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- PDB-6ppj: Cryo-EM structure of AdnA(D934A)-AdnB(D1014A) in complex with AMPPNP -
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Basic information
Entry | Database: PDB / ID: 6ppj | ||||||
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Title | Cryo-EM structure of AdnA(D934A)-AdnB(D1014A) in complex with AMPPNP | ||||||
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![]() | DNA BINDING PROTEIN | ||||||
Function / homology | ![]() DNA helicase complex / DNA 3'-5' helicase / recombinational repair / exonuclease activity / 3'-5' DNA helicase activity / DNA helicase activity / DNA helicase / hydrolase activity / DNA repair / DNA binding ...DNA helicase complex / DNA 3'-5' helicase / recombinational repair / exonuclease activity / 3'-5' DNA helicase activity / DNA helicase activity / DNA helicase / hydrolase activity / DNA repair / DNA binding / ATP binding / cytosol Similarity search - Function | ||||||
Biological species | ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å | ||||||
![]() | Jia, N. / Unciuleac, M. / Shuman, S. / Patel, D.J. | ||||||
![]() | ![]() 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. | ||||||
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-Validation report
Summary document | ![]() | 873.5 KB | Display | ![]() |
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Full document | ![]() | 891 KB | Display | |
Data in XML | ![]() | 51.6 KB | Display | |
Data in CIF | ![]() | 81.3 KB | Display | |
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-Related structure data
Related structure data | ![]() 20440MC ![]() 6pprC ![]() 6ppuC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Assembly
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Components
#1: Protein | Mass: 114735.938 Da / Num. of mol.: 1 / Mutation: D1014A Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() References: UniProt: A0A0D6HIW1, UniProt: A0QTS0*PLUS, DNA helicase |
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#2: Protein | Mass: 110899.562 Da / Num. of mol.: 1 / Mutation: D934A Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() References: UniProt: A0A0D6HKQ2, UniProt: A0QTR9*PLUS, DNA helicase |
#3: Chemical | ChemComp-SF4 / |
#4: Chemical | ChemComp-ANP / |
Has ligand of interest | Y |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: AdnAB-D934A-D1014A mutant in complex with AMPPNP / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT |
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Molecular weight | Value: 0.2 MDa / Experimental value: YES |
Source (natural) | Organism: ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 7.5 / Details: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl |
Buffer component | Formula: Tris |
Specimen | Conc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Instrument: 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 |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 2.16 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
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Processing
EM software | Name: RELION / Version: 2.1 / Category: 3D reconstruction |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION |
3D reconstruction | Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 80396 / Symmetry type: POINT |