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Yorodumi- EMDB-8140: 3.87 Angstrom structure of the nucleosome core particle obtained ... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-8140 | |||||||||
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Title | 3.87 Angstrom structure of the nucleosome core particle obtained by phase-plate cryo-EM | |||||||||
Map data | None | |||||||||
Sample |
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Biological species | Xenopus laevis (African clawed frog) / synthetic construct (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.87 Å | |||||||||
Authors | Chua EYD / Vogirala VK / Inian O / Wong ASW / Plitzko JM / Danev R / Sandin S | |||||||||
Funding support | Singapore, 1 items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_8140.map.gz | 14.6 MB | EMDB map data format | |
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Header (meta data) | emd-8140-v30.xml emd-8140.xml | 18.6 KB 18.6 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_8140_fsc.xml | 5.7 KB | Display | FSC data file |
Images | emd_8140.png | 21.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-8140 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-8140 | HTTPS FTP |
-Validation report
Summary document | emd_8140_validation.pdf.gz | 78.4 KB | Display | EMDB validaton report |
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Full document | emd_8140_full_validation.pdf.gz | 77.5 KB | Display | |
Data in XML | emd_8140_validation.xml.gz | 494 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-8140 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-8140 | HTTPS FTP |
-Related structure data
Similar structure data |
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-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_8140.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | None | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.38 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Nucleosome core particle with 145 base pairs of the Widom 601 seq...
Entire | Name: Nucleosome core particle with 145 base pairs of the Widom 601 sequence. |
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Components |
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-Supramolecule #1: Nucleosome core particle with 145 base pairs of the Widom 601 seq...
Supramolecule | Name: Nucleosome core particle with 145 base pairs of the Widom 601 sequence. type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Xenopus laevis (African clawed frog) |
Molecular weight | Experimental: 200 KDa |
-Supramolecule #2: Widom 601 DNA, 145 base pairs
Supramolecule | Name: Widom 601 DNA, 145 base pairs / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Experimental: 90 KDa |
-Supramolecule #3: Histone octamer
Supramolecule | Name: Histone octamer / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#5 |
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Source (natural) | Organism: Xenopus laevis (African clawed frog) |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant plasmid: pUC57 |
Molecular weight | Experimental: 110 KDa |
-Macromolecule #1: Widom 601 DNA, 145 base pairs
Macromolecule | Name: Widom 601 DNA, 145 base pairs / type: dna / ID: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Sequence | String: ATCAGAATCC CGGTGCCGAG GCCGCTCAAT TGGTCGTAGA CAGCTCTAGC ACCGCTTAAA CGCACGTACG CGCTGTCCCC CGCGTTTTAA CCGCCAAGGG GATTACTCCC TAGTCTCCAG GCACGTGTCA GATATATACA TCGAT |
-Macromolecule #2: Histone 2A
Macromolecule | Name: Histone 2A / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Xenopus laevis (African clawed frog) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: SGRGKQGGKT RAKAKTRSSR AGLQFPVGRV HRLLRKGNYA ERVGAGAPVY LAAVLEYLTA EILELAGNAA RDNKKTRIIP RHLQLAVRND EELNKLLGRV TIAQGGVLPN IQSVLLPKKT ESSKSKSK |
-Macromolecule #3: Histone 2B
Macromolecule | Name: Histone 2B / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Xenopus laevis (African clawed frog) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: PEPAKSAPAP KKGSKKAVTK TQKKDGKKRR KTRKESYAIY VYKVLKQVHP DTGISSKAMS IMNSFVNDVF ERIAGEASRL AHYNKRSTIT SREIQTAVRL LLPGELAKHA VSEGTKAVTK YTSAK |
-Macromolecule #4: Histone 3
Macromolecule | Name: Histone 3 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO |
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Source (natural) | Organism: Xenopus laevis (African clawed frog) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: ARTKQTARKS TGGKAPRKQL ATKAARKSAP ATGGVKKPHR YRPGTVALRE IRRYQKSTEL LIRKLPFQRL VREIAQDFKT DLRFQSSAVM ALQEASEAYL VALFEDTNLC AIHAKRVTIM PKDIQLARRI RGERA |
-Macromolecule #5: Histone 4
Macromolecule | Name: Histone 4 / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO |
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Source (natural) | Organism: Xenopus laevis (African clawed frog) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: SGRGKGGKGL GKGGAKRHRK VLRDNIQGIT KPAIRRLARR GGVKRISGLI YEETRGVLKV FLENVIRDAV TYTEHAKRKT VTAMDVVYAL KRQGRTLYGF GG |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 2.5 mg/mL | ||||||||||||
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Buffer | pH: 7.5 Component:
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Grid | Model: Quantifoil R2/2 or R1/4 / Material: COPPER/RHODIUM / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR | ||||||||||||
Vitrification | Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK III / Details: 3-4 seconds blotting time. | ||||||||||||
Details | This sample was monodispersed |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Phase plate: VOLTA PHASE PLATE |
Image recording | Film 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 beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model | PDB ID: |
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Refinement | Space: REAL / Protocol: OTHER / Overall B value: 78.96 |