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- EMDB-44141: Cryo-EM structure of yeast (Nap1)2-H2A-H2B-Kap114-RanGTP -

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Basic information

Entry
Database: EMDB / ID: EMD-44141
TitleCryo-EM structure of yeast (Nap1)2-H2A-H2B-Kap114-RanGTP
Map dataComposite map.
Sample
  • Complex: Complex of Kap114 bound to Ran GTPase Gsp1, H2A-H2B and Nap1
    • Protein or peptide: KAP114 isoform 1
    • Protein or peptide: Histone H2A
    • Protein or peptide: Histone H2B
    • Protein or peptide: GTP-binding nuclear protein
    • Protein or peptide: NAP1 isoform 1
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
KeywordsHistone / Chaperone / Import / Nucleosome Assembly / TRANSPORT PROTEIN
Function / homology: / : / : / : / :
Function and homology information
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.88 Å
AuthorsFung HYJ / Jiou J / Chook YM
Funding support United States, 5 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM141461 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM069909 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32GM008203 United States
Welch FoundationI-1532 United States
Cancer Prevention and Research Institute of Texas (CPRIT)RP220582 United States
CitationJournal: J Cell Biol / Year: 2025
Title: Nap1 and Kap114 co-chaperone H2A-H2B and facilitate targeted histone release in the nucleus.
Authors: Ho Yee Joyce Fung / Jenny Jiou / Ashley B Niesman / Natalia E Bernardes / Yuh Min Chook /
Abstract: Core histones, synthesized and processed in the cytoplasm, must be chaperoned as they are transported into the nucleus for nucleosome assembly. The importin Kap114 transports H2A-H2B into the yeast ...Core histones, synthesized and processed in the cytoplasm, must be chaperoned as they are transported into the nucleus for nucleosome assembly. The importin Kap114 transports H2A-H2B into the yeast nucleus, where RanGTP facilitates histone release. Kap114 and H2A-H2B also bind the histone chaperone Nap1, but how Nap1 and Kap114 cooperate in transport and nucleosome assembly remains unclear. Here, biochemical and structural analyses show that Kap114, H2A-H2B, and a Nap1 dimer (Nap12) associate in the absence and presence of RanGTP to form equimolar complexes. A previous study had shown that RanGTP reduces Kap114's ability to chaperone H2A-H2B, but a new cryo-EM structure of the Nap12•H2A-H2B•Kap114•RanGTP complex explains how both Kap114 and Nap12 interact with H2A-H2B, restoring its chaperoning within the assembly while effectively depositing it into nucleosomes. Together, our results suggest that Kap114 and Nap12 provide a sheltered path that facilitates the transfer of H2A-H2B from Kap114 to Nap12, ultimately directing its specific deposition into nucleosomes.
History
DepositionMar 19, 2024-
Header (metadata) releaseNov 27, 2024-
Map releaseNov 27, 2024-
UpdateDec 11, 2024-
Current statusDec 11, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44141.png

Downloads & links

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Map

FileDownload / File: emd_44141.map.gz / Format: CCP4 / Size: 527.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite map.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.74 Å/pix.
x 517 pix.
= 381.546 Å		0.74 Å/pix.
x 517 pix.
= 381.546 Å		0.74 Å/pix.
x 517 pix.
= 381.546 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.738 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.03740375 - 0.700464
Average (Standard dev.)0.00036833077 (±0.0054988614)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-2-2-2
Dimensions517517517
Spacing517517517
CellA=B=C: 381.546 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Complex of Kap114 bound to Ran GTPase Gsp1, H2A-H2B and Nap1

EntireName: Complex of Kap114 bound to Ran GTPase Gsp1, H2A-H2B and Nap1
Components
  • Complex: Complex of Kap114 bound to Ran GTPase Gsp1, H2A-H2B and Nap1
    • Protein or peptide: KAP114 isoform 1
    • Protein or peptide: Histone H2A
    • Protein or peptide: Histone H2B
    • Protein or peptide: GTP-binding nuclear protein
    • Protein or peptide: NAP1 isoform 1
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: Complex of Kap114 bound to Ran GTPase Gsp1, H2A-H2B and Nap1

