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- EMDB-44136: Cryo-EM structure of yeast (Nap1)2-H2A-H2B-Kap114 -

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Basic information

Entry
Database: EMDB / ID: EMD-44136
TitleCryo-EM structure of yeast (Nap1)2-H2A-H2B-Kap114
Map dataComposite map of consensus and locally refined map of Nap1 and histone region.
Sample
  • Complex: Crosslinked mixture of Kap114 with Nap1 and H2A-H2B
    • Protein or peptide: NAP1 isoform 1
    • Protein or peptide: KAP114 isoform 1
    • Protein or peptide: Histone H2A
    • Protein or peptide: Histone H2B
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: 3.54 Å
AuthorsJiou J / Fung HYJ / 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_44136.png

Downloads & links

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Map

FileDownload / File: emd_44136.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite map of consensus and locally refined map of Nap1 and histone region.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 480 pix.
= 398.4 Å		0.83 Å/pix.
x 480 pix.
= 398.4 Å		0.83 Å/pix.
x 480 pix.
= 398.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0688
Minimum - Maximum-0.06855512 - 0.499007
Average (Standard dev.)0.0007003397 (±0.008213085)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 398.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Crosslinked mixture of Kap114 with Nap1 and H2A-H2B

EntireName: Crosslinked mixture of Kap114 with Nap1 and H2A-H2B
Components
  • Complex: Crosslinked mixture of Kap114 with Nap1 and H2A-H2B
    • Protein or peptide: NAP1 isoform 1
    • Protein or peptide: KAP114 isoform 1
    • Protein or peptide: Histone H2A
    • Protein or peptide: Histone H2B

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Supramolecule #1: Crosslinked mixture of Kap114 with Nap1 and H2A-H2B

SupramoleculeName: Crosslinked mixture of Kap114 with Nap1 and H2A-H2B / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Nap1 and H2A-H2B were pre-complexed with excess histones, and Kap114 was added before mild crosslinking and SEC separation.
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 72 KDa

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Macromolecule #1: NAP1 isoform 1

MacromoleculeName: NAP1 isoform 1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 36.182355 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MLGSLVGQDS GYVGGLPKNV KEKLLSLKTL QSELFEVEKE FQVEMFELEN KFLQKYKPIW EQRSRIISG QEQPKPEQIA KGQEIVESLN ETELLVDEEE KAQNDSEEEQ VKGIPSFWLT ALENLPIVCD TITDRDAEVL E YLQDIGLE ...String:
MGSSHHHHHH SSGLVPRGSH MLGSLVGQDS GYVGGLPKNV KEKLLSLKTL QSELFEVEKE FQVEMFELEN KFLQKYKPIW EQRSRIISG QEQPKPEQIA KGQEIVESLN ETELLVDEEE KAQNDSEEEQ VKGIPSFWLT ALENLPIVCD TITDRDAEVL E YLQDIGLE YLTDGRPGFK LLFRFDSSAN PFFTNDILCK TYFYQKELGY SGDFIYDHAE GCEISWKDNA HNVTVDLEMR KQ RNKTTKQ VRTIEKITPI ESFFNFFDPP KIQNEDQDEE LEEDLEERLA LDYSIGEQLK DKLIPRAVDW FTGAAL

UniProtKB: UNIPROTKB: A0A8H4BY55

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Macromolecule #2: KAP114 isoform 1

MacromoleculeName: KAP114 isoform 1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 114.220867 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GGSMDINELI IGAQSADKHT REVAETQLLQ WCDSDASQVF KALANVALQH EASLESRQFA LLSLRKLITM YWSPGFESYR STSNVEIDV KDFIREVLLK LCLNDNENTK IKNGASYCIV QISAVDFPDQ WPQLLTVIYD AISHQHSLNA MSLLNEIYDD V VSEEMFFE ...String:
GGSMDINELI IGAQSADKHT REVAETQLLQ WCDSDASQVF KALANVALQH EASLESRQFA LLSLRKLITM YWSPGFESYR STSNVEIDV KDFIREVLLK LCLNDNENTK IKNGASYCIV QISAVDFPDQ WPQLLTVIYD AISHQHSLNA MSLLNEIYDD V VSEEMFFE GGIGLATMEI VFKVLNTETS TLIAKIAALK LLKACLLQMS SHNEYDEASR KSFVSQCLAT SLQILGQLLT LN FGNVDVI SQLKFKSIIY ENLVFIKNDF SRKHFSSELQ KQFKIMAIQD LENVTHINAN VETTESEPLL ETVHDCSIYI VEF LTSVCT LQFSVEEMNK IITSLTILCQ LSSETREIWT SDFNTFVSKE TGLAASYNVR DQANEFFTSL PNPQLSLIFK VVSN DIEHS TCNYSTLESL LYLLQCILLN DDEITGENID QSLQILIKTL ENILVSQEIP ELILARAILT IPRVLDKFID ALPDI KPLT SAFLAKSLNL ALKSDKELIK SATLIAFTYY CYFAELDSVL GPEVCSETQE KVIRIINQVS SDAEEDTNGA LMEVLS QVI SYNPKEPHSR KEILQAEFHL VFTISSEDPA NVQVVVQSQE CLEKLLDNIN MDNYKNYIEL CLPSFINVLD SNNANNY RY SPLLSLVLEF ITVFLKKKPN DGFLPDEINQ YLFEPLAKVL AFSTEDETLQ LATEAFSYLI FNTDTRAMEP RLMDIMKV L ERLLSLEVSD SAAMNVGPLV VAIFTRFSKE IQPLIGRILE AVVVRLIKTQ NISTEQNLLS VLCFLTCNDP KQTVDFLSS FQIDNTDALT LVMRKWIEAF EVIRGEKRIK ENIVALSNLF FLNDKRLQKV VVNGNLIPYE GDLIITRSMA KKMPDRYVQV PLYTKIIKL FVSELSFQSK QPNPEQLITS DIKQEVVNAN KDDDNDDWED VDDVLDYDKL KEYIDDDVDE EADDDSDDIT G LMDVKESV VQLLVRFFKE VASKDVSGFH CIYETLSDSE RKVLSEALL

UniProtKB: UNIPROTKB: A0A8H4BZV8

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Macromolecule #3: Histone H2A

MacromoleculeName: Histone H2A / type: protein_or_peptide / ID: 3 / 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 #4: Histone H2B

MacromoleculeName: Histone H2B / type: protein_or_peptide / ID: 4 / 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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.2 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
20.0 mMTris
150.0 mMsodium chlorideNaCl
2.0 mMTCEP
0.003125 %Triton X-100
GridModel: Quantifoil / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY
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: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 1080 / Average exposure time: 5.4 sec. / Average electron dose: 59.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 105000
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: 4314112
Details: Blob picking on 100 micrographs and then further template picking.
Startup modelType of model: NONE / Details: Ab-initio reconstruction.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC
Details: Composite map was generated by first multiplying the consensus volume with the particle subtraction mask using vop multiply, and composited with the locally refined map covering the Nap1 and ...Details: Composite map was generated by first multiplying the consensus volume with the particle subtraction mask using vop multiply, and composited with the locally refined map covering the Nap1 and histone area by vop maximum in ChimeraX.
Number images used: 113011
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 N-terminal region of Kap114 and H2A-H2B.

9b23

source_name: PDB, initial_model_type: experimental modelFor Nap1.

7-f1

chain_id: A, source_name: AlphaFold, initial_model_type: in silico modelFor C-terminal region of Kap114
RefinementProtocol: OTHER
Output model

PDB-9b3f:
Cryo-EM structure of yeast (Nap1)2-H2A-H2B-Kap114

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