1. Max Planck Institute of Biochemistry, D-82152 Martinsried, Germany
2. Present address: The Department of Life Sciences, The Ben Gurion University and the NIBN, Beer Sheva 84105, Israel.

Correspondence to: Wolfgang Baumeister¹Ohad Medalia^1,2 Correspondence and requests for materials should be addressed to W.B. (Email: baumeist@biochem.mpg.de) or O.M. (Email: omedalia@bgu.ac.il).

Nuclear pore complexes reside in the nuclear envelope of eukaryotic cells and mediate the nucleocytoplasmic exchange of macromolecules¹. Traffic is regulated by mobile transport receptors that target their cargo to the central translocation channel, where phenylalanine-glycine-rich repeats serve as binding sites². The structural analysis of the nuclear pore is a formidable challenge given its size, its location in a membranous environment and its dynamic nature. Here we have used cryo-electron tomography³ to study the structure of nuclear pore complexes in their functional environment, that is, in intact nuclei of Dictyostelium discoideum. A new image-processing strategy compensating for deviations of the asymmetric units (protomers) from a perfect eight-fold symmetry enabled us to refine the structure and to identify new features. Furthermore, the superposition of a large number of tomograms taken in the presence of cargo, which was rendered visible by gold nanoparticles, has yielded a map outlining the trajectories of import cargo. Finally, we have performed single-molecule Monte Carlo simulations of nuclear import to interpret the experimentally observed cargo distribution in the light of existing models for nuclear import.

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