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- PDB-7ai7: MutS in Intermediate state -

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Basic information

Entry
Database: PDB / ID: 7ai7
TitleMutS in Intermediate state
Components
  • DNA (5'-D(P*CP*TP*TP*AP*GP*CP*TP*TP*AP*GP*GP*AP*TP*C)-3')
  • DNA (5'-D(P*GP*AP*TP*CP*CP*TP*AP*AP*CP*TP*AP*AP*G)-3')
  • DNA mismatch repair protein MutS
KeywordsDNA BINDING PROTEIN / DNA Mismatch Repair MutS
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 / DNA / DNA (> 10) / DNA mismatch repair protein MutS
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsFernandez-Leiro, R. / Bhairosing-Kok, D. / Sixma, T.K. / Lamers, M.H.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)U105197143 United Kingdom
CitationJournal: Nat Struct Mol Biol / Year: 2021
Title: The selection process of licensing a DNA mismatch for repair.
Authors: Rafael Fernandez-Leiro / Doreth Bhairosing-Kok / Vladislav Kunetsky / Charlie Laffeber / Herrie H Winterwerp / Flora Groothuizen / Alexander Fish / Joyce H G Lebbink / Peter Friedhoff / ...Authors: Rafael Fernandez-Leiro / Doreth Bhairosing-Kok / Vladislav Kunetsky / Charlie Laffeber / Herrie H Winterwerp / Flora Groothuizen / Alexander Fish / Joyce H G Lebbink / Peter Friedhoff / Titia K Sixma / Meindert H Lamers /
Abstract: DNA mismatch repair detects and removes mismatches from DNA by a conserved mechanism, reducing the error rate of DNA replication by 100- to 1,000-fold. In this process, MutS homologs scan DNA, ...DNA mismatch repair detects and removes mismatches from DNA by a conserved mechanism, reducing the error rate of DNA replication by 100- to 1,000-fold. In this process, MutS homologs scan DNA, recognize mismatches and initiate repair. How the MutS homologs selectively license repair of a mismatch among millions of matched base pairs is not understood. Here we present four cryo-EM structures of Escherichia coli MutS that provide snapshots, from scanning homoduplex DNA to mismatch binding and MutL activation via an intermediate state. During scanning, the homoduplex DNA forms a steric block that prevents MutS from transitioning into the MutL-bound clamp state, which can only be overcome through kinking of the DNA at a mismatch. Structural asymmetry in all four structures indicates a division of labor between the two MutS monomers. Together, these structures reveal how a small conformational change from the homoduplex- to heteroduplex-bound MutS acts as a licensing step that triggers a dramatic conformational change that enables MutL binding and initiation of the repair cascade.
History
DepositionSep 26, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 31, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 21, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Apr 28, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-11793
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Structure viewerMolecule:
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Assembly

Deposited unit
A: DNA mismatch repair protein MutS
B: DNA mismatch repair protein MutS
C: DNA (5'-D(P*GP*AP*TP*CP*CP*TP*AP*AP*CP*TP*AP*AP*G)-3')
D: DNA (5'-D(P*CP*TP*TP*AP*GP*CP*TP*TP*AP*GP*GP*AP*TP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)200,2106
Polymers199,3554
Non-polymers8542
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area13250 Å2
ΔGint-87 kcal/mol
Surface area61820 Å2
MethodPISA

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Components

#1: Protein DNA mismatch repair protein MutS /


Mass: 95397.898 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
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: DNA chain DNA (5'-D(P*GP*AP*TP*CP*CP*TP*AP*AP*CP*TP*AP*AP*G)-3')


Mass: 4288.818 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(P*CP*TP*TP*AP*GP*CP*TP*TP*AP*GP*GP*AP*TP*C)-3')


Mass: 4270.789 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
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
1MutS loaded on matched DNA in the presence of ATPCOMPLEX#1-#30MULTIPLE SOURCES
2DNA mismatch repair protein MutSCOMPLEX#11RECOMBINANT
3DNACOMPLEX#2-#31RECOMBINANT
Molecular weightValue: 0.190 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (strain K12) (bacteria)83333
23synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23synthetic construct (others)32630
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
120 mMHepes1
250 mMPotasium Glutamate1
32 mMTEMEDTetramethylethylenediamine1
41 mMATPAdenosine triphosphate1
50.006 w/vTween-201
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Protein sample was purified over a gel filtration column and mixed with DNA+ATP prior to grid preparation
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot for 3 seconds before plunging

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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 magnification: 105000 X / Calibrated defocus min: 500 nm / Calibrated defocus max: 4000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 12 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2351
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4GctfCTF correction
7Coot0.91model fitting
9REFMAC5model refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 104000
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 67679 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: RECIPROCAL
Details: Initial Jelly Body refinement Final refinement with proSmart restraints
Atomic model buildingPDB-ID: 1E3M

1e3m

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