The endosomal sorting complex required for transport (ESCRT) is a highly conserved protein machinery that drives a divers set of physiological and pathological membrane remodeling processes. However, the structural basis of ESCRT-III polymers stabilizing, constricting and cleaving negatively curved membranes is yet unknown. Here we present cryo-EM structures of membrane-coated CHMP2A–CHMP3 filaments from Homo sapiens of two different diameters at 3.3 and 3.6 Å resolution. The structures reveal helical filaments assembled by CHMP2A–CHMP3 heterodimers in the open ESCRT-III conformation, which generates a partially positive charged membrane interaction surface, positions short N-terminal motifs for membrane interaction and the C-terminal VPS4 target sequence toward the tube interior. Inter-filament interactions are electrostatic, which may facilitate filament sliding upon VPS4-mediated polymer remodeling. Fluorescence microscopy as well as high-speed atomic force microscopy imaging corroborate that VPS4 can constrict and cleave CHMP2A–CHMP3 membrane tubes. We therefore conclude that CHMP2A–CHMP3–VPS4 act as a minimal membrane fission machinery.
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Cryo-EM maps and models were deposited to the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB) with the following codes: membrane-bound CHMP2A–CHMP3, 430 Å diameter (PDB ID 7ZCG, EMD-14630) and membrane-bound CHMP2A–CHMP3, 410 Å diameter (PDB ID 7ZCH, EMD-14631). Raw gels are provided as source data. Source data are provided with this paper.
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This research was funded by the ANR (grant nos. ANR-14-CE09-0003-01 and ANR-19-CE11-0002-02 to W.W.). W.W. acknowledges support from the Institut Universitaire de France and access to the platforms of the Grenoble Instruct-ERIC center (IBS and ISBG; grant no. UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology, with support from FRISBI (grant no. ANR-10-INBS-05-02) and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (grant no. ANR-17-EURE-0003). The IBS electron microscope facility is supported by the Auvergne-Rhône-Alpes Region, the Fondation pour la Recherche Medicale, the FEDER/ERDF fund (European Regional Development Fund) and the GIS-IBiSA (Infrastructures en Biologie, Sante et Agronomie). We acknowledge the provision of in-house experimental time from the CM01 facility at the European Synchrotron Radiation Facility and we thank L. Estrozi for extensive discussion and help with helical image analysis. We further thank the HIV Reagent Program, Division of AIDS, NIAID, NIH for providing the Anti-Human Immunodeficiency Virus 1 (HIV-1) p24 Monoclonal (183-H12-5C), ARP-3537, contributed by B. Chesebro and K. Wehrly.
The authors declare no competing interests.
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Extended Data Fig. 1 Cryo-EM data processing of CHMP2A-CHMP3 membrane-coated tubes and helical symmetry analyses.
(a) Representative cryo-electron micrograph of CHMP2A-CHMP3 membrane tubes; arrow pointing to the lipid bilayer. Scale bar, 50 nm. (b) Selected 2D class averages of manually picked datasets; tube diameter range from 380 to 490 Å as indicated. (c), (d) Helical symmetry determination and representation of the elementary and biological helices for the 430 Å and 410 Å diameter tubes. Left panel, the sum of the 2D power spectra of segments corresponding to one class-average show in both cases a maximum on or near the meridian corresponding to the pitch (Bessel order n = 1 for C1 helix; n = 2 for C2 helix). The left half of the sum of the power spectra show the calculated position from helixplorer of the two first maxima of each Bessel function. The right half of the sum of the power spectra highlight for each symmetry the layer line corresponding to the pitch and the layer line spacing corresponding to the repeat distance. Middle panel, six asymmetric units of the elementary helix are represented; CHMP2A-CHMP3 dimers; in red, symmetry-related protomers in light red. Right panel, side view of the elementary helix with turns (red, yellow, green, blue and aqua) indicated with a black dashed line. The gray dashed line follows one turn of the biological helix. The central grey line (in c) highlights the C2 symmetry axis. Symmetry parameters of both the elementary and biological helices are indicated. (e, f) Central slices looking down the helical axis. The red arrows in the right zoom-in images indicate the density of the N-terminal region prone to insert into the lipid bilayer. (g) Representative of a cryoEM image showing protein-free vesicles, generated during the membrane coating protocol. 257 particles were picked and the diameter determined from 2-D class averages. (h) Average membrane thickness of protein-free vesicle compared to the membrane coated onto CHMP2A-CHMP3 polymers was determined from 30 measurements in each group from one preparation. The boxes show the lower quartile (25th percentile), the median, and the upper quartile (75th percentile). The smallest and largest values are indicated by the small horizontal bars at the end of the whiskers. The statistical significance was assessed using two-tailed t test.
