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- PDB-7mw0: Crystal structure of Homo sapiens NUP93 solenoid (residues 174-819) -

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Entry
Database: PDB / ID: 7mw0
TitleCrystal structure of Homo sapiens NUP93 solenoid (residues 174-819)
ComponentsNuclear pore complex protein Nup93
KeywordsTRANSPORT PROTEIN / nuclear pore complex / nucleocytoplasmic transport / alpha-helical solenoid / nuclear pore
Function / homology
Function and homology information


nuclear envelope organization / nuclear pore complex assembly / Nuclear Pore Complex (NPC) Disassembly / Transport of Ribonucleoproteins into the Host Nucleus / Regulation of Glucokinase by Glucokinase Regulatory Protein / Defective TPR may confer susceptibility towards thyroid papillary carcinoma (TPC) / Transport of the SLBP independent Mature mRNA / Transport of the SLBP Dependant Mature mRNA / NS1 Mediated Effects on Host Pathways / SUMOylation of SUMOylation proteins ...nuclear envelope organization / nuclear pore complex assembly / Nuclear Pore Complex (NPC) Disassembly / Transport of Ribonucleoproteins into the Host Nucleus / Regulation of Glucokinase by Glucokinase Regulatory Protein / Defective TPR may confer susceptibility towards thyroid papillary carcinoma (TPC) / Transport of the SLBP independent Mature mRNA / Transport of the SLBP Dependant Mature mRNA / NS1 Mediated Effects on Host Pathways / SUMOylation of SUMOylation proteins / Transport of Mature mRNA Derived from an Intronless Transcript / Rev-mediated nuclear export of HIV RNA / structural constituent of nuclear pore / SUMOylation of RNA binding proteins / Nuclear import of Rev protein / Transport of Mature mRNA derived from an Intron-Containing Transcript / NEP/NS2 Interacts with the Cellular Export Machinery / tRNA processing in the nucleus / Postmitotic nuclear pore complex (NPC) reformation / nucleocytoplasmic transport / Viral Messenger RNA Synthesis / poly(A)+ mRNA export from nucleus / SUMOylation of ubiquitinylation proteins / Vpr-mediated nuclear import of PICs / positive regulation of SMAD protein signal transduction / SUMOylation of DNA replication proteins / Regulation of HSF1-mediated heat shock response / SUMOylation of DNA damage response and repair proteins / nuclear pore / SUMOylation of chromatin organization proteins / nuclear periphery / HCMV Late Events / Transcriptional regulation by small RNAs / ISG15 antiviral mechanism / HCMV Early Events / protein import into nucleus / nuclear envelope / snRNP Assembly / nuclear membrane / centrosome / SARS-CoV-2 activates/modulates innate and adaptive immune responses / membrane / cytosol
Similarity search - Function
Nucleoporin interacting component Nup93/Nic96 / Nup93/Nic96
Similarity search - Domain/homology
Nuclear pore complex protein Nup93
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2 Å
AuthorsPetrovic, S. / Samanta, D. / Perriches, T. / Bley, C.J. / Thierbach, K. / Brown, B. / Nie, S. / Mobbs, G.W. / Stevens, T.A. / Liu, X. ...Petrovic, S. / Samanta, D. / Perriches, T. / Bley, C.J. / Thierbach, K. / Brown, B. / Nie, S. / Mobbs, G.W. / Stevens, T.A. / Liu, X. / Tomaleri, G.P. / Schaus, L. / Hoelz, A.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM117360 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM111461 United States
Howard Hughes Medical Institute (HHMI)55108534 United States
Heritage Medical Research Institute
CitationJournal: Science / Year: 2022
Title: Architecture of the linker-scaffold in the nuclear pore.
Authors: Stefan Petrovic / Dipanjan Samanta / Thibaud Perriches / Christopher J Bley / Karsten Thierbach / Bonnie Brown / Si Nie / George W Mobbs / Taylor A Stevens / Xiaoyu Liu / Giovani Pinton ...Authors: Stefan Petrovic / Dipanjan Samanta / Thibaud Perriches / Christopher J Bley / Karsten Thierbach / Bonnie Brown / Si Nie / George W Mobbs / Taylor A Stevens / Xiaoyu Liu / Giovani Pinton Tomaleri / Lucas Schaus / André Hoelz /
