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- EMDB-25408: Mfd DNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-25408
TitleMfd DNA complex
Map data
Sample
  • Complex: mfd complex with DNA
    • Protein or peptide: Transcription-repair-coupling factor
    • DNA: DNA (5'-D(P*TP*TP*GP*CP*CP*TP*CP*GP*CP*TP*GP*CP*CP*A)-3')
    • DNA: DNA (5'-D(P*TP*GP*GP*CP*GP*GP*CP*GP*AP*GP*GP*C)-3')
KeywordsDNA TRANSLOCASE / TRANSCRIPTION-COUPLED DNA REPAIR / HELICASE / ATPASE / DNA BINDING PROTEIN / HYDROLASE-DNA COMPLEX / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


transcription-coupled nucleotide-excision repair, DNA damage recognition / RNA polymerase core enzyme binding / nucleotide-excision repair, preincision complex assembly / DNA translocase activity / 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 / nucleotide-excision repair, preincision complex assembly / DNA translocase activity / 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
Transcription-repair-coupling factor
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.2 Å
AuthorsOakley AJ / Xu Z-Q
Funding support United States, 2 items
OrganizationGrant numberCountry
Australian Research Council (ARC)DP210100365 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1RM1GM130450 United States
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Mechanism of transcription modulation by the transcription-repair coupling factor.
Authors: Bishnu P Paudel / Zhi-Qiang Xu / Slobodan Jergic / Aaron J Oakley / Nischal Sharma / Simon H J Brown / James C Bouwer / Peter J Lewis / Nicholas E Dixon / Antoine M van Oijen / Harshad Ghodke /
Abstract: Elongation by RNA polymerase is dynamically modulated by accessory factors. The transcription-repair coupling factor (TRCF) recognizes paused/stalled RNAPs and either rescues transcription or ...Elongation by RNA polymerase is dynamically modulated by accessory factors. The transcription-repair coupling factor (TRCF) recognizes paused/stalled RNAPs and either rescues transcription or initiates transcription termination. Precisely how TRCFs choose to execute either outcome remains unclear. With Escherichia coli as a model, we used single-molecule assays to study dynamic modulation of elongation by Mfd, the bacterial TRCF. We found that nucleotide-bound Mfd converts the elongation complex (EC) into a catalytically poised state, presenting the EC with an opportunity to restart transcription. After long-lived residence in this catalytically poised state, ATP hydrolysis by Mfd remodels the EC through an irreversible process leading to loss of the RNA transcript. Further, biophysical studies revealed that the motor domain of Mfd binds and partially melts DNA containing a template strand overhang. The results explain pathway choice determining the fate of the EC and provide a molecular mechanism for transcription modulation by TRCF.
History
DepositionNov 11, 2021-
Header (metadata) releaseMay 25, 2022-
Map releaseMay 25, 2022-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25408.png

Downloads & links

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Map

FileDownload / File: emd_25408.map.gz / Format: CCP4 / Size: 70.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.74 Å
Density
Contour LevelBy AUTHOR: 0.0621
Minimum - Maximum-0.20870614 - 0.7557957
Average (Standard dev.)-0.00043559808 (±0.016569737)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions264264264
Spacing264264264
CellA=B=C: 195.36 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : mfd complex with DNA

EntireName: mfd complex with DNA
Components
  • Complex: mfd complex with DNA
    • Protein or peptide: Transcription-repair-coupling factor
    • DNA: DNA (5'-D(P*TP*TP*GP*CP*CP*TP*CP*GP*CP*TP*GP*CP*CP*A)-3')
    • DNA: DNA (5'-D(P*TP*GP*GP*CP*GP*GP*CP*GP*AP*GP*GP*C)-3')

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Supramolecule #1: mfd complex with DNA

SupramoleculeName: mfd complex with DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 130 KDa

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Macromolecule #1: Transcription-repair-coupling factor

