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- PDB-8ag6: human MutSalpha (MSH2/MSH6) binding to DNA with a GT mismatch -

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Basic information

Entry
Database: PDB / ID: 8ag6
Titlehuman MutSalpha (MSH2/MSH6) binding to DNA with a GT mismatch
Components
  • (DNA (50-MER)) x 2
  • (DNA mismatch repair protein ...) x 2
KeywordsDNA BINDING PROTEIN / DNA mismatch repair / MMR / MutSa / Lynch Syndrome
Function / homology
Function and homology information


somatic recombination of immunoglobulin genes involved in immune response / MutSbeta complex / Defective Mismatch Repair Associated With MSH3 / MutSalpha complex / Defective Mismatch Repair Associated With MSH2 / Defective Mismatch Repair Associated With MSH6 / guanine/thymine mispair binding / somatic recombination of immunoglobulin gene segments / maintenance of DNA repeat elements / B cell mediated immunity ...somatic recombination of immunoglobulin genes involved in immune response / MutSbeta complex / Defective Mismatch Repair Associated With MSH3 / MutSalpha complex / Defective Mismatch Repair Associated With MSH2 / Defective Mismatch Repair Associated With MSH6 / guanine/thymine mispair binding / somatic recombination of immunoglobulin gene segments / maintenance of DNA repeat elements / B cell mediated immunity / positive regulation of helicase activity / positive regulation of isotype switching to IgA isotypes / meiotic mismatch repair / centromeric DNA binding / mitotic recombination / positive regulation of isotype switching to IgG isotypes / mismatched DNA binding / negative regulation of DNA recombination / Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta) / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / isotype switching / postreplication repair / oxidative phosphorylation / response to UV-B / mitotic intra-S DNA damage checkpoint signaling / ATP-dependent DNA damage sensor activity / germ cell development / response to X-ray / mismatch repair / intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / somatic hypermutation of immunoglobulin genes / ATP-dependent activity, acting on DNA / response to UV / protein localization to chromatin / methylated histone binding / intrinsic apoptotic signaling pathway / B cell differentiation / determination of adult lifespan / TP53 Regulates Transcription of DNA Repair Genes / male gonad development / intrinsic apoptotic signaling pathway in response to DNA damage / double-strand break repair / spermatogenesis / negative regulation of neuron apoptotic process / in utero embryonic development / damaged DNA binding / chromosome, telomeric region / DNA repair / intracellular membrane-bounded organelle / chromatin binding / chromatin / Golgi apparatus / enzyme binding / ATP hydrolysis activity / protein homodimerization activity / DNA binding / nucleoplasm / ATP binding / membrane / nucleus / cytosol
Similarity search - Function
DNA mismatch repair Msh2-type / DNA mismatch repair protein Msh2 / 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 ...DNA mismatch repair Msh2-type / DNA mismatch repair protein Msh2 / 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 / domain with conserved PWWP motif / PWWP domain / PWWP domain profile. / PWWP domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / DNA / DNA (> 10) / DNA mismatch repair protein Msh2 / DNA mismatch repair protein Msh6
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsBruekner, S.R. / Sixma, T.K.
Funding support Netherlands, European Union, 2items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)NOW-TOP 714.016.002 Netherlands
European CommissionH2020-MSCA-ITN-2016 722433 DNAREPAIRMANEuropean Union
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Unexpected moves: a conformational change in MutSα enables high-affinity DNA mismatch binding.
Authors: Susanne R Bruekner / Wietske Pieters / Alexander Fish / A Manuel Liaci / Serge Scheffers / Emily Rayner / Daphne Kaldenbach / Lisa Drost / Marleen Dekker / Sandrine van Hees-Stuivenberg / ...Authors: Susanne R Bruekner / Wietske Pieters / Alexander Fish / A Manuel Liaci / Serge Scheffers / Emily Rayner / Daphne Kaldenbach / Lisa Drost / Marleen Dekker / Sandrine van Hees-Stuivenberg / Elly Delzenne-Goette / Charlotte de Konink / Hellen Houlleberghs / Hendrikus Jan Dubbink / Abeer AlSaegh / Niels de Wind / Friedrich Förster / Hein Te Riele / Titia K Sixma /
Abstract: The DNA mismatch repair protein MutSα recognizes wrongly incorporated DNA bases and initiates their correction during DNA replication. Dysfunctions in mismatch repair lead to a predisposition to ...The DNA mismatch repair protein MutSα recognizes wrongly incorporated DNA bases and initiates their correction during DNA replication. Dysfunctions in mismatch repair lead to a predisposition to cancer. Here, we study the homozygous mutation V63E in MSH2 that was found in the germline of a patient with suspected constitutional mismatch repair deficiency syndrome who developed colorectal cancer before the age of 30. Characterization of the mutant in mouse models, as well as slippage and repair assays, shows a mildly pathogenic phenotype. Using cryogenic electron microscopy and surface plasmon resonance, we explored the mechanistic effect of this mutation on MutSα function. We discovered that V63E disrupts a previously unappreciated interface between the mismatch binding domains (MBDs) of MSH2 and MSH6 and leads to reduced DNA binding. Our research identifies this interface as a 'safety lock' that ensures high-affinity DNA binding to increase replication fidelity. Our mechanistic model explains the hypomorphic phenotype of the V63E patient mutation and other variants in the MBD interface.
History
DepositionJul 19, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 25, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 8, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Mar 1, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA mismatch repair protein Msh2
B: DNA mismatch repair protein Msh6
E: DNA (50-MER)
F: DNA (50-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)294,9026
Polymers294,4514
Non-polymers4522
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, gel filtration was performed to purify the MutSalpha complex, surface plasmon resonance, SPR was performed to validate DNA binding
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14590 Å2
ΔGint-130 kcal/mol
Surface area90940 Å2

