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- EMDB-13255: The structure of E. coli MutL bound to a 3' resected DNA end -

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Basic information

Entry
Database: EMDB / ID: EMD-13255
TitleThe structure of E. coli MutL bound to a 3' resected DNA end
Map data
Sample
  • Complex: E. coli MutL N-terminal dimer bound to a 3' resected DNA end
    • Complex: 3' resected DNA end
      • DNA: Template strandTranscription (biology)
      • DNA: Primer strand
    • Complex: E. coli MutL N-terminal dimer
      • Protein or peptide: DNA mismatch repair protein MutL
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
Function / homology
Function and homology information


single-stranded DNA-dependent ATP-dependent DNA helicase complex / mismatch repair involved in maintenance of fidelity involved in DNA-dependent DNA replication / mismatch repair complex / regulation of DNA recombination / nucleotide-excision repair, DNA duplex unwinding / mismatched DNA binding / ATP-dependent DNA damage sensor activity / mismatch repair / ATP hydrolysis activity / DNA binding ...single-stranded DNA-dependent ATP-dependent DNA helicase complex / mismatch repair involved in maintenance of fidelity involved in DNA-dependent DNA replication / mismatch repair complex / regulation of DNA recombination / nucleotide-excision repair, DNA duplex unwinding / mismatched DNA binding / ATP-dependent DNA damage sensor activity / mismatch repair / ATP hydrolysis activity / DNA binding / ATP binding / identical protein binding
Similarity search - Function
DNA mismatch repair protein, MutL / MutL, C-terminal domain, regulatory subdomain / MutL C terminal dimerisation domain / MutL, C-terminal, dimerisation / MutL, C-terminal domain superfamily / MutL, C-terminal domain, dimerisation subdomain / MutL C terminal dimerisation domain / DNA mismatch repair protein family, N-terminal / DNA mismatch repair protein, S5 domain 2-like / DNA mismatch repair, conserved site ...DNA mismatch repair protein, MutL / MutL, C-terminal domain, regulatory subdomain / MutL C terminal dimerisation domain / MutL, C-terminal, dimerisation / MutL, C-terminal domain superfamily / MutL, C-terminal domain, dimerisation subdomain / MutL C terminal dimerisation domain / DNA mismatch repair protein family, N-terminal / DNA mismatch repair protein, S5 domain 2-like / DNA mismatch repair, conserved site / DNA mismatch repair protein MutL/Mlh/Pms / DNA mismatch repair protein, C-terminal domain / DNA mismatch repair proteins mutL / hexB / PMS1 signature. / DNA mismatch repair protein, C-terminal domain / Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase / Histidine kinase/HSP90-like ATPase superfamily / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold
Similarity search - Domain/homology
DNA mismatch repair protein MutL
Similarity search - Component
Biological speciesDNA molecule (others) / Escherichia coli K-12 (bacteria) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsBorsellini A / Lamers MH
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
Marie Sklodowska-Curie Actions, FragNET ITNEuropean Union
CitationJournal: Nucleic Acids Res / Year: 2022
Title: MutL binds to 3' resected DNA ends and blocks DNA polymerase access.
Authors: Alessandro Borsellini / Joyce H G Lebbink / Meindert H Lamers /
Abstract: DNA mismatch repair removes mis-incorporated bases after DNA replication and reduces the error rate a 100-1000-fold. After recognition of a mismatch, a large section of up to a thousand nucleotides ...DNA mismatch repair removes mis-incorporated bases after DNA replication and reduces the error rate a 100-1000-fold. After recognition of a mismatch, a large section of up to a thousand nucleotides is removed from the daughter strand followed by re-synthesis. How these opposite activities are coordinated is poorly understood. Here we show that the Escherichia coli MutL protein binds to the 3' end of the resected strand and blocks access of Pol I and Pol III. The cryo-EM structure of an 85-kDa MutL-DNA complex, determined to 3.7 Å resolution, reveals a unique DNA binding mode that positions MutL at the 3' end of a primer-template, but not at a 5' resected DNA end or a blunt DNA end. Hence, our work reveals a novel role for MutL in the final stages of mismatch repair by preventing premature DNA synthesis during removal of the mismatched strand.
History
DepositionJul 23, 2021-
Header (metadata) releaseJun 29, 2022-
Map releaseJun 29, 2022-
UpdateAug 31, 2022-
Current statusAug 31, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13255.png

