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- EMDB-43004: Cryo-EM structure of doubly-bound SNF2h-nucleosome complex (confo... -

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Basic information

Entry
Database: EMDB / ID: EMD-43004
TitleCryo-EM structure of doubly-bound SNF2h-nucleosome complex (conformation 1)
Map datadoubly-bound SNF2h nucleosome complex variability component 2A (cryoSPARC local refinement)
Sample
  • Complex: ATP-dependent chromatin remodeler SNF2h in complex with nucleosome
    • Complex: Nucleosome with Xenopus laevis histones
      • Protein or peptide: Histone H3.2
      • Protein or peptide: Histone H4
      • Protein or peptide: Histone H2A type 1
      • Protein or peptide: Histone H2B 1.1
      • DNA: Widom 601 DNA (147-mer) with 60 base pairs flanking DNA (forward strand)
      • DNA: Widom 601 DNA (147-mer) with 60 base pairs flanking DNA (reverse strand)
    • Complex: SNF2h
      • Protein or peptide: SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5
Keywordsnucleosome / chromatin remodeler / ISWI / SNF2h / DNA BINDING PROTEIN
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 / Helicase conserved C-terminal domain / Histone H3 signature 1. / 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
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 speciesHomo sapiens (human) / Xenopus laevis (African clawed frog) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsChio US / Palovcak E / Armache JP / Narlikar GJ / Cheng Y
Funding support United States, 4 items
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
DepositionDec 4, 2023-
Header (metadata) releaseMar 20, 2024-
Map releaseMar 20, 2024-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43004.png

Downloads & links

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Map

FileDownload / File: emd_43004.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationdoubly-bound SNF2h nucleosome complex variability component 2A (cryoSPARC local refinement)
Voxel sizeX=Y=Z: 0.8468 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-0.6205537 - 1.704843
Average (Standard dev.)0.00073082093 (±0.053413253)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 325.1712 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A

Fileemd_43004_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_43004_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ATP-dependent chromatin remodeler SNF2h in complex with nucleosome

EntireName: ATP-dependent chromatin remodeler SNF2h in complex with nucleosome
Components
  • Complex: ATP-dependent chromatin remodeler SNF2h in complex with nucleosome
    • Complex: Nucleosome with Xenopus laevis histones
      • Protein or peptide: Histone H3.2
      • Protein or peptide: Histone H4
      • Protein or peptide: Histone H2A type 1
      • Protein or peptide: Histone H2B 1.1
      • DNA: Widom 601 DNA (147-mer) with 60 base pairs flanking DNA (forward strand)
      • DNA: Widom 601 DNA (147-mer) with 60 base pairs flanking DNA (reverse strand)
    • Complex: SNF2h
      • Protein or peptide: SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5

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Supramolecule #1: ATP-dependent chromatin remodeler SNF2h in complex with nucleosome

SupramoleculeName: ATP-dependent chromatin remodeler SNF2h in complex with nucleosome
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Details: Human SNF2h recombinantly expressed and purified from E. coli. Nucleosome reconstituted with Xenopus laevis histones.
Molecular weightTheoretical: 300 KDa

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Supramolecule #2: Nucleosome with Xenopus laevis histones

SupramoleculeName: Nucleosome with Xenopus laevis histones / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#6
Details: Histones recombinantly expressed in E. coli. DNA generated by PCR.
Molecular weightTheoretical: 200 KDa

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Supramolecule #3: SNF2h

SupramoleculeName: SNF2h / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #7
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 100 KDa

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Macromolecule #1: Histone H3.2

MacromoleculeName: Histone H3.2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
ARTKQTARKS TGGKAPRKQL ATKAARKSAP ATGGVKKPHR YRPGTVALRE IRRYQKSTE LLIRKLPFQR LVREIAQDFK TDLRFQSSAV MALQEASEAY LVALFEDTNL AAIHAKRVTI MPKDIQLAR RIRGERA

UniProtKB: Histone H3.2

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Macromolecule #2: Histone H4

MacromoleculeName: Histone H4 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
SGRGKGGKGL GKGGAKRHRK VLRDNIQGIT KPAIRRLARR GGVKRISGLI YEETRGVLK VFLENVIRDA VTYTEHAKRK TVTAMDVVYA LKRQGRTLYG FGG

UniProtKB: Histone H4

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Macromolecule #3: Histone H2A type 1

MacromoleculeName: Histone H2A type 1 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
SGRGKQGGKT RAKAKTRSSR AGLQFPVGRV HRLLRKGNYA ERVGAGAPVY LAAVLEYLT AEILELAGNA ARDNKKTRII PRHLQLAVRN DEELNKLLGR VTIAQGGVLP NIQSVLLPKK TESSKSAKS K

UniProtKB: Histone H2A type 1

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Macromolecule #4: Histone H2B 1.1

MacromoleculeName: Histone H2B 1.1 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
AKSAPAPKKG SKKAVTKTQK KDGKKRRKTR KESYAIYVYK VLKQVHPDTG ISSKAMSIM NSFVNDVFER IAGEASRLAH YNKRSTITSR EIQTAVRLLL PGELAKHAVS EGTKAVTKYT SAK

UniProtKB: Histone H2B 1.1

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Macromolecule #7: SWI/SNF-related matrix-associated actin-dependent regulator of ch...

