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- EMDB-13071: The structure of MutS bound to two molecules of ADP-Vanadate -

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Basic information

Entry
Database: EMDB / ID: EMD-13071
TitleThe structure of MutS bound to two molecules of ADP-Vanadate
Map data
Sample
  • Organelle or cellular component: DNA mismatch repair protein MutS
    • Protein or peptide: DNA mismatch repair protein MutS
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: VANADATE ION
Function / homology
Function and homology information


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 ...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 / ATP binding / identical protein binding / cytosol
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 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA mismatch repair protein MutS
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsLamers MH / Borsellini A / Friedhoff P / Kunetsky V
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
Marie Sklodowska-Curie Actions, FragNET ITNEuropean Union
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Cryogenic electron microscopy structures reveal how ATP and DNA binding in MutS coordinates sequential steps of DNA mismatch repair.
Authors: Alessandro Borsellini / Vladislav Kunetsky / Peter Friedhoff / Meindert H Lamers /
Abstract: DNA mismatch repair detects and corrects mismatches introduced during DNA replication. The protein MutS scans for mismatches and coordinates the repair cascade. During this process, MutS undergoes ...DNA mismatch repair detects and corrects mismatches introduced during DNA replication. The protein MutS scans for mismatches and coordinates the repair cascade. During this process, MutS undergoes multiple conformational changes in response to ATP binding, hydrolysis and release, but how ATP induces the various MutS conformations is incompletely understood. Here we present four cryogenic electron microscopy structures of Escherichia coli MutS at sequential stages of the ATP hydrolysis cycle that reveal how ATP binding and hydrolysis induce closing and opening of the MutS dimer, respectively. Biophysical analysis demonstrates how DNA binding modulates the ATPase cycle by prevention of hydrolysis during scanning and mismatch binding, while preventing ADP release in the sliding clamp state. Nucleotide release is achieved when MutS encounters single-stranded DNA that is produced during removal of the daughter strand. The combination of ATP binding and hydrolysis and its modulation by DNA enables MutS to adopt the different conformations needed to coordinate the sequential steps of the mismatch repair cascade.
History
DepositionJun 10, 2021-
Header (metadata) releaseJan 12, 2022-
Map releaseJan 12, 2022-
UpdateOct 26, 2022-
Current statusOct 26, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.029
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.029
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ou0
  • Surface level: 0.029
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13071.png

Downloads & links

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Map

FileDownload / File: emd_13071.map.gz / Format: CCP4 / Size: 3.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.32 Å
Density
Contour LevelBy AUTHOR: 0.029 / Movie #1: 0.029
Minimum - Maximum-0.058120873 - 0.12236884
Average (Standard dev.)0.0016994565 (±0.009344618)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin464854
Dimensions1129786
Spacing9711286
CellA: 128.04001 Å / B: 147.84001 Å / C: 113.520004 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z9711286
origin x/y/z0.0000.0000.000
length x/y/z128.040147.840113.520
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS484654
NC/NR/NS9711286
D min/max/mean-0.0580.1220.002

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Supplemental data

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Sample components

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Entire : DNA mismatch repair protein MutS

EntireName: DNA mismatch repair protein MutS
Components
  • Organelle or cellular component: DNA mismatch repair protein MutS
    • Protein or peptide: DNA mismatch repair protein MutS
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: VANADATE ION

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Supramolecule #1: DNA mismatch repair protein MutS

SupramoleculeName: DNA mismatch repair protein MutS / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: MutS bound to two molecules of AMPPNP
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 190 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Macromolecule #1: DNA mismatch repair protein MutS

MacromoleculeName: DNA mismatch repair protein MutS / type: protein_or_peptide / ID: 1
Details: ADP Vanadate ATP hydrolysis transition state analogue.
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 90.433234 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: HHHHHHMSAI ENFDAHTPMM QQYLRLKAQH PEILLFYRMG DFYELFYDDA KRASQLLDIS LTKRGASAGE PIPMAGIPYH AVENYLAKL VNQGESVAIC EQIGDPATSK GPVERKVVRI VTPGTISDEA LLQERQDNLL AAIWQDSKGF GYATLDISSG R FRLSEPAD ...String:
HHHHHHMSAI ENFDAHTPMM QQYLRLKAQH PEILLFYRMG DFYELFYDDA KRASQLLDIS LTKRGASAGE PIPMAGIPYH AVENYLAKL VNQGESVAIC EQIGDPATSK GPVERKVVRI VTPGTISDEA LLQERQDNLL AAIWQDSKGF GYATLDISSG R FRLSEPAD RETMAAELQR TNPAELLYAE DFAEMSLIEG RRGLRRRPLW EFEIDTARQQ LNLQFGTRDL VGFGVENAPR GL CAAGCLL QYAKDTQRTT LPHIRSITME REQDSIIMDA ATRRNLEITQ NLAGGAENTL ASVLDCTVTP MGSRMLKRWL HMP VRDTRV LLERQQTIGA LQDFTAGLQP VLRQVGDLER ILARLALRTA RPRDLARMRH AFQQLPELRA QLETVDSAPV QALR EKMGE FAELRDLLER AIIDTPPVLV RDGGVIASGY NEELDEWRAL ADGATDYLER LEVRERERTG LDTLKVGFNA VHGYY IQIS RGQSHLAPIN YMRRQTLKNA ERYIIPELKE YEDKVLTSKG KALALEKQLY EELFDLLLPH LEALQQSASA LAELDV LVN LAERAYTLNY TCPTFIDKPG IRITEGRHPV VEQVLNEPFI ANPLNLSPQR RMLIITGPNM GGKSTYMRQT ALIALMA YI GSYVPAQKVE IGPIDRIFTR VGAADDLASG RSTFMVEMTE TANILHNATE YSLVLMDEIG RGTSTYDGLS LAWACAEN L ANKIKALTLF ATHYFELTQL PEKMEGVANV HLDALEHGDT IAFMHSVQDG AASKSYGLAV AALAGVPKEV IKRARQKLR ELESIS

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 2 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #4: VANADATE ION

MacromoleculeName: VANADATE ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: VO4
Molecular weightTheoretical: 114.939 Da
Chemical component information

ChemComp-VN3:
VANADATE ION / Vanadate

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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

Concentration0.95 mg/mL
BufferpH: 8.5
Component:
ConcentrationFormulaName
20.0 mM2-Amino-2-hydroxymethyl-propane-1,3-dioltris buffer
150.0 mMNaClSodium chloridesodium chloride
2.0 mM1,4-DithiothreitolDTT
5.0 mMMgCl2magnesium chloride
0.01 %C58H114O26Tween20
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.0004 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 76 % / Chamber temperature: 4 K / Instrument: LEICA PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-50 / Number real images: 4835 / Average electron dose: 57.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 496416
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: OTHER
Details: density map coming from MutS bound to two molecules of AMPPNP. density map reported together with this one in the same manuscript.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: REFMAC / Number images used: 81316
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1e3m

RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Overall B value: 70
Output model

PDB-7ou0:
The structure of MutS bound to two molecules of ADP-Vanadate

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