Architecture of the nuclear pore complex coat

Stuwe, T.; Correia, Ana Luísa; Lin, Daniel H.; Paduch, Marcin; Lu, Vincent; Kossiakoff, Anthony A.; Hoelz, André

doi:10.1126/science.aaa4136

Cited by 104 publications

(154 citation statements)

References 28 publications

Supporting

Mentioning

146

Contrasting

Unclassified

Order By: Relevance

“…By combining validated crosslinks from the two chemistries they obtained a set of 59 and 47 intermolecular crosslinks, respectively. The resulting model is consistent with the recently published crystal structure [63]. A combination of two flavors of 3D EM -cryo-electron tomography and single-particle EM -and XL-MS was employed by the Beck group to define restraints within the purified Nup87 complex and between different copies of the same complex within the assembled nuclear pore complex [64].…”

Section: The Role Of Xl-ms In Integrative Structural Biologysupporting

confidence: 78%

Crosslinking and Mass Spectrometry: An Integrated Technology to Understand the Structure and Function of Molecular Machines

Leitner

Faini

Stengel

et al. 2016

Trends in Biochemical Sciences

349

285

View full text Add to dashboard Cite

In recent years, chemical crosslinking of protein complexes and the identification of crosslinked residues by mass spectrometry (XL-MS; sometimes abbreviated as CX-MS) has become an important technique bridging mass spectrometry (MS) and structural biology. By now, XL-MS is well established and supported by publicly available resources as a convenient and versatile part of the structural biologist's toolbox. The combination of XL-MS with cryoelectron microscopy (cryo-EM) and/or integrative modeling is particularly promising to study the topology and structure of large protein assemblies. Among the targets studied so far are proteasomes, ribosomes, polymerases, chromatin remodelers, and photosystem complexes. Here we provide an overview of recent advances in XL-MS, the current state of the field, and a cursory outlook on future challenges. Chemical Crosslinking as a Tool for Structural BiologyStructural biology makes use of many different techniques to elucidate the 3D structures of proteins and protein complexes. While high-resolution structures have traditionally been obtained by X-ray crystallography, cryo-EM is increasingly able to also generate (near) atomic-resolution models. In recent years, techniques and applications of MS have also rapidly progressed. Earlier studies were largely focused on the large-scale identification and quantification of proteins, whereas recent methods also support queries into the composition, stoichiometry, and spatial arrangement of subunits in a complex. These developments have now further progressed toward generating information that contributes, as part of hybrid structural strategies, to the structure elucidation of large molecular assemblies including protein complexes that perform essential processes in the cell. XL-MS is a particularly powerful mass spectrometric technique in this respect, because it provides several layers of information. Identifying protein-protein contacts through XL-MS confirms physical proximity between subunits because the proteins must be close enough in space to be crosslinked. Localizing the side chains that are connected restricts this proximity to certain regions (e.g., domains or even single helices or loops). Finally, the structure of the connected side chains and the crosslinker moiety impart a distance restraint that can be used for molecular modeling purposes because an upper Trends Chemical crosslinking followed by the mass spectrometric analysis of cross linked peptides (XL MS) identifies con tact sites between residues within a single or between multiple proteins.The application of XL MS to many bio logically relevant molecular machines has been shown, with a rapidly growing number of successful studies reported in the past 2 3 years.Crosslinking data are useful in integra tive modeling workflows by providing distance restraints on the surface of folded proteins and complexes. XL MS has been shown to be particularly powerful in combination with 3D cryo electron microscopy. Sciences ; 41 (2016), 1. -S. 20-32 https://dx.doi.org/10.1016...

show abstract

Section: The Role Of Xl-ms In Integrative Structural Biologysupporting

confidence: 78%

Crosslinking and Mass Spectrometry: An Integrated Technology to Understand the Structure and Function of Molecular Machines

