Cryo-EM structures of the ATP-bound Vps4E233Q hexamer and its complex with Vta1 at near-atomic resolution

The cellular ESCRT-III (endosomal sorting complex required for transport-III) and Vps4 (vacuolar protein sorting 4) comprise a common machinery that mediates a variety of membrane remodelling events. Vps4 is essential for the machinery function by using the energy from ATP hydrolysis to disassemble the ESCRT-III polymer into individual proteins. Here, we report the structures of the ATP-bound Vps4E233Q hexamer and its complex with the cofactor Vta1 (vps twenty associated 1) at resolutions of 3.9 and 4.2 Å, respectively, determined by electron cryo-microscopy. Six Vps4E233Q subunits in both assemblies exhibit a spiral-shaped ring-like arrangement. Locating at the periphery of the hexameric ring, Vta1 dimer bridges two adjacent Vps4 subunits by two different interaction modes to promote the formation of the active Vps4 hexamer during ESCRT-III filament disassembly. The structural findings, together with the structure-guided biochemical and single-molecule analyses, provide important insights into the process of the ESCRT-III polymer disassembly by Vps4.

The molecular weights of the two major peaks were determined as ~300 kDa and ~100 kDa, corresponding to Vps4 E233Q hexamer and dimer, respectively. (b) A representative electron micrograph of the Vps4 E233Q hexamer low pass filtered to 5 Å. A few typical protein particles of top views and side views are marked by red and blue circles, respectively. (c) Typical good reference-free 2-D class averages from the single particle images of the Vps4 E233Q hexamer. (d) Euler angle distribution of particles contributing to the final reconstruction of the Vps4 E233Q hexamer. Each cylinder represents one view and the size of the cylinder is proportional to the number of particles for that view. (e) Examples of the densities in the cryo-EM map of the Vps4 E233Q hexamer, including an α helix (residues 249-260) and the β sheet (residues 168-172 for strand 1, 193-198 for strand 2, 227-232 for strand 3, 270-276 for strand 4, 293-295 for strand 5) from subunit C. The atomic models are displayed in stick representation, and the density maps were showed as blue mesh. Segmented density maps are displayed at ~ 6 σ contour level. (f) The EM density map of the Vps4 E233Q hexamer color-coded to show the local resolution as estimated by ResMap. (g) Gold-standard FSC curves of the reconstruction of the Vps4 E233Q hexamer. The reconstruction gives an overall resolution of 4.1 Å, and 3.9 Å after applying a soft mask around the rigid part (excluding the subunit A). (h) FSC curves for the cross-validation of the atomic models of the Vps4 E233Q hexamer. The lack of significant separation between work and free FSC curves suggested that the models were not overfitted. See Methods for details. Figure 2 Docking of the atomic models into the density maps of the ATP-bound Vps4 E233Q hexamer and its complex with Vta1.

Supplementary
Docking of the atomic models into the density maps of the ATP-bound Vps4 E233Q hexamer (a) and the ATP-bound Vps4 E233Q -Vta1 complex (b). The interface areas between Vps4 E233Q subunits in the ATP-bound Vps4 E233Q hexamer (a) and in the ATPbound Vps4 E233Q -Vta1 complex (b) are also shown. The coloring scheme is the same as in Fig. 1c,e. The interface areas of subunits A and its neighboring subunits (~1000 Å 2 buried surface), which are only about half of those of other pairs of subunits (~2000 Å 2 buried surface), are underlined.

Supplementary Figure 3 Superimposition of Vps4 E233Q subunits and Vta1 CTD dimers.
(a) Comparison of the twelve Vps4 E233Q subunits from the Vta1-free Vps4 E233Q hexamer and the Vta1-bound Vps4 E233Q hexamer. The coloring scheme is the same as in Fig. 1c,e. Note that the large ATPase domains of subunits A (red arrows) from both the Vta1-free Vps4 E233Q hexamer and the Vta1-bound Vps4 E233Q hexamer exhibit different positions relative to the small ATPase domains, compared to other subunits. (b) Alignment of the four Vta1 CTD dimers from the ATP-bound Vps4 E233Q -Vta1 complex. The coloring scheme is the same as in Fig. 1e. All of the CTD dimers are well aligned.

Supplementary Figure 4 Nucleotide binding sites of the Vps4 E233Q subunits.
(a) Zoomed-in views of the ATPase active centers for all six Vps4 E233Q subunits from the ATP-bound Vps4 E233Q hexamer. The density maps are contoured at the 5.8 σ level for subunits B-F, and the 5.0 σ level for subunit A. The coloring scheme is the same as in Fig. 1c. (b) Zoomed-in views of the ATPase active centers for all six Vps4 E233Q subunits from the ATP-bound Vps4 E233Q -Vta1 complex. The density maps are contoured at the 6.8 σ level for subunits B-F, and the 5.6 σ level for subunit A. The coloring scheme is the same as in Fig. 1e. The EM densities are shown together with the built atomic model with protein in cartoon representation and nucleotide in stick representation. The positions of the nucleotide in subunit A are marked by red dashed circles. Five out of the six Vps4 E233Q subunits of both the ATP-bound Vps4 E233Q hexamer and the ATP-bound Vps4 E233Q -Vta1 complex show clear densities for the nucleotide.

Supplementary Figure 5 Purification and cryo-EM analysis of the ATP-bound
Vps4 E233Q -Vta1 complex.
(a) SEC of the formation of the ATP-bound Vps4 E233Q -Vta1 complex (left panel) and the SDS-PAGE gel (13% resolving gel) of the corresponding fractions from the chromatogram of the Vps4 E233Q -Vta1 mixture in the presence of ATP (right panel). (b) A representative electron micrograph of the ATP-bound Vps4 E233Q -Vta1 complex low pass filtered to 5 Å. A few typical protein particles of top views and side views are marked by red and blue circles, respectively. (c) Typical good reference-free 2D class averages from the single particle images. The additional rod-like densities compared with the 2D class averages of the Vps4 E233Q hexamer are highlighted by red dashed circles. (d) Euler angle distribution of particles contributing to the final reconstruction. Each cylinder represents one view and the size of the cylinder is proportional to the number of particles for that view. (e) Examples of the densities in the cryo-EM map of the ATP-bound Vps4 E233Q -Vta1 complex, including an a helix (residues 249-260) and the b sheet (residues 168-172 for strand 1, 193-198 for strand 2, 227-232 for strand 3, 270-276 for strand 4, 293-295 for strand 5) from subunit C. The atomic models are displayed in stick representation, and the density maps were showed as blue mesh. Segmented density maps are displayed at ~ 7 σ contour level. (f) The EM density map color-coded to show the local resolution as estimated by ResMap. (g) Gold-standard FSC curves of the reconstruction. The reconstruction gives an overall resolution of 4.3 Å, and 4.2 Å after applying a soft mask around the rigid part (excluding the subunit A and all bound Vta1 proteins). (h) FSC curves for the cross-validation of the atomic models of the ATP-bound Vps4 E233Q -Vta1 complex.
(a) Different views of the EM density map of the ATP-bound Vps4 E233Q -Vta1 complex at 6.8 σ contour level to show the ATP-bound Vps4 E233Q hexamer. The coloring scheme is the same as in Fig. 1d. (b) Docking of the atomic model into the cryo-EM density map. The density map is contoured at the 6.8 σ level, and only the models of the Vps4 E233Q subunits are shown. The coloring scheme is the same as in Fig. 1e. (c) Different views of the helix α3 and C-terminal helix in the Vta1-bound Vps4 E233Q hexamer to show the spiral arrangement of Vps4 E233Q subunits.