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- EMDB-44095: Cryo-EM structure of Nap1 core -

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Basic information

Entry
Database: EMDB / ID: EMD-44095
TitleCryo-EM structure of Nap1 core
Map dataMain map
Sample
  • Complex: Complex of Kap114 bound to Nap1 and histone H2A-H2B
    • Protein or peptide: NAP1 isoform 1
KeywordsHistone / Chaperone
Function / homology:
Function and homology information
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 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: 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, 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_44095.png

Downloads & links

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Map

FileDownload / File: emd_44095.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 280 pix.
= 232.4 Å		0.83 Å/pix.
x 280 pix.
= 232.4 Å		0.83 Å/pix.
x 280 pix.
= 232.4 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: 0.06
Minimum - Maximum-0.21768728 - 0.37529027
Average (Standard dev.)0.000150311 (±0.009153976)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 232.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half B

Fileemd_44095_half_map_1.map
AnnotationHalf B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half A

Fileemd_44095_half_map_2.map
AnnotationHalf A
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 Nap1 and histone H2A-H2B

EntireName: Complex of Kap114 bound to Nap1 and histone H2A-H2B
Components
  • Complex: Complex of Kap114 bound to Nap1 and histone H2A-H2B
    • Protein or peptide: NAP1 isoform 1

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Supramolecule #1: Complex of Kap114 bound to Nap1 and histone H2A-H2B

SupramoleculeName: Complex of Kap114 bound to Nap1 and histone H2A-H2B / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: crosslinked sample.
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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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 / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
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: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 230210
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Details: AlphaFold Multimer was used to generate initial model.
RefinementProtocol: OTHER
Output model

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

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