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- PDB-9np6: Cryo-EM structure of AdnA(D934A)-AdnB(D1014A) in complex with AMP... -

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Basic information

Entry
Database: PDB / ID: 9np6
TitleCryo-EM structure of AdnA(D934A)-AdnB(D1014A) in complex with AMPPNP and blunt end DNA
Components
  • (DNA 3'-5' helicase) x 2
  • DNA (30-MER)
KeywordsISOMERASE/DNA / ATPase / helicase / DNA / ISOMERASE-DNA complex
Function / homology
Function and homology information


DNA helicase complex / recombinational repair / exonuclease activity / 3'-5' DNA helicase activity / DNA 3'-5' helicase / DNA binding / ATP binding / cytosol
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 / PD-(D/E)XK endonuclease-like domain superfamily / UvrD/REP helicase N-terminal domain / UvrD-like DNA helicase ATP-binding domain profile. / DNA helicase, UvrD/REP type ...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 / PD-(D/E)XK endonuclease-like domain superfamily / UvrD/REP helicase N-terminal domain / UvrD-like DNA helicase ATP-binding domain profile. / DNA helicase, UvrD/REP type / UvrD-like helicase, ATP-binding domain / Restriction endonuclease type II-like / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / IRON/SULFUR CLUSTER / DNA / DNA (> 10) / DNA 3'-5' helicase / DNA 3'-5' helicase
Similarity search - Component
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsWarren, G.M. / De la Cruz, M.J. / Goldgur, Y. / Shuman, S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID) United States
CitationJournal: Nucleic Acids Res / Year: 2026
Title: Structure of an initiation complex of mycobacterial helicase-nuclease AdnAB at a blunt double-strand break reveals local melting that engages the 3' tracking strand at the ratchet pawl of the helicase motor.
Authors: Garrett M Warren / M Jason de la Cruz / Yehuda Goldgur / Stewart Shuman /
Abstract: Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting double-strand breaks. The AdnB subunit hydrolyzes ATP to drive single-nucleotide steps of ...Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting double-strand breaks. The AdnB subunit hydrolyzes ATP to drive single-nucleotide steps of 3'-to-5' translocation of AdnAB on the tracking DNA strand via a ratchet-like mechanism. AdnB Trp325, which makes a π stack on a nucleobase 5' of a flipped-out nucleoside, is the ratchet pawl without which ATP hydrolysis is mechanically futile. Here we report a cryo-EM (cryogenic electron microscopy) structure of AdnAB in complex with a blunt-ended DNA. AdnAB forms an initiation complex by melting five terminal base pairs and threading the splayed out 5' and 3' single strands into the AdnA nuclease domain and the AdnB motor domain, respectively. The melted 5-nucleotide 3' tracking strand is in-gear with respect to the ratchet and pawl of the AdnB motor. An analogous π-stacking aromatic pawl is present in the motor subunit of the bacterial end-resection enzymes Bacillus AddAB and Escherichia coli RecBCD. Our results highlight a common theme whereby binding of the motor-nuclease to a blunt DSB end, in the absence of ATP hydrolysis, is coupled to local melting of the terminal base pairs that suffices to engage the "clutch" of the motor on the tracking strand.
History
DepositionMar 11, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 19, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA 3'-5' helicase
B: DNA 3'-5' helicase
X: DNA (30-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)248,5486
Polymers247,1843
Non-polymers1,3643
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein DNA 3'-5' helicase / AdnA


Mass: 110986.648 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Gene: MSMEG_1941 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: A0QTR9, DNA 3'-5' helicase
#2: Protein DNA 3'-5' helicase / AdnB


Mass: 118084.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Gene: MSMEI_1900 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: I7FZ56, DNA 3'-5' helicase
#3: DNA chain DNA (30-MER)


Mass: 18112.617 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#5: Chemical ChemComp-SF4 / 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
Has protein modificationN

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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: AdnA(D934A)-AdnB(D1014A) in complex with AMPPNP and blunt end DNA
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria) / Strain: MC2 155
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Image recordingElectron dose: 52.9 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20_4459 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 104148 / Symmetry type: POINT
RefinementHighest resolution: 3.4 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00412684
ELECTRON MICROSCOPYf_angle_d0.65517477
ELECTRON MICROSCOPYf_dihedral_angle_d14.5682072
ELECTRON MICROSCOPYf_chiral_restr0.0422120
ELECTRON MICROSCOPYf_plane_restr0.0052134

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