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- EMDB-28899: Cryo-EM structure of Importin-9 bound to RanGTP -

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Basic information

Entry
Database: EMDB / ID: EMD-28899
TitleCryo-EM structure of Importin-9 bound to RanGTP
Map data
Sample
  • Complex: Importin-9 bound to Gsp1
    • Protein or peptide: Importin-9
    • Protein or peptide: GTP-binding nuclear protein GSP1/CNR1
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
KeywordsNuclear Import / Importin / RanGTP / PROTEIN TRANSPORT
Function / homology
Function and homology information


proteasome localization / regulation of cell cycle phase transition / histone chaperone activity / Postmitotic nuclear pore complex (NPC) reformation / exonucleolytic trimming to generate mature 3'-end of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / regulation of nucleocytoplasmic transport / nuclear import signal receptor activity / poly(A)+ mRNA export from nucleus / nucleus organization / ribosomal subunit export from nucleus ...proteasome localization / regulation of cell cycle phase transition / histone chaperone activity / Postmitotic nuclear pore complex (NPC) reformation / exonucleolytic trimming to generate mature 3'-end of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / regulation of nucleocytoplasmic transport / nuclear import signal receptor activity / poly(A)+ mRNA export from nucleus / nucleus organization / ribosomal subunit export from nucleus / small GTPase binding / protein import into nucleus / nuclear envelope / histone binding / GTPase activity / GTP binding / membrane / nucleus / cytosol / cytoplasm
Similarity search - Function
small GTPase Ran family profile. / Ran GTPase / Importin-beta N-terminal domain profile. / Importin-beta N-terminal domain / Importin-beta N-terminal domain / Importin-beta, N-terminal domain / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase ...small GTPase Ran family profile. / Ran GTPase / Importin-beta N-terminal domain profile. / Importin-beta N-terminal domain / Importin-beta N-terminal domain / Importin-beta, N-terminal domain / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / Armadillo-like helical / Small GTP-binding protein domain / Armadillo-type fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GTP-binding nuclear protein GSP1/CNR1 / Importin-9
Similarity search - Component
Biological speciesHomo sapiens (human) / Saccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.78 Å
AuthorsBernardes NE / Chook YM
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM141461-02 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Mechanism of RanGTP priming H2A-H2B release from Kap114 in an atypical RanGTP•Kap114•H2A-H2B complex.
Authors: Jenny Jiou / Joy M Shaffer / Natalia E Bernades / Ho Yee Joyce Fung / Juliana Kikumoto Dias / Sheena D'Arcy / Yuh Min Chook /
Abstract: Previously, we showed that the nuclear import receptor Importin-9 wraps around the H2A-H2B core to chaperone and transport it from the cytoplasm to the nucleus. However, unlike most nuclear import ...Previously, we showed that the nuclear import receptor Importin-9 wraps around the H2A-H2B core to chaperone and transport it from the cytoplasm to the nucleus. However, unlike most nuclear import systems where RanGTP dissociates cargoes from their importins, RanGTP binds stably to the Importin-9•H2A-H2B complex, and formation of the ternary RanGTP•Importin-9•H2A-H2B complex facilitates H2A-H2B release to the assembling nucleosome. It was unclear how RanGTP and the cargo H2A-H2B can bind simultaneously to an importin, and how interactions of the three components position H2A-H2B for release. Here, we show cryo-EM structures of Importin-9•RanGTP and of its yeast homolog Kap114, including Kap114•RanGTP, Kap114•H2A-H2B, and RanGTP•Kap114•H2A-H2B, to explain how the conserved Kap114 binds H2A-H2B and RanGTP simultaneously and how the GTPase primes histone transfer to the nucleosome. In the ternary complex, RanGTP binds to the N-terminal repeats of Kap114 in the same manner as in the Kap114/Importin-9•RanGTP complex, and H2A-H2B binds via its acidic patch to the Kap114 C-terminal repeats much like in the Kap114/Importin-9•H2A-H2B complex. Ran binds to a different conformation of Kap114 in the ternary RanGTP•Kap114•H2A-H2B complex. Here, Kap114 no longer contacts the H2A-H2B surface proximal to the H2A docking domain that drives nucleosome assembly, positioning it for transfer to the assembling nucleosome or to dedicated H2A-H2B chaperones in the nucleus.
History
DepositionNov 18, 2022-
Header (metadata) releaseJul 26, 2023-
Map releaseJul 26, 2023-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28899.png

Downloads & links

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Map

FileDownload / File: emd_28899.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.837 Å
Density
Contour LevelBy AUTHOR: 0.08
Minimum - Maximum-0.5161266 - 0.8857766
Average (Standard dev.)0.0007740198 (±0.016340587)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 251.1 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_28899_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_28899_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Importin-9 bound to Gsp1

