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- EMDB-15518: human MutSalpha mutant (MSH2-V63E/MSH6) on DNA containing a GT mi... -

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Basic information

Entry
Database: EMDB / ID: EMD-15518
Titlehuman MutSalpha mutant (MSH2-V63E/MSH6) on DNA containing a GT mismatch
Map data
Sample
  • Complex: human MutSalpha mutant (MSH2-V63E/MSH6) on mismatched DNA
    • Complex: MSH2 V63E
      • Protein or peptide: MSH2 V63E
    • 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: 4.0 Å
AuthorsBruekner SR / Sixma TK
Funding support Netherlands, European Union, 2 items
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 MutSalpha enables high affinity DNA mismatch binding
Authors: Bruekner SR / Pieters W / Fish A / Liaci AM / Scheffers S / Rayner E / Kaldenbach D / Drost L / Dekker M / van Hees-Stuivenberg S / Delzenne-Goette E / de Konink C / Houllleberghs H / ...Authors: Bruekner SR / Pieters W / Fish A / Liaci AM / Scheffers S / Rayner E / Kaldenbach D / Drost L / Dekker M / van Hees-Stuivenberg S / Delzenne-Goette E / de Konink C / Houllleberghs H / Dubbink HJ / AlSaegh A / de Wind N / Forster F / te Riele H / Sixma TK
History
DepositionAug 1, 2022-
Header (metadata) releaseJan 25, 2023-
Map releaseJan 25, 2023-
UpdateJan 25, 2023-
Current statusJan 25, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15518.png

Downloads & links

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Map

FileDownload / File: emd_15518.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 360 pix.
= 300.96 Å		0.84 Å/pix.
x 360 pix.
= 300.96 Å		0.84 Å/pix.
x 360 pix.
= 300.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.836 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0043
Minimum - Maximum-0.0044029006 - 0.01793054
Average (Standard dev.)3.2245065e-05 (±0.00064370444)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 300.96002 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_15518_additional_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Additional map: #2

Fileemd_15518_additional_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15518_half_map_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_15518_half_map_2.map
Projections & Slices
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Sample components

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Entire : human MutSalpha mutant (MSH2-V63E/MSH6) on mismatched DNA

EntireName: human MutSalpha mutant (MSH2-V63E/MSH6) on mismatched DNA
Components
  • Complex: human MutSalpha mutant (MSH2-V63E/MSH6) on mismatched DNA
    • Complex: MSH2 V63E
      • Protein or peptide: MSH2 V63E
    • Complex: MSH6
      • Protein or peptide: MSH6
    • Complex: DNA (50-mer)
      • DNA: DNA (50-mer)
      • DNA: DNA (50-mer)

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Supramolecule #1: human MutSalpha mutant (MSH2-V63E/MSH6) on mismatched DNA

SupramoleculeName: human MutSalpha mutant (MSH2-V63E/MSH6) on mismatched DNA
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Details: MSH2 has a valine to glutamate mutation at position 63
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 31 KDa

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Supramolecule #2: MSH2 V63E

SupramoleculeName: MSH2 V63E / type: complex / ID: 2 / Chimera: Yes / Parent: 1 / Macromolecule list: #1
Details: MSH2 has a valine to glutamate mutation at postition 63
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 / Details: MSH6 has an N-terminal 10His-TwinStrep tag
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 V63E

MacromoleculeName: MSH2 V63E / type: protein_or_peptide / ID: 1 / Details: valine 63 is mutated to glutamate / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAVQPKETLQ LESAAEVGFV RFFQGMPEKP TTTVRLFDRG DFYTAHGEDA LLAAREVFKT QGEIKYMGPA GAKNLQSVVL SKMNFESFVK DLLLVRQYRV EVYKNRAGNK ASKENDWYLA YKASPGNLSQ FEDILFGNND MSASIGVVGV KMSAVDGQRQ VGVGYVDSIQ ...String:
MAVQPKETLQ LESAAEVGFV RFFQGMPEKP TTTVRLFDRG DFYTAHGEDA LLAAREVFKT QGEIKYMGPA 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: 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

Concentration0.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMHEPESHEPES
150.0 mMKClpotassium chloride
5.0 mMMgCl2magnesium chloride
1.0 mMTCEPTCEP
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.01 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 2 s blotting time with force 10.
Details2 uM MutSalpha V63E with 2-fold excess mismatched DNA

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsData collected on Krios 1 at the Netherlands Center for Electron Nanoscopy
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 4456 / Average exposure time: 1.9 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4470125
Startup modelType of model: OTHER
Details: Map from MutSalpha WT was used as a starting model to select complete MutSalpha particles and remove broken particles and single subunits. The actual reconstruction on the cleaned particle ...Details: Map from MutSalpha WT was used as a starting model to select complete MutSalpha particles and remove broken particles and single subunits. The actual reconstruction on the cleaned particle set was started ab initio
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.1)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 4.1)
Details: Final classification of full particles was done giving 3 models which were generated using cryoDRGN: 1) closed MSH2/MSH6 dimer 2) open MSH2 clamp 3) junk Resulting maps from 1) and 2) are ...Details: Final classification of full particles was done giving 3 models which were generated using cryoDRGN: 1) closed MSH2/MSH6 dimer 2) open MSH2 clamp 3) junk Resulting maps from 1) and 2) are deposited as D_1292124425_em-additional-volume_P1.map.V2 and D_1292124425_em-additional-volume_P2.map.V2 respectively.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.1)
Final reconstructionNumber classes used: 3 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.1)
Details: This particle stack was further classified, seperating closed MSH2/MSH6 dimer and open MSH2 clamp particles (see final classification).
Number images used: 67182
FSC plot (resolution estimation)

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