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- EMDB-22146: Structure of Mfd bound to dsDNA -

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Basic information

Entry
Database: EMDB / ID: EMD-22146
TitleStructure of Mfd bound to dsDNA
Map data
Samplecomplex of Mfd bound to dsDNA in the presence of transition state analog ADP-AlFx:
Transcription-repair-coupling factor / (nucleic-acidNucleic acid) x 2
Function / homology
Function and homology information


transcription-coupled nucleotide-excision repair, DNA damage recognition / RNA polymerase core enzyme binding / DNA translocase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / transcription-coupled nucleotide-excision repair / damaged DNA binding / hydrolase activity / DNA repair / cellular response to DNA damage stimulus / regulation of transcription, DNA-templated ...transcription-coupled nucleotide-excision repair, DNA damage recognition / RNA polymerase core enzyme binding / DNA translocase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / transcription-coupled nucleotide-excision repair / damaged DNA binding / hydrolase activity / DNA repair / cellular response to DNA damage stimulus / regulation of transcription, DNA-templated / DNA binding / ATP binding / cytosol
Similarity search - Function
Transcription-repair-coupling factor, C-terminal domain / TRCF / TRCF domain / Transcription-repair coupling factor / TRCF-like, C-terminal D7 domai / UvrB interaction domain / UvrB, interaction domain / CarD-like/TRCF domain / CarD-like/TRCF domain / CarD-like/TRCF domain ...Transcription-repair-coupling factor, C-terminal domain / TRCF / TRCF domain / Transcription-repair coupling factor / TRCF-like, C-terminal D7 domai / UvrB interaction domain / UvrB, interaction domain / CarD-like/TRCF domain / CarD-like/TRCF domain / CarD-like/TRCF domain / CarD-like/TRCF domain superfamily / 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. / Helicase, C-terminal / DEAD-like helicases superfamily / 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) / Escherichia coli (strain K12) (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.5 Å
AuthorsZhang C / Lyumkis D
Funding support United States, 2 items
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, 2020-
Header (metadata) releaseAug 19, 2020-
Map releaseAug 19, 2020-
UpdateAug 19, 2020-
Current statusAug 19, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.7
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 2.7
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6xeo
  • Surface level: 2.7
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_22146.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 240 pix.
= 276. Å
1.15 Å/pix.
x 240 pix.
= 276. Å
1.15 Å/pix.
x 240 pix.
= 276. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.15 Å
Density
Contour LevelBy AUTHOR: 1.5 / Movie #1: 2.7
Minimum - Maximum-4.0549855 - 10.698584
Average (Standard dev.)0.004318794 (±0.45324782)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 276.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z276.000276.000276.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ510510510
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-4.05510.6990.004

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Supplemental data

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Half map: half map 1

Fileemd_22146_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: half map 2

Fileemd_22146_half_map_2.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire complex of Mfd bound to dsDNA in the presence of transition state...

EntireName: complex of Mfd bound to dsDNA in the presence of transition state analog ADP-AlFx
Number of Components: 4

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Component #1: protein, complex of Mfd bound to dsDNA in the presence of transit...

ProteinName: complex of Mfd bound to dsDNA in the presence of transition state analog ADP-AlFx
Recombinant expression: No
MassTheoretical: 150 kDa
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, Transcription-repair-coupling factor

ProteinName: Transcription-repair-coupling factor / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 132.524062 kDa
SourceSpecies: Escherichia coli (strain K12) (bacteria) / Strain: K12
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: nucleic-acid, DNA (5'-D(P*AP*GP*GP*AP*TP*AP*CP*TP*TP*AP*CP*AP*GP*...

nucleic acidName: DNA (5'-D(P*AP*GP*GP*AP*TP*AP*CP*TP*TP*AP*CP*AP*GP*CP*CP*AP*TP*C)-3')
Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DA)(DT)(DA)(DG)(DG)(DA)(DT)(DA)(DC)(DT) (DT)(DA)(DC)(DA)(DG)(DC)(DC)(DA)(DT)(DC) (DG)
MassTheoretical: 6.431186 kDa
SourceSpecies: synthetic construct (others)

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Component #4: nucleic-acid, DNA (5'-D(P*GP*AP*TP*GP*GP*CP*TP*GP*TP*AP*AP*GP*TP*...

nucleic acidName: DNA (5'-D(P*GP*AP*TP*GP*GP*CP*TP*GP*TP*AP*AP*GP*TP*AP*TP*CP*CP*T)-3')
Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DG)(DA)(DT)(DG)(DG)(DC)(DT)(DG)(DT)(DA) (DA)(DG)(DT)(DA)(DT)(DC)(DC)(DT)
MassTheoretical: 5.5466 kDa
SourceSpecies: synthetic construct (others)

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Experimental details

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Sample preparation

SpecimenSpecimen State: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 1 mg/mL / pH: 8
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen Name: ETHANE / 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
ImagingMicroscope: FEI TALOS ARCTICA
Electron gunElectron Source: FIELD EMISSION GUN / Accelerating Voltage: 200 kV / Electron Dose: 24 e/Å2 / Illumination Mode: FLOOD BEAM
LensMagnification: 36000 X (nominal) / Cs: 2.7 mm / Imaging Mode: BRIGHT FIELD / Defocus: 1.9 - 3.5 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of Digital Images: 652

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Image processing

ProcessingMethod: single particle reconstruction / Applied Symmetry: C1 (asymmetric) / Number of Projections: 9822
3D reconstructionAlgorithm: FOURIER SPACE / Software: cisTEM / Resolution: 5.5 Å / Resolution Method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Target Criteria: Correlation coefficient / Refinement space: REAL
Input PDB model: 2EYQ
Chain ID: A
Output model

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