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- EMDB-15519: human MutSalpha (MSH2/MSH6) on DNA containing a GT mismatch in th... -

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Basic information

Entry
Database: EMDB / ID: EMD-15519
Titlehuman MutSalpha (MSH2/MSH6) on DNA containing a GT mismatch in the presence of ADP
Map data
Sample
  • Complex: MutSalpha on mismatched DNA in the presence of ADP
    • Complex: MSH2
      • Protein or peptide: MSH2
    • Complex: MSH6
      • Protein or peptide: MSH6
    • Complex: DNA (50-mer)
      • DNA: DNA (50-mer)
      • DNA: DNA (50-mer)
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsBruekner SR / Liaci AM / Sixma TK
Funding support Netherlands, European Union, 2 items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)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
DepositionAug 1, 2022-
Header (metadata) releaseJan 25, 2023-
Map releaseJan 25, 2023-
UpdateMar 1, 2023-
Current statusMar 1, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15519.png

Downloads & links

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Map

FileDownload / File: emd_15519.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 256 pix.
= 266.496 Å		1.04 Å/pix.
x 256 pix.
= 266.496 Å		1.04 Å/pix.
x 256 pix.
= 266.496 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.041 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.021
Minimum - Maximum-0.055528723 - 0.10174949
Average (Standard dev.)0.0003432529 (±0.0032034111)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 266.496 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_15519_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_15519_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : MutSalpha on mismatched DNA in the presence of ADP

EntireName: MutSalpha on mismatched DNA in the presence of ADP
Components
  • Complex: MutSalpha on mismatched DNA in the presence of ADP
    • Complex: MSH2
      • Protein or peptide: MSH2
    • Complex: MSH6
      • Protein or peptide: MSH6
    • Complex: DNA (50-mer)
      • DNA: DNA (50-mer)
      • DNA: DNA (50-mer)

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Supramolecule #1: MutSalpha on mismatched DNA in the presence of ADP

SupramoleculeName: MutSalpha on mismatched DNA in the presence of ADP / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 31 KDa

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Supramolecule #2: MSH2

SupramoleculeName: MSH2 / type: complex / ID: 2 / Chimera: Yes / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: MSH6

SupramoleculeName: MSH6 / type: complex / ID: 3 / Chimera: Yes / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: DNA (50-mer)

SupramoleculeName: DNA (50-mer) / type: complex / ID: 4 / Chimera: Yes / Parent: 1 / Macromolecule list: #3-#4 / Details: contains a GT mismatch at position 25
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: MSH2

MacromoleculeName: MSH2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAVQPKETLQ LESAAEVGFV RFFQGMPEKP TTTVRLFDRG DFYTAHGEDA LLAAREVFKT QGVIKYMGPA GAKNLQSVVL SKMNFESFVK DLLLVRQYRV EVYKNRAGNK ASKENDWYLA YKASPGNLSQ FEDILFGNND MSASIGVVGV KMSAVDGQRQ VGVGYVDSIQ ...String:
MAVQPKETLQ LESAAEVGFV RFFQGMPEKP TTTVRLFDRG DFYTAHGEDA LLAAREVFKT QGVIKYMGPA GAKNLQSVVL SKMNFESFVK DLLLVRQYRV EVYKNRAGNK ASKENDWYLA YKASPGNLSQ FEDILFGNND MSASIGVVGV KMSAVDGQRQ VGVGYVDSIQ RKLGLCEFPD NDQFSNLEAL LIQIGPKECV LPGGETAGDM GKLRQIIQRG GILITERKKA DFSTKDIYQD LNRLLKGKKG EQMNSAVLPE MENQVAVSSL SAVIKFLELL SDDSNFGQFE LTTFDFSQYM KLDIAAVRAL NLFQGSVEDT TGSQSLAALL NKCKTPQGQR LVNQWIKQPL MDKNRIEERL NLVEAFVEDA ELRQTLQEDL LRRFPDLNRL AKKFQRQAAN LQDCYRLYQG INQLPNVIQA LEKHEGKHQK LLLAVFVTPL TDLRSDFSKF QEMIETTLDM DQVENHEFLV KPSFDPNLSE LREIMNDLEK KMQSTLISAA RDLGLDPGKQ IKLDSSAQFG YYFRVTCKEE KVLRNNKNFS TVDIQKNGVK FTNSKLTSLN EEYTKNKTEY EEAQDAIVKE IVNISSGYVE PMQTLNDVLA QLDAVVSFAH VSNGAPVPYV RPAILEKGQG RIILKASRHA CVEVQDEIAF IPNDVYFEKD KQMFHIITGP NMGGKSTYIR QTGVIVLMAQ IGCFVPCESA EVSIVDCILA RVGAGDSQLK GVSTFMAEML ETASILRSAT KDSLIIIDEL GRGTSTYDGF GLAWAISEYI ATKIGAFCMF ATHFHELTAL ANQIPTVNNL HVTALTTEET LTMLYQVKKG VCDQSFGIHV AELANFPKHV IECAKQKALE LEEFQYIGES QGYDIMEPAA KKCYLEREQG EKIIQEFLSK VKQMPFTEMS EENITIKLKQ LKAEVIAKNN SFVNEIISRI KVTT

