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Entry
Database: EMDB / ID: EMD-24057
TitleSingle particle cryo-EM structure of the Chaetomium thermophilum Nup192-Nic96-Nup53-Nup145N complex (Nup192 residues 1-1756; Nic96 residues 240-301; Nup53 31-67; Nup145N 616-683)
Map dataUnsharpened map generated with cryoSPARC NU refine
Sample
  • Complex: Nup192-Nic96-Nup53-Nup145N heterotetramer
    • Protein or peptide: Nucleoporin NUP192
    • Protein or peptide: Nucleoporin NIC96
    • Protein or peptide: Nucleoporin NUP53
    • Protein or peptide: Nucleoporin NUP145N
Keywordsnuclear pore complex / nucleocytoplasmic transport / alpha-helical solenoid / nuclear pore / TRANSPORT PROTEIN
Function / homology
Function and homology information


nuclear pore inner ring / telomere tethering at nuclear periphery / nuclear pore organization / nuclear pore cytoplasmic filaments / post-transcriptional tethering of RNA polymerase II gene DNA at nuclear periphery / structural constituent of nuclear pore / RNA export from nucleus / poly(A)+ mRNA export from nucleus / nuclear localization sequence binding / Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases ...nuclear pore inner ring / telomere tethering at nuclear periphery / nuclear pore organization / nuclear pore cytoplasmic filaments / post-transcriptional tethering of RNA polymerase II gene DNA at nuclear periphery / structural constituent of nuclear pore / RNA export from nucleus / poly(A)+ mRNA export from nucleus / nuclear localization sequence binding / Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases / mRNA transport / nuclear pore / protein import into nucleus / protein transport / nuclear membrane / hydrolase activity / RNA binding
Similarity search - Function
Nucleoporin Nup186/Nup192/Nup205 / Nuclear pore complex scaffold, nucleoporins 186/192/205 / Nucleoporin interacting component Nup93/Nic96 / Nup93/Nic96 / Nucleoporin FG repeat / Nucleoporin FG repeat region / Nuclear pore complex protein NUP96, C-terminal domain / Nuclear protein 96 / Nuclear pore complex protein Nup98-Nup96-like, autopeptidase S59 domain / Nuclear pore complex protein Nup98-Nup96-like, autopeptidase S59 domain superfamily ...Nucleoporin Nup186/Nup192/Nup205 / Nuclear pore complex scaffold, nucleoporins 186/192/205 / Nucleoporin interacting component Nup93/Nic96 / Nup93/Nic96 / Nucleoporin FG repeat / Nucleoporin FG repeat region / Nuclear pore complex protein NUP96, C-terminal domain / Nuclear protein 96 / Nuclear pore complex protein Nup98-Nup96-like, autopeptidase S59 domain / Nuclear pore complex protein Nup98-Nup96-like, autopeptidase S59 domain superfamily / Nucleoporin autopeptidase / NUP C-terminal domain profile. / Nucleoporin peptidase S59-like / Nucleotide-binding alpha-beta plait domain superfamily
Similarity search - Domain/homology
Nucleoporin NIC96 / Nucleoporin NUP53 / Nucleoporin NUP192 / Nucleoporin NUP145
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: 3.22 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-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_24057.png

Downloads & links

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Map

FileDownload / File: emd_24057.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened map generated with cryoSPARC NU refine
Voxel sizeX=Y=Z: 1.4208 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.4355282 - 1.9388669
Average (Standard dev.)-0.001523027 (±0.048745774)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 363.7248 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_24057_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Map sharpened with PHENIX Autosharpen model based local sharpening

Fileemd_24057_additional_1.map
AnnotationMap sharpened with PHENIX Autosharpen model based local sharpening
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: Half-dataset map generated with cryoSPARC

Fileemd_24057_half_map_1.map
AnnotationHalf-dataset map generated with cryoSPARC
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: Half-dataset map generated with cryoSPARC

Fileemd_24057_half_map_2.map
AnnotationHalf-dataset map generated with cryoSPARC
Projections & Slices
AxesZYX

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Sample components

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Entire : Nup192-Nic96-Nup53-Nup145N heterotetramer

EntireName: Nup192-Nic96-Nup53-Nup145N heterotetramer
Components
  • Complex: Nup192-Nic96-Nup53-Nup145N heterotetramer
    • Protein or peptide: Nucleoporin NUP192
    • Protein or peptide: Nucleoporin NIC96
    • Protein or peptide: Nucleoporin NUP53
    • Protein or peptide: Nucleoporin NUP145N

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Supramolecule #1: Nup192-Nic96-Nup53-Nup145N heterotetramer

SupramoleculeName: Nup192-Nic96-Nup53-Nup145N heterotetramer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)
Molecular weightTheoretical: 215.7 KDa

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Macromolecule #1: Nucleoporin NUP192

