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- PDB-9b1d: Cryo-EM structure of native SWR1 bound to DNA (composite structure) -

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Basic information

Entry
Database: PDB / ID: 9b1d
TitleCryo-EM structure of native SWR1 bound to DNA (composite structure)
Components
  • (DNA (147-MER)) x 2
  • (RuvB-like protein ...) x 2
  • (Vacuolar protein sorting-associated protein ...) x 2
  • Actin-like protein ARP6
  • Helicase SWR1
KeywordsGENE REGULATION / Chromatin Remodeler / Snf2 family ATPase / histone exchange / H2A.Z
Function / homology
Function and homology information


TTT Hsp90 cochaperone complex / R2TP complex / protein targeting to vacuole / Swr1 complex / Ino80 complex / box C/D snoRNP assembly / 3'-5' DNA helicase activity / NuA4 histone acetyltransferase complex / nucleosome binding / nuclear periphery ...TTT Hsp90 cochaperone complex / R2TP complex / protein targeting to vacuole / Swr1 complex / Ino80 complex / box C/D snoRNP assembly / 3'-5' DNA helicase activity / NuA4 histone acetyltransferase complex / nucleosome binding / nuclear periphery / DNA helicase activity / rRNA processing / 5'-3' DNA helicase activity / histone binding / DNA helicase / protein stabilization / chromatin remodeling / DNA repair / regulation of DNA-templated transcription / regulation of transcription by RNA polymerase II / chromatin / ATP hydrolysis activity / zinc ion binding / ATP binding / nucleus / cytoplasm
Similarity search - Function
Vps71/ZNHIT1 / Zinc finger HIT-type profile. / Vps72/YL1, N-terminal / YL1 nuclear protein / Zinc finger, HIT-type / Vps72/YL1, C-terminal / YL1 nuclear protein C-terminal domain / YL1 nuclear protein C-terminal domain / RuvB-like / RuvB-like, AAA-lid domain ...Vps71/ZNHIT1 / Zinc finger HIT-type profile. / Vps72/YL1, N-terminal / YL1 nuclear protein / Zinc finger, HIT-type / Vps72/YL1, C-terminal / YL1 nuclear protein C-terminal domain / YL1 nuclear protein C-terminal domain / RuvB-like / RuvB-like, AAA-lid domain / RuvBL1/2, DNA/RNA binding domain / TIP49 P-loop domain / TIP49 AAA-lid domain / TIP49, P-loop domain / Actin / Actin family / Actin / ATPase, nucleotide binding domain / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / DNA (> 100) / Vacuolar protein sorting-associated protein 72 / Vacuolar protein sorting-associated protein 71 / RuvB-like protein 2 / Actin-like protein ARP6
Similarity search - Component
Biological speciesSaccharomyces cerevisiae W303 (yeast)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsLouder, R.K. / Park, G. / Wu, C.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM149291 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM125831 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32GM133151 United States
CitationJournal: Cell / Year: 2024
Title: Molecular basis of global promoter sensing and nucleosome capture by the SWR1 chromatin remodeler.
Authors: Robert K Louder / Giho Park / Ziyang Ye / Justin S Cha / Anne M Gardner / Qin Lei / Anand Ranjan / Eva Höllmüller / Florian Stengel / B Franklin Pugh / Carl Wu /
Abstract: The SWR1 chromatin remodeling complex is recruited to +1 nucleosomes downstream of transcription start sites of eukaryotic promoters, where it exchanges histone H2A for the specialized variant H2A.Z. ...The SWR1 chromatin remodeling complex is recruited to +1 nucleosomes downstream of transcription start sites of eukaryotic promoters, where it exchanges histone H2A for the specialized variant H2A.Z. Here, we use cryoelectron microscopy (cryo-EM) to resolve the structural basis of the SWR1 interaction with free DNA, revealing a distinct open conformation of the Swr1 ATPase that enables sliding from accessible DNA to nucleosomes. A complete structural model of the SWR1-nucleosome complex illustrates critical roles for Swc2 and Swc3 subunits in oriented nucleosome engagement by SWR1. Moreover, an extended DNA-binding α helix within the Swc3 subunit enables sensing of nucleosome linker length and is essential for SWR1-promoter-specific recruitment and activity. The previously unresolved N-SWR1 subcomplex forms a flexible extended structure, enabling multivalent recognition of acetylated histone tails by reader domains to further direct SWR1 toward the +1 nucleosome. Altogether, our findings provide a generalizable mechanism for promoter-specific targeting of chromatin and transcription complexes.
History
DepositionMar 13, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 9, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update
Revision 1.2Dec 11, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Helicase SWR1
B: Vacuolar protein sorting-associated protein 72
C: Actin-like protein ARP6
D: Vacuolar protein sorting-associated protein 71
E: RuvB-like protein 1
F: RuvB-like protein 2
G: RuvB-like protein 1
H: RuvB-like protein 2
I: RuvB-like protein 1
J: RuvB-like protein 2
Y: DNA (147-MER)
Z: DNA (147-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)875,07830
Polymers870,95112
Non-polymers4,12718
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 2 molecules AC

