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- PDB-9b23: Cryo-EM structure of Nap1 core -

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Basic information

Entry
Database: PDB / ID: 9b23
TitleCryo-EM structure of Nap1 core
ComponentsNAP1 isoform 1
KeywordsCHAPERONE / Histone
Function / homology:
Function and homology information
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 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
Citation
Journal: 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.
#1: Journal: bioRxiv / Year: 2024
Title: Nap1 and Kap114 co-chaperone H2A-H2B and facilitate targeted histone release in the nucleus.
Authors: Ho Yee Joyce Fung / Ashley B Neisman / Natalia E Bernardes / Jenny Jiou / Yuh Min Chook
Abstract: Core histones are synthesized and processed in the cytoplasm before transport into the nucleus for assembly into nucleosomes; however, they must also be chaperoned as free histones are toxic. The ...Core histones are synthesized and processed in the cytoplasm before transport into the nucleus for assembly into nucleosomes; however, they must also be chaperoned as free histones are toxic. The importin Kap114 binds and transports histone H2A-H2B into the yeast nucleus, where RanGTP facilitates H2A-H2B release. Kap114 and H2A-H2B also bind the Nap1 histone chaperone, which is found in both the cytoplasm and the nucleus, but how Nap1 and Kap114 cooperate in H2A-H2B processing and nucleosome assembly has been unclear. To understand these mechanisms, we used biochemical and structural analyses to reveal how Nap1, Kap114, H2A-H2B and RanGTP interact. We show that Kap114, H2A-H2B and a Nap1 dimer (Nap1 ) assemble into a 1:1:1 ternary complex. Cryogenic electron microscopy revealed two distinct Kap114/Nap1 /H2A-H2B structures: one of H2A-H2B sandwiched between Nap1 and Kap114, and another in which Nap1 bound to the Kap114·H2A-H2B complex without contacting H2A-H2B. Another Nap1 ·H2A-H2B·Kap114·Ran structure reveals the nuclear complex. Mutagenesis revealed shared critical interfaces in all three structures. Consistent with structural findings, DNA competition experiments demonstrated that Kap114 and Nap1 together chaperone H2A-H2B better than either protein alone. When RanGTP is present, Kap114's chaperoning activity diminishes. However, the presence of Nap1 within the Nap1 ·H2A-H2B·Kap114·Ran quaternary complex restores its ability to chaperone H2A-H2B. This complex effectively deposits H2A-H2B into nucleosomes. Together, these findings suggest that Kap114 and Nap12 provide a sheltered path from cytoplasm to nucleus, facilitating the transfer of H2A-H2B from Kap114 to Nap1 , ultimately directing its specific deposition into nucleosomes.
History
DepositionMar 14, 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


Theoretical massNumber of molelcules
Total (without water)72,3652
Polymers72,3652
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: Known to be homodimer.
TypeNameSymmetry operationNumber
identity operation1_5551

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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
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: Complex of Kap114 bound to Nap1 and histone H2A-H2B / Type: COMPLEX / Details: crosslinked sample. / 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 mesh size: 300 divisions/in. / 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: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 230210 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Atomic model buildingDetails: AlphaFold Multimer was used to generate initial model.
Source name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0034764
ELECTRON MICROSCOPYf_angle_d0.5626433
ELECTRON MICROSCOPYf_dihedral_angle_d5.992604
ELECTRON MICROSCOPYf_chiral_restr0.039690
ELECTRON MICROSCOPYf_plane_restr0.006851

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