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- PDB-6vz4: Cryo-EM structure of Sth1-Arp7-Arp9-Rtt102 bound to the nucleosom... -

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Basic information

Entry
Database: PDB / ID: 6vz4
TitleCryo-EM structure of Sth1-Arp7-Arp9-Rtt102 bound to the nucleosome in ADP Beryllium Fluoride state
Components
  • (DNA (185-MER)) x 2
  • Actin-like protein ARP9
  • Actin-related protein 7
  • Histone H2A
  • Histone H2B
  • Histone H3
  • Histone H4
  • Nuclear protein STH1/NPS1
  • Regulator of Ty1 transposition protein 102
KeywordsMOTOR PROTEIN / Chromatin remodeling / Nucleosome / Gene Regulation
Function / homology
Function and homology information


RHO GTPases activate IQGAPs / RHO GTPases Activate WASPs and WAVEs / Regulation of actin dynamics for phagocytic cup formation / chromatin remodeling at centromere / Platelet degranulation / DNA translocase activity / RSC-type complex / SWI/SNF complex / nucleosome disassembly / ATP-dependent chromatin remodeler activity ...RHO GTPases activate IQGAPs / RHO GTPases Activate WASPs and WAVEs / Regulation of actin dynamics for phagocytic cup formation / chromatin remodeling at centromere / Platelet degranulation / DNA translocase activity / RSC-type complex / SWI/SNF complex / nucleosome disassembly / ATP-dependent chromatin remodeler activity / NuA4 histone acetyltransferase complex / chromosome, centromeric region / ATP-dependent activity, acting on DNA / cytoskeleton organization / histone reader activity / meiotic cell cycle / chromosome segregation / helicase activity / transcription elongation by RNA polymerase II / lysine-acetylated histone binding / base-excision repair / structural constituent of chromatin / nucleosome / double-strand break repair / nucleosome assembly / chromatin organization / DNA helicase / chromatin remodeling / protein heterodimerization activity / chromatin binding / regulation of DNA-templated transcription / chromatin / regulation of transcription by RNA polymerase II / structural molecule activity / positive regulation of transcription by RNA polymerase II / ATP hydrolysis activity / DNA binding / ATP binding / nucleus
Similarity search - Function
Transcription regulator protein Rtt102 / Rtt102p-like transcription regulator protein / Snf2-ATP coupling, chromatin remodelling complex / Snf2, ATP coupling domain / Snf2-ATP coupling, chromatin remodelling complex / HSA domain / Helicase/SANT-associated domain / HSA domain profile. / : / SNF2-like, N-terminal domain superfamily ...Transcription regulator protein Rtt102 / Rtt102p-like transcription regulator protein / Snf2-ATP coupling, chromatin remodelling complex / Snf2, ATP coupling domain / Snf2-ATP coupling, chromatin remodelling complex / HSA domain / Helicase/SANT-associated domain / HSA domain profile. / : / SNF2-like, N-terminal domain superfamily / SNF2, N-terminal / SNF2-related domain / Actin / Actin family / Actin / 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 H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / Histone H2A / Histone 2A / 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 / ATPase, nucleotide binding domain / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Histone-fold / Bromodomain, conserved site / Bromodomain signature. / Bromodomain / Bromodomain profile. / bromo domain / Bromodomain / Bromodomain-like superfamily / 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 / ADENOSINE-5'-TRIPHOSPHATE / BERYLLIUM TRIFLUORIDE ION / DNA / DNA (> 10) / DNA (> 100) / Histone H2B 1.1 / Histone H2A type 1 / Nuclear protein STH1/NPS1 / Regulator of Ty1 transposition protein 102 ...ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / BERYLLIUM TRIFLUORIDE ION / DNA / DNA (> 10) / DNA (> 100) / Histone H2B 1.1 / Histone H2A type 1 / Nuclear protein STH1/NPS1 / Regulator of Ty1 transposition protein 102 / Histone H4 / Actin-like protein ARP9 / Actin-related protein 7 / Histone H2A / Histone H2B / Histone H3
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
synthetic construct (others)
Saccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsLeschziner, A.E. / Baker, R.W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R01GM092895-08 United States
CitationJournal: Nat Struct Mol Biol / Year: 2021
Title: Structural insights into assembly and function of the RSC chromatin remodeling complex.
Authors: Richard W Baker / Janice M Reimer / Peter J Carman / Bengi Turegun / Tsutomu Arakawa / Roberto Dominguez / Andres E Leschziner /
Abstract: SWI/SNF chromatin remodelers modify the position and spacing of nucleosomes and, in humans, are linked to cancer. To provide insights into the assembly and regulation of this protein family, we ...SWI/SNF chromatin remodelers modify the position and spacing of nucleosomes and, in humans, are linked to cancer. To provide insights into the assembly and regulation of this protein family, we focused on a subcomplex of the Saccharomyces cerevisiae RSC comprising its ATPase (Sth1), the essential actin-related proteins (ARPs) Arp7 and Arp9 and the ARP-binding protein Rtt102. Cryo-EM and biochemical analyses of this subcomplex shows that ARP binding induces a helical conformation in the helicase-SANT-associated (HSA) domain of Sth1. Surprisingly, the ARP module is rotated 120° relative to the full RSC about a pivot point previously identified as a regulatory hub in Sth1, suggesting that large conformational changes are part of Sth1 regulation and RSC assembly. We also show that a conserved interaction between Sth1 and the nucleosome acidic patch enhances remodeling. As some cancer-associated mutations dysregulate rather than inactivate SWI/SNF remodelers, our insights into RSC complex regulation advance a mechanistic understanding of chromatin remodeling in disease states.
History
DepositionFeb 27, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 2, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 23, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jan 27, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
Revision 1.3Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Histone H3
B: Histone H4
C: Histone H2A
D: Histone H2B
E: Histone H3
F: Histone H4
G: Histone H2A
H: Histone H2B
I: DNA (185-MER)
J: DNA (185-MER)
K: Nuclear protein STH1/NPS1
L: Actin-related protein 7
M: Actin-like protein ARP9
N: Regulator of Ty1 transposition protein 102
hetero molecules


