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- EMDB-8140: 3.87 Angstrom structure of the nucleosome core particle obtained ... -

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Basic information

Entry
Database: EMDB / ID: EMD-8140
Title3.87 Angstrom structure of the nucleosome core particle obtained by phase-plate cryo-EM
Map dataNone
Sample
  • Complex: Nucleosome core particle with 145 base pairs of the Widom 601 sequence.
    • Complex: Widom 601 DNA, 145 base pairs
      • DNA: Widom 601 DNA, 145 base pairs
    • Complex: Histone octamer
      • Protein or peptide: Histone 2A
      • Protein or peptide: Histone 2B
      • Protein or peptide: Histone 3
      • Protein or peptide: Histone 4
Biological speciesXenopus laevis (African clawed frog) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.87 Å
AuthorsChua EYD / Vogirala VK / Inian O / Wong ASW / Plitzko JM / Danev R / Sandin S
Funding support Singapore, 1 items
OrganizationGrant numberCountry
Ministry of Education Singapore
CitationJournal: Nucleic Acids Res / Year: 2016
Title: 3.9 Å structure of the nucleosome core particle determined by phase-plate cryo-EM.
Authors: Eugene Y D Chua / Vinod K Vogirala / Oviya Inian / Andrew S W Wong / Lars Nordenskiöld / Juergen M Plitzko / Radostin Danev / Sara Sandin /
Abstract: The Volta phase plate is a recently developed electron cryo-microscopy (cryo-EM) device that enables contrast enhancement of biological samples. Here we have evaluated the potential of combining ...The Volta phase plate is a recently developed electron cryo-microscopy (cryo-EM) device that enables contrast enhancement of biological samples. Here we have evaluated the potential of combining phase-plate imaging and single particle analysis to determine the structure of a small protein-DNA complex. To test the method, we made use of a 200 kDa Nucleosome Core Particle (NCP) reconstituted with 601 DNA for which a high-resolution X-ray crystal structure is known. We find that the phase plate provides a significant contrast enhancement that permits individual NCPs and DNA to be clearly identified in amorphous ice. The refined structure from 26,060 particles has an overall resolution of 3.9 Å and the density map exhibits structural features consistent with the estimated resolution, including clear density for amino acid side chains and DNA features such as the phosphate backbone. Our results demonstrate that phase-plate cryo-EM promises to become an important method to determine novel near-atomic resolution structures of small and challenging samples, such as nucleosomes in complex with nucleosome-binding factors.
History
DepositionMar 31, 2016-
Header (metadata) releaseJun 8, 2016-
Map releaseSep 7, 2016-
UpdateNov 18, 2020-
Current statusNov 18, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.027
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.027
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8140.png

Downloads & links

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Map

FileDownload / File: emd_8140.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.38 Å/pix.
x 160 pix.
= 220.8 Å		1.38 Å/pix.
x 160 pix.
= 220.8 Å		1.38 Å/pix.
x 160 pix.
= 220.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.38 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.027 / Movie #1: 0.027
Minimum - Maximum-0.056169275 - 0.11585718
Average (Standard dev.)0.00018258722 (±0.0066085593)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 220.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.381.381.38
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z220.800220.800220.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ256256256
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.0560.1160.000

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Supplemental data

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Sample components

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Entire : Nucleosome core particle with 145 base pairs of the Widom 601 seq...

EntireName: Nucleosome core particle with 145 base pairs of the Widom 601 sequence.
Components
  • Complex: Nucleosome core particle with 145 base pairs of the Widom 601 sequence.
    • Complex: Widom 601 DNA, 145 base pairs
      • DNA: Widom 601 DNA, 145 base pairs
    • Complex: Histone octamer
      • Protein or peptide: Histone 2A
      • Protein or peptide: Histone 2B
      • Protein or peptide: Histone 3
      • Protein or peptide: Histone 4

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Supramolecule #1: Nucleosome core particle with 145 base pairs of the Widom 601 seq...

SupramoleculeName: Nucleosome core particle with 145 base pairs of the Widom 601 sequence.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightExperimental: 200 KDa

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Supramolecule #2: Widom 601 DNA, 145 base pairs

SupramoleculeName: Widom 601 DNA, 145 base pairs / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: synthetic construct (others)
Molecular weightExperimental: 90 KDa

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Supramolecule #3: Histone octamer

SupramoleculeName: Histone octamer / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#5
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pUC57
Molecular weightExperimental: 110 KDa

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Macromolecule #1: Widom 601 DNA, 145 base pairs

MacromoleculeName: Widom 601 DNA, 145 base pairs / type: dna / ID: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
ATCAGAATCC CGGTGCCGAG GCCGCTCAAT TGGTCGTAGA CAGCTCTAGC ACCGCTTAAA CGCACGTACG CGCTGTCCCC CGCGTTTTAA CCGCCAAGGG GATTACTCCC TAGTCTCCAG GCACGTGTCA GATATATACA TCGAT

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Macromolecule #2: Histone 2A

MacromoleculeName: Histone 2A / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SGRGKQGGKT RAKAKTRSSR AGLQFPVGRV HRLLRKGNYA ERVGAGAPVY LAAVLEYLTA EILELAGNAA RDNKKTRIIP RHLQLAVRND EELNKLLGRV TIAQGGVLPN IQSVLLPKKT ESSKSKSK

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Macromolecule #3: Histone 2B

MacromoleculeName: Histone 2B / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
PEPAKSAPAP KKGSKKAVTK TQKKDGKKRR KTRKESYAIY VYKVLKQVHP DTGISSKAMS IMNSFVNDVF ERIAGEASRL AHYNKRSTIT SREIQTAVRL LLPGELAKHA VSEGTKAVTK YTSAK

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Macromolecule #4: Histone 3

MacromoleculeName: Histone 3 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
ARTKQTARKS TGGKAPRKQL ATKAARKSAP ATGGVKKPHR YRPGTVALRE IRRYQKSTEL LIRKLPFQRL VREIAQDFKT DLRFQSSAVM ALQEASEAYL VALFEDTNLC AIHAKRVTIM PKDIQLARRI RGERA

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Macromolecule #5: Histone 4

MacromoleculeName: Histone 4 / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SGRGKGGKGL GKGGAKRHRK VLRDNIQGIT KPAIRRLARR GGVKRISGLI YEETRGVLKV FLENVIRDAV TYTEHAKRKT VTAMDVVYAL KRQGRTLYGF GG

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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

Concentration2.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
1.0 mMC10H16N2O8EDTA
1.0 mMC4H10O2S2DTT
GridModel: Quantifoil R2/2 or R1/4 / Material: COPPER/RHODIUM / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK III / Details: 3-4 seconds blotting time.
DetailsThis sample was monodispersed

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-20 / Average exposure time: 10.0 sec. / Average electron dose: 31.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsThe recorded micrographs were clipped to remove dust and the movie frames were motion-corrected.
Particle selectionNumber selected: 38409
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3lz0


Details: Initial model was made from 3LZ0 and low pass filtered to 80 Angstroms.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.87 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Details: RELION 1.4 was used for the final reconstruction. / Number images used: 26060
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.4)
Final 3D classificationNumber classes: 4 / Avg.num./class: 7000 / Software - Name: RELION (ver. 1.4)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3lz0

RefinementSpace: REAL / Protocol: OTHER / Overall B value: 78.96

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