title: In situ structural analysis of the human nuclear pore complex.
abstract: Nuclear pore complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of approximately 30 nucleoporins (Nups), 15 are structured and form the Y and inner-ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ~60 nm in diameter. The scaffold is decorated with transport-channel Nups that often contain phenylalanine-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here we combine cryo-electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modelling to generate, to our knowledge, the most comprehensive architectural model of the human nuclear pore complex to date. Our data suggest previously unknown protein interfaces across Y complexes and to inner-ring complex members. We show that the transport-channel Nup358 (also known as Ranbp2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport-channel Nups. We conclude that, similar to coated vesicles, several copies of the same structural building block--although compositionally identical--engage in different local sets of interactions and conformations.
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: von Appen
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Kosinski
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Lenore
authors.initials: L
authors.lastname: Sparks
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Ori
authors.affiliation: Department of Chemistry, Chemical Biology and Biomedical Engineering, Stevens Institute of Technology, 507 River St., Hoboken NJ 07030, USA.
authors.forename: Amanda L
authors.initials: AL
authors.lastname: DiGuilio
authors.affiliation: Friedrich Miescher Laboratory of the Max Planck Society, Spemannstrasse 39, 72076 Tübingen, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Vollmer
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Marie-Therese
authors.initials: MT
authors.lastname: Mackmull
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Niccolo
authors.initials: N
authors.lastname: Banterle
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Luca
authors.initials: L
authors.lastname: Parca
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Kastritis
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Buczak
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Shyamal
authors.initials: S
authors.lastname: Mosalaganti
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Wim
authors.initials: W
authors.lastname: Hagen
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Amparo
authors.initials: A
authors.lastname: Andres-Pons
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Edward A
authors.initials: EA
authors.lastname: Lemke
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Peer
authors.initials: P
authors.lastname: Bork
authors.affiliation: Friedrich Miescher Laboratory of the Max Planck Society, Spemannstrasse 39, 72076 Tübingen, Germany.
authors.forename: Wolfram
authors.initials: W
authors.lastname: Antonin
authors.affiliation: Department of Chemistry, Chemical Biology and Biomedical Engineering, Stevens Institute of Technology, 507 River St., Hoboken NJ 07030, USA.
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Glavy
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany.|Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada.
authors.forename: Khanh Huy
authors.initials: KH
authors.lastname: Bui
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Beck
mesh_terms: D001665:Binding Sites; D020285:Cryoelectron Microscopy; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D009685:Nuclear Envelope / Q000378:metabolism; D022022:Nuclear Pore / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D011487:Protein Conformation; D055503:Protein Multimerization; D055550:Protein Stability
chemical_list: D018832:Molecular Chaperones; D028861:Nuclear Pore Complex Proteins; C094493:ran-binding protein 2