(A) A dimer of human Crm1 extracted from a unit cell of a Crm1-Snurportin1 complex that lacks RanGTP (PDB 3GB8) was fit into the EM reconstruction (grey envelope) of Crm1 and RanGTP bound to Rev-RRE (Figure 2—figure supplement 2). Residues that differ between murine and human Crm1 that enhance Rev-RRE activity (Sherer et al., 2011; Elinav et al., 2012) are shown in gold and form an interface between two Crm1 monomers (detailed in panel B and Figure 2—figure supplement 3). Two NES binding sites poised to engage two Rev NES peptides are shown in cyan. (C–D) Human Crm1 recognizes Rev-RRE with higher affinity and increased cooperativity compared to its murine ortholog, shown by gel mobility shift assays with radiolabeled RRE, a molar excess of Rev, and increasing concentrations of Crm1-RanGTP. The Hill equation or a version of the McGhee-von Hippel model (Epstein, 1978) was fit to the quantified data from three independent experiments (R2 > 0.99 for all models compared to average values) as shown in (D) and described in ‘Materials and methods’. Asterisks denote *p < 0.05 and **p < 0.01 from one-tailed t-tests. A schematic model of the complex is shown to highlight the physical meaning of the McGhee-von Hippel parameters. (E) Size-exclusion chromatograms of Rev-RRE complexes assembled with murine Crm1 or human Crm1 show that murine Crm1 elutes with a larger apparent mass, presumably due to a larger hydrodynamic radius in the absence of Crm1-Crm1 interactions. (F) Class averages of Rev-RRE complexes bound to a dimer of human (426 particles) or murine (617 particles) Crm1 from negatively-stained micrographs of particles from (E). Murine Crm1 particles showed a wider range of sizes than the human particles, with most being larger, and the murine particles showed different features, such as an hourglass shape with fewer details in the center of the particle than human Crm1 particles. Scale bars show 10 nm.