In-cell architecture of the nuclear pore and snapshots of its turnover

Allegretti, Matteo; Zimmerli, Christian E.; Rantos, Vasileios; Wilfling, Florian; Ronchi, Paolo; Fung, Herman K H; Lee, Chia-Wei; Hagen, Wim J. H.; Turoňová, Beata; Karius, Kai; Börmel, Mandy; Zhang, Xiaojie; Müller, Christoph W.; Schwab, Yannick; Mahamid, Julia; Pfander, Boris; Kosiński, Jan; Beck, Martin

doi:10.1038/s41586-020-2670-5

Cited by 165 publications

(260 citation statements)

References 71 publications

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“…d , View from the periplasm of the EM density corresponding to the central pore. The top scoring Rosetta model is shown e , Analysis of the pore diameter from 100 Rosetta models with HOLE 39 , median pore diameter (black) and 90% confidence interval (grey). Side view of EccC 5 pore helices of a top scoring model showing the pore diameter analysis.…”

Section: Mainmentioning

confidence: 99%

Structure of the mycobacterial ESX-5 Type VII Secretion System hexameric pore complex

Beckham

Ritter

Chojnowski

et al. 2020

Preprint

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To establish an infection, pathogenic mycobacteria use the Type VII secretion or ESX system to secrete virulence proteins across their cell envelope. The five ESX systems (ESX-1 to ESX-5) have evolved diverse functions in the cell, with the ESX-5 found almost exclusively in pathogens. Here we present a high-resolution cryo-electron microscopy structure of the hexameric ESX-5 Type VII secretion system. This 2.1 MDa membrane protein complex is built by a total of 30 subunits from six protomeric units, which are composed of the core components EccB5, EccC5, two copies of EccD5, and EccE5. The hexameric assembly of the overall ESX-5 complex is defined by specific inter-protomer interactions mediated by EccB5 and EccC5. The central transmembrane pore is formed by six pairs of EccC5 transmembrane helices that adopt a closed conformation in the absence of substrate in our structure. On the periplasmic face of the ESX-5 complex, we observe an extended arrangement of the six EccB5 subunits around a central cleft. Our structural findings provide molecular details of ESX-5 assembly and observations of the central secretion pore, which reveal insights into possible gating mechanisms used to regulate the transport of substrates.

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Section: Mainmentioning

confidence: 99%

Structure of the mycobacterial ESX-5 Type VII Secretion System hexameric pore complex

Beckham

Ritter

Chojnowski

et al. 2020

Preprint

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“…In eukaryotic cells, the presence of a physical separation between the nucleus and the cytoplasm in the form of a double membrane assures a physical and temporal separation of the transcription and translation processes but creates the need for a selective and efficient transport of thousands of macromolecules across the nuclear envelope ( Görlich and Kutay, 1999 ; Conti and Izaurralde, 2001 ; Fried and Kutay, 2003 ; Cook et al, 2007 ). This continual ballet, with controlled bidirectional flows, is orchestrated by nuclear transport receptors (NTRs) that carry their cargoes from one compartment to the other by crossing the nuclear envelope at the level of the nuclear pore complexes (NPCs) ( Tran and Wente, 2006 ; Wente and Rout, 2010 ; Allegretti et al, 2020 ). Transport receptors of the karyopherin-β (Kapβ) family account for the vast majority of the cargo flow through the NPC.…”

Section: Introductionmentioning

confidence: 99%

Transportin-1: A Nuclear Import Receptor with Moonlighting Functions

Mboukou

Rajendra

Kleinová

et al. 2021

Front. Mol. Biosci.

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Transportin-1 (Trn1), also known as karyopherin-β2 (Kapβ2), is probably the best-characterized nuclear import receptor of the karyopherin-β family after Importin-β, but certain aspects of its functions in cells are still puzzling or are just recently emerging. Since the initial identification of Trn1 as the nuclear import receptor of hnRNP A1 ∼25 years ago, several molecular and structural studies have unveiled and refined our understanding of Trn1-mediated nuclear import. In particular, the understanding at a molecular level of the NLS recognition by Trn1 made a decisive step forward with the identification of a new class of NLSs called PY-NLSs, which constitute the best-characterized substrates of Trn1. Besides PY-NLSs, many Trn1 cargoes harbour NLSs that do not resemble the archetypical PY-NLS, which complicates the global understanding of cargo recognition by Trn1. Although PY-NLS recognition is well established and supported by several structures, the recognition of non-PY-NLSs by Trn1 is far less understood, but recent reports have started to shed light on the recognition of this type of NLSs. Aside from its principal and long-established activity as a nuclear import receptor, Trn1 was shown more recently to moonlight outside nuclear import. Trn1 has for instance been caught in participating in virus uncoating, ciliary transport and in modulating the phase separation properties of aggregation-prone proteins. Here, we focus on the structural and functional aspects of Trn1-mediated nuclear import, as well as on the moonlighting activities of Trn1.

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“…No reuse allowed without permission. Finally, p-values are calculated from the cross-correlation scores of alternative fits as published before 7,21,24,29,31,32 . Briefly, the cross-correlation scores are first transformed to z-scores (Fisher's z-transform 33 ) and centered, from which two-sided p-values are computed using standard deviation derived from an empirical null distribution (derived from all obtained unique fits and fitted using fdrtool R-package 34 ).…”

Section: Calculation Of Fit Librariesmentioning

confidence: 99%

“…The copyright holder for this preprint (which this version posted April 6, 2021. ; https://doi.org/10.1101/2021.04.06.438590 doi: bioRxiv preprint traj/ directory in the output folder from modelling and displaying it with UCSF Chimera. An example trajectory of the best scoring model from CR Y-complex (from the in-cell ScNPC model 7 ) can be inspected in Supplementary Video 1.…”

Section: Troubleshooting Critical Stepmentioning

confidence: 99%

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Integrative structural modelling of macromolecular complexes using Assembline

Rantos

Karius

Kosiński

2021

Preprint

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Integrative modelling enables structure determination of macromolecular complexes by combining data from multiple experimental sources such as X-ray crystallography, electron microscopy (EM), or crosslinking mass spectrometry (XL-MS). It is particularly useful for complexes not amenable to high-resolution EM-complexes that are flexible, heterogenous, or imaged in cells with cryo-electron tomography. We have recently developed an integrative modelling protocol that allowed us to model multi-megadalton complexes as large as the nuclear pore complex. Here, we describe the Assembline software package, which combines multiple programs and libraries with our own algorithms in a streamlined modelling pipeline. Assembline builds ensembles of models satisfying data from atomic structures or homology models, EM maps and other experimental data, and provides tools for their analysis. Comparing to other methods, Assembline enables efficient sampling of conformational space through a multi-step procedure, provides new modeling restraints, and includes a unique configuration system for setting up the modelling project. Our protocol achieves exhaustive sampling in less than 100 - 1,000 CPU-hours even for complexes in the megadalton range. For larger complexes, resources available in institutional or public computer clusters are needed and sufficient to run the protocol. We also provide step-by-step instructions for preparing the input, running the core modelling steps, and assessing modelling performance at any stage.

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In-cell architecture of the nuclear pore and snapshots of its turnover

Cited by 165 publications

References 71 publications

Structure of the mycobacterial ESX-5 Type VII Secretion System hexameric pore complex

Structure of the mycobacterial ESX-5 Type VII Secretion System hexameric pore complex

Transportin-1: A Nuclear Import Receptor with Moonlighting Functions

Integrative structural modelling of macromolecular complexes using Assembline

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