SupramoleculeName: Complex of Kap114 bound to Ran GTPase Gsp1, H2A-H2B and Nap1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Details: Complex was formed and dialyzed overnight, then mildly crosslinked and separated by size-exclusion chromatography.
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: KAP114 isoform 1

MacromoleculeName: KAP114 isoform 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 114.833539 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSPNSRVDMD INELIIGAQS ADKHTREVAE TQLLQWCDSD ASQVFKALAN VALQHEASLE SRQFALLSLR KLITMYWSPG FESYRSTSN VEIDVKDFIR EVLLKLCLND NENTKIKNGA SYCIVQISAV DFPDQWPQLL TVIYDAISHQ HSLNAMSLLN E IYDDVVSE ...String:
GSPNSRVDMD INELIIGAQS ADKHTREVAE TQLLQWCDSD ASQVFKALAN VALQHEASLE SRQFALLSLR KLITMYWSPG FESYRSTSN VEIDVKDFIR EVLLKLCLND NENTKIKNGA SYCIVQISAV DFPDQWPQLL TVIYDAISHQ HSLNAMSLLN E IYDDVVSE EMFFEGGIGL ATMEIVFKVL NTETSTLIAK IAALKLLKAC LLQMSSHNEY DEASRKSFVS QCLATSLQIL GQ LLTLNFG NVDVISQLKF KSIIYENLVF IKNDFSRKHF SSELQKQFKI MAIQDLENVT HINANVETTE SEPLLETVHD CSI YIVEFL TSVCTLQFSV EEMNKIITSL TILCQLSSET REIWTSDFNT FVSKETGLAA SYNVRDQANE FFTSLPNPQL SLIF KVVSN DIEHSTCNYS TLESLLYLLQ CILLNDDEIT GENIDQSLQI LIKTLENILV SQEIPELILA RAILTIPRVL DKFID ALPD IKPLTSAFLA KSLNLALKSD KELIKSATLI AFTYYCYFAE LDSVLGPEVC SETQEKVIRI INQVSSDAEE DTNGAL MEV LSQVISYNPK EPHSRKEILQ AEFHLVFTIS SEDPANVQVV VQSQECLEKL LDNINMDNYK NYIELCLPSF INVLDSN NA NNYRYSPLLS LVLEFITVFL KKKPNDGFLP DEINQYLFEP LAKVLAFSTE DETLQLATEA FSYLIFNTDT RAMEPRLM D IMKVLERLLS LEVSDSAAMN VGPLVVAIFT RFSKEIQPLI GRILEAVVVR LIKTQNISTE QNLLSVLCFL TCNDPKQTV DFLSSFQIDN TDALTLVMRK WIEAFEVIRG EKRIKENIVA LSNLFFLNDK RLQKVVVNGN LIPYEGDLII TRSMAKKMPD RYVQVPLYT KIIKLFVSEL SFQSKQPNPE QLITSDIKQE VVNANKDDDN DDWEDVDDVL DYDKLKEYID DDVDEEADDD S DDITGLMD VKESVVQLLV RFFKEVASKD VSGFHCIYET LSDSERKVLS EALL

UniProtKB: UNIPROTKB: A0A8H4BZV8

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Macromolecule #2: Histone H2A

MacromoleculeName: Histone H2A / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 13.88198 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SGGKGGKAGS AAKASQSRSA KAGLTFPVGR VHRLLRRGNY AQRIGSGAPV YLTAVLEYLA AEILELAGNA ARDNKKTRII PRHLQLAIR NDDELNKLLG NVTIAQGGVL PNIHQNLLPK KSAKTAKASQ EL

UniProtKB: UNIPROTKB: A0A6A5Q402

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Macromolecule #3: Histone H2B

