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- EMDB-28788: Cryo-EM structure of Kap114 bound to Gsp1 (RanGTP) -

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Basic information

Entry
Database: EMDB / ID: EMD-28788
TitleCryo-EM structure of Kap114 bound to Gsp1 (RanGTP)
Map datasharpened
Sample
  • Complex: Complex of Kap114 bound to Gsp1 (RanGTP)
    • Protein or peptide: Importin subunit beta-5
    • Protein or peptide: GTP-binding nuclear protein GSP1/CNR1
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
KeywordsKaryopherin Beta / Nuclear Transport / GTPase / PROTEIN TRANSPORT-NUCLEAR PROTEIN complex
Function / homology
Function and homology information


regulation of cell cycle phase transition / 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 / poly(A)+ mRNA export from nucleus / nucleus organization / nuclear import signal receptor activity / NLS-bearing protein import into nucleus / ribosomal subunit export from nucleus / mRNA transport ...regulation of cell cycle phase transition / 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 / poly(A)+ mRNA export from nucleus / nucleus organization / nuclear import signal receptor activity / NLS-bearing protein import into nucleus / ribosomal subunit export from nucleus / mRNA transport / nuclear pore / small GTPase binding / protein import into nucleus / nuclear envelope / GTPase activity / GTP binding / 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 subunit beta-5
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.49 Å
AuthorsJiou J / 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 4, 2022-
Header (metadata) releaseJul 26, 2023-
Map releaseJul 26, 2023-
UpdateJul 26, 2023-
Current statusJul 26, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28788.png

Downloads & links

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Map

FileDownload / File: emd_28788.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened
Voxel sizeX=Y=Z: 1.059 Å
Density
Contour LevelBy AUTHOR: 0.23
Minimum - Maximum-1.3627895 - 2.0416124
Average (Standard dev.)0.0048790635 (±0.054231353)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 211.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half-A

Fileemd_28788_half_map_1.map
Annotationhalf-A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-B

Fileemd_28788_half_map_2.map
Annotationhalf-B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of Kap114 bound to Gsp1 (RanGTP)

EntireName: Complex of Kap114 bound to Gsp1 (RanGTP)
Components
  • Complex: Complex of Kap114 bound to Gsp1 (RanGTP)
    • Protein or peptide: Importin subunit beta-5
    • Protein or peptide: GTP-binding nuclear protein GSP1/CNR1
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Complex of Kap114 bound to Gsp1 (RanGTP)

SupramoleculeName: Complex of Kap114 bound to Gsp1 (RanGTP) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)

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Macromolecule #1: Importin subunit beta-5

MacromoleculeName: Importin subunit beta-5 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 114.019695 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MDINELIIGA QSADKHTREV AETQLLQWCD SDASQVFKAL ANVALQHEAS LESRQFALLS LRKLITMYWS PGFESYRSTS NVEIDVKDF IREVLLKLCL NDNENTKIKN GASYCIVQIS AVDFPDQWPQ LLTVIYDAIS HQHSLNAMSL LNEIYDDVVS E EMFFEGGI ...String:
MDINELIIGA QSADKHTREV AETQLLQWCD SDASQVFKAL ANVALQHEAS LESRQFALLS LRKLITMYWS PGFESYRSTS NVEIDVKDF IREVLLKLCL NDNENTKIKN GASYCIVQIS AVDFPDQWPQ LLTVIYDAIS HQHSLNAMSL LNEIYDDVVS E EMFFEGGI GLATMEIVFK VLNTETSTLI AKIAALKLLK ACLLQMSSHN EYDEASRKSF VSQCLATSLQ ILGQLLTLNF GN VDVISQL KFKSIIYENL VFIKNDFSRK HFSSELQKQF KIMAIQDLEN VTHINANVET TESEPLLETV HDCSIYIVEF LTS VCTLQF SVEEMNKIIT SLTILCQLSS ETREIWTSDF NTFVSKETGL AASYNVRDQA NEFFTSLPNP QLSLIFKVVS NDIE HSTCN YSTLESLLYL LQCILLNDDE ITGENIDQSL QILIKTLENI LVSQEIPELI LARAILTIPR VLDKFIDALP DIKPL TSAF LAKSLNLALK SDKELIKSAT LIAFTYYCYF AELDSVLGPE VCSETQEKVI RIINQVSSDA EEDTNGALME VLSQVI SYN PKEPHSRKEI LQAEFHLVFT ISSEDPANVQ VVVQSQECLE KLLDNINMDN YKNYIELCLP SFINVLDSNN ANNYRYS PL LSLVLEFITV FLKKKPNDGF LPDEINQYLF EPLAKVLAFS TEDETLQLAT EAFSYLIFNT DTRAMEPRLM DIMKVLER L LSLEVSDSAA MNVGPLVVAI FTRFSKEIQP LIGRILEAVV VRLIKTQNIS TEQNLLSVLC FLTCNDPKQT VDFLSSFQI DNTDALTLVM RKWIEAFEVI RGEKRIKENI VALSNLFFLN DKRLQKVVVN GNLIPYEGDL IITRSMAKKM PDRYVQVPLY TKIIKLFVS ELSFQSKQPN PEQLITSDIK QEVVNANKDD DNDDWEDVDD VLDYDKLKEY IDDDVDEEAD DDSDDITGLM D VKESVVQL LVRFFKEVAS KDVSGFHCIY ETLSDSERKV LSEALL

UniProtKB: Importin subunit beta-5

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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 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 20.41759 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString:
MSAPAANGEV PTFKLVLVGD GGTGKTTFVK RHLTGEFEKK YIATIGVEVH PLSFYTNFGE IKFDVWDTAG LEKFGGLRDG YYINAQCAI IMFDVTSRIT YKNVPNWHRD LVRVCENIPI VLCGNKVDVK ERKVKAKTIT FHRKKNLQYY DISAKSNYNF E KPFLWLAR KLAGNPQLEF V

UniProtKB: GTP-binding nuclear protein GSP1/CNR1

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Macromolecule #3: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / 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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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration4.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mM(HOCH2)3CNH2Trisaminomethane
300.0 mMNaClSodium chlorideSodium Chloride
2.0 mMMgCl2Magnesium Chloride
1.0 mMC9H15O6PTris(2-carboxyethyl)phosphine hydrochloride
0.1 %H(C2H4O)nO(C6H4)C9H19Nonyl phenoxypolyethoxylethanol

Details: 20 mM Tris pH 7.5, 300 mM NaCl, 2 mM MgCl2, 1 mM TCEP, and 0.1% NP-40
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 4 K / Instrument: FEI VITROBOT MARK IV
Details1 Kap114 to 1.2 Gsp1 (RanGTP) ratio

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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
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 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:

6aho

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.49 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 277305

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