Extended Data Fig. 2 Cryo-EM image processing workflow and structure determination.
Basic image processing strategy used for helical 3D reconstruction and refinement of 430 and 410 Å diameter tubes is shown. Helical filaments were segmented and classified based on the tube diameter. Segment subsets were subjected to symmetry determination (https://rico.ibs.fr/helixplorer) and initial 3D model generation in SPRING, followed by symmetry refinement and final 3D structure refinement in RELION. A complete description of the processing workflow is provided in ‘Materials and Methods’ section.
Extended Data Fig. 3 Fourier shell correlation (FSC) curves, local resolution maps and atomic model fitting.
(a) FSC curves for the 430 Å (black) and 410 Å (red) diameter tube maps, with the resolutions at the FSC cut-off of 0.143 are indicated. Model versus map FSC curves, with the resolutions at the FSC cut-off of 0.5 are indicated for the 430 Å (blue) and 410 Å (pink) diameter tube maps. Local resolution estimates are mapped onto the 430 Å (b), and 410 Å (c) diameter tube cryo-EM density maps and the color keys (right) highlight the local resolution values in Å. (d) The refined atomic model of CHMP2A-CHMP3 dimer was fit into the corresponding cryo-EM density map of the 430 Å diameter tube. The inset (below) represents the zoomed-in view of the fitted model, indicating CHMP2A and CHMP3 helices and the corresponding map.
Extended Data Fig. 4 Structure-based mutagenesis of CHMP2A-CHMP3 heterodimer formation and polymerization in vitro.
(a) Close-up views of the pairs of residues (black) mutated to cysteine to induce the formation of disulfide-linked CHMP2A (light blue) - CHMP3 (pink) heterodimers upon polymerization. (b) Cysteine cross-linking of the CHMP2A-CHMP3 heterodimer. Mutant CHMP2A_D57C was incubated with CHMP3_S75C and CHMP2A_N18C with CHMP3_V110C to induce polymerization as reported for wild-type CHMP2A and CHMP3. SDS-PAGE analysis showing that both CHMP2A_D57C-CHMP3_S75C and CHMP2A_N18C-CHMP3_V110C formed disulfide-linked dimers under non-reducing SDS PAGE conditions. (c) Negative staining electron micrographs showing regular tube formation for CHMP2A_N18C-CHMP3_V110C (right), while CHMP2A_D57C-CHMP3_S75C (left) produced only shorter tubes. Scale bar, 100 nm. (d) Close-up views of the CHMP3 interface residues A96, A82 and M89E tested for heterodimer formation and polymerization. (e) Negative staining electron micrographs of CHMP2A-CHMP3 wild-type and mutants (CHMP3_A96E, CHMP3_A82E, CHMP3-A82E_A96E and CHMP3_M89E). Scale bar, 200 nm. Experiments shown in b, c and e have been repeated three times.
Extended Data Fig. 5 ESCRT-III sequence alignment.
(a) Sequence alignment of CHMP3 (AF219226), S. cerevisiae Vps24 (QHB09957), S. cerevisiae Vps2 (P36108.2) and CHMP2A (NM_198426.3). Secondary structure elements are shown for CHMP3 above the sequence and for CHMP2A below the sequence alignment. Blue triangles indicate basic residues of CHMP3 (above) and CHMP2A (below) exposed at the membrane binding interface. Blue rectangles show basic residues and red squares conserved acidic residues exposed at the interface between filaments. (b) Sequence alignment of S. cerevisiae Snf7 (Z73197.1) and its secondary structure (pdb 5FD9), CHMP4A (NM_014169.5), CHMP4B (NM_176812.5) CHMP4C (NM_152284), CHMP5 (NM_016410.6) and CHMP6 (NM_024591.5). Conserved acidic residues implicated in inter-filament interactions in the CHMP2A-CHMP3 polymer are indicated as red squares.
