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- PDB-6ppu: Cryo-EM structure of AdnAB-AMPPNP-DNA complex -

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Basic information

Entry
Database: PDB / ID: 6ppu
TitleCryo-EM structure of AdnAB-AMPPNP-DNA complex
Components
  • ATP-dependent DNA helicase (UvrD/REP)
  • DNA (29-MER)
  • UvrD/REP helicase
KeywordsDNA BINDING PROTEIN/DNA / DNA / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


DNA 3'-5' helicase / DNA helicase activity / DNA helicase / hydrolase activity / DNA repair / DNA binding / ATP binding
Similarity search - Function
: / PD-(D/E)XK endonuclease-like domain, AddAB-type / PD-(D/E)XK nuclease superfamily / DExx box DNA helicase domain superfamily / UvrD-like DNA helicase C-terminal domain profile. / UvrD-like DNA helicase, C-terminal / 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. ...: / PD-(D/E)XK endonuclease-like domain, AddAB-type / PD-(D/E)XK nuclease superfamily / DExx box DNA helicase domain superfamily / UvrD-like DNA helicase C-terminal domain profile. / UvrD-like DNA helicase, C-terminal / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
IRON/SULFUR CLUSTER / DNA / DNA (> 10) / DNA helicase / DNA 3'-5' helicase / DNA 3'-5' helicase
Similarity search - Component
Biological speciesMycobacterium smegmatis (bacteria)
Mycolicibacterium smegmatis (bacteria)
Bacillus subtilis subsp. subtilis str. 168 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsJia, N. / Unciuleac, M. / Shuman, S. / Patel, D.J.
CitationJournal: Proc Natl Acad Sci U S A / Year: 2019
Title: Structures and single-molecule analysis of bacterial motor nuclease AdnAB illuminate the mechanism of DNA double-strand break resection.
Authors: Ning Jia / Mihaela C Unciuleac / Chaoyou Xue / Eric C Greene / Dinshaw J Patel / Stewart Shuman /
Abstract: Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N- ...Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Here we report cryoelectron microscopy (cryo-EM) structures of AdnAB in three functional states: in the absence of DNA and in complex with forked duplex DNAs before and after cleavage of the 5' single-strand DNA (ssDNA) tail by the AdnA nuclease. The structures reveal the path of the 5' ssDNA through the AdnA nuclease domain and the mechanism of 5' strand cleavage; the path of the 3' tracking strand through the AdnB motor and the DNA contacts that couple ATP hydrolysis to mechanical work; the position of the AdnA iron-sulfur cluster subdomain at the Y junction and its likely role in maintaining the split trajectories of the unwound 5' and 3' strands. Single-molecule DNA curtain analysis of DSB resection reveals that AdnAB is highly processive but prone to spontaneous pausing at random sites on duplex DNA. A striking property of AdnAB is that the velocity of DSB resection slows after the enzyme experiences a spontaneous pause. Our results highlight shared as well as distinctive properties of AdnAB vis-à-vis the RecBCD and AddAB clades of bacterial DSB-resecting motor nucleases.
History
DepositionJul 8, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 20, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 4, 2019Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Dec 18, 2019Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Mar 20, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
B: UvrD/REP helicase
A: ATP-dependent DNA helicase (UvrD/REP)
X: DNA (29-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)216,0325
Polymers215,6563
Non-polymers3762
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14110 Å2
ΔGint-130 kcal/mol
Surface area69420 Å2

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Components

#1: Protein UvrD/REP helicase


Mass: 118128.547 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155 / Gene: MSMEI_1900 / Production host: Escherichia coli (E. coli) / References: UniProt: I7FZ56
#2: Protein ATP-dependent DNA helicase (UvrD/REP)


Mass: 76049.312 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis (bacteria), (gene. exp.) Mycobacterium smegmatis (bacteria)
Gene: pcrA_1, ERS451418_01973 / Production host: Escherichia coli (E. coli)
References: UniProt: A0A0D6HKQ2, UniProt: A0QTR9*PLUS, DNA helicase
#3: DNA chain DNA (29-MER)


Mass: 21477.703 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Bacillus subtilis subsp. subtilis str. 168 (bacteria)
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER / Iron–sulfur cluster


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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

Component
IDNameTypeEntity IDParent-IDSource
1AdnAB-AMPPNP-DNA complexCOMPLEX#1-#30RECOMBINANT
2UvrD/REP helicaseCOMPLEX1RECOMBINANT
3ATP-dependent DNA helicase (UvrD/REP)COMPLEX1RECOMBINANT
4DNACOMPLEX1RECOMBINANT
Molecular weightValue: 0.2 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Mycolicibacterium smegmatis (bacteria)246196
23Mycolicibacterium smegmatis (bacteria)1772
34Bacillus subtilis subsp. subtilis str. 168 (bacteria)224308
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23Escherichia coli (E. coli)562
Buffer solutionpH: 7.5 / Details: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl
Buffer componentFormula: Tris
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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
Image recordingElectron dose: 2.16 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM softwareName: RELION / Version: 2.1 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 60108 / Symmetry type: POINT

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