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- PDB-6fhs: CryoEM Structure of INO80core -

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Basic information

Entry
Database: PDB / ID: 6fhs
TitleCryoEM Structure of INO80core
Components
  • (RuvB-like helicase) x 2
  • Arp5
  • Ino80
  • les2
  • les6
KeywordsDNA BINDING PROTEIN
Function / homology
Function and homology information


DASH complex / protein transport along microtubule to mitotic spindle pole body / mitotic sister chromatid biorientation / attachment of spindle microtubules to kinetochore / Ino80 complex / attachment of mitotic spindle microtubules to kinetochore / ATP-dependent activity, acting on DNA / helicase activity / mitotic spindle / kinetochore ...DASH complex / protein transport along microtubule to mitotic spindle pole body / mitotic sister chromatid biorientation / attachment of spindle microtubules to kinetochore / Ino80 complex / attachment of mitotic spindle microtubules to kinetochore / ATP-dependent activity, acting on DNA / helicase activity / mitotic spindle / kinetochore / chromatin organization / DNA helicase / chromatin remodeling / DNA repair / ATP hydrolysis activity / ATP binding / nucleus
Similarity search - Function
DASH complex subunit Dad4 / DASH complex subunit Dad4 / INO80 complex subunit B-like conserved region / INO80 complex, subunit Ies2 / INO80 complex, subunit Ies6 / PAPA-1-like conserved region / PAPA-1 / Vps72/YL1, C-terminal / YL1 nuclear protein C-terminal domain / YL1 nuclear protein C-terminal domain ...DASH complex subunit Dad4 / DASH complex subunit Dad4 / INO80 complex subunit B-like conserved region / INO80 complex, subunit Ies2 / INO80 complex, subunit Ies6 / PAPA-1-like conserved region / PAPA-1 / 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 / ADENOSINE-5'-TRIPHOSPHATE / INO80 complex subunit B-like conserved region domain-containing protein / RuvB-like helicase / RuvB-like helicase / Uncharacterized protein / Vps72/YL1 C-terminal domain-containing protein
Similarity search - Component
Biological speciesChaetomium thermophilum var. thermophilum DSM 1495 (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.754 Å
AuthorsEustermann, S. / Schall, K. / Kostrewa, D. / Strauss, M. / Hopfner, K.
Funding support Germany, 3items
OrganizationGrant numberCountry
European Molecular Biology OrganizationEMBO ALTF 1098-2012 Germany
European Research CouncilATMMACHINE Germany
German Research FoundationCRC1064, GRK1721 Germany
CitationJournal: Nature / Year: 2018
Title: Structural basis for ATP-dependent chromatin remodelling by the INO80 complex.
Authors: Sebastian Eustermann / Kevin Schall / Dirk Kostrewa / Kristina Lakomek / Mike Strauss / Manuela Moldt / Karl-Peter Hopfner /
Abstract: In the eukaryotic nucleus, DNA is packaged in the form of nucleosomes, each of which comprises about 147 base pairs of DNA wrapped around a histone protein octamer. The position and histone ...In the eukaryotic nucleus, DNA is packaged in the form of nucleosomes, each of which comprises about 147 base pairs of DNA wrapped around a histone protein octamer. The position and histone composition of nucleosomes is governed by ATP-dependent chromatin remodellers such as the 15-subunit INO80 complex . INO80 regulates gene expression, DNA repair and replication by sliding nucleosomes, the exchange of histone H2A.Z with H2A, and the positioning of + 1 and -1 nucleosomes at promoter DNA. The structures and mechanisms of these remodelling reactions are currently unknown. Here we report the cryo-electron microscopy structure of the evolutionarily conserved core of the INO80 complex from the fungus Chaetomium thermophilum bound to a nucleosome, at a global resolution of 4.3 Å and with major parts at 3.7 Å. The INO80 core cradles one entire gyre of the nucleosome through multivalent DNA and histone contacts. An Rvb1/Rvb2 AAA ATPase heterohexamer is an assembly scaffold for the complex and acts as a 'stator' for the motor and nucleosome-gripping subunits. The Swi2/Snf2 ATPase motor binds to nucleosomal DNA at superhelical location -6, unwraps approximately 15 base pairs, disrupts the H2A-DNA contacts and is poised to pump entry DNA into the nucleosome. Arp5 and Ies6 bind superhelical locations -2 and -3 to act as a counter grip for the motor, on the other side of the H2A-H2B dimer. The Arp5 insertion domain forms a grappler element that binds the nucleosome dyad, connects the Arp5 actin-fold and entry DNA over a distance of about 90 Å and packs against histone H2A-H2B near the 'acidic patch'. Our structure together with biochemical data suggests a unified mechanism for nucleosome sliding and histone editing by INO80. The motor is part of a macromolecular ratchet, persistently pumping entry DNA across the H2A-H2B dimer against the Arp5 grip until a large nucleosome translocation step occurs. The transient exposure of H2A-H2B by motor activity as well as differential recognition of H2A.Z and H2A may regulate histone exchange.
History
DepositionJan 15, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 25, 2018Provider: repository / Type: Initial release
Revision 1.1May 2, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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Assembly

