Negative staining was carried out using protein complexes deposited on glow-discharged, 200-square-mesh carbon-coated copper grids (Electron Microscopy Sciences). Gel filtration–purified samples containing Kap121 or its complex with 53C, 53core, or 53core and Nic96 (all mixed at equimolar ratios) were diluted up to 10-fold before they were stained with 2% (w/v) uranyl acetate and analyzed. Micrographs were collected on the JEOL 1400 Plus Transmission Electron Microscope operating at 120 kV, with a Gatan 2K × 2K digital camera at 40,000× nominal magnification (2.5 Å/pixel) and a defocus range of −0.8 to −1.5 μm. Particle picking and image analysis were performed using RELION v2.1 (55). A small subset of particles was initially picked manually to generate 2D class averages by reference-free classification. These 2D classes were subsequently low pass–filtered to a resolution of 50 Å and used as references for automated particle picking. Particles that were picked incorrectly or were not well centered in the class averages were removed from subsequent stages of the analysis. A final set of autopicked particles was subjected to three rounds of 2D classification and grouped into 50 to 100 classes, which were sufficient to represent different particle orientations. For optimal 3D classification, these particles were first subjected to 3D refinement using the crystal structure of Kap121 (PDB code: 3W3T), low pass–filtered to 60 Å, as a reference. The map from a 3D refinement was then used in 3D classification, which was carried out by setting the regularization parameter T to 4 and gradually fine-tuning the image alignment sampling to a final setting of 35 iterations with an angular sampling interval of 7.5°, offset search range set to 5 pixels, and step to 1 pixel. The final refined 3D maps were calculated using 17,014 particles for Kap121, 30,941 for the Kap121 complex with 53C, 54,055 for 53core·Kap121 complexes, and 16,055 for the 2:2 ternary complex containing Kap121, 53core, and Nic96. The quality of the obtained maps was sufficient to distinguish various conformational states of the Kap121-containing complexes from the unbound/dissociated Kap121 particles present, to varying degrees, in all tested samples. To visualize in detail the stem structure linking two Kap121 molecules within the 2:2 assembly of the 53core·Kap121 and Kap121·53core·Nic96 complexes, additional particle alignment was performed with a binary mask encompassing exclusively this region. Crystal structures of Kap121 (PDB code: 3W3T), Nic96 (PDB code: 2QX5), and the Nup53 RRM domain (PDB code: 5UAZ) were fitted into the final density maps with high cross-correlation scores (between 0.8 and 1.0) in UCSF (University of California, San Francisco) Chimera (56). However, the unique orientation of the RRM domain within the stem region of the 53core·Kap121 complex could not be unambiguously determined at the resolution of the NS-EM map.

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