Figure 1. NPCs imaged and probed by AFM.

a, Tapping-mode AFM image of the cytoplasmic side of an isolated nuclear envelope. Inset: Electron microscopy image of a supersharp AFM tip. b, AFM-image representing the un-indented cytoplasmic surface of a single NPC, reconstructed from a force spectroscopy measurement. The red line marks the cytoplasmic ring structure, and yellow lines define shells that are concentric with it. c, Rotationally symmetrized surface (grey scale) based on the image in b. The blue-to-red colour scale represents a vertical cross section showing the rotationally averaged local stiffness −∂F(r, z)/∂z that was experienced by the probe on and in the NPC. The plain grey area denotes indentation depths for which insufficient force data were available. d, Force (grey dots) as a function of tip-sample separation for lateral positions with radii r < 0.2 R inside the NPC, where R is the average radius of the cytoplasmatic ring. The red curve is the average of the force data, and the arrow indicates the contact point. The blue and black dashed lines correspond to fits with indentation models for a spherical and a conical tip, respectively (see Supplementary Eqs. 2-3). e, Stiffness as a function of tip-sample separation for radial positions r < 0.2 R inside the NPC.