Abstract: INTRODUCTION In eukaryotic cells, the selective bidirectional transport of macromolecules between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC). Embedded in nuclear envelope ...INTRODUCTION In eukaryotic cells, the selective bidirectional transport of macromolecules between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC). Embedded in nuclear envelope pores, the ~110-MDa human NPC is an ~1200-Å-wide and ~750-Å-tall assembly of ~1000 proteins, collectively termed nucleoporins. Because of the NPC's eightfold rotational symmetry along the nucleocytoplasmic axis, each of the ~34 different nucleoporins occurs in multiples of eight. Architecturally, the NPC's symmetric core is composed of an inner ring encircling the central transport channel and two outer rings anchored on both sides of the nuclear envelope. Because of its central role in the flow of genetic information from DNA to RNA to protein, the NPC is commonly targeted in viral infections and its nucleoporin constituents are associated with a plethora of diseases. RATIONALE Although the arrangement of most scaffold nucleoporins in the NPC's symmetric core was determined by quantitative docking of crystal structures into cryo-electron tomographic (cryo-ET) maps of intact NPCs, the topology and molecular details of their cohesion by multivalent linker nucleoporins have remained elusive. Recently, in situ cryo-ET reconstructions of NPCs from various species have indicated that the NPC's inner ring is capable of reversible constriction and dilation in response to variations in nuclear envelope membrane tension, thereby modulating the diameter of the central transport channel by ~200 Å. We combined biochemical reconstitution, high-resolution crystal and single-particle cryo-electron microscopy (cryo-EM) structure determination, docking into cryo-ET maps, and physiological validation to elucidate the molecular architecture of the linker-scaffold interaction network that not only is essential for the NPC's integrity but also confers the plasticity and robustness necessary to allow and withstand such large-scale conformational changes. RESULTS By biochemically mapping scaffold-binding regions of all fungal and human linker nucleoporins and determining crystal and single-particle cryo-EM structures of linker-scaffold complexes, we completed the characterization of the biochemically tractable linker-scaffold network and established its evolutionary conservation, despite considerable sequence divergence. We determined a series of crystal and single-particle cryo-EM structures of the intact Nup188 and Nup192 scaffold hubs bound to their Nic96, Nup145N, and Nup53 linker nucleoporin binding regions, revealing that both proteins form distinct question mark-shaped keystones of two evolutionarily conserved hetero‑octameric inner ring complexes. Linkers bind to scaffold surface pockets through short defined motifs, with flanking regions commonly forming additional disperse interactions that reinforce the binding. Using a structure‑guided functional analysis in , we confirmed the robustness of linker‑scaffold interactions and established the physiological relevance of our biochemical and structural findings. The near-atomic composite structures resulting from quantitative docking of experimental structures into human and cryo-ET maps of constricted and dilated NPCs structurally disambiguated the positioning of the Nup188 and Nup192 hubs in the intact fungal and human NPC and revealed the topology of the linker-scaffold network. The linker-scaffold gives rise to eight relatively rigid inner ring spokes that are flexibly interconnected to allow for the formation of lateral channels. Unexpectedly, we uncovered that linker‑scaffold interactions play an opposing role in the outer rings by forming tight cross-link staples between the eight nuclear and cytoplasmic outer ring spokes, thereby limiting the dilatory movements to the inner ring. CONCLUSION We have substantially advanced the structural and biochemical characterization of the symmetric core of the and human NPCs and determined near-atomic composite structures. The composite structures uncover the molecular mechanism by which the evolutionarily conserved linker‑scaffold establishes the NPC's integrity while simultaneously allowing for the observed plasticity of the central transport channel. The composite structures are roadmaps for the mechanistic dissection of NPC assembly and disassembly, the etiology of NPC‑associated diseases, the role of NPC dilation in nucleocytoplasmic transport of soluble and integral membrane protein cargos, and the anchoring of asymmetric nucleoporins. [Figure: see text].
History
DepositionMay 15, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 15, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 22, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_id_CSD / _citation.journal_volume ..._citation.journal_id_CSD / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
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Assembly