Deposited unit
A: RuvB-like helicase
B: RuvB-like helicase
C: RuvB-like helicase
D: RuvB-like helicase
E: RuvB-like helicase
F: RuvB-like helicase
G: Ino80
H: les2
I: les6
J: Arp5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)605,75317
Polymers602,68310
Non-polymers3,0707
Water0
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area89490 Å2
ΔGint-417 kcal/mol
Surface area132940 Å2
MethodPISA

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Components

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Protein , 6 types, 10 molecules ABCDEFGHIJ

#1: Protein RuvB-like helicase


Mass: 50451.848 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)
Strain: DSM 1495 / CBS 144.50 / IMI 039719 / Cell line: High Five Cells (BTI-TN-5B1-4) / Gene: CTHT_0006820
Cell line (production host): High Five Cells (BTI-TN-5B1-4)
Production host: Trichoplusia ni (cabbage looper) / References: UniProt: G0RYI5, DNA helicase
#2: Protein RuvB-like helicase


Mass: 53212.746 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)
Strain: DSM 1495 / CBS 144.50 / IMI 039719 / Gene: CTHT_0006170 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: G0RYC2, DNA helicase
#3: Protein Ino80


Mass: 127529.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: C-terminal double FlagTag
Source: (gene. exp.) Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)
Production host: Trichoplusia ni (cabbage looper)
#4: Protein les2


Mass: 53258.902 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)
Strain: DSM 1495 / CBS 144.50 / IMI 039719 / Gene: CTHT_0004910 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: G0RY01
#5: Protein les6


Mass: 23127.523 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)
Strain: DSM 1495 / CBS 144.50 / IMI 039719 / Gene: CTHT_0032670 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: G0S590
#6: Protein Arp5


Mass: 87773.086 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)
Strain: DSM 1495 / CBS 144.50 / IMI 039719 / Gene: CTHT_0032660 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: G0S589

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Non-polymers , 2 types, 7 molecules

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


Mass: 427.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#8: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine 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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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

ComponentName: INO80core masked reconstruction / Type: COMPLEX
Details: 3xRvb1, 3x Rvb2, Ino80insert, Arp5, Ies2, Ies6 other parts of complex are masked out
Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightValue: 0.6 MDa / Experimental value: NO
Source (natural)Organism: Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 8
Details: 20 mM HEPES pH 8, 60 mM KCl, 0.5% glycerol, 0.25 mM CaCl2, 20 uM ZnCl2, 0.25 mM DTT, 0.05% Octyl-beta-glucoside
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Monodisperse sample: INO80core complex reconstituted with nucleosomal substrate was purified by gelfiltration. Addition of nucleotides or crosslinking was not required.
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 281 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: 3500 nm / Calibrated defocus min: 1300 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 59.6 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3992
Details: Images were collected in movie mode with 4 frames per second and 10s total aquisition
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
1RELION2.1.1bparticle selection
2SerialEM3.6image acquisition
4CTFFIND4CTF correction
7Coot0.8.9model fitting
9cryoSPARCinitial Euler assignment
10RELION2.1.1bfinal Euler assignment
12RELION2.1.1b3D reconstruction
13PHENIX1.12model refinement
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 1 Å / B: 1 Å / C: 1 Å / Space group name: P1 / Space group num: 1
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 251692
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.754 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 144278 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00527842
ELECTRON MICROSCOPYf_angle_d0.81937648
ELECTRON MICROSCOPYf_dihedral_angle_d7.82317044
ELECTRON MICROSCOPYf_chiral_restr0.0534279
ELECTRON MICROSCOPYf_plane_restr0.0074878

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