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- PDB-7sjr: Cryo-EM structure of AdnA-AdnB(W325A) in complex with DNA and AMPPNP -

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Basic information

Entry
Database: PDB / ID: 7sjr
TitleCryo-EM structure of AdnA-AdnB(W325A) in complex with DNA and AMPPNP
Components
  • (DNA helicase) x 2
  • DNA (70-MER)
KeywordsDNA BINDING PROTEIN/DNA / homologous recombination / DNA end resection / cryoelectron microscopy / DNA curtain / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


DNA 3'-5' helicase / exonuclease activity / DNA helicase activity / isomerase 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 / PD-(D/E)XK endonuclease-like domain superfamily / 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 / PD-(D/E)XK endonuclease-like domain superfamily / 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 helicase / DNA 3'-5' helicase
Similarity search - Component
Biological speciesMycolicibacterium smegmatis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsWang, J. / Warren, G.M. / Shuman, S. / Patel, D.J.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)P30CA008748 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI64693 United States
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Structure-activity relationships at a nucleobase-stacking tryptophan required for chemomechanical coupling in the DNA resecting motor-nuclease AdnAB.
Authors: Garrett M Warren / Aviv Meir / Juncheng Wang / Dinshaw J Patel / Eric C Greene / Stewart Shuman /
Abstract: Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks. The AdnB subunit hydrolyzes ATP to drive single-nucleotide ...Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA 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. Trp325 in AdnB motif III, which intercalates into the tracking strand and makes a π stack on a nucleobase 5' of a flipped-out nucleoside, is the putative ratchet pawl without which ATP hydrolysis is mechanically futile. Here, we report that AdnAB mutants wherein Trp325 was replaced with phenylalanine, tyrosine, histidine, leucine, or alanine retained activity in ssDNA-dependent ATP hydrolysis but displayed a gradient of effects on DSB resection. The resection velocities of Phe325 and Tyr325 mutants were 90% and 85% of the wild-type AdnAB velocity. His325 slowed resection rate to 3% of wild-type and Leu325 and Ala325 abolished DNA resection. A cryo-EM structure of the DNA-bound Ala325 mutant revealed that the AdnB motif III peptide was disordered and the erstwhile flipped out tracking strand nucleobase reverted to a continuous base-stacked arrangement with its neighbors. We conclude that π stacking of Trp325 on a DNA nucleobase triggers and stabilizes the flipped-out conformation of the neighboring nucleoside that underlies formation of a ratchet pawl.
History
DepositionOct 18, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 22, 2021Provider: repository / Type: Initial release
Revision 1.1Jan 12, 2022Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Feb 9, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID
Revision 1.3Jun 5, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-25164
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Structure viewerMolecule:
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Assembly

Deposited unit
B: DNA helicase
A: DNA helicase
C: DNA (70-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)251,4046
Polymers250,5223
Non-polymers8823
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules BA

#1: Protein DNA helicase


Mass: 118013.406 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis (bacteria) / Gene: pcrA, BIN_B_03433 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A653FJ17, DNA helicase
#2: Protein DNA helicase


Mass: 111030.656 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis (bacteria) / Gene: pcrA_1, ERS451418_01973 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A0D6HKQ2, DNA helicase

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DNA chain , 1 types, 1 molecules C

#3: DNA chain DNA (70-MER)


Mass: 21477.703 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mycolicibacterium smegmatis (bacteria)

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Non-polymers , 3 types, 3 molecules

#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


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM

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Details

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

ComponentName: Cryo-EM map of AdnA-AdnB(W325A) in complex with DNA and AMPPNP
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R1.2/1.3
VitrificationCryogen 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 FIELD / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 53 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 98408 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6PPJ

6ppj


Accession code: 6PPJ / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00514001
ELECTRON MICROSCOPYf_angle_d0.70419215
ELECTRON MICROSCOPYf_dihedral_angle_d17.3422207
ELECTRON MICROSCOPYf_chiral_restr0.0442261
ELECTRON MICROSCOPYf_plane_restr0.0052377

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