#1: Protein Helicase SWR1


Mass: 178058.172 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Fusion protein with C-terminal 3xFLAG / Source: (natural) Saccharomyces cerevisiae W303 (yeast) / Variant: W1558-4C / References: DNA helicase
#3: Protein Actin-like protein ARP6


Mass: 50100.582 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae W303 (yeast) / Variant: W1558-4C / References: UniProt: Q12509

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Vacuolar protein sorting-associated protein ... , 2 types, 2 molecules BD

#2: Protein Vacuolar protein sorting-associated protein 72 / SWR complex protein 2


Mass: 90709.008 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae W303 (yeast) / Variant: W1558-4C / References: UniProt: Q03388
#4: Protein Vacuolar protein sorting-associated protein 71 / SWR complex protein 6


Mass: 32073.479 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae W303 (yeast) / Variant: W1558-4C / References: UniProt: Q03433

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RuvB-like protein ... , 2 types, 6 molecules EGIFHJ

#5: Protein RuvB-like protein 1


Mass: 91413.242 Da / Num. of mol.: 3 / Source method: isolated from a natural source
Details: Fusion protein with C-terminal maltose-binding protein
Source: (natural) Saccharomyces cerevisiae W303 (yeast) / Variant: W1558-4C / References: DNA helicase
#6: Protein RuvB-like protein 2 / RUVBL2 / TIP49-homology protein 2 / TIP49b homolog


Mass: 51673.488 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae W303 (yeast) / Variant: W1558-4C / References: UniProt: Q12464, DNA helicase

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DNA chain , 2 types, 2 molecules YZ

#7: DNA chain DNA (147-MER)


Mass: 45145.754 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#8: DNA chain DNA (147-MER)


Mass: 45604.047 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 4 types, 18 molecules

#9: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#10: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg
#11: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#12: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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Details

Has ligand of interestN
Has protein modificationN

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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
1Native SWR1 bound to DNA.COMPLEXEndogenously purified yeast SWR1 complex bound to 147-bp dsDNA fragment in the presence of ATPgS.#1-#80NATURAL
2Native SWR1 complexCOMPLEXEndogenously purified yeast SWR1 complex#1-#61NATURAL
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
111.28 MDaNO
211.19 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
21Saccharomyces cerevisiae (brewer's yeast)4932W303
32Saccharomyces cerevisiae (brewer's yeast)4932W303
Buffer solutionpH: 7.6
Details: 20 mM HEPES pH 7.6, 0.2 mM EDTA, 2 mM MgCl2, 100 mM NaCl, 0.01% IGEPAL CA-630, 3.5% glycerol, and 0.25 mM TCEP, 1 mM ATP-gamma-s, 0.05% glutaraldehyde.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
20.2 mMEDTA1
32 mMmagnesium chlorideMgCl21
4100 mMsodium chlorideNaCl1
50.01 %IGEPAL CA-6301
63.5 %glycerol1
70.25 mMTCEP1
81 mMATP-gamma-s1
90.05 %glutaraldehyde1
SpecimenConc.: 0.125 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 6 second blot time and blot force of 12.

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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 FIELD / Calibrated magnification: 48543 X / Nominal defocus max: 3600 nm / Nominal defocus min: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4 sec. / Electron dose: 54 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5379
Details: Each micrograph was fractionated into 64 frames within a 4 second exposure.

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Processing

EM software
IDNameVersionCategory
1RELION3.1.3particle selection
2SerialEM3.7.6image acquisition
4GctfCTF correction
7ISOLDE1.7model fitting
9RELION3.1.3initial Euler assignment
10RELION3.1.3final Euler assignment
11RELION3.1.3classification
12RELION3.1.33D reconstruction
13PHENIX1.21_5207model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 386667
Details: 2D classification was used to remove graphene edges.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 101246 / Algorithm: FOURIER SPACE
Details: Refined maps were post-processed using DeepEMhancer
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 84 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16GEJ

6gej

16GEJ1PDBexperimental model
21AlphaFoldin silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 83.88 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003233757
ELECTRON MICROSCOPYf_angle_d0.490845847
ELECTRON MICROSCOPYf_chiral_restr0.0415344
ELECTRON MICROSCOPYf_plane_restr0.00345653
ELECTRON MICROSCOPYf_dihedral_angle_d14.27995012

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