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- PDB-7mr3: Cryo-EM structure of RecBCD-DNA complex with docked RecBNuc and s... -

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Basic information

Entry
Database: PDB / ID: 7mr3
TitleCryo-EM structure of RecBCD-DNA complex with docked RecBNuc and stabilized RecD
Components
  • (DNA (60-MER)) x 2
  • RecBCD enzyme subunit RecB
  • RecBCD enzyme subunit RecC
  • RecBCD enzyme subunit RecD
KeywordsHYDROLASE/DNA / SF1 helicase / complex / DNA repair / motor protein / HYDROLASE-DNA complex
Function / homology
Function and homology information


exodeoxyribonuclease V / exodeoxyribonuclease V activity / exodeoxyribonuclease V complex / DNA 5'-3' helicase / DNA 3'-5' helicase / clearance of foreign intracellular DNA / DNA translocase activity / single-stranded DNA helicase activity / recombinational repair / 3'-5' DNA helicase activity ...exodeoxyribonuclease V / exodeoxyribonuclease V activity / exodeoxyribonuclease V complex / DNA 5'-3' helicase / DNA 3'-5' helicase / clearance of foreign intracellular DNA / DNA translocase activity / single-stranded DNA helicase activity / recombinational repair / 3'-5' DNA helicase activity / ATP-dependent activity, acting on DNA / DNA helicase activity / DNA endonuclease activity / helicase activity / double-strand break repair via homologous recombination / response to radiation / DNA recombination / DNA damage response / magnesium ion binding / ATP hydrolysis activity / DNA binding / ATP binding / cytosol
Similarity search - Function
RecBCD enzyme subunit RecB / RecBCD enzyme subunit RecD / RecBCD enzyme subunit RecC / RecC, C-terminal / RecBCD enzyme subunit RecD, N-terminal domain / : / Exodeoxyribonuclease V, gamma subunit / RecC C-terminal domain / RecBCD enzyme subunit RecD, N-terminal domain / PD-(D/E)XK endonuclease-like domain, AddAB-type ...RecBCD enzyme subunit RecB / RecBCD enzyme subunit RecD / RecBCD enzyme subunit RecC / RecC, C-terminal / RecBCD enzyme subunit RecD, N-terminal domain / : / Exodeoxyribonuclease V, gamma subunit / RecC C-terminal domain / RecBCD enzyme subunit RecD, N-terminal domain / PD-(D/E)XK endonuclease-like domain, AddAB-type / PD-(D/E)XK nuclease superfamily / DExx box DNA helicase domain superfamily / AAA domain / UvrD-like DNA helicase C-terminal domain profile. / UvrD-like DNA helicase, C-terminal / UvrD-like helicase C-terminal domain / UvrD-like helicase C-terminal domain / UvrD-like helicase C-terminal domain / UvrD/REP helicase N-terminal domain / UvrD-like helicase, ATP-binding domain / UvrD-like DNA helicase ATP-binding domain profile. / DNA helicase, UvrD/REP type / PD-(D/E)XK endonuclease-like domain superfamily / Restriction endonuclease type II-like / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA / DNA (> 10) / RecBCD enzyme subunit RecD / RecBCD enzyme subunit RecC / RecBCD enzyme subunit RecB
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsHao, L. / Zhang, R. / Lohman, T.M.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM045948 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM136632 United States
CitationJournal: J Mol Biol / Year: 2021
Title: Heterogeneity in E. coli RecBCD Helicase-DNA Binding and Base Pair Melting.
Authors: Linxuan Hao / Rui Zhang / Timothy M Lohman /
Abstract: E. coli RecBCD, a helicase/nuclease involved in double stranded (ds) DNA break repair, binds to a dsDNA end and melts out several DNA base pairs (bp) using only its binding free energy. We examined ...E. coli RecBCD, a helicase/nuclease involved in double stranded (ds) DNA break repair, binds to a dsDNA end and melts out several DNA base pairs (bp) using only its binding free energy. We examined RecBCD-DNA initiation complexes using thermodynamic and structural approaches. Measurements of enthalpy changes for RecBCD binding to DNA ends possessing pre-melted ssDNA tails of increasing length suggest that RecBCD interacts with ssDNA as long as 17-18 nucleotides and can melt at least 10-11 bp upon binding a blunt DNA end. Cryo-EM structures of RecBCD alone and in complex with a blunt-ended dsDNA show significant conformational heterogeneities associated with the RecB nuclease domain (RecB) and the RecD subunit. In the absence of DNA, 56% of RecBCD molecules show no density for the RecB nuclease domain, RecB, and all RecBCD molecules show only partial density for RecD. DNA binding reduces these conformational heterogeneities, with 63% of the molecules showing density for both RecD and RecB. This suggests that the RecB domain is dynamic and influenced by DNA binding. The major RecBCD-DNA structural class in which RecB is docked onto RecC shows melting of at least 11 bp from a blunt DNA end, much larger than previously observed. A second structural class in which RecB is not docked shows only four bp melted suggesting that RecBCD complexes transition between states with different extents of DNA melting and that the extent of melting regulates initiation of helicase activity.
History
DepositionMay 7, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 28, 2021Provider: repository / Type: Initial release
Revision 1.1Aug 11, 2021Group: Database references / Category: citation / database_2
Item: _citation.journal_volume / _citation.title ..._citation.journal_volume / _citation.title / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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  • Deposited structure unit
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Assembly

