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- PDB-8vxy: Structure of HamA(E138A,K140A)B-plasmid DNA complex from the Esch... -

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Basic information

Entry
Database: PDB / ID: 8vxy
TitleStructure of HamA(E138A,K140A)B-plasmid DNA complex from the Escherichia coli Hachiman defense system
Components
  • (Plasmid DNA) x 2
  • HamA
  • HamB
KeywordsIMMUNE SYSTEM / Nuclease-helicase complex / antiphage defense system
Function / homology
Function and homology information


helicase activity / nucleic acid binding / hydrolase activity / ATP binding
Similarity search - Function
Anti-bacteriophage protein A/HamA, C-terminal domain / HamA / : / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal ...Anti-bacteriophage protein A/HamA, C-terminal domain / HamA / : / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DEAD/DEAH box helicase / DUF1837 domain-containing protein
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.19 Å
AuthorsTuck, O.T. / Hu, J.J. / Doudna, J.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States) United States
CitationJournal: bioRxiv / Year: 2024
Title: Hachiman is a genome integrity sensor.
Authors: Owen T Tuck / Benjamin A Adler / Emily G Armbruster / Arushi Lahiri / Jason J Hu / Julia Zhou / Joe Pogliano / Jennifer A Doudna /
Abstract: Hachiman is a broad-spectrum antiphage defense system of unknown function. We show here that Hachiman comprises a heterodimeric nuclease-helicase complex, HamAB. HamA, previously a protein of unknown ...Hachiman is a broad-spectrum antiphage defense system of unknown function. We show here that Hachiman comprises a heterodimeric nuclease-helicase complex, HamAB. HamA, previously a protein of unknown function, is the effector nuclease. HamB is the sensor helicase. HamB constrains HamA activity during surveillance of intact dsDNA. When the HamAB complex detects DNA damage, HamB helicase activity liberates HamA, unleashing nuclease activity. Hachiman activation degrades all DNA in the cell, creating 'phantom' cells devoid of both phage and host DNA. We demonstrate Hachiman activation in the absence of phage by treatment with DNA-damaging agents, suggesting that Hachiman responds to aberrant DNA states. Phylogenetic similarities between the Hachiman helicase and eukaryotic enzymes suggest this bacterial immune system has been repurposed for diverse functions across all domains of life.
History
DepositionFeb 6, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 13, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 20, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: Plasmid DNA
D: Plasmid DNA
A: HamA
B: HamB
hetero molecules


Theoretical massNumber of molelcules
Total (without water)179,2635
Polymers178,7564
Non-polymers5071
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: DNA chain Plasmid DNA


Mass: 9385.020 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: unknown sequence / Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli)
#2: DNA chain Plasmid DNA


Mass: 9664.453 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: unknown sequence / Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli)
#3: Protein HamA / VrlR-like protein / DUF1837 domain-containing protein


Mass: 28807.664 Da / Num. of mol.: 1 / Mutation: E138A,K140A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: ECOR31 / Gene: HamA / Plasmid: pGEX / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): AI / References: UniProt: Q6XGE5
#4: Protein HamB


Mass: 130898.453 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: ECOR31 / Gene: HamB / Plasmid: pGEX / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): AI / References: UniProt: A0A426EXV0
#5: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
Has ligand of interestY
Sequence detailsThe DNA sequence is unknown.

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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: HamA(E138A,K140A)B-plasmid DNA complex / Type: COMPLEX / Entity ID: #1, #3-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: ECOR31
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21-AI / Plasmid: pGEX
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2100 mMpotassium chlorideKCl1
31 mMTCEPC9H15O6P1
40.5 %glycerolC3H8O31
52 mMmagnesium chlorideMgCl21
61 mMadenosine triphosphateC10H16N5O13P31
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3724

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.2.1particle selection
2SerialEM4.0.19image acquisition
4cryoSPARC4.2.1CTF correctionpatch ctf
7Coot0.9.8.7model fitting
9PHENIX1.19.2-4158model refinement
10cryoSPARC4.2.1initial Euler assignment
11cryoSPARC4.2.1final Euler assignment
12cryoSPARC4.2.1classification
13cryoSPARC4.2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 16398369
3D reconstructionResolution: 3.19 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 29904 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model buildingDetails: v1.4.0 / Source name: AlphaFold / Type: in silico model

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