MacromoleculeName: Nucleoporin NUP192 / 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: 200.047484 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GPLMSGLNDI FEAQKIEWHE GSAGGSGHMT DLRKLEALQA LHAELVAVRQ HRFEGLQVLE TLLEEQTDAF KALIAKPARD TKDREALGK EPKKLKIGEE EYSLNEDFVN DCLKLADELD LNEKESARIL IDCDAEGDVE TQSRPLWECG VIRFHQERKY L LDCMRLIL ...String:
GPLMSGLNDI FEAQKIEWHE GSAGGSGHMT DLRKLEALQA LHAELVAVRQ HRFEGLQVLE TLLEEQTDAF KALIAKPARD TKDREALGK EPKKLKIGEE EYSLNEDFVN DCLKLADELD LNEKESARIL IDCDAEGDVE TQSRPLWECG VIRFHQERKY L LDCMRLIL EIAADEDIDA GLQESFGVAA EDKIFGIPPP WERNKENQPT QVKKFIPRCM EAMKGVRSML QCMADKANAR NM LQQASLV RPLDNQETLD FSRLSLVEQH ECLASILHAA VQRHHATIAD FQDFIKILRK WDKYDHFLIH LIPVLAAYIT EFG SPEGMG DLQQARRLND FICKGGDEDS WALPVLGAAV RAWWIAEHNG FYLDDTVQDL RGINLDEEDE QRTKQFLDAL KEGA FDFIL SVAADCKAQE WQDPSQLGAR QWLQRKIPSL PSEPFPFSHF LQHSLMVHLE GFVDATISNL PDVLRKLRTE EDEQR QLRP NHEQDMDLER FLIIISYAYE GRPDAAMSFW EDPDSNLAGF LQWASRRAST PLVSAFCEML RCLADNEECA TAAHNF LLD EGHQASGKMK RSQSLTWSQI FKELEYFTTK VCSERPNPPQ ASMHRPGRPG ADPAEIEPES ALMLECYLRL IAKLATE SE IARKRLIMDE DFNLVDTILK LSVGVIPHRL RACIFYVLKA LMIRKTHEEL DAMWRWVEAW MTNPFSSLPG SQGAPQRI S FLGQTPGPQE CMEMMFREFG TGFEQSNAFI QLLTTLLVPP EGLNSLNDSV PFPEWLGSSI RTLGIEPYVD FVFDVFANR TKDISDPSQL RILRLSCLDF VMVCLVTFNE DLIVLGHESN ISIDDAMAAT NLATYVRLHP FSRVMEWLFN EKVITSLINT IHQDPISLG SASPDSPLVV SILRAIQVMI KALELQETYL HLVRPEVLRY QGEAGVRRKP VANAAYSAFE DGILSHLSLV V DLGKYCNL GHAELTLACL KLLEKISTSS RILSAWSPDS GRLGHRNKAI VQLERNGEGE TISASLSASI MATLDPALAA SG ENYRVKL AILDFLYACL RATPDQPTIA HQLLGFHCEL SKLGIEPKGP FDMQKSLFHS LLNVLITLTV SEEEQGMRGY LVT LKYRVL RILQLLWKSP LSASLVMDEL RATNFLFHML LREVQIQPQL PWDGQLVTGC EFLLSDASLA YIDYLASRAA IFEY IGKEL CSVSQNRIPS IKRQIFDALN GQIFVDEEAP LTIPSIFDFF DFINTDYKWE EIPSPHFTYL KDLDLGPCIL EHKYA GVHY DIRKAQEILA LKRKEYEHSQ LATPEFLETV ELEEKVLIEW LTVRNRANLL LTARLNLLQA WANLLLVMIE SNDFKS TPK MAFLLQALQA ILPTLEAFSS LKSDEAFELA RVAKVLLWKL DFSQDSDAGL DREQFTVGNL IGDKLFQLFQ LCLSAIS QC SGTPELRSLY YSICYRYLTA VVDNDATVAA TPASSTIGPT RSVTNARART LKAITLYGDR LLNVICDDAY GSDTTCQT A AMILLNALVH TSRASSAAGV SPADVDCPII DALNRLNFIG VLVDSLKEIL NEWLAPSSTF DPSLSTNASP SLPIPASPS QQYTSAKLAL LLQLCQTRQG AKYVLQANLF RALEQSGVFA ADPELVEVDS ESGVPRVVAL ERHYALLVAL ARVVGAAVTA RGAHNIVQG RKFLTQHRGL VVHVLKKNAG IGGGVVGNSL ASSINGGSTA TMTRRDEILA QQALEERIEE LAEAFMLLIT A TGFLEYES EQVPSEQPRA HTTFFH

UniProtKB: Nucleoporin NUP192

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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

UniProtKB: Nucleoporin NIC96

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Macromolecule #3: Nucleoporin NUP53

MacromoleculeName: Nucleoporin NUP53 / type: protein_or_peptide / ID: 3 / 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: 4.173613 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SQQDGSLRSR KANLETGAFG KSTRRTRSKA ATPAKRED

UniProtKB: Nucleoporin NUP53

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Macromolecule #4: Nucleoporin NUP145N

MacromoleculeName: Nucleoporin NUP145N / type: protein_or_peptide / ID: 4 / 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: 7.077983 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SATPLSGKAK VKSRSILPMY KLSPANASRL VTTPQKRAYG FSFSAYGSPT SPSSSASSTP GAFGQSILS

UniProtKB: Nucleoporin NUP145

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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
150.0 mMNaClsodium chloride
20.0 mM(HOCH2)3CNH2tris(hydroxymethyl)aminomethane
5.0 mMC4H10O2S2dithiothreitol
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 4319 / Average electron dose: 100.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2286858
Startup modelType of model: OTHER
Details: ab initio stochastic gradient descent reconstruction
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.22 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 484910
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-7mvv:
Single particle cryo-EM structure of the Chaetomium thermophilum Nup192-Nic96-Nup53-Nup145N complex (Nup192 residues 1-1756; Nic96 residues 240-301; Nup53 31-67; Nup145N 616-683)

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Atomic model buiding 2

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-7mvv:
Single particle cryo-EM structure of the Chaetomium thermophilum Nup192-Nic96-Nup53-Nup145N complex (Nup192 residues 1-1756; Nic96 residues 240-301; Nup53 31-67; Nup145N 616-683)

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