Leitner

Faini

Stengel

et al. 2016

Trends in Biochemical Sciences

349

285

View full text Add to dashboard Cite

show abstract

“…In contrast, the crown-trunk domain of scNup145C (aa 178 -555) interacted with scNup84 (31,32). The scNup85 tail (aa 533-744) also interacted directly with full-length scNup145C, as previously shown for various species (9,10,33). Four additional complexes for which crystal structures exist tested positive in our screen: scNsp1-Nup57; scNup57-Nup49; scNup82-Nup159; scNup82-Nup116 (34,35) (Fig.…”

Section: Comprehensive High-resolution Y2h Ppi Screening Forsupporting

confidence: 74%

“…Because of the resolution gap between the crystal structures and the tomographic reconstructions and an incomplete set of available crystal structures, obtained from a variety of evolutionarily distant species, docking approaches are still incomplete (9,10,18). This is further complicated by the paucity of intersubcomplex interaction data (12, 18 -21).…”

mentioning

confidence: 99%

Systematic Protein–Protein Interaction Analysis Reveals Intersubcomplex Contacts in the Nuclear Pore Complex

Apelt

Knockenhauer

Leksa

et al. 2016

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

The nuclear pore complex (NPC) enables transport across the nuclear envelope. It is one of the largest multiprotein assemblies in the cell, built from about 30 proteins called nucleoporins (Nups), organized into distinct subcomplexes. Structure determination of the NPC is a major research goal. The assembled ϳ40 -112 MDa NPC can be visualized by cryoelectron tomography (cryo-ET), while Nup subcomplexes are studied crystallographically. Docking the crystal structures into the cryo-ET maps is difficult because of limited resolution. Further, intersubcomplex contacts are not well characterized. Here, we systematically investigated direct interactions between Nups. In a comprehensive, structurebased, yeast two-hybrid interaction matrix screen, we mapped protein-protein interactions in yeast and human. Benchmarking against crystallographic and coaffinity purification data from the literature demonstrated the high coverage and accuracy of the data set. Novel intersubcomplex interactions were validated biophysically in microscale thermophoresis experiments and in intact cells through protein fragment complementation. These intersubcomplex interaction data provide direct experimental evidence toward possible structural arrangements of architectural elements within the assembled NPC, or they may point to assembly intermediates. Our data favors an assembly model in which major architectural elements of the NPC, notably the Y-complex, exist in different structural contexts within the scaffold. Molecular & Cellular

show abstract

“…1): the nucleoplasmic and cytoplasmic rings, and the inner ring (for a review, see Grossman et al, 2012). This arrangement and the nucleoporins creating these structures are similarly found in yeast (Hoelz et al, 2011;Stuwe et al, 2015;Lin et al, 2016). However, here, we will primarily focus our discussion on vertebrate NPCs.…”

Section: Npc Architecturementioning

confidence: 98%

Perforating the nuclear boundary – how nuclear pore complexes assemble

Weberruß

Antonin

2016

Journal of Cell Science

View full text Add to dashboard Cite

The nucleus is enclosed by the nuclear envelope, a double membrane which creates a selective barrier between the cytoplasm and the nuclear interior. Its barrier and transport characteristics are determined by nuclear pore complexes (NPCs) that are embedded within the nuclear envelope, and control molecular exchange between the cytoplasm and nucleoplasm. In this Commentary, we discuss the biogenesis of these huge protein assemblies from approximately one thousand individual proteins. We will summarize current knowledge about distinct assembly modes in animal cells that are characteristic for different cell cycle phases and their regulation.

show abstract

Architecture of the nuclear pore complex coat

Cited by 104 publications

References 28 publications

Crosslinking and Mass Spectrometry: An Integrated Technology to Understand the Structure and Function of Molecular Machines

Crosslinking and Mass Spectrometry: An Integrated Technology to Understand the Structure and Function of Molecular Machines

Systematic Protein–Protein Interaction Analysis Reveals Intersubcomplex Contacts in the Nuclear Pore Complex

Perforating the nuclear boundary – how nuclear pore complexes assemble

Contact Info

Product

Resources

About