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- EMDB-24056: Single particle cryo-EM structure of the Chaetomium thermophilum ... -

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Entry
Database: EMDB / ID: EMD-24056
TitleSingle particle cryo-EM structure of the Chaetomium thermophilum Nup192-Nic96 complex (Nup192 residues 1-1756; Nic96 residues 240-301)
Map dataUnsharpened map generated with cryoSPARC
Sample
  • Complex: Nup192-Nic96 heterodimer
    • Protein or peptide: Nucleoporin NUP192
    • 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
Nucleoporin Nup186/Nup192/Nup205 / Nuclear pore complex scaffold, nucleoporins 186/192/205 / Nucleoporin interacting component Nup93/Nic96 / Nup93/Nic96
Similarity search - Domain/homology
Nucleoporin NIC96 / Nucleoporin NUP192
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: 3.77 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_24056.png

Downloads & links

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Map

FileDownload / File: emd_24056.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened map generated with cryoSPARC
Voxel sizeX=Y=Z: 1.668 Å
Density
Contour LevelBy AUTHOR: 0.12
Minimum - Maximum-0.28408742 - 0.7741276
Average (Standard dev.)-0.0012572175 (±0.02512269)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 320.25598 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_24056_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: Map sharpened with PHENIX Autosharpen

Fileemd_24056_additional_1.map
AnnotationMap sharpened with PHENIX Autosharpen
Projections & Slices
AxesZYX

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Histogram

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Half map: #1

Fileemd_24056_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: Half-dataset map generated with cryoSPARC

Fileemd_24056_half_map_2.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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Sample components

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Entire : Nup192-Nic96 heterodimer

EntireName: Nup192-Nic96 heterodimer
Components
  • Complex: Nup192-Nic96 heterodimer
    • Protein or peptide: Nucleoporin NUP192
    • Protein or peptide: Nucleoporin NIC96

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Supramolecule #1: Nup192-Nic96 heterodimer

SupramoleculeName: Nup192-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: 204.5 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

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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
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: 4.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 165000
Specialist opticsEnergy filter - Name: GIF Quantum LS
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number grids imaged: 1 / Number real images: 1082 / Average electron dose: 54.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 719840
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: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.77 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 176609
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-7mvu:
Single particle cryo-EM structure of the Chaetomium thermophilum Nup192-Nic96 complex (Nup192 residues 1-1756; Nic96 residues 240-301)

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Atomic model buiding 2

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-7mvu:
Single particle cryo-EM structure of the Chaetomium thermophilum Nup192-Nic96 complex (Nup192 residues 1-1756; Nic96 residues 240-301)

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