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- PDB-8v4y: Cryo-EM structure of singly-bound SNF2h-nucleosome complex with S... -

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Basic information

Entry
Database: PDB / ID: 8v4y
TitleCryo-EM structure of singly-bound SNF2h-nucleosome complex with SNF2h at inactive SHL2 (conformation 1)
Components
  • (Widom 601 DNA (147-mer) with 60 base pairs flanking DNA ...) x 2
  • Histone H2A type 1
  • Histone H2B
  • Histone H3.2
  • Histone H4
  • SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5
KeywordsDNA BINDING PROTEIN/DNA / nucleosome / chromatin remodeler / ISWI / SNF2h / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


histone octamer slider activity / RSF complex / ACF complex / WICH complex / negative regulation of mitotic chromosome condensation / CHRAC / NoRC complex / : / : / B-WICH complex ...histone octamer slider activity / RSF complex / ACF complex / WICH complex / negative regulation of mitotic chromosome condensation / CHRAC / NoRC complex / : / : / B-WICH complex / NURF complex / rDNA heterochromatin formation / chromatin silencing complex / : / : / ATP-dependent chromatin remodeler activity / positive regulation of transcription by RNA polymerase III / negative regulation of transcription by RNA polymerase I / positive regulation of transcription by RNA polymerase I / regulation of DNA replication / nucleosome binding / ATP-dependent activity, acting on DNA / pericentric heterochromatin / heterochromatin formation / condensed chromosome / Deposition of new CENPA-containing nucleosomes at the centromere / cellular response to leukemia inhibitory factor / positive regulation of DNA replication / helicase activity / DNA-templated transcription initiation / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / NoRC negatively regulates rRNA expression / B-WICH complex positively regulates rRNA expression / fibrillar center / nucleosome assembly / structural constituent of chromatin / nucleosome / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / site of double-strand break / chromatin organization / chromatin remodeling / protein heterodimerization activity / DNA repair / DNA damage response / nucleolus / regulation of DNA-templated transcription / regulation of transcription by RNA polymerase II / ATP hydrolysis activity / positive regulation of transcription by RNA polymerase II / DNA binding / nucleoplasm / ATP binding / nucleus
Similarity search - Function
ISWI, HAND domain / SLIDE domain / ISWI, HAND domain superfamily / HAND / SLIDE / SANT domain profile. / SANT domain / : / SNF2-like, N-terminal domain superfamily / SNF2, N-terminal ...ISWI, HAND domain / SLIDE domain / ISWI, HAND domain superfamily / HAND / SLIDE / SANT domain profile. / SANT domain / : / SNF2-like, N-terminal domain superfamily / SNF2, N-terminal / SNF2-related domain / SANT SWI3, ADA2, N-CoR and TFIIIB'' DNA-binding domains / SANT/Myb domain / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone H2A / Histone 2A / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H3 signature 1. / Helicase conserved C-terminal domain / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Homeobox-like domain superfamily / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Histone-fold / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / BERYLLIUM TRIFLUORIDE ION / DNA / DNA (> 10) / DNA (> 100) / SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 / Histone H2B 1.1 / Histone H2A type 1 / Histone H4 / Histone H3.2
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
Homo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsChio, U.S. / Palovcak, E. / Armache, J.P. / Narlikar, G.J. / Cheng, Y.
Funding support United States, 4items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM140847 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM127020 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM137463 United States
CitationJournal: Nat Commun / Year: 2024
Title: Functionalized graphene-oxide grids enable high-resolution cryo-EM structures of the SNF2h-nucleosome complex without crosslinking.
Authors: Un Seng Chio / Eugene Palovcak / Anton A A Smith / Henriette Autzen / Elise N Muñoz / Zanlin Yu / Feng Wang / David A Agard / Jean-Paul Armache / Geeta J Narlikar / Yifan Cheng /
Abstract: Single-particle cryo-EM is widely used to determine enzyme-nucleosome complex structures. However, cryo-EM sample preparation remains challenging and inconsistent due to complex denaturation at the ...Single-particle cryo-EM is widely used to determine enzyme-nucleosome complex structures. However, cryo-EM sample preparation remains challenging and inconsistent due to complex denaturation at the air-water interface (AWI). Here, to address this issue, we develop graphene-oxide-coated EM grids functionalized with either single-stranded DNA (ssDNA) or thiol-poly(acrylic acid-co-styrene) (TAASTY) co-polymer. These grids protect complexes between the chromatin remodeler SNF2h and nucleosomes from the AWI and facilitate collection of high-quality micrographs of intact SNF2h-nucleosome complexes in the absence of crosslinking. The data yields maps ranging from 2.3 to 3 Å in resolution. 3D variability analysis reveals nucleotide-state linked conformational changes in SNF2h bound to a nucleosome. In addition, the analysis provides structural evidence for asymmetric coordination between two SNF2h protomers acting on the same nucleosome. We envision these grids will enable similar detailed structural analyses for other enzyme-nucleosome complexes and possibly other protein-nucleic acid complexes in general.
History
DepositionNov 29, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 20, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Histone H3.2
B: Histone H4
C: Histone H2A type 1
D: Histone H2B
E: Histone H3.2
F: Histone H4
G: Histone H2A type 1
H: Histone H2B
I: Widom 601 DNA (147-mer) with 60 base pairs flanking DNA (reverse strand)
J: Widom 601 DNA (147-mer) with 60 base pairs flanking DNA (forward strand)
W: SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)358,51014
Polymers357,99311
Non-polymers5183
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 5 types, 9 molecules AEBFCGDHW

