β2 appendage is critical for endocytosis and that the β3 appendage cannot substitute for this activity. Secondly, hooks containing a clathrin-box motif are not sufficient for vesicle formation. This suggested to us that the β2 appendage is active in clathrin polymerization. Structure of clathrin-β2HA minicoat cages If the β2 appendage contributes to clathrin polymerization, the nature of its interaction with assembled clathrin is of particular interest. In order to investigate this, we analysed cryo-electron micrographs of clathrin assembled in the presence of β2HA (Fig EV1A–G). Saturation of β2HA binding sites on clathrin was achieved using a 60-fold molar excess of β2HA (Fig EV1A and B). Of the 57,528 particles analysed, 29% of the total particle data set (16,641 particles) was occupied by the minicoat class of cages (Fig EV1C–G). Subsequent extensive supervised and unsupervised 3D classifications identified the particles most stably associated with the minicoat cage architecture (Appendix Figs S1 and S2). These 13 983 minicoat particles were refined to a gold standard resolution of 9.1 Å (Appendix Fig S3). Click here to expand this figure. Figure EV1. Clathrin-β2HA reconstitution, data collection and processing Clathrin triskelia (3 µM) were assembled in the presence of increasing concentrations (3–240 µM) of β2-adaptin616-951 (β2HA). Clathrin assemblies were pelleted and analysed by SDS–PAGE to determine the amount of clathrin (CHC and CLCa/b) and β2HA in the pellet (P) and supernatant (S) fractions. Densitometry of gels in A shows that increasing amounts of β2HA pelleted with clathrin during the reconstitution experiments, with a 60-fold excess of adaptor protein yielding the maximum amount of clathrin-binding. Negative stain TEM analysis of clathrin cages reconstituted with a 60-fold excess (240 µM) of β2HA. Scale bar = 200 nm. A representative cryo-electron micrograph (left) at −1.4 µm defocus and the corresponding power spectrum indicating the information content at high spatial frequencies (right). 2D class averages of classes that were selected for 3D classification in RELION. Particle occupancy of the 10 classes obtained with supervised, asymmetric 3D classification in RELION. 3D surface representations of the 3 clathrin cages generated from the supervised, asymmetric 3D classification of clathrin cage particles. Their colour corresponds to the class occupancy data shown in panel F. Only the orange cage was reconstructed in full, enabling its cage geometry to be confirmed as minicoat. Download figure Download PowerPoint In order to locate β2HA within the map density, we compared the β2HA-clathrin map to a map of clathrin cages assembled in the absence of β2HA. While a difference map did reveal density in a location just above the terminal domains, it was not well-defined (Fig 3A and B). We therefore conducted a voxel-by-voxel comparison between the two maps to locate statistically significant differences (Young et al, 2013). This method allows the location of differences to be determined with confidence but does not define the shape of difference density. This enabled us to evaluate the entire minicoat particle data set globally for potential β2HA binding locations. The results of this analysis confirmed a significant difference just above the terminal domains (Fig 3C). We also noted significant differences in some other areas, away from β2HA, that may be related to triskelion leg movements or other conformational changes upon β2HA binding. Figure 3. Global difference analysis of clathrin-β2HA compared to clathrin alone Unsharpened cryo-EM map of clathrin-β2HA minicoat cage architecture at 9.1 Å resolution. Difference map of clathrin-β2HA minicoat and clathrin-only minicoat. Differences in density are shown in orange. Clathrin-β2HA and clathrin-only minicoat maps. Statistically significant differences are shown on a rainbow colour scheme (see inserted panel) with red, orange, yellow and green being the areas of significant difference. The light blue and dark blue areas indicate regions where the significance is below our threshold, or there is no significant difference between the two maps. The regions with the most significant difference density at P < 0.0005 (in red) were interpreted as the binding site of β2HA. Other regions show significant differences due to conformation changes related to binding. The contour level of all maps is 3.0 times sigma above the mean of the map. All images were created in UCSF Chimera (Pettersen et al, 2004). Download figure Download PowerPoint Finding β2HA in clathrin-β2HA minicoats Our global difference analysis suggested that the β2HA was indeed bound to the cages but not well-resolved. Association of β2HA with clathrin cages may increase sample heterogeneity either through effects on the cage structure itself or through variations in mode of binding, ultimately affecting resolution. In addition, clathrin terminal domain flexibility may result in weaker density in the terminal domain and linker region (Fotin et al, 2004; Morris et al, 2019). We therefore used signal subtraction to reduce the dominance of the strong features of the outer clathrin cage in order to classify the weaker terminal domain signal more precisely (Bai et al, 2015) (Appendix Fig S4). 