Before immobilization, the ESCRT tubes were treated for 30 min with 1 mM tobacco etch virus protease to remove the MBP tag from CHMP2AΔC. After removal of MBP, 2 μl of ESCRT tubes was incubated on the previously prepared lipid bilayer for 15 min. After the incubation time, the surface was cleaned gently with recording buffer. The glass rod was then attached to the piezo head of the HS-AFM with a small amount of wax. All HS-AFM data were taken in amplitude modulation mode using a sample scanning HS-AFM [Research Institute of Biomolecule Metrology (RIBM), Japan] (47, 56). Short cantilevers (USC-F1.2-k0.15, NanoWorld, Switzerland) with spring constant of 0.15 N/m, resonance frequency around 0.6 MHz, and a quality factor of ∼2 in buffer were used. The cantilever-free amplitude is 3 nm, and the set-point amplitude for the cantilever oscillation was set around 2.7 nm. Images were taken at 0.2 to 1 frame/s depending on the size of the image. Unless mentioned, all the HS-AFM recordings were performed in recording buffer. For all experiments with VPS4B and/or ATP Mg2+, the indicated concentrations of VPS4B ranging from 1 to 10 μM (dissolved in recording buffer) were applied directly to the sample chamber during continuous imaging. The ESCRT-III CHMP2A-CHMP3 tubes were first imaged in recording buffer, and then, VPS4B was added at indicated concentrations and incubated for 3 to 4 min (to provide sufficient time for diffusion of VPS4B inside the tubes) before the addition of ATP Mg2+. Because of the perturbation by the added liquid, sometimes tip-sample interactions were briefly affected, but never longer than 30 s. The time mentioned in the figure panels represents time starting at the first image, that is, within 30 s from the moment of VPS4B/ATP addition.

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