Extended Data Fig. 6 Structure-based mutagenesis of CHMP2A-CHMP3 polymer formation.
(a) Negative staining electron micrograph showing regular tube formation by CHMP2A-CHMP3_K112A, K119A, K132A, K136A polymerization. Scale bar, 200 nm. (b) The substitution of four basic residues in CHMP3(1–150)-GFP (4KA: K112A, K119A, K132A, K136A) does not diminish its dominant-negative effect on HIV-1 budding. Western blot analyses of Gag released from Gag expressing cells as Gag-VLPs (upper panel) and detection of Gag in total cell extracts (lower panel): lane 1, control transfected with pcDNA; lane 2, Gag expression; lane 3, Gag and GFP-VPS4A E228Q expression; lane 4, Gag and CHMP3(1-150)-GFP expression; lane 5, Gag and CHMP3(1-150)-4KA-GFP (4KA) expression. (c) Representative fluorescence images of HeLa cells transfected with Gag/mCherry-Gag and CHMP3(1-150)-GFP or 4KA (CHMP3(1-150)4KA-GFP). Cellular distribution of wild-type and mutant 4KA indicates predominantly plasma membrane and intracellular localization as well as co-localization with mCherry-Gag. Scale bar, 10 µm. (d) Negative staining electron micrographs showing no polymer formation of CHMP3 mutants (upper left panel, close-up of a ribbon diagram illustrating the interface residues) R24A, K25A, R28A, R32A (upper middle panel) and R24E, K25E, R28E, R32E (upper right panel) with CHMP2A. (Lower panel) CHMP2A mutants (lower left panel, close-up of a ribbon diagram illustrating the interface residues) R16A, R20A, R24A, R31A (lower middle panel) and R16E, R20E, R24E, R31E (lower right panel) did not polymerize with CHMP3 in vitro. Scale bar, 200 nm. Experiments shown in a and d have been repeated two times and experiments shown in b and c one times.
Extended Data Fig. 7 High ionic strength unwinds the CHMP2A-CHMP3 filaments.
(a) Negative staining electron micrographs showing CHMP2A-CHMP3 wild-type polymers after treatment with 1 M NaCl (b, c) and 1 M KCl (d). (e) Close-up of cryo-EM images shows unwinding of ~20 nm wide filaments corresponding to the six-start helix observed in the structure. Single and multi-stranded unwound filaments are indicated by arrows. Experiments shown in a-e have been repeated three times.
Extended Data Fig. 8 Incorporation of VPS4B and ATP into CHMP2A-CHMP3 membrane tubes induces their disassembly.
(a) SDS-PAGE analyses of purified CHMP2A-CHMP3 polymers; lane 1, CHMP2A-CHMP3 polymers cleaved with TEV and coated with a lipid bilayer; lane 2 CHMP2A-CHMP3 polymers, TEV cleaved and incorporation of VPS4B prior to lipid bilayer coating. Negative staining electron micrographs of CHMP2A-CHMP3-VPS4B membrane-coated polymers before (b) and after (c) incubation with ATP and Mg2+. Red arrows point to membrane vesicles resulting from tube cleavage. Scale bar, 200 nm. Experiments have been repeated three times.
Extended Data Fig. 9 Imaging of VPS4B and ATP induced cleavage of CHMP2A-CHMP3 membrane coated tubes.
(a) A CHMP2A-CHMP3-caged ATP membrane coated tube was activated at 365 nm (365 nm 30%, 100 ms at each time point) to uncage ATP and imaged over 282 s, which indicated that uncaging did not change the tube structure (snapshots from Supplementary Video 2). (b) The kymograph of the tube (yellow line) shows that the tube stays intact over the imaging time. (c) Imaging of a CHMP2A-CHMP3-VPS4B-caged ATP membrane-coated tube following ATP uncaging (365 nm, 30%, 100 ms at each time point) reveals cleavage of the tube at several sites over the imaging time (snapshots from Supplementary Video 5). Scale Bar, 1 µm. (d) The kymograph of the tube (yellow line) indicates tube cleavage (arrows) over the imaging time. Scale bars are 1 μm.
Extended Data Fig. 10 Height distribution of CHMP2A-CHMP3 tubes with and without membrane coating.