Deposited unit
A: Nuclear pore complex protein Nup93
hetero molecules


Theoretical massNumber of molelcules
Total (without water)76,8932
Polymers76,8311
Non-polymers621
Water3,459192
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, SEC-MALS
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)74.870, 66.070, 79.180
Angle α, β, γ (deg.)90.000, 116.275, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

#1: Protein Nuclear pore complex protein Nup93 / 93 kDa nucleoporin / Nucleoporin Nup93


Mass: 76831.352 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NUP93, KIAA0095 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8N1F7
#2: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6O2
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 192 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.29 Å3/Da / Density % sol: 46.18 %
Crystal growTemperature: 294 K / Method: vapor diffusion, hanging drop / pH: 8.5 / Details: 0.075 M TRIS pH 8.5, 19 % (w/v) PEG 20000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL12-2 / Wavelength: 0.97946 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Dec 18, 2018
RadiationMonochromator: Liquid nitrogen-cooled double crystal Si(111)
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97946 Å / Relative weight: 1
ReflectionResolution: 2→40 Å / Num. obs: 46714 / % possible obs: 99.09 % / Redundancy: 20.9 % / Biso Wilson estimate: 42 Å2 / CC1/2: 1 / CC star: 1 / Rmerge(I) obs: 0.088 / Rpim(I) all: 0.02 / Rrim(I) all: 0.09 / Net I/σ(I): 26
Reflection shellResolution: 2→2.071 Å / Redundancy: 21.5 % / Rmerge(I) obs: 2.537 / Mean I/σ(I) obs: 1.54 / Num. unique obs: 4598 / CC1/2: 0.733 / CC star: 0.92 / Rpim(I) all: 0.555 / Rrim(I) all: 2.598 / % possible all: 98.27

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Processing

Software
NameVersionClassification
Blu-Icedata collection
XDSdata reduction
XDSdata scaling
PHASERphasing
PHENIX1.19.1_4122refinement
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5HB3

5hb3


Resolution: 2→40 Å / SU ML: 0.3001 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 31.3705
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2544 2002 4.29 %
Rwork0.2249 44712 -
obs0.2262 46714 99.03 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 57.45 Å2
Refinement stepCycle: LAST / Resolution: 2→40 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5062 0 4 192 5258
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00245159
X-RAY DIFFRACTIONf_angle_d0.45186965
X-RAY DIFFRACTIONf_chiral_restr0.0334778
X-RAY DIFFRACTIONf_plane_restr0.0025903
X-RAY DIFFRACTIONf_dihedral_angle_d11.9711949
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2-2.050.40461340.393094X-RAY DIFFRACTION97.08
2.05-2.10.37521420.34273150X-RAY DIFFRACTION98.24
2.1-2.160.37421470.3253116X-RAY DIFFRACTION98.55
2.16-2.230.30791390.29413198X-RAY DIFFRACTION98.61
2.23-2.310.28591460.28783150X-RAY DIFFRACTION98.74
2.31-2.410.27421400.27543209X-RAY DIFFRACTION98.94
2.41-2.520.2941470.27423166X-RAY DIFFRACTION98.98
2.52-2.650.29351450.26543191X-RAY DIFFRACTION99.2
2.65-2.820.28681360.25013199X-RAY DIFFRACTION99.32
2.82-3.030.30141430.25233223X-RAY DIFFRACTION99.53
3.03-3.340.331440.25743215X-RAY DIFFRACTION99.58
3.34-3.820.24741460.20693222X-RAY DIFFRACTION99.76
3.82-4.810.18291450.17453251X-RAY DIFFRACTION99.97
4.81-37.410.22031480.18613328X-RAY DIFFRACTION99.89

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