MacromoleculeName: Transcription-repair-coupling factor / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Molecular weightTheoretical: 130.152422 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MPEQYRYTLP VKAGEQRLLG ELTGAACATL VAEIAERHAG PVVLIAPDMQ NALRLHDEIS QFTDQMVMNL ADWETLPYDS FSPHQDIIS SRLSTLYQLP TMQRGVLIVP VNTLMQRVCP HSFLHGHALV MKKGQRLSRD ALRTQLDSAG YRHVDQVMEH G EYATRGAL ...String:
MPEQYRYTLP VKAGEQRLLG ELTGAACATL VAEIAERHAG PVVLIAPDMQ NALRLHDEIS QFTDQMVMNL ADWETLPYDS FSPHQDIIS SRLSTLYQLP TMQRGVLIVP VNTLMQRVCP HSFLHGHALV MKKGQRLSRD ALRTQLDSAG YRHVDQVMEH G EYATRGAL LDLFPMGSEL PYRLDFFDDE IDSLRVFDVD SQRTLEEVEA INLLPAHEFP TDKAAIELFR SQWRDTFEVK RD PEHIYQQ VSKGTLPAGI EYWQPLFFSE PLPPLFSYFP ANTLLVNTGD LETSAERFQA DTLARFENRG VDPMRPLLPP QSL WLRVDE LFSELKNWPR VQLKTEHLPT KAANANLGFQ KLPDLAVQAQ QKAPLDALRK FLETFDGPVV FSVESEGRRE ALGE LLARI KIAPQRIMRL DEASDRGRYL MIGAAEHGFV DTVRNLALIC ESDLLGERVA RRRQDSRRTI NPDTLIRNLA ELHIG QPVV HLEHGVGRYA GMTTLEAGGI TGEYLMLTYA NDAKLYVPVS SLHLISRYAG GAEENAPLHK LGGDAWSRAR QKAAEK VRD VAAELLDIYA QRAAKEGFAF KHDREQYQLF CDSFPFETTP DQAQAINAVL SDMCQPLAMD RLVCGDVGFG KTEVAMR AA FLAVDNHKQV AVLVPTTLLA QQHYDNFRDR FANWPVRIEM ISRFRSAKEQ TQILAEVAEG KIDILIGTHK LLQSDVKF K DLGLLIVDEE HRFGVRHKER IKAMRANVDI LTLTATPIPR TLNMAMSGMR DLSIIATPPA RRLAVKTFVR EYDSMVVRE AILREILRGG QVYYLYNDVE NIQKAAERLA ELVPEARIAI GHGQMREREL ERVMNDFHHQ RFNVLVCTTI IETGIDIPTA NTIIIERAD HFGLAQLHQL RGRVGRSHHQ AYAWLLTPHP KAMTTDAQKR LEAIASLEDL GAGFALATHD LEIRGAGELL G EEQSGSME TIGFSLYMEL LENAVDALKA GREPSLEDLT SQQTEVELRM PSLLPDDFIP DVNTRLSFYK RIASAKTENE LE EIKVELI DRFGLLPDPA RTLLDIARLR QQAQKLGIRK LEGNEKGGVI EFAEKNHVNP AWLIGLLQKQ PQHYRLDGPT RLK FIQDLS ERKTRIEWVR QFMRELEENA IA

UniProtKB: Transcription-repair-coupling factor

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Macromolecule #2: DNA (5'-D(P*TP*TP*GP*CP*CP*TP*CP*GP*CP*TP*GP*CP*CP*A)-3')

MacromoleculeName: DNA (5'-D(P*TP*TP*GP*CP*CP*TP*CP*GP*CP*TP*GP*CP*CP*A)-3')
type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 17.597205 KDa
SequenceString: (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT) (DG)(DC)(DC)(DT)(DC)(DG) ...String:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT) (DG)(DC)(DC)(DT)(DC)(DG)(DC)(DT) (DG)(DC)(DC)(DG)(DT)(DC)(DG)(DC)(DC)(DA)

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Macromolecule #3: DNA (5'-D(P*TP*GP*GP*CP*GP*GP*CP*GP*AP*GP*GP*C)-3')

MacromoleculeName: DNA (5'-D(P*TP*GP*GP*CP*GP*GP*CP*GP*AP*GP*GP*C)-3') / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 5.607617 KDa
SequenceString:
(DT)(DG)(DG)(DC)(DG)(DA)(DC)(DG)(DG)(DC) (DA)(DG)(DC)(DG)(DA)(DG)(DG)(DC)

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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

Concentration1.6 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
20.0 mMTris/HCl
40.0 mMpotassium chlorideKCl
5.0 mMmagnesium chlorideMgCl2
1.0 mMbeta-mercaptoethanol
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 279 K / Instrument: FEI VITROBOT MARK IV
DetailsADP, NaF and AlCl3 were added to final concentrations of 2, 5 and 0.5 mM, respectively.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 472 / Average exposure time: 50.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.9000000000000001 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 110562
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6xeo

Final reconstructionNumber classes used: 3 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 5.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 31718
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial modelPDB ID:

6xeo


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Correlation coeficient
Output model

PDB-7ssg:
Mfd DNA complex

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