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

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Basic information

Entry
Database: PDB / ID: 9b3f
TitleCryo-EM structure of yeast (Nap1)2-H2A-H2B-Kap114
Components
  • Histone H2A
  • Histone H2B
  • KAP114 isoform 1
  • NAP1 isoform 1
KeywordsTRANSPORT PROTEIN / Histone / Chaperone / Import / Nucleosome Assembly
Function / homology: / : / : / :
Function and homology information
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.54 Å
AuthorsJiou, J. / Fung, H.Y.J. / Chook, Y.M.
Funding support United States, 5items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 27, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 11, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed ..._citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: NAP1 isoform 1
E: NAP1 isoform 1
A: KAP114 isoform 1
B: Histone H2A
C: Histone H2B


Theoretical massNumber of molelcules
Total (without water)214,6015
Polymers214,6015
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein NAP1 isoform 1


Mass: 36182.355 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: NAP1, GI527_G0003692 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A8H4BY55
#2: Protein KAP114 isoform 1


Mass: 114220.867 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: KAP114, GI527_G0002177 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A8H4BZV8
#3: Protein Histone H2A


Mass: 13881.980 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: HTA2, GI527_G0000202 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A6A5Q402
#4: Protein Histone H2B


Mass: 14133.145 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: HTB2, GI527_G0000203 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A6A5Q1U6
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Crosslinked mixture of Kap114 with Nap1 and H2A-H2B / Type: COMPLEX
Details: Nap1 and H2A-H2B were pre-complexed with excess histones, and Kap114 was added before mild crosslinking and SEC separation.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.072 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris1
2150 mMsodium chlorideNaCl1
32 mMTCEP1
40.003125 %Triton X-1001
SpecimenConc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Crosslinked sample.
Specimen supportGrid material: COPPER / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 280 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 900 nm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5.4 sec. / Electron dose: 59 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1080
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
7UCSF ChimeraXmodel fittingISOLDE was used for modeling
8Cootmodel fitting
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
14PHENIX1.19.1_4122:model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4314112
Details: Blob picking on 100 micrographs and then further template picking.
3D reconstructionResolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 113011
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.
Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Atomic model building

3D fitting-ID: 1

IDPDB-IDAccession codeDetailsInitial refinement model-IDSource nameTypeChain-ID
18F1E

8f1e

8F1EFor N-terminal region of Kap114 and H2A-H2B.1PDBexperimental model
29B23

9b23

9B23For Nap1.2PDBexperimental model
3AF-P53067-F1For C-terminal region of Kap1143AlphaFoldin silico modelA
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00213632
ELECTRON MICROSCOPYf_angle_d0.47318438
ELECTRON MICROSCOPYf_dihedral_angle_d4.781771
ELECTRON MICROSCOPYf_chiral_restr0.0352137
ELECTRON MICROSCOPYf_plane_restr0.0032370

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