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- PDB-8vhi: NPM2-H1.8 isolated from Xenopus egg extract -

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ID or keywords:

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Basic information

Entry
Database: PDB / ID: 8vhi
TitleNPM2-H1.8 isolated from Xenopus egg extract
ComponentsNucleoplasmin isoform X1
KeywordsCHAPERONE / H1 / Xenopus egg extract / MagIC-cryo-EM / Histone chaperone
Function / homology
Function and homology information


single fertilization / positive regulation of DNA replication / histone binding / chromatin remodeling / chromatin binding / nucleolus / RNA binding / nucleoplasm / cytoplasm
Similarity search - Function
Nucleoplasmin core domain / Nucleoplasmin core domain superfamily / Nucleoplasmin/nucleophosmin domain / Nucleoplasmin family
Similarity search - Domain/homology
Biological speciesXenopus laevis (African clawed frog)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5 Å
AuthorsArimura, Y. / Funabiki, H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
Citation
Journal: Elife / Year: 2025
Title: MagIC-Cryo-EM, structural determination on magnetic beads for scarce macromolecules in heterogeneous samples.
Authors: Yasuhiro Arimura / Hide A Konishi / Hironori Funabiki /
Abstract: Cryo-EM single-particle analyses typically require target macromolecule concentration at 0.05~5.0 mg/ml, which is often difficult to achieve. Here, we devise netic solation and oncentration (MagIC)- ...Cryo-EM single-particle analyses typically require target macromolecule concentration at 0.05~5.0 mg/ml, which is often difficult to achieve. Here, we devise netic solation and oncentration (MagIC)-cryo-EM, a technique enabling direct structural analysis of targets captured on magnetic beads, thereby reducing the targets' concentration requirement to <0.0005 mg/mL. Adapting MagIC-cryo-EM to a Chromatin Immunoprecipitation protocol, we characterized structural variations of the linker histone H1.8-associated nucleosomes that were isolated from interphase and metaphase chromosomes in egg extract. Combining plicated election o xclude ubbish particles (DuSTER), a particle curation method that excludes low signal-to-noise ratio particles, we also resolved the 3D cryo-EM structures of nucleoplasmin NPM2 co-isolated with the linker histone H1.8 and revealed distinct open and closed structural variants. Our study demonstrates the utility of MagIC-cryo-EM for structural analysis of scarce macromolecules in heterogeneous samples and provides structural insights into the cell cycle-regulation of H1.8 association to nucleosomes.
#1: Journal: Elife / Year: 2024
Title: MagIC-Cryo-EM: Structural determination on magnetic beads for scarce macromolecules in heterogeneous samples
Authors: Arimura, Y. / Konishi, H.A. / Funabiki, H.
History
DepositionJan 2, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 11, 2024Provider: repository / Type: Initial release
Revision 1.0Dec 11, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Dec 11, 2024Data content type: Additional map / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Dec 11, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Dec 11, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 11, 2024Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 11, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 11, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jun 4, 2025Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / em_software
Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Nucleoplasmin isoform X1
B: Nucleoplasmin isoform X1
C: Nucleoplasmin isoform X1
D: Nucleoplasmin isoform X1
E: Nucleoplasmin isoform X1


Theoretical massNumber of molelcules
Total (without water)109,7475
Polymers109,7475
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Nucleoplasmin isoform X1


Mass: 21949.467 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: A0A1L8H245
Has protein modificationN

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: NPM2 complexed with H1.8 / Type: CELL / Entity ID: all / Source: NATURAL
Source (natural)Organism: Xenopus laevis (African clawed frog)
Buffer solutionpH: 7.4
Details: 10mM HEPES-KOH (pH 7.4), 30 mM KCl, 1 mM EGTA, 10 ng/microL leupeptin, 10 ng/microL pepstatin, 10 ng/microL chymostatin, 1 mM sodium butyrate, 1 mM beta-glycerophosphate, trehalose, and 1,6-hexanediol
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: The grid was incubated on the 40 x 20 mm N52 neodymium disc magnets for 5 minutes and vitrified using the Vitrobot Mark IV (FEI) with a 2-second blotting time at room temperature under 100% humidity conditions.
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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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 defocus max: 3500 nm / Nominal defocus min: 1500 nm
Image recording
IDImaging-IDElectron dose (e/Å2)Film or detector model
1145.3GATAN K3 BIOQUANTUM (6k x 4k)
2151.92GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
8PHENIXmodel refinement
10cryoSPARC4.4initial Euler assignment
11cryoSPARC4.4final Euler assignment
13cryoSPARC4.43D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 92428 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0064370
ELECTRON MICROSCOPYf_angle_d0.6825915
ELECTRON MICROSCOPYf_dihedral_angle_d5.419570
ELECTRON MICROSCOPYf_chiral_restr0.052675
ELECTRON MICROSCOPYf_plane_restr0.009770

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