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- EMDB-24058: Single particle cryo-EM structure of the Chaetomium thermophilum ... -

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Entry
Database: EMDB / ID: EMD-24058
TitleSingle particle cryo-EM structure of the Chaetomium thermophilum Nup188-Nic96 complex (Nup188 residues 1-1858; Nic96 residues 240-301)
Map dataUnsharpened map generated with Relion Auto-refine
Sample
  • Complex: Nup188-Nic96 heterodimer
    • Protein or peptide: Nucleoporin NUP188
    • Protein or peptide: Nucleoporin NIC96
Function / homology
Function and homology information


structural constituent of nuclear pore / mRNA transport / nuclear pore / protein transport / nuclear membrane
Similarity search - Function
Nup188 SH3-like domain / Nuclear pore protein Nup188, C-terminal / Nuclear pore protein NUP188 C-terminal domain / Nucleoporin Nup188, N-terminal / Nucleoporin Nup188, N-terminal / : / Nucleoporin Nup188, N-terminal subdomain III / Nucleoporin Nup188 / Nucleoporin interacting component Nup93/Nic96 / Nup93/Nic96
Similarity search - Domain/homology
Nucleoporin NIC96 / Nucleoporin NUP188
Similarity search - Component
Biological speciesChaetomium thermophilum var. thermophilum DSM 1495 (fungus) / Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.39 Å
AuthorsPetrovic S / Samanta D / Perriches T / Bley CJ / Thierbach K / Brown B / Nie S / Mobbs GW / Stevens TA / Liu X ...Petrovic S / Samanta D / Perriches T / Bley CJ / Thierbach K / Brown B / Nie S / Mobbs GW / Stevens TA / Liu X / Tomaleri GP / Schaus L / Hoelz A
Funding support United States, 4 items
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 United States
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, 2021-
Header (metadata) releaseJun 15, 2022-
Map releaseJun 15, 2022-
UpdateJun 22, 2022-
Current statusJun 22, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_24058.png

Downloads & links

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Map

FileDownload / File: emd_24058.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened map generated with Relion Auto-refine
Voxel sizeX=Y=Z: 0.972 Å
Density
Contour LevelBy AUTHOR: 0.015
Minimum - Maximum-0.05277618 - 0.10985778
Average (Standard dev.)9.2104403e-07 (±0.001981255)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 330.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_24058_msk_1.map
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Additional map: LocScale locally sharpened map (model B-factor based scaling)

Fileemd_24058_additional_1.map
AnnotationLocScale locally sharpened map (model B-factor based scaling)
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Additional map: Sharpened map generated with Relion PostProcess

Fileemd_24058_additional_2.map
AnnotationSharpened map generated with Relion PostProcess
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Half map: Half-dataset map generated with Relion Auto-refine

Fileemd_24058_half_map_1.map
AnnotationHalf-dataset map generated with Relion Auto-refine
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Half map: Half-dataset map generated with Relion Auto-refine

Fileemd_24058_half_map_2.map
AnnotationHalf-dataset map generated with Relion Auto-refine
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Sample components

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Entire : Nup188-Nic96 heterodimer

EntireName: Nup188-Nic96 heterodimer
Components
  • Complex: Nup188-Nic96 heterodimer
    • Protein or peptide: Nucleoporin NUP188
    • Protein or peptide: Nucleoporin NIC96

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Supramolecule #1: Nup188-Nic96 heterodimer

SupramoleculeName: Nup188-Nic96 heterodimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 211.2 KDa

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Macromolecule #1: Nucleoporin NUP188

