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- PDB-7ou0: The structure of MutS bound to two molecules of ADP-Vanadate -

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Basic information

Entry
Database: PDB / ID: 7ou0
TitleThe structure of MutS bound to two molecules of ADP-Vanadate
ComponentsDNA mismatch repair protein MutS
KeywordsDNA BINDING PROTEIN / DNA mismatch repair protein
Function / homology
Function and homology information


adenine/cytosine mispair binding / MutS complex / mismatch repair complex / regulation of DNA recombination / mismatched DNA binding / DNA binding, bending / ATP-dependent DNA damage sensor activity / mismatch repair / ADP binding / damaged DNA binding ...adenine/cytosine mispair binding / MutS complex / mismatch repair complex / regulation of DNA recombination / mismatched DNA binding / DNA binding, bending / ATP-dependent DNA damage sensor activity / mismatch repair / ADP binding / damaged DNA binding / DNA damage response / ATP hydrolysis activity / ATP binding / identical protein binding / cytosol
Similarity search - Function
DNA mismatch repair protein MutS / DNA mismatch repair protein MutS/MSH / DNA mismatch repair protein MutS-like, N-terminal / DNA mismatch repair protein MutS, connector domain / DNA mismatch repair protein MutS, clamp / DNA mismatch repair protein MutS, N-terminal / MutS, connector domain superfamily / MutS domain I / MutS domain II / MutS family domain IV ...DNA mismatch repair protein MutS / DNA mismatch repair protein MutS/MSH / DNA mismatch repair protein MutS-like, N-terminal / DNA mismatch repair protein MutS, connector domain / DNA mismatch repair protein MutS, clamp / DNA mismatch repair protein MutS, N-terminal / MutS, connector domain superfamily / MutS domain I / MutS domain II / MutS family domain IV / MutS domain III / DNA mismatch repair MutS family / DNA mismatch repair protein MutS, C-terminal / DNA mismatch repair protein MutS, core / DNA mismatch repair protein MutS, core domain superfamily / MutS domain V / DNA mismatch repair proteins mutS family signature. / DNA-binding domain of DNA mismatch repair MUTS family / ATPase domain of DNA mismatch repair MUTS family / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / VANADATE ION / DNA mismatch repair protein MutS
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsLamers, M.H. / Borsellini, A. / Friedhoff, P. / Kunetsky, V.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
Marie Sklodowska-Curie Actions, FragNET ITNEuropean Union
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Cryogenic electron microscopy structures reveal how ATP and DNA binding in MutS coordinates sequential steps of DNA mismatch repair.
Authors: Alessandro Borsellini / Vladislav Kunetsky / Peter Friedhoff / Meindert H Lamers /
Abstract: DNA mismatch repair detects and corrects mismatches introduced during DNA replication. The protein MutS scans for mismatches and coordinates the repair cascade. During this process, MutS undergoes ...DNA mismatch repair detects and corrects mismatches introduced during DNA replication. The protein MutS scans for mismatches and coordinates the repair cascade. During this process, MutS undergoes multiple conformational changes in response to ATP binding, hydrolysis and release, but how ATP induces the various MutS conformations is incompletely understood. Here we present four cryogenic electron microscopy structures of Escherichia coli MutS at sequential stages of the ATP hydrolysis cycle that reveal how ATP binding and hydrolysis induce closing and opening of the MutS dimer, respectively. Biophysical analysis demonstrates how DNA binding modulates the ATPase cycle by prevention of hydrolysis during scanning and mismatch binding, while preventing ADP release in the sliding clamp state. Nucleotide release is achieved when MutS encounters single-stranded DNA that is produced during removal of the daughter strand. The combination of ATP binding and hydrolysis and its modulation by DNA enables MutS to adopt the different conformations needed to coordinate the sequential steps of the mismatch repair cascade.
History
DepositionJun 10, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 12, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 26, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jul 17, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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 / _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
B: DNA mismatch repair protein MutS
A: DNA mismatch repair protein MutS
hetero molecules


Theoretical massNumber of molelcules
Total (without water)181,9998
Polymers180,8662
Non-polymers1,1336
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9640 Å2
ΔGint-89 kcal/mol
Surface area52690 Å2
MethodPISA

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Components

#1: Protein DNA mismatch repair protein MutS


Mass: 90433.234 Da / Num. of mol.: 2 / Mutation: R840E
Source method: isolated from a genetically manipulated source
Details: ADP Vanadate ATP hydrolysis transition state analogue.
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: mutS, fdv, b2733, JW2703 / Production host: Escherichia coli (E. coli) / References: UniProt: P23909
#2: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#3: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#4: Chemical ChemComp-VO4 / VANADATE ION


Mass: 114.939 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: VO4 / Feature type: SUBJECT OF INVESTIGATION
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: DNA mismatch repair protein MutS / Type: ORGANELLE OR CELLULAR COMPONENT / Details: MutS bound to two molecules of AMPPNP / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.19 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 8.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMtris buffer2-Amino-2-hydroxymethyl-propane-1,3-diol1
2150 mMsodium chlorideNaCl1
32 mMDTT1,4-Dithiothreitol1
45 mMmagnesium chlorideMgCl21
50.01 %Tween20C58H114O261
SpecimenConc.: 0.95 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA PLUNGER / Cryogen name: ETHANE / Humidity: 76 % / Chamber temperature: 4 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 FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 57 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 4835
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 50 / Used frames/image: 1-50

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4Gctf1.06CTF correction
7Coot0.9.2model fitting
9REFMACrefmac5model refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13REFMAC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 496416
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 81316 / Symmetry type: POINT
Atomic model buildingB value: 70 / Protocol: FLEXIBLE FIT / Space: RECIPROCAL
Atomic model buildingPDB-ID: 1E3M

1e3m


Accession code: 1E3M / Source name: PDB / Type: experimental model

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