Deposited unit
B: RecBCD enzyme subunit RecB
C: RecBCD enzyme subunit RecC
D: RecBCD enzyme subunit RecD
X: DNA (60-MER)
A: DNA (60-MER)


Theoretical massNumber of molelcules
Total (without water)367,0595
Polymers367,0595
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: assay for oligomerization
TypeNameSymmetry operationNumber
identity operation1_5551
MethodPISA

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Components

#1: Protein RecBCD enzyme subunit RecB / Exodeoxyribonuclease V 135 kDa polypeptide / Exodeoxyribonuclease V beta chain / Exonuclease V ...Exodeoxyribonuclease V 135 kDa polypeptide / Exodeoxyribonuclease V beta chain / Exonuclease V subunit RecB / ExoV subunit RecB / Helicase/nuclease RecBCD subunit RecB


Mass: 134110.641 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: recB, ior, rorA, b2820, JW2788 / Production host: Escherichia coli (E. coli) / References: UniProt: P08394, exodeoxyribonuclease V
#2: Protein RecBCD enzyme subunit RecC / Exodeoxyribonuclease V 125 kDa polypeptide / Exodeoxyribonuclease V gamma chain / Exonuclease V ...Exodeoxyribonuclease V 125 kDa polypeptide / Exodeoxyribonuclease V gamma chain / Exonuclease V subunit RecC / ExoV subunit RecC


Mass: 128974.102 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: recC, b2822, JW2790 / Production host: Escherichia coli (E. coli) / References: UniProt: P07648, exodeoxyribonuclease V
#3: Protein RecBCD enzyme subunit RecD / Exodeoxyribonuclease V 67 kDa polypeptide / Exodeoxyribonuclease V alpha chain / Exonuclease V ...Exodeoxyribonuclease V 67 kDa polypeptide / Exodeoxyribonuclease V alpha chain / Exonuclease V subunit RecD / ExoV subunit RecD / Helicase/nuclease RecBCD subunit RecD


Mass: 66990.367 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: recD, hopE, b2819, JW2787 / Production host: Escherichia coli (E. coli) / References: UniProt: P04993, exodeoxyribonuclease V
#4: DNA chain DNA (60-MER)


Mass: 18482.836 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Synthetic construct (others)
#5: DNA chain DNA (60-MER)


Mass: 18500.861 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: RecBCD heterotrimer in complex with 60bp blunt-ended dsDNA
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Source (natural)Organism: Escherichia coli K12 (bacteria) / Strain: K12
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-hydrochlorideTris-HClTris1
250 mMSodium chlorideNaClSodium chloride1
34 mMMagnesium chlorideMgCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm
Image recordingElectron dose: 49.5 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k)
Image scansWidth: 3838 / Height: 3710

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
EM software
IDNameCategory
4RELIONCTF correction
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 45202 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00524261
ELECTRON MICROSCOPYf_angle_d0.8233099
ELECTRON MICROSCOPYf_dihedral_angle_d12.5433558
ELECTRON MICROSCOPYf_chiral_restr0.053635
ELECTRON MICROSCOPYf_plane_restr0.0064204

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