13,983 particles of the inner region of the minicoat cage were classified into 20 classes, with occupancy ranging from 1.4 to 12.2%, reflecting the heterogeneity of this cage region. Particles belonging to each class were refined individually to a higher resolution (Fig EV2). The outputs of the individual refinements (each at contour level σ3) varied in the quality and completeness of the terminal domain density. However in two classes, 15 and 18, distinct density consistent with bound β2HA was observed. Click here to expand this figure. Figure EV2. Unsupervised, masked 3D classification of signal-subtracted minicoat cage particles Output of unsupervised, masked 3D classification of signal-subtracted minicoat cage particles. Particles were separated into 20 classes: hexagonal faces (representative of average class quality for all remaining polygonal faces) are shown for each class with the corresponding refinement (at 3σ contour level) shown below. Download figure Download PowerPoint In the case of class 15, these densities were in a different location to that shown by our global analysis, on alternate terminal domains within a polyhedral face (Fig EV3A–D). Comparison of equivalent positions in a minicoat cage without adaptor bound demonstrated that the densities present at the terminal domains were a consequence of β2HA binding (Fig EV3A and B). Looking at adjacent polyhedral faces, for a given hub region where 3 terminal domains (from separate triskelia) converge, two terminal domains are engaged in an interaction with a single β2-appendage leaving one terminal domain unoccupied (Fig EV3C). Interestingly, β2HA density was not present at any of the 4 hubs in the minicoat cage where 3 pentagonal faces join. This class was refined further using localized reconstruction (described below). Click here to expand this figure. Figure EV3. Locating and sub-classifying β2HA density in class 15 of masked, 3D classification output A. Representative hexagonal and pentagonal faces for class 15 3D auto refinement. Solid blue ellipses highlight new density seen following masked, 3D classification within a given polygonal face. Dashed ellipses highlight densities connecting adjacent polygonal faces. B. Representative hexagonal and pentagonal faces for clathrin-only minicoat cage (low pass filtered to 20 Å). Equivalent positions to those in column A are marked in ellipses, highlighting the lack of density in these regions. C, D. Density cross-linking terminal domains from two, adjacent pentagonal faces (denoted P-P) is marked in blue ellipse. Density cross-linking terminal domains from adjacent hexagonal and pentagonal faces (denoted P-H) is marked in blue ellipse. The geometric context of P-P and P-H densities is depicted in D. Download figure Download PowerPoint For class 18, density could be seen on every terminal domain in all the hexagonal faces of the minicoat volume, but was less well-resolved (Fig 4A and B). In contrast to class 15 these densities lay parallel to the terminal domain beta-propeller and did not contact neighbouring terminal domains. We used localized reconstruction (Ilca et al, 2015; Morris et al, 2019) to improve the resolution of the hexagonal faces from this class of minicoat particles. Rigid-body fitting of the clathrin terminal domain atomic structure revealed surplus density on either side of the beta-propeller structure (Fig 4C). The location of this density is consistent with our earlier global difference analysis. The surplus density at either side of the terminal domain was large enough to accommodate the atomic structure of the β2-appendage (Fig 4C) but could not support an unambiguous fit of this structure. Figure 4. Unattributed density in hexagonal faces of clathrin minicoats Close-up view of the unattributed density on each terminal domain in a given hexagonal face of the clathrin-β2HA minicoat cage that was resolved from particles belonging to class 18. Views from outside and inside a given hexagonal face are shown (left and right, respectively). Example densities are highlighted in dashed red ellipses. Using localized reconstruction (Ilca et al, 2015), all hexagonal faces from the minicoat cage shown in panel A were extracted and averaged resulting in a 19 Å map. A single terminal domain and connecting linker and ankle region, is highlighted in cyan, with the atomic model of clathrin terminal domain β-propeller structure (PDB 1BPO) rigid body fitted into the density (shown in dark blue). Rigid-body fitting of the clathrin terminal domain atomic structure (shown in dark blue) revealed surplus density on either side of the β-propeller structure, which was large enough to accommodate the atomic structure of the β2-adaptin appendage (PDB 1E42, shown as pink and orange wireframes) but not sufficiently defined to support an unambiguous fit to the density. All images were created, and rigid-body fitting was conducted, in UCSF Chimera (Pettersen et al, 2004). Download figure Download PowerPoint Resolving β2HA in the minicoat hub substructure Having established through our analysis of whole cages that β2HA has at least two different binding locations on assembled clathrin, we next improved the resolution of the most defined density for β2HA by making use of the local symmetry present within the cages. We extracted and refined the hub regions from each vertex of the minicoat cage particles belonging to Class 15 (Fig EV2), using localized reconstruction within RELION (Ilca et al, 2015). Using this approach we refined a total of 26,624 minicoat hub regions to a global resolution of 9.6 Å (Fig EV4A–I). This resulted in a considerable improvement in resolution when compared to the whole-cage particles of Class 15 which refined to 19.8 Å (Fig EV2). Hubs surrounded by three pentagonal faces, which did not show additional density, were excluded from this refinement. A difference map and statistical comparison confirmed the presence of density due to β2HA (Fig 5A–D). We also found that separating the hubs according to whether the β2HA density linked terminal domains emerging from two pentagonal faces or from one pentagonal face and one hexagonal face resulted in improved map definition (Fig EV3C and D). These two classes were refined separately to global resolutions of ∼10 Å (10.5 Å for P-P hubs and 10.1 Å for H-P hubs, Fig EV4B and C), consistent with the reduced number of particles in each subset. Despite this slightly lower resolution, the β2HA density in these maps was more clearly defined than in previous maps, with an intensity equal to the adjoining terminal domains (at contour level σ3), and a 1:2 β2HA:terminal domain binding ratio for both hub volumes (Fig EV4A–C). Click here to expand this figure. Figure EV4. Localized reconstruction of minicoat cage particles A. Asymmetric 3D auto refinement of hub regions for all 26,624 minicoat particles from class 15 yielded a 9.7 Å resolution volume. 180° rotation of this asymmetric unit revealed a single β2-appendage connecting two or t
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