EntireName: Importin-9 bound to Gsp1
Components
  • Complex: Importin-9 bound to Gsp1
    • Protein or peptide: Importin-9
    • Protein or peptide: GTP-binding nuclear protein GSP1/CNR1
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate

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Supramolecule #1: Importin-9 bound to Gsp1

SupramoleculeName: Importin-9 bound to Gsp1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Importin-9

MacromoleculeName: Importin-9 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 116.06232 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAAAAAAGAA SGLPGPVAQG LKEALVDTLT GILSPVQEVR AAAEEQIKVL EVTEEFGVHL AELTVDPQGA LAIRQLASVI LKQYVETHW CAQSEKFRPP ETTERAKIVI RELLPNGLRE SISKVRSSVA YAVSAIAHWD WPEAWPQLFN LLMEMLVSGD L NAVHGAMR ...String:
MAAAAAAGAA SGLPGPVAQG LKEALVDTLT GILSPVQEVR AAAEEQIKVL EVTEEFGVHL AELTVDPQGA LAIRQLASVI LKQYVETHW CAQSEKFRPP ETTERAKIVI RELLPNGLRE SISKVRSSVA YAVSAIAHWD WPEAWPQLFN LLMEMLVSGD L NAVHGAMR VLTEFTREVT DTQMPLVAPV ILPEMYKIFT MAEVYGIRTR SRAVEIFTTC AHMICNMEEL EKGAAKVLIF PV VQQFTEA FVQALQIPDG PTSDSGFKME VLKAVTALVK NFPKHMVSSM QQILPIVWNT LTESAAFYVR TEVNYTEEVE DPV DSDGEV LGFENLVFSI FEFVHALLEN SKFKSTVKKA LPELIYYIIL YMQITEEQIK VWTANPQQFV EDEDDDTFSY TVRI AAQDL LLAVATDFQN ESAAALAAAA TRHLQEAEQT KNSGTEHWWK IHEACMLALG SVKAIITDSV KNGRIHFDMH GFLTN VILA DLNLSVSPFL LGRALWAASR FTVAMSPELI QQFLQATVSG LHETQPPSVR ISAVRAIWGY CDQLKVSEST HVLQPF LPS ILDGLIHLAA QFSSEVLNLV METLCIVCTV DPEFTASMES KICPFTIAIF LKYSNDPVVA SLAQDIFKEL SQIEACQ GP MQMRLIPTLV SIMQAPADKI PAGLCATAID ILTTVVRNTK PPLSQLLICQ AFPAVAQCTL HTDDNATMQN GGECLRAY V SVTLEQVAQW HDEQGHNGLW YVMQVVSQLL DPRTSEFTAA FVGRLVSTLI SKAGRELGEN LDQILRAILS KMQQAETLS VMQSLIMVFA HLVHTQLEPL LEFLCSLPGP TGKPALEFVM AEWTSRQHLF YGQYEGKVSS VALCKLLQHG INADDKRLQD IRVKGEEIY SMDEGIRTRS KSAKNPERWT NIPLLVKILK LIINELSNVM EANAARQATP AEWSQDDSND MWEDQEEEEE E EEDGLAGQ LLSDILATSK YEEDYYEDDE EDDPDALKDP LYQIDLQAYL TDFLCQFAQQ PCYIMFSGHL NDNERRVLQT IG I

UniProtKB: Importin-9

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Macromolecule #2: GTP-binding nuclear protein GSP1/CNR1

MacromoleculeName: GTP-binding nuclear protein GSP1/CNR1 / type: protein_or_peptide / ID: 2 / Details: Mutation Q71L / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 24.825357 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSAPAANGEV PTFKLVLVGD GGTGKTTFVK RHLTGEFEKK YIATIGVEVH PLSFYTNFGE IKFDVWDTAG LEKFGGLRDG YYINAQCAI IMFDVTSRIT YKNVPNWHRD LVRVCENIPI VLCGNKVDVK ERKVKAKTIT FHRKKNLQYY DISAKSNYNF E KPFLWLAR ...String:
MSAPAANGEV PTFKLVLVGD GGTGKTTFVK RHLTGEFEKK YIATIGVEVH PLSFYTNFGE IKFDVWDTAG LEKFGGLRDG YYINAQCAI IMFDVTSRIT YKNVPNWHRD LVRVCENIPI VLCGNKVDVK ERKVKAKTIT FHRKKNLQYY DISAKSNYNF E KPFLWLAR KLAGNPQLEF VASPALAPPE VQVDEQLMQQ YQQEMEQATA LPLPDEDDAD L

UniProtKB: GTP-binding nuclear protein GSP1/CNR1

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM / Guanosine triphosphate

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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

BufferpH: 7.5
GridModel: Quantifoil / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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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 / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 52.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6n1z


Details: CHAIN A
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.78 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 232798

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-8f7a:
Cryo-EM structure of Importin-9 bound to RanGTP

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