MacromoleculeName: SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5
type: protein_or_peptide / ID: 7 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSSAAEPPPP PPPESAPSKP AASIASGGSN SSNKGGPEGV AAQAVASAAS AGPADAEMEE IFDDASPGKQ KEIQEPDPTY EEKMQTDRAN RFEYLLKQTE LFAHFIQPAA QKTPTSPLKM KPGRPRIKKD EKQNLLSVGD YRHRRTEQEE DEELLTESSK ATNVCTRFED ...String:
MSSAAEPPPP PPPESAPSKP AASIASGGSN SSNKGGPEGV AAQAVASAAS AGPADAEMEE IFDDASPGKQ KEIQEPDPTY EEKMQTDRAN RFEYLLKQTE LFAHFIQPAA QKTPTSPLKM KPGRPRIKKD EKQNLLSVGD YRHRRTEQEE DEELLTESSK ATNVCTRFED SPSYVKWGKL RDYQVRGLNW LISLYENGIN GILADEMGLG KTLQTISLLG YMKHYRNIPG PHMVLVPKST LHNWMSEFKR WVPTLRSVCL IGDKEQRAAF VRDVLLPGEW DVCVTSYEML IKEKSVFKKF NWRYLVIDEA HRIKNEKSKL SEIVREFKTT NRLLLTGTPL QNNLHELWSL LNFLLPDVFN SADDFDSWFD TNNCLGDQKL VERLHMVLRP FLLRRIKADV EKSLPPKKEV KIYVGLSKMQ REWYTRILMK DIDILNSAGK MDKMRLLNIL MQLRKCCNHP YLFDGAEPGP PYTTDMHLVT NSGKMVVLDK LLPKLKEQGS RVLIFSQMTR VLDILEDYCM WRNYEYCRLD GQTPHDERQD SINAYNEPNS TKFVFMLSTR AGGLGINLAT ADVVILYDSD WNPQVDLQAM DRAHRIGQTK TVRVFRFITD NTVEERIVER AEMKLRLDSI VIQQGRLVDQ NLNKIGKDEM LQMIRHGATH VFASKESEIT DEDIDGILER GAKKTAEMNE KLSKMGESSL RNFTMDTESS VYNFEGEDYR EKQKIAFTEW IEPPKRERKA NYAVDAYFRE ALRVSEPKAP KAPRPPKQPN VQDFQFFPPR LFELLEKEIL FYRKTIGYKV PRNPELPNAA QAQKEEQLKI DEAESLNDEE LEEKEKLLTQ GFTNWNKRDF NQFIKANEKW GRDDIENIAR EVEGKTPEEV IEYSAVFWER CNELQDIEKI MAQIERGEAR IQRRISIKKA LDTKIGRYKA PFHQLRISYG TNKGKNYTEE EDRFLICMLH KLGFDKENVY DELRQCIRNS PQFRFDWFLK SRTAMELQRR CNTLITLIER ENMELEEKEK AEKKKRGPKP STQKRKMDGA PDGRGRKKKL KL

UniProtKB: SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5

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Macromolecule #5: Widom 601 DNA (147-mer) with 60 base pairs flanking DNA (forward ...

MacromoleculeName: Widom 601 DNA (147-mer) with 60 base pairs flanking DNA (forward strand)
type: dna / ID: 5 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString: CTGGAGAATC CCGGTGCCGa gGCCGCTCAA TTGGTCGTAG ACAGCTCTAG CACCGCTTAA ACGCACGTAC GCGCTGTCCC CCGCGTTTTA ACCGCCAAGG GGATTACTCC CTAGTCTCCA GGCACGTGTC AGATATATAC ATCCTGTGCA TGTATTGAAC AGCGACCTTG ...String:
CTGGAGAATC CCGGTGCCGa gGCCGCTCAA TTGGTCGTAG ACAGCTCTAG CACCGCTTAA ACGCACGTAC GCGCTGTCCC CCGCGTTTTA ACCGCCAAGG GGATTACTCC CTAGTCTCCA GGCACGTGTC AGATATATAC ATCCTGTGCA TGTATTGAAC AGCGACCTTG CCGGTGCCAG TCGGATAGTG TTCCGAGCTC CCACTCT

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Macromolecule #6: Widom 601 DNA (147-mer) with 60 base pairs flanking DNA (reverse ...

MacromoleculeName: Widom 601 DNA (147-mer) with 60 base pairs flanking DNA (reverse strand)
type: dna / ID: 6 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString: AGAGTGGGAG CTCGGAACAC TATCCGACTG GCACCGGCAA GGTCGCTGTT CAATACATGC ACAGGATGTA TATATCTGAC ACGTGCCTG GAGACTAGGG AGTAATCCCC TTGGCGGTTA AAACGCGGGG GACAGCGCGT ACGTGCGTTT AAGCGGTGCT A GAGCTGTC ...String:
AGAGTGGGAG CTCGGAACAC TATCCGACTG GCACCGGCAA GGTCGCTGTT CAATACATGC ACAGGATGTA TATATCTGAC ACGTGCCTG GAGACTAGGG AGTAATCCCC TTGGCGGTTA AAACGCGGGG GACAGCGCGT ACGTGCGTTT AAGCGGTGCT A GAGCTGTC TACGACCAAT TGAGCGGCct CGGCACCGGG ATTCTCCAG

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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

BufferpH: 7.5
Component:
ConcentrationNameFormula
12.5 mMN-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
60.0 mMpotassium chlorideKCl
5.0 mMmagnesium chlorideMgCl2
2.0 mMadenosine diphosphate
2.0 mMberyllium sulfateBeSO4
10.0 mMsodium fluorideNaF
1.5 %glycerol

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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details100 nM nucleosome with 500 nM SNF2h

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 66.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 8509125
Details: Particles picked using cryoSPARC's template picker with templates generated from a nucleosome.
Startup modelType of model: OTHER / Details: cryoSPARC ab initio model
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Software - details: Local refinement
Details: cryoSPARC local refinement was used for the reconstruction. Gold standard FSC determined by cryoSPARC using auto FSC mask generation and tightening.
Number images used: 16330
FSC plot (resolution estimation)

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