+Open data
-Basic information
Entry | Database: PDB / ID: 8qbl | ||||||
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Title | Retron-Eco1 filament with inactive effector (E106A, 2 segments) | ||||||
Components |
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Keywords | IMMUNE SYSTEM / retron / N-glycosidase / DNA-RNA-Protein complex | ||||||
Function / homology | Function and homology information RNA-directed DNA polymerase / RNA-directed DNA polymerase activity / defense response to virus / RNA binding / metal ion binding Similarity search - Function | ||||||
Biological species | Escherichia coli BL21 (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.66 Å | ||||||
Authors | Carabias del Rey, A. / Montoya, G. | ||||||
Funding support | Denmark, 1items
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Citation | Journal: Mol Cell / Year: 2024 Title: Retron-Eco1 assembles NAD-hydrolyzing filaments that provide immunity against bacteriophages. Authors: Arturo Carabias / Sarah Camara-Wilpert / Mario Rodríguez Mestre / Blanca Lopéz-Méndez / Ivo A Hendriks / Ruiliang Zhao / Tillmann Pape / Anders Fuglsang / Sean Hoi-Ching Luk / Michael