MacromoleculeName: Nucleoporin NUP188 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus)
Strain: DSM 1495 / CBS 144.50 / IMI 039719
Molecular weightTheoretical: 204.429719 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GPHNMATLTD RTYLPPLEDC LTGRTVILSW RLVASALEDA DLARLTSPAL STFLRDGFVH ELLKHPARVF EPKDLKQEFE TKTSSIQTV APGVDTIKKD ALWLADAVAI NQVAALRIVL IEYQTRAHSH LVLPLSTQDV ANIQEAAGVG DAHASSILSL L NPASAVDA ...String:
GPHNMATLTD RTYLPPLEDC LTGRTVILSW RLVASALEDA DLARLTSPAL STFLRDGFVH ELLKHPARVF EPKDLKQEFE TKTSSIQTV APGVDTIKKD ALWLADAVAI NQVAALRIVL IEYQTRAHSH LVLPLSTQDV ANIQEAAGVG DAHASSILSL L NPASAVDA ETMWCDFETE ARRRERILAT YLSERRSFTA AVDALVTFLL HSAPGQHKDL DSLRRALLKD AFAFDEDLDV PD RSKLLTM APTYMNLVED CIARAQALPA KLGESFKTEA FELDWLRTAI TEAVHSLSIA FQALDLDTPY FAPHELLSEW FEL MNSSLF LESILGFEVV ADLAMPARSL VSAICLKMLN IDRTIQFLHD FDYPDGEEPY LLSSQTLNKI HTAVTNAVNS GVAA SLPVA FAWSLIVHQM HLGYQERAER RDLLVNQRAQ AGFELEFQPS ASTPNRRRRN SAGSIVSLEA SPYDDFLREQ RLDND IAPV EQIAMLATSR GQVYQVMSEM ALCLGTTHEA AFRPAVGARA RLVFQDLLKR SAYLIPYQDE PVFSLLAILA TGRQYW DVT DALSASSLNQ VYTDMLDDET LFTQFTMQAI NRFPYEFNPF SVLCRVLAAA LITNKDKADV VTGWLWRTPT LTVDWNP AW DRSYELCFED ENTNSFRLTR DVDLFGSASP ARPRHLAAEE RFIIPEGTLG RFVTDVGRTA RLEFEHSALA LLGKRLEV K AAEEICDSGM APLDVDEQAE AVAMLATVLR AESLKSTAKG GDPEAPLKFL KEASRLLPHN KDILTVISDT IDGLVEKEL LELDGPQIAV LASCLQFLHA ALAVCPGRVW AYMSRCALIA GDARPGRLSR ITGSLDMYAE RFDLLSSAVK LFAALIDSAA CSAVQRRAG STALVSVRSA VENPWLGTSE KILSRVALAI AQAALDVYES TTTWRFRSEL DRSILVRDVV GLMHKLVVHA H TLSSHLTS TLSPAAAHII SSFLTPPPSA SSLRFQPLLG TLLVALITPR ATLYPGQSRI LAERVTSVLA FCTSLLRAAD FL GQTHIPL QTHLFQSACL LARLPAANAV YRAPVLELLR ALVEVAGRAA NGSGEPPSLL GYLGSHAARS FISLVEGIDK PFG RVEHAV VTWRFFAAVI RNRQQWMAGC LLTGRTPREA LKGGGEQKIE RKVGEGSVLA AAMERLREVK SLDVQEAVAV MDFV VSAQN YWPWTIFAVR KEKEVVDALR GYVRGLKAPG MVMKTDGAAA AAFQARIAAY VAETFAMQLY HMRQMRQAEK FAGEL VADL DYFLREGVMV WGYNASLHGN FARNFAKRFP GVEVDDFKRT MWLPRELGKG YYYALEVAEQ MLGFDAGWGG VKQSGF RKE METANLNLSL VEAQVSLFHA WEYLLLELTL SLLPKKENAA FARQVLQVVE QCLEANQRSQ PPENIFVVLG HARAGLA LT LLQRLADANQ LPRDVTHLLA LVSSAIHAVE NPFGANDLPY FRTLLKILFV VLRAAKQGTA KPGESNVAIT QQVLTILD R VVARCFRALA ALVHEQQQNA TDGTTTAPED LALITAILQA CLSVPGIEQC QVQVLNIMAA HDVFQVAVAL FSWADRLLP ANPSPASSST STSATNPASG DPVYGELALL FLLELSALPA LAEHLACDGL LGHLAAARLA GYMRRTNVGP FAENAGAARC YAIWAKCLL PLLLNILAAL GSTVAPEVAW VLNQFPNLLQ SSVERIEPPG FSRPTLSLAS TPPRQKFISL LEISEIHSLA L LTRVLAAC RAQNARDVPE VTWDGAKVLE CVEYWLRGRK VLRERLVPLG PREVEWRGMV ATGGVVGVAG DGGEGCENRL EE KAVGLLV GVREVLEGGL EGEGE

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Macromolecule #2: Nucleoporin NIC96

MacromoleculeName: Nucleoporin NIC96 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus)
Strain: DSM 1495 / CBS 144.50 / IMI 039719
Molecular weightTheoretical: 7.173938 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SGTGLGEVDV DTYLSNLQTK TTLSMIADGL ERSARDFDAF LEENVTLEWE AQRKRIYQHF GIK

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
100.0 mMNaClSodium chloridesodium chloride
20.0 mM(HOCH2)3CNH2tris(hydroxymethyl)aminomethane
5.0 mMC4H10O2S2dithiothreitol
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Bioquantum
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 10740 / Average exposure time: 2.0 sec. / Average electron dose: 103.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6317697
CTF correctionSoftware - Name: cryoSPARC
Startup modelType of model: OTHER
Details: ab initio stochastic gradient descent reconstruction
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.39 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 709123
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-7mvy:
Single particle cryo-EM structure of the Chaetomium thermophilum Nup188-Nic96 complex (Nup188 residues 1-1858; Nic96 residues 240-301)

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Atomic model buiding 2

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-7mvy:
Single particle cryo-EM structure of the Chaetomium thermophilum Nup188-Nic96 complex (Nup188 residues 1-1858; Nic96 residues 240-301)

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