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Macromolecule #2: MSH6

MacromoleculeName: MSH6 / type: protein_or_peptide / ID: 2 / Details: contains an N-terminal 10His-TwinStrep tag / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAHHHHHHHH HHGSAASWSH PQFEKGGGSG GGSGGGSWSH PQFEKGALEV LFQGPSRQST LYSFFPKSPA LSDANKASAR ASREGGRAAA APEASPSPGG DAAWSEAGPG PRPLARSASP PKAKNLNGGL RRSVAPAAPT SCDFSPGDLV WAKMEGYPWW PCLVYNHPFD ...String:
MAHHHHHHHH HHGSAASWSH PQFEKGGGSG GGSGGGSWSH PQFEKGALEV LFQGPSRQST LYSFFPKSPA LSDANKASAR ASREGGRAAA APEASPSPGG DAAWSEAGPG PRPLARSASP PKAKNLNGGL RRSVAPAAPT SCDFSPGDLV WAKMEGYPWW PCLVYNHPFD GTFIREKGKS VRVHVQFFDD SPTRGWVSKR LLKPYTGSKS KEAQKGGHFY SAKPEILRAM QRADEALNKD KIKRLELAVC DEPSEPEEEE EMEVGTTYVT DKSEEDNEIE SEEEVQPKTQ GSRRSSRQIK KRRVISDSES DIGGSDVEFK PDTKEEGSSD EISSGVGDSE SEGLNSPVKV ARKRKRMVTG NGSLKRKSSR KETPSATKQA TSISSETKNT LRAFSAPQNS ESQAHVSGGG DDSSRPTVWY HETLEWLKEE KRRDEHRRRP DHPDFDASTL YVPEDFLNSC TPGMRKWWQI KSQNFDLVIC YKVGKFYELY HMDALIGVSE LGLVFMKGNW AHSGFPEIAF GRYSDSLVQK GYKVARVEQT ETPEMMEARC RKMAHISKYD RVVRREICRI ITKGTQTYSV LEGDPSENYS KYLLSLKEKE EDSSGHTRAY GVCFVDTSLG KFFIGQFSDD RHCSRFRTLV AHYPPVQVLF EKGNLSKETK TILKSSLSCS LQEGLIPGSQ FWDASKTLRT LLEEEYFREK LSDGIGVMLP QVLKGMTSES DSIGLTPGEK SELALSALGG CVFYLKKCLI DQELLSMANF EEYIPLDSDT VSTTRSGAIF TKAYQRMVLD AVTLNNLEIF LNGTNGSTEG TLLERVDTCH TPFGKRLLKQ WLCAPLCNHY AINDRLDAIE DLMVVPDKIS EVVELLKKLP DLERLLSKIH NVGSPLKSQN HPDSRAIMYE ETTYSKKKII DFLSALEGFK VMCKIIGIME EVADGFKSKI LKQVISLQTK NPEGRFPDLT VELNRWDTAF DHEKARKTGL ITPKAGFDSD YDQALADIRE NEQSLLEYLE KQRNRIGCRT IVYWGIGRNR YQLEIPENFT TRNLPEEYEL KSTKKGCKRY WTKTIEKKLA NLINAEERRD VSLKDCMRRL FYNFDKNYKD WQSAVECIAV LDVLLCLANY SRGGDGPMCR PVILLPEDTP PFLELKGSRH PCITKTFFGD DFIPNDILIG CEEEEQENGK AYCVLVTGPN MGGKSTLMRQ AGLLAVMAQM GCYVPAEVCR LTPIDRVFTR LGASDRIMSG ESTFFVELSE TASILMHATA HSLVLVDELG RGTATFDGTA IANAVVKELA ETIKCRTLFS THYHSLVEDY SQNVAVRLGH MACMVENECE DPSQETITFL YKFIKGACPK SYGFNAARLA NLPEEVIQKG HRKAREFEKM NQSLRLFREV CLASERSTVD AEAVHKLLTL IKEL

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Macromolecule #3: DNA (50-mer)

MacromoleculeName: DNA (50-mer) / type: dna / ID: 3 / Details: contains a GT mismatch / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
CTTAGCTTAG GATCATCGAG GATCGAGCTC GGTGCAATTC AGCGGTACCC

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Macromolecule #4: DNA (50-mer)

MacromoleculeName: DNA (50-mer) / type: dna / ID: 4 / Details: contains a GT mismatch / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
GGGTACCGCT GAATTGCACC GAGCTTGATC CTCGATGATC CTAAGCTAAG

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.25 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMHEPESHEPES
150.0 mMKClpotassium chloride
5.0 mMMgCl2magnesium chloride
2.0 mMDTTdithiothreitol
1.0 mMADPAdenosine diphosphateadenosine diphosphate
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.023 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 2 s blotting with force 0.
Details1 uM MutSalpha with 2-fold excess mismatched DNA and 1 mM ADP

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 30.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Detailscollected at 200 kV Talos Arctica at Utrecht University, the Netherlands
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 2608 / Average exposure time: 7.8 sec. / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1707083 / Details: Relion Autopick
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 5 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 111096
FSC plot (resolution estimation)

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