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- PDB-7aic: MutS-MutL in clamp state (kinked clamp domain) -

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Basic information

Entry
Database: PDB / ID: 7aic
TitleMutS-MutL in clamp state (kinked clamp domain)
Components
  • (DNA (30-MER)) x 2
  • DNA mismatch repair protein MutL
  • DNA mismatch repair protein MutS
KeywordsDNA BINDING PROTEIN / DNA Mismatch Repair MutS
Function / homology
Function and homology information


single-stranded DNA-dependent ATP-dependent DNA helicase complex / mismatch repair involved in maintenance of fidelity involved in DNA-dependent DNA replication / 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 ...single-stranded DNA-dependent ATP-dependent DNA helicase complex / mismatch repair involved in maintenance of fidelity involved in DNA-dependent DNA replication / 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 / DNA binding / ATP binding / identical protein binding / cytosol
Similarity search - Function
DNA mismatch repair protein, MutL / MutL, C-terminal domain, regulatory subdomain / MutL C terminal dimerisation domain / DNA mismatch repair protein MutS / MutL, C-terminal, dimerisation / MutL, C-terminal domain superfamily / MutL, C-terminal domain, dimerisation subdomain / MutL C terminal dimerisation domain / DNA mismatch repair protein family, N-terminal / DNA mismatch repair protein, S5 domain 2-like ...DNA mismatch repair protein, MutL / MutL, C-terminal domain, regulatory subdomain / MutL C terminal dimerisation domain / DNA mismatch repair protein MutS / MutL, C-terminal, dimerisation / MutL, C-terminal domain superfamily / MutL, C-terminal domain, dimerisation subdomain / MutL C terminal dimerisation domain / DNA mismatch repair protein family, N-terminal / DNA mismatch repair protein, S5 domain 2-like / DNA mismatch repair, conserved site / DNA mismatch repair protein MutS/MSH / DNA mismatch repair protein MutL/Mlh/Pms / DNA mismatch repair protein, C-terminal domain / DNA mismatch repair proteins mutL / hexB / PMS1 signature. / DNA mismatch repair protein, C-terminal domain / 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 / Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase / Histidine kinase/HSP90-like ATPase superfamily / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / DNA mismatch repair protein MutL / DNA mismatch repair protein MutS
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5 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
Revision 1.3May 1, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _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

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Structure visualization

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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 mismatch repair protein MutL
D: DNA (30-MER)
E: DNA (30-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)249,2487
Polymers248,2365
Non-polymers1,0122
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15640 Å2
ΔGint-93 kcal/mol
Surface area73800 Å2
MethodPISA

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Components

#1: Protein DNA mismatch repair protein MutS


Mass: 95269.625 Da / Num. of mol.: 2
Mutation: D825R, C93A, C235S, C239A, C297S, C569S, C711V, D246C
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: Protein DNA mismatch repair protein MutL


Mass: 39248.535 Da / Num. of mol.: 1 / Mutation: N131C,C61S,C216L,C256F,C276Y
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: mutL, b4170, JW4128 / Production host: Escherichia coli (E. coli) / References: UniProt: P23367
#3: DNA chain DNA (30-MER)


Mass: 9303.989 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (30-MER)


Mass: 9143.893 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*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 DNA substrate with one central mismatched basepair in the presence of AMP-PNPCOMPLEX#1-#40MULTIPLE SOURCES
2DNA mismatch repair protein MutS and DNA mismatch repair endonuclease MutLCOMPLEX#1-#21RECOMBINANT
3DNACOMPLEX#3-#41RECOMBINANT
Molecular weightValue: 0.280 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (strain K12) (bacteria)83333
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
33synthetic construct (others)32630
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
120 mMHepes1
250 mMPotasium Glutamate1
32 mMTEMED1
41 mMATP1
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+AMP-PNP 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 FIELD / Nominal magnification: 64000 X / Calibrated defocus min: 1500 nm / Calibrated defocus max: 3000 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: 290291
3D reconstructionResolution: 5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 32308 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: RECIPROCAL
Details: Initial Jelly Body refinement Final refinement with proSmart restraints
Atomic model buildingPDB-ID: 5AKB

5akb


Accession code: 5AKB / Source name: PDB / Type: experimental model

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