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- PDB-7ai5: MutS in Scanning state -

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Basic information

Entry
Database: PDB / ID: 7ai5
TitleMutS in Scanning state
Components
  • DNA (5'-D(P*CP*GP*GP*TP*AP*CP*CP*CP*AP*AP*TP*TP*CP*GP*CP*CP*CP*TP*AP*TP*AP*G)-3')
  • DNA (5'-D(P*CP*TP*AP*TP*AP*GP*GP*GP*CP*GP*AP*AP*TP*TP*GP*GP*GP*TP*AP*CP*CP*G)-3')
  • DNA mismatch repair protein MutS
KeywordsDNA BINDING PROTEIN / DNA Mismatch Repair MutS
Function / homology
Function and homology information


mismatched DNA binding / mismatch repair / damaged DNA binding / ATP binding
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 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'-TRIPHOSPHATE / 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: 4.4 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
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  • Simplified surface model + fitted atomic model
  • EMDB-11791
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  • Superimposition on EM map
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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*CP*TP*AP*TP*AP*GP*GP*GP*CP*GP*AP*AP*TP*TP*GP*GP*GP*TP*AP*CP*CP*G)-3')
D: DNA (5'-D(P*CP*GP*GP*TP*AP*CP*CP*CP*AP*AP*TP*TP*CP*GP*CP*CP*CP*TP*AP*TP*AP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)205,3156
Polymers204,3014
Non-polymers1,0142
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10200 Å2
ΔGint-76 kcal/mol
Surface area74470 Å2
MethodPISA
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1010A2 - 800
2010B2 - 800

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Components

#1: Protein DNA mismatch repair protein MutS /


Mass: 95397.898 Da / Num. of mol.: 2 / Mutation: D835R
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: mutS, ACU57_10490, AUQ13_17300, BANRA_01250, BANRA_04372, BMA87_05210, BvCms2454_03957, BvCmsKSP026_01123, BvCmsSINP011_00857, C9Z39_17590, CI693_21650, D2185_15190, D3821_16315, D4638_12960, ...Gene: mutS, ACU57_10490, AUQ13_17300, BANRA_01250, BANRA_04372, BMA87_05210, BvCms2454_03957, BvCmsKSP026_01123, BvCmsSINP011_00857, C9Z39_17590, CI693_21650, D2185_15190, D3821_16315, D4638_12960, D9D20_12730, D9D44_11420, DAH34_11930, DJ503_05760, DL326_19385, DT034_19915, DTL43_09115, E2119_10690, E2127_10890, E2128_08030, EAI52_13070, EC3234A_48c00420, ECTO6_01123, ED307_14565, EEP23_04760, EI041_14570, EL75_0954, EL79_0966, EL80_0968, ELT20_17315, EPT01_07440, FORC82_1112, FV293_17550, GHR40_09850, GKF74_12505, GKF86_14440, GKF89_15940, NCTC10963_01107, NCTC13216_00777, NCTC8500_01160, NCTC9045_01268, NCTC9062_02087, RK56_022910, SAMEA3484427_01823, SAMEA3484429_01685, SAMEA3752559_02626, SAMEA3753300_03556
Production host: Escherichia coli (E. coli) / References: UniProt: A0A037YGY1
#2: DNA chain DNA (5'-D(P*CP*TP*AP*TP*AP*GP*GP*GP*CP*GP*AP*AP*TP*TP*GP*GP*GP*TP*AP*CP*CP*G)-3')


Mass: 6832.414 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Plasmid DNA molecule (pRC1765), sequence in structure unidentified
Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(P*CP*GP*GP*TP*AP*CP*CP*CP*AP*AP*TP*TP*CP*GP*CP*CP*CP*TP*AP*TP*AP*G)-3')


Mass: 6672.318 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Plasmid DNA molecule (pRC1765), sequence in structure unidentified
Source: (synth.) synthetic construct (others)
#4: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, 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 (E. coli)562
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
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil
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

SoftwareName: REFMAC / Version: 5.8.0258 / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4GctfCTF correction
7Coot0.91model fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13REFMAC5model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 229664
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 36682 / Algorithm: BACK PROJECTION / 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

RefinementResolution: 5→126.36 Å / Cor.coef. Fo:Fc: 0.944 / SU B: 186.237 / SU ML: 2.008
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.37776 --
obs0.37776 29780 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 117.105 Å2
Baniso -1Baniso -2Baniso -3
1-10.94 Å213.05 Å26.21 Å2
2---1.31 Å22.86 Å2
3----9.63 Å2
Refinement stepCycle: 1 / Total: 13316
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0080.01313636
ELECTRON MICROSCOPYr_bond_other_d00.01712498
ELECTRON MICROSCOPYr_angle_refined_deg1.3691.60918646
ELECTRON MICROSCOPYr_angle_other_deg1.3761.62928914
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.23851572
ELECTRON MICROSCOPYr_dihedral_angle_2_deg29.26821.185726
ELECTRON MICROSCOPYr_dihedral_angle_3_deg15.791152244
ELECTRON MICROSCOPYr_dihedral_angle_4_deg15.16215126
ELECTRON MICROSCOPYr_chiral_restr0.0710.21792
ELECTRON MICROSCOPYr_gen_planes_refined0.0070.0214732
ELECTRON MICROSCOPYr_gen_planes_other0.0030.022904
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it3.34512.4186300
ELECTRON MICROSCOPYr_mcbond_other3.34512.4186299
ELECTRON MICROSCOPYr_mcangle_it5.98418.6247868
ELECTRON MICROSCOPYr_mcangle_other5.98318.6257869
ELECTRON MICROSCOPYr_scbond_it2.17912.8567336
ELECTRON MICROSCOPYr_scbond_other2.17912.8567335
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other4.2519.21610778
ELECTRON MICROSCOPYr_long_range_B_refined13.75332762
ELECTRON MICROSCOPYr_long_range_B_other13.75332762
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Ens-ID: 1 / Number: 49696 / Refine-ID: ELECTRON MICROSCOPY / Type: interatomic distance / Rms dev position: 0.05 Å / Weight position: 0.05

Dom-IDAuth asym-ID
1A
2B
LS refinement shellResolution: 5→5.13 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.438 2249 -
obs--100 %

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