Downloads & links

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Map

FileDownload / File: emd_13255.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.859 Å
Density
Contour LevelBy AUTHOR: 0.004
Minimum - Maximum-0.03163788 - 0.06550156
Average (Standard dev.)9.9354096e-05 (±0.0013708891)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 219.904 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : E. coli MutL N-terminal dimer bound to a 3' resected DNA end

EntireName: E. coli MutL N-terminal dimer bound to a 3' resected DNA end
Components
  • Complex: E. coli MutL N-terminal dimer bound to a 3' resected DNA end
    • Complex: 3' resected DNA end
      • DNA: Template strandTranscription (biology)
      • DNA: Primer strand
    • Complex: E. coli MutL N-terminal dimer
      • Protein or peptide: DNA mismatch repair protein MutL
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: E. coli MutL N-terminal dimer bound to a 3' resected DNA end

SupramoleculeName: E. coli MutL N-terminal dimer bound to a 3' resected DNA end
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: 3' resected DNA end

SupramoleculeName: 3' resected DNA end / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: DNA molecule (others)
Recombinant expressionOrganism: synthetic construct (others)

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Supramolecule #3: E. coli MutL N-terminal dimer

SupramoleculeName: E. coli MutL N-terminal dimer / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: DNA mismatch repair protein MutL

MacromoleculeName: DNA mismatch repair protein MutL / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 68.005508 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPIQVLPPQL ANQIAAGEVV ERPASVVKEL VENSLDAGAT RIDIDIERGG AKLIRIRDNG CGIKKDELAL ALARHATSKI ASLDDLEAI ISLGFRGEAL ASISSVSRLT LTSRTAEQQE AWQAYAEGRD MNVTVKPAAH PVGTTLEVLD LFYNTPARRK F LRTEKTEF ...String:
MPIQVLPPQL ANQIAAGEVV ERPASVVKEL VENSLDAGAT RIDIDIERGG AKLIRIRDNG CGIKKDELAL ALARHATSKI ASLDDLEAI ISLGFRGEAL ASISSVSRLT LTSRTAEQQE AWQAYAEGRD MNVTVKPAAH PVGTTLEVLD LFYNTPARRK F LRTEKTEF NHIDEIIRRI ALARFDVTIN LSHNGKIVRQ YRAVPEGGQK ERRLGAICGT AFLEQALAIE WQHGDLTLRG WV ADPNHTT PALAEIQYCY VNGRMMRDRL INHAIRQACE DKLGADQQPA FVLYLEIDPH QVDVNVHPAK HEVRFHQSRL VHD FIYQGV LSVLQQQLET PLPLDDEPQP APRSIPENRV AAGRNHFAEP AAREPVAPRY TPAPASGSRP AAPWPNAQPG YQKQ QGEVY RQLLQTPAPM QKLKAPEPQE PALAANSQSF GRVLTIVHSD CALLERDGNI SLLSLPVAER WLRQAQLTPG EAPVC AQPL LIPLRLKVSA EEKSALEKAQ SALAELGIDF QSDAQHVTIR AVPLPLRQQN LQILIPELIG YLAKQSVFEP GNIAQW IAR NLMSEHAQWS MAQAITLLAD VERLCPQLVK TPPGGLLQSV DLHPAIKALK DE

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Macromolecule #2: Template strand

MacromoleculeName: Template strand / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 6.841428 KDa
SequenceString:
(DG)(DC)(DT)(DG)(DG)(DA)(DG)(DG)(DC)(DT) (DA)(DA)(DG)(DC)(DT)(DA)(DA)(DG)(DC)(DT) (DG)(DA)

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Macromolecule #3: Primer strand

MacromoleculeName: Primer strand / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 3.941584 KDa
SequenceString:
(DT)(DC)(DA)(DG)(DC)(DT)(DT)(DA)(DG)(DC) (DT)(DT)(DA)

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Macromolecule #4: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 2 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration0.4 mg/mL
BufferpH: 8.5
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3tris
150.0 mMNaClSodium chloridesodium chloride
5.0 mMMgCl2magnesium chloride
2.0 mMC4H10O2S2Dithiothreitol
0.01 %C58H114O26tween20
GridModel: Quantifoil R0.6/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.0002 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 76 % / Chamber temperature: 277.15 K / Instrument: LEICA PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 105000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 2 / Number real images: 539000 / Average electron dose: 54.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 539000
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: INSILICO MODEL
In silico model: model generated with initial model job type in RELION
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 149000
FSC plot (resolution estimation)

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