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Components

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DNA mismatch repair protein ... , 2 types, 2 molecules AB

#1: Protein DNA mismatch repair protein Msh2 / / hMSH2 / MutS protein homolog 2


Mass: 104861.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MSH2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P43246
#2: Protein DNA mismatch repair protein Msh6 / / hMSH6 / G/T mismatch-binding protein / GTBP / GTMBP / MutS protein homolog 6 / MutS-alpha 160 kDa ...hMSH6 / G/T mismatch-binding protein / GTBP / GTMBP / MutS protein homolog 6 / MutS-alpha 160 kDa subunit / p160


Mass: 158767.312 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MSH6, GTBP / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P52701

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DNA chain , 2 types, 2 molecules EF

#3: DNA chain DNA (50-MER)


Mass: 15418.874 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (50-MER)


Mass: 15402.874 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 2 molecules

#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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Details

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-IDSourceDetails
1MutSalpha on mismatched DNACOMPLEX#1-#40RECOMBINANT
2MSH2COMPLEX#11RECOMBINANThas ADP+Mg bound
3MSH6COMPLEX#21RECOMBINANTContains N-terminal 10His-TwinStrep tag removable by 3C
4DNA containing a G/T mismatchCOMPLEX#3-#41MULTIPLE SOURCES
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.294 MDaNO
210.105 MDaNO
310.159 MDaNO
410.031 MDaNO
54
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
54Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDPlasmid
21Spodoptera frugiperda (fall armyworm)7108pFastBac Dual
32Spodoptera frugiperda (fall armyworm)7108
43Spodoptera frugiperda (fall armyworm)7108
54Spodoptera frugiperda (fall armyworm)7108
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESHEPES1
2150 mMpotassium chlorideKCl1
35 mMmagnesium chlorideMgCl21
41 mMTCEPTCEP1
SpecimenConc.: 0.53 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 2 uM MutSa with 2-fold excess DNA oligo
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 3 s blotting with blot force 10

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: collected on Krios 1 at Netherlands Center for Electron Nanoscopy (NeCEN)
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm / C2 aperture diameter: 50.000000001 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.9 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4821

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Processing

EM software
IDNameVersionCategory
2EPU2.10.0image acquisition
4RELION4CTF correction
7UCSF ChimeraXmodel fitting
9cryoSPARCinitial Euler assignment
10RELION4final Euler assignment
11RELION4classification
12RELION43D reconstruction
13PHENIX1.20.1model refinement
14Coot0.9.7model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5412220
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 289289 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Details: PDB-REDO webserver: https://pdb-redo.eu/
Atomic model buildingPDB-ID: 2O8B

2o8b

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