(a) AFM image of CHMP2A-CHMP3 tubes without membrane. (b) AFM image of CHMP2A-CHMP3 tubes coated with membrane. (c) Cross-section of AFM images of CHMP2A-CHMP3 in panel a (blue dotted line) and panel b (red dotted line). (d) Height histogram of CHMP2A-CHMP3 tubes with (red, n = 84) and without (blue, n = 98) membrane coating with respect to the surface. (e) Snapshots HS-AFM images (from Supplementary Video 7) of membrane-coated tubes loaded with 10 mM caged ATP initially taken without and later with UV irradiation. The UV was turned on from 700 s onwards. No VPS4B is present. Scale bar, 200 nm.
Supplementary Video 1
Fluorescence microscopy imaging of membrane-coated tubes containing caged ATP (example 1). A CHMP2A–CHMP3-caged ATP membrane-coated tube was activated at 365 nm for 10 s to uncage ATP and imaged over 291 s.
Supplementary Video 2
Fluorescence microscopy imaging of membrane-coated tubes containing caged ATP (example 2). Another dataset of CHMP2A–CHMP3-caged ATP membrane-coated tube activated at 365 nm for 100 ms at each time point to uncage ATP and imaged over 300 s.
Supplementary Video 3
Fluorescence microscopy imaging of membrane-coated tubes containing VPS4B. A CHMP2A–CHMP3–VPS4B membrane-coated tube was activated at 365 nm for 10 s to uncage ATP and imaged over 251 s.
Supplementary Video 4
Fluorescence microscopy imaging of membrane-coated tubes containing VPS4 and caged ATP (example 1). Imaging of a CHMP2A–CHMP3–VPS4B-caged ATP membrane-coated tube following ATP uncaging (365 nm, 10 s). This demonstrates tube fission followed by a shrinking event from both sides.
Supplementary Video 5
Fluorescence microscopy imaging of membrane-coated tubes containing VPS4B-caged ATP and caged ATP (example 2). Another dataset of CHMP2A–CHMP3–VPS4B-caged ATP membrane-coated tube following ATP uncaging (365 nm, 100 ms at each time point) reveals cleavage of the tube at several sites over the imaging time.
Supplementary Video 6
Fluorescence microscopy imaging of membrane-coated tubes containing VPS4B and caged ATP (example 3). Imaging of a CHMP2A–CHMP3–VPS4B-caged ATP membrane-coated tube following ATP uncaging (365 nm) showing at 36 s a shrinking event from the end of a tube and at 51 s a cleavage of the tube.
Supplementary Video 7
HS-AFM imaging of membrane-coated tubes with caged ATP, without VPS4B, UV on 700 s. HS-AFM video of membrane-coated CHMP2A–CHMP3 tubes loaded with 10 mM caged ATP, taken before and after UV (365 nm) irradiation. Imaging time 5 s per frame.
Supplementary Video 8
HS-AFM imaging of membrane-coated tubes with VPS4B and caged ATP, UV off. HS-AFM video of membrane-coated CHMP2A–CHMP3 tubes loaded with 5 µM VPS4B and 10 mM caged ATP, taken without UV irradiation. Imaging time 3 s per frame.
Supplementary Video 9
HS-AFM imaging of membrane-coated tubes with VPS4B and caged ATP, UV on. HS-AFM video of membrane-coated CHMP2A–CHMP3 tubes loaded with 5 µM VPS4B and 10 mM caged ATP, taken with 365 nm UV irradiation. UV was switched on <5 s before the start of the imaging (corresponds to 0 s). Imaging time 2 s per frame.
Source Data Fig. 1
Source Data Extended Data Fig. 4
Source Data Extended Data Fig. 6
Unprocessed western blot, upper panel (VLPs) Extended Data Fig. 6b.
Source Data Extended Data Fig. 6
Unprocessed western blot, lower panel (cells) Extended Data Fig. 6b.
Source Data Extended Data Fig. 8
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Azad, K., Guilligay, D., Boscheron, C. et al. Structural basis of CHMP2A–CHMP3 ESCRT-III polymer assembly and membrane cleavage. Nat Struct Mol Biol 30, 81–90 (2023). https://doi.org/10.1038/s41594-022-00867-8