MacromoleculeName: Histone H2B / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 14.133145 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SSAAEKKPAS KAPAEKKPAA KKTSTSVDGK KRSKVRKETY SSYIYKVLKQ THPDTGISQK SMSILNSFVN DIFERIATEA SKLAAYNKK STISAREIQT AVRLILPGEL AKHAVSEGTR AVTKYSSSTQ A

UniProtKB: UNIPROTKB: A0A6A5Q1U6

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Macromolecule #4: GTP-binding nuclear protein

MacromoleculeName: GTP-binding nuclear protein / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 21.28352 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MASAPAANGE VPTFKLVLVG DGGTGKTTFV KRHLTGEFEK KYIATIGVEV HPLSFYTNFG EIKFDVWDTA GLEKFGGLRD GYYINAQCA IIMFDVTSRI TYKNVPNWHR DLVRVCENIP IVLCGNKVDV KERKVKAKTI TFHRKKNLQY YDISAKSNYN F EKPFLWLA RKLAGNPQLE FVENLYFQ

UniProtKB: UNIPROTKB: A0A6A5PUD0

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Macromolecule #5: NAP1 isoform 1

MacromoleculeName: NAP1 isoform 1 / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 48.035375 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSMGTDPIRT KPKSSMQIDN APTPHNTPAS VLNPSYLKNG NPVRAQAQEQ DDKIGTINEE DILANQPLLL QSIQDRLGSL VGQDSGYVG GLPKNVKEKL LSLKTLQSEL FEVEKEFQVE MFELENKFLQ KYKPIWEQRS RIISGQEQPK PEQIAKGQEI V ESLNETEL ...String:
GSMGTDPIRT KPKSSMQIDN APTPHNTPAS VLNPSYLKNG NPVRAQAQEQ DDKIGTINEE DILANQPLLL QSIQDRLGSL VGQDSGYVG GLPKNVKEKL LSLKTLQSEL FEVEKEFQVE MFELENKFLQ KYKPIWEQRS RIISGQEQPK PEQIAKGQEI V ESLNETEL LVDEEEKAQN DSEEEQVKGI PSFWLTALEN LPIVADTITD RDAEVLEYLQ DIGLEYLTDG RPGFKLLFRF DS SANPFFT NDILAKTYFY QKELGYSGDF IYDHAEGAEI SWKDNAHNVT VDLEMRKQRN KTTKQVRTIE KITPIESFFN FFD PPKIQN EDQDEELEED LEERLALDYS IGEQLKDKLI PRAVDWFTGA ALEFEFEEDE EEADEDEDEE DDDDHGLEDD DGES AEEQD DFAGRPEQAP ECKQS

UniProtKB: UNIPROTKB: A0A8H4BY55

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 7 / Number of copies: 1 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

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

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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

Concentration1.5 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
20.0 mMTris
150.0 mMsodium chlorideNaCl
2.0 mMTCEP
0.003125 %Tyloxapol
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 80 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 280 K / Instrument: FEI VITROBOT MARK IV
DetailsCrosslinked sample.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 9331 / Average exposure time: 3.6 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1381753 / Details: Blob picker then followed by Topaz picking.
Startup modelType of model: NONE / Details: Ab-initio reconstruction.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.88 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC
Details: Composite map was generated by first applying vop multiply to the particle subtraction mask and the consensus volume (covering Kap114 and RanGTP), then composited with locally refined region ...Details: Composite map was generated by first applying vop multiply to the particle subtraction mask and the consensus volume (covering Kap114 and RanGTP), then composited with locally refined region for the Nap1 and Histone region by vop maximum in Chimera X.
Number images used: 133516
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC

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Atomic model buiding 1

Initial model
PDB IDChainDetails

8f1e

source_name: PDB, initial_model_type: experimental modelFor Kap114 and RanGTP.

9b3f

source_name: PDB, initial_model_type: experimental modelFor Nap1, histone and C-terminal end of Kap114.
RefinementProtocol: OTHER
Output model

PDB-9b3i:
Cryo-EM structure of yeast (Nap1)2-H2A-H2B-Kap114-RanGTP

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