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- PDB-8oo9: CryoEM Structure INO80core Hexasome complex ATPase-DNA refinement... -

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Basic information

Entry
Database: PDB / ID: 8oo9
TitleCryoEM Structure INO80core Hexasome complex ATPase-DNA refinement state1
Components
  • Chromatin-remodeling ATPase INO80
  • DNA strand 1
  • DNA strand 2
KeywordsDNA BINDING PROTEIN / ATP-dependent chromatin remodeler
Function / homologyADENOSINE-5'-DIPHOSPHATE / TETRAFLUOROALUMINATE ION / DNA / DNA (> 10) / DNA (> 100)
Function and homology information
Biological speciesThermochaetoides thermophila (fungus)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsZhang, M. / Jungblut, A. / Hoffmann, T. / Eustermann, S.
Funding support Germany, European Union, 4items
OrganizationGrant numberCountry
EIPOD fellowship under Marie Sklodowska-Curie Actions COFUND847543 Germany
European Research Council (ERC)833613European Union
German Research Foundation (DFG)CRC136 Germany
German Research Foundation (DFG)CRC1064 Germany
Citation
Journal: Science / Year: 2023
Title: Hexasome-INO80 complex reveals structural basis of noncanonical nucleosome remodeling.
Authors: Min Zhang / Anna Jungblut / Franziska Kunert / Luis Hauptmann / Thomas Hoffmann / Olga Kolesnikova / Felix Metzner / Manuela Moldt / Felix Weis / Frank DiMaio / Karl-Peter Hopfner / Sebastian Eustermann /
Abstract: Loss of H2A-H2B histone dimers is a hallmark of actively transcribed genes, but how the cellular machinery functions in the context of noncanonical nucleosomal particles remains largely elusive. In ...Loss of H2A-H2B histone dimers is a hallmark of actively transcribed genes, but how the cellular machinery functions in the context of noncanonical nucleosomal particles remains largely elusive. In this work, we report the structural mechanism for adenosine 5'-triphosphate-dependent chromatin remodeling of hexasomes by the INO80 complex. We show how INO80 recognizes noncanonical DNA and histone features of hexasomes that emerge from the loss of H2A-H2B. A large structural rearrangement switches the catalytic core of INO80 into a distinct, spin-rotated mode of remodeling while its nuclear actin module remains tethered to long stretches of unwrapped linker DNA. Direct sensing of an exposed H3-H4 histone interface activates INO80, independently of the H2A-H2B acidic patch. Our findings reveal how the loss of H2A-H2B grants remodelers access to a different, yet unexplored layer of energy-driven chromatin regulation.
#1: Journal: Acta Crystallogr D Struct Biol / Year: 2018
Title: Real-space refinement in PHENIX for cryo-EM and crystallography.
Authors: Pavel V Afonine / Billy K Poon / Randy J Read / Oleg V Sobolev / Thomas C Terwilliger / Alexandre Urzhumtsev / Paul D Adams /
Abstract: This article describes the implementation of real-space refinement in the phenix.real_space_refine program from the PHENIX suite. The use of a simplified refinement target function enables very fast ...This article describes the implementation of real-space refinement in the phenix.real_space_refine program from the PHENIX suite. The use of a simplified refinement target function enables very fast calculation, which in turn makes it possible to identify optimal data-restraint weights as part of routine refinements with little runtime cost. Refinement of atomic models against low-resolution data benefits from the inclusion of as much additional information as is available. In addition to standard restraints on covalent geometry, phenix.real_space_refine makes use of extra information such as secondary-structure and rotamer-specific restraints, as well as restraints or constraints on internal molecular symmetry. The re-refinement of 385 cryo-EM-derived models available in the Protein Data Bank at resolutions of 6 Å or better shows significant improvement of the models and of the fit of these models to the target maps.
#2: Journal: Acta Crystallogr D Biol Crystallogr / Year: 2010
Title: Features and development of Coot.
Authors: P Emsley / B Lohkamp / W G Scott / K Cowtan /
Abstract: Coot is a molecular-graphics application for model building and validation of biological macromolecules. The program displays electron-density maps and atomic models and allows model manipulations ...Coot is a molecular-graphics application for model building and validation of biological macromolecules. The program displays electron-density maps and atomic models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are 'discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behaviour (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallographic community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.
History
DepositionApr 4, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 26, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 2, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Jul 24, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin
Item: _em_3d_fitting_list.initial_refinement_model_id / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
G: Chromatin-remodeling ATPase INO80
K: DNA strand 1
L: DNA strand 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)271,0136
Polymers270,4583
Non-polymers5543
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5340 Å2
ΔGint-53 kcal/mol
Surface area25100 Å2

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Components

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Protein , 1 types, 1 molecules G

#1: Protein Chromatin-remodeling ATPase INO80


Mass: 130887.656 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Ino80 N-terminal truncation C-terminal 2xFlagTag / Source: (gene. exp.) Thermochaetoides thermophila (fungus) / Gene: TT172_LOCUS3790 / Production host: Trichoplusia ni (cabbage looper)

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DNA chain , 2 types, 2 molecules KL

#2: DNA chain DNA strand 1


Mass: 69527.195 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA strand 2


Mass: 70043.562 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 3 molecules

#4: Chemical ChemComp-ADP / ADENOSINE-5'-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
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-ALF / TETRAFLUOROALUMINATE ION


Mass: 102.975 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: AlF4 / 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

ComponentName: INO80 core module in complex with hexasome / Type: COMPLEX
Details: 11-subunit ct INO80 contains two modules (core and Arp8 module) Each module was picked and analyzed separately The core module + hexasome has an overall weight of 0.861MDa The 11-subunit ct ...Details: 11-subunit ct INO80 contains two modules (core and Arp8 module) Each module was picked and analyzed separately The core module + hexasome has an overall weight of 0.861MDa The 11-subunit ct INO80 + hexasome has an overall weight of 1.1MDa Ino80, Ies2, Ies6, Ies4,Arp6, Rvb1, Rvb2, Arp8, Arp4, Actin, Taf14 Hexasome DNA, 2xH3, 2xH4, H2A, H2B
Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.861 MDa / Experimental value: NO
Source (natural)Organism: Thermochaetoides thermophila (fungus)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.5
Details: 30mM HEPES, pH7.5 50mM NaCl 0.25mM CaCl2 0.25mM DTT 2mM ADP 3.3mM MgCl2 10mM NaF 2mM AlCl3 0.05% octyl-beta-glucoside
SpecimenConc.: 0.88 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 11-subunit ctINO80 reconstituted with hexasome
Specimen supportDetails: 10% Oxygene 90% Argone / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K
Details: wait time of 5s, blot force at 3, and a blot time of 2s with Whatman blotting paper (Cytiva, CAT No. 10311807)

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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 / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50.36 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 15384

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4.1.14CTF correction
7ISOLDE1.4model fitting
8Coot0.9.7model fitting
10RELION4initial Euler assignment
13RELION43D reconstruction
20PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2137460
Details: Particles were initially picked by WARP to generate an initial model, which was subsequently used for the 3D template picking
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 72400 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
13I62

3i62

13I621PDBexperimental model
28AV6

8av6

18AV62PDBexperimental model

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