#1: Protein Histone H3.2


Mass: 15271.863 Da / Num. of mol.: 2 / Mutation: G102A, C110A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): pLysS / References: UniProt: P84233
#2: Protein Histone H4 /


Mass: 11263.231 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): pLysS / References: UniProt: P62799
#3: Protein Histone H2A type 1


Mass: 13978.241 Da / Num. of mol.: 2 / Mutation: G99R, A123S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): pLysS / References: UniProt: P06897
#4: Protein Histone H2B / / H2B1.1


Mass: 13524.752 Da / Num. of mol.: 2 / Mutation: S29T
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): pLysS / References: UniProt: P02281
#7: Protein SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 / SWI/SNF-related matrix-associated actin-dependent regulator of chromatin A5 / Sucrose nonfermenting ...SWI/SNF-related matrix-associated actin-dependent regulator of chromatin A5 / Sucrose nonfermenting protein 2 homolog / hSNF2H


Mass: 122089.336 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SMARCA5, SNF2H, WCRF135 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta
References: UniProt: O60264, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement

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Widom 601 DNA (147-mer) with 60 base pairs flanking DNA ... , 2 types, 2 molecules IJ

#5: DNA chain Widom 601 DNA (147-mer) with 60 base pairs flanking DNA (reverse strand)


Mass: 64200.895 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: DNA chain Widom 601 DNA (147-mer) with 60 base pairs flanking DNA (forward strand)


Mass: 63626.480 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 3 molecules

#8: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#9: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#10: Chemical ChemComp-BEF / BERYLLIUM TRIFLUORIDE ION


Mass: 66.007 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: BeF3 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1ATP-dependent chromatin remodeler SNF2h in complex with nucleosomeCOMPLEXHuman SNF2h recombinantly expressed and purified from E. coli. Nucleosome reconstituted with Xenopus laevis histones.#1-#70MULTIPLE SOURCES
2Nucleosome with Xenopus laevis histonesCOMPLEXHistones recombinantly expressed in E. coli. DNA generated by PCR.#1-#61MULTIPLE SOURCES
3SNF2hCOMPLEX#71RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.3 MDaNO
220.2 MDaNO
330.1 MDaNO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: Rosetta(DE3)
Buffer solutionpH: 7.5
Details: 12.5 mM HEPES-KOH, pH 7.5, 60 mM KCl, 5 mM MgCl2, 2 mM ADP, 2 mM BeSO4, 10 mM NaF, 1.5% glycerol
Buffer component
IDConc.NameFormulaBuffer-ID
112.5 mMN-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid1
260 mMpotassium chlorideKCl1
35 mMmagnesium chlorideMgCl21
42 mMadenosine diphosphate1
52 mMberyllium sulfateBeSO41
610 mMsodium fluorideNaF1
71.5 %glycerol1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 100 nM nucleosome with 500 nM SNF2h
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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 FIELDBright-field microscopy / Nominal defocus max: 1800 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Image recordingElectron dose: 66 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategoryDetails
1cryoSPARCparticle selectionTemplate picker
2SerialEMimage acquisition
4cryoSPARCCTF correctionPatch CTF estimation
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstructionNon-uniform refinement
CTF correctionDetails: cryoSPARC Patch CTF estimation / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 8509125
Details: Particles picked using cryoSPARC's template picker with templates generated from a nucleosome.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 146702
Details: cryoSPARC non-uniform refinement. Gold-standard FSC determined by cryoSPARC using auto mask generation and tightening.
Symmetry type: POINT

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