Theoretical massNumber of molelcules
Total (without water)444,62518
Polymers443,60114
Non-polymers1,0254
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: cross-linking, Complex assembly was performed using the GRAFIX crosslinking protocol.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area69570 Å2
ΔGint-469 kcal/mol
Surface area140100 Å2
Number of models10

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Components

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Protein , 8 types, 12 molecules AEBFCGDHKLMN

#1: Protein Histone H3


Mass: 15407.075 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: hist1h3g, h3c8, H3l / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21 Rosetta / References: UniProt: Q92133
#2: Protein Histone H4


Mass: 11394.426 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 (bacteria) / Strain (production host): BL21 Rosetta / References: UniProt: P62799
#3: Protein Histone H2A


Mass: 14109.436 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: hist1h2aj, LOC494591, XELAEV_18003602mg / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21 Rosetta / References: UniProt: Q6AZJ8, UniProt: P06897*PLUS
#4: Protein Histone H2B


Mass: 13810.048 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: H2B, XELAEV_18028547mg / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21 Rosetta / References: UniProt: Q92130, UniProt: P02281*PLUS
#7: Protein Nuclear protein STH1/NPS1 / ATP-dependent helicase STH1 / Chromatin structure-remodeling complex protein STH1 / SNF2 homolog


Mass: 95105.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: STH1, NPS1, YIL126W / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21 Rosetta / References: UniProt: P32597, DNA helicase
#8: Protein Actin-related protein 7 / Actin-like protein ARP7 / Chromatin structure-remodeling complex protein ARP7 / SWI/SNF complex component ARP7


Mass: 53863.016 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: ARP7, SWP61, YPR034W, YP9367.14 / Production host: Escherichia coli (E. coli) / References: UniProt: Q12406
#9: Protein Actin-like protein ARP9 / Chromatin structure-remodeling complex protein ARP9 / SWI/SNF complex component ARP9


Mass: 53131.930 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: ARP9 / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21 Rosetta / References: UniProt: Q05123
#10: Protein Regulator of Ty1 transposition protein 102


Mass: 17817.615 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: RTT102, YGR275W, G9378 / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21 Rosetta / References: UniProt: P53330

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain DNA (185-MER)


Mass: 56840.203 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli) / Strain (production host): DH5 alpha
#6: DNA chain DNA (185-MER)


Mass: 57400.555 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli) / Strain (production host): DH5 alpha

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Non-polymers , 4 types, 4 molecules

#11: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#12: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#13: Chemical ChemComp-BEF / BERYLLIUM TRIFLUORIDE ION


Mass: 66.007 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: BeF3
#14: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM

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Details

Has ligand of interestN

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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
IDNameTypeEntity IDParent-IDSource
1cryo-EM structure of Sth1-Arp7-Arp9-Rtt102 bound to the nucleosome in ADP Beryllium Fluoride stateCOMPLEX#1-#100RECOMBINANT
2Histone H3, Histone H4, Histone H2A, Histone H2BCOMPLEX#1-#41RECOMBINANT
3DNACOMPLEX#5-#61RECOMBINANT
4Nuclear protein STH1/NPS1, Actin-related protein 7, Actin-like protein ARP9, Regulator of Ty1 transposition protein 102COMPLEX#7-#101RECOMBINANT
Molecular weightValue: 0.44 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
14Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)559292
22Xenopus laevis (African clawed frog)8355
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
14Escherichia coli (E. coli)562BL21 Rosetta
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample was crosslinked using the GRAFIX protocol
Specimen supportDetails: Glow discharge for 30 seconds at 25 mAmp / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 4 second blot time, blot force 20

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 36000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 7 sec. / Electron dose: 53 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 45

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Processing

EM software
IDNameVersionCategory
2Leginon3.3image acquisition
4CTFFIND4CTF correction
7UCSF Chimeramodel fitting
12RELION33D reconstruction
13Rosettamodel refinement
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 293940 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: Initial model docking was done in Chimera. Phenix.real_space_refine was used to refine the nucleosome model. Sth1-Arp7-Arp9-Rtt102 were refined in Rosetta and the top ten models were deposited.
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
15Z3U

5z3u

15Z3U1PDBexperimental model
24I6M

4i6m

14I6M2PDBexperimental model
35TGC

5tgc

15TGC3PDBexperimental model

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