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- PDB-6xeo: Structure of Mfd bound to dsDNA -

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Basic information

Entry
Database: PDB / ID: 6xeo
TitleStructure of Mfd bound to dsDNA
Components
  • DNA (5'-D(P*AP*GP*GP*AP*TP*AP*CP*TP*TP*AP*CP*AP*GP*CP*CP*AP*TP*C)-3')
  • DNA (5'-D(P*GP*AP*TP*GP*GP*CP*TP*GP*TP*AP*AP*GP*TP*AP*TP*CP*CP*T)-3')
  • Transcription-repair-coupling factor
KeywordsDNA BINDING PROTEIN / Hydrolase/DNA / DNA translocase / transcription-coupled DNA repair / helicase / ATPase / Hydrolase-DNA complex
Function / homology
Function and homology information


transcription-coupled nucleotide-excision repair, DNA damage recognition / RNA polymerase core enzyme binding / DNA translocase activity / nucleotide-excision repair, preincision complex assembly / DNA repair complex / transcription-coupled nucleotide-excision repair / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / damaged DNA binding / hydrolase activity / DNA repair ...transcription-coupled nucleotide-excision repair, DNA damage recognition / RNA polymerase core enzyme binding / DNA translocase activity / nucleotide-excision repair, preincision complex assembly / DNA repair complex / transcription-coupled nucleotide-excision repair / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / damaged DNA binding / hydrolase activity / DNA repair / DNA damage response / regulation of DNA-templated transcription / DNA binding / ATP binding / cytosol
Similarity search - Function
: / MFD, D3 domain / : / Transcription-repair coupling factor / Transcription-repair-coupling factor, C-terminal domain / TRCF-like, C-terminal D7 domain / TRCF domain / TRCF / UvrB, interaction domain / UvrB interaction domain ...: / MFD, D3 domain / : / Transcription-repair coupling factor / Transcription-repair-coupling factor, C-terminal domain / TRCF-like, C-terminal D7 domain / TRCF domain / TRCF / UvrB, interaction domain / UvrB interaction domain / CarD-like/TRCF, RNAP-interacting domain / CarD-like/TRCF, RNAP-interacting domain superfamily / CarD-like/TRCF RID domain / CarD-like/TRCF domain / DEAD/DEAH box helicase / DEAD/DEAH box helicase domain / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA / DNA (> 10) / Transcription-repair-coupling factor
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.5 Å
AuthorsBrugger, C. / Deaconescu, A.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)DP5 OD021396 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM121975 United States
CitationJournal: Nat Commun / Year: 2020
Title: Molecular determinants for dsDNA translocation by the transcription-repair coupling and evolvability factor Mfd.
Authors: Christiane Brugger / Cheng Zhang / Margaret M Suhanovsky / David D Kim / Amy N Sinclair / Dmitry Lyumkis / Alexandra M Deaconescu /
Abstract: Mfd couples transcription to nucleotide excision repair, and acts on RNA polymerases when elongation is impeded. Depending on impediment severity, this action results in either transcription ...Mfd couples transcription to nucleotide excision repair, and acts on RNA polymerases when elongation is impeded. Depending on impediment severity, this action results in either transcription termination or elongation rescue, which rely on ATP-dependent Mfd translocation on DNA. Due to its role in antibiotic resistance, Mfd is also emerging as a prime target for developing anti-evolution drugs. Here we report the structure of DNA-bound Mfd, which reveals large DNA-induced structural changes that are linked to the active site via ATPase motif VI. These changes relieve autoinhibitory contacts between the N- and C-termini and unmask UvrA recognition determinants. We also demonstrate that translocation relies on a threonine in motif Ic, widely conserved in translocases, and a family-specific histidine near motif IVa, reminiscent of the "arginine clamp" of RNA helicases. Thus, Mfd employs a mode of DNA recognition that at its core is common to ss/ds translocases that act on DNA or RNA.
History
DepositionJun 12, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 19, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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  • EMDB-22146
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Transcription-repair-coupling factor
B: DNA (5'-D(P*AP*GP*GP*AP*TP*AP*CP*TP*TP*AP*CP*AP*GP*CP*CP*AP*TP*C)-3')
C: DNA (5'-D(P*GP*AP*TP*GP*GP*CP*TP*GP*TP*AP*AP*GP*TP*AP*TP*CP*CP*T)-3')


Theoretical massNumber of molelcules
Total (without water)144,5023
Polymers144,5023
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Transcription-repair-coupling factor / TRCF


Mass: 132524.062 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: mfd, b1114, JW1100 / Production host: Escherichia coli (E. coli)
References: UniProt: P30958, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#2: DNA chain DNA (5'-D(P*AP*GP*GP*AP*TP*AP*CP*TP*TP*AP*CP*AP*GP*CP*CP*AP*TP*C)-3')


Mass: 6431.186 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(P*GP*AP*TP*GP*GP*CP*TP*GP*TP*AP*AP*GP*TP*AP*TP*CP*CP*T)-3')


Mass: 5546.600 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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

ComponentName: complex of Mfd bound to dsDNA in the presence of transition state analog ADP-AlFx
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.15 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMsodium chlorideNaClSodium chloride1
220 mMmagnesium chlorideMgCl21
32 mMTCEP1
420 mMTris-HClTris1
51
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Chamber temperature: 277 K
Details: 2.5ul Mfd-DNA complex at 1.0 mg/mL was applied to UltrAuFoil R1.2/1.3 300 mesh grids (Quantifoil) that were previously plasma-cleaned using a Gatan Solarus (75% argon/25% oxygen atmosphere, ...Details: 2.5ul Mfd-DNA complex at 1.0 mg/mL was applied to UltrAuFoil R1.2/1.3 300 mesh grids (Quantifoil) that were previously plasma-cleaned using a Gatan Solarus (75% argon/25% oxygen atmosphere, 15 W for 7s), then manually blotted with a Whatman No. 1 filter paper in a cold room with >80% humidity, and plunged into liquid ethane using a manual plunger

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 36000 X / Nominal defocus max: 3.5 nm / Nominal defocus min: 1.9 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 24 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 652
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 60 / Used frames/image: 1-60

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Processing

EM software
IDNameVersionCategory
1Warpparticle selection
2Leginonimage acquisition
4MotionCorr2CTF correction
7PHENIX1.15.2_3472model fitting
9RELIONinitial Euler assignment
10cisTEMfinal Euler assignment
12cisTEM3D reconstruction
13PHENIX1.15.2_3472model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 5.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 9822 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 2EYQ

2eyq


Pdb chain-ID: A / Accession code: 2EYQ / Pdb chain residue range: 5-1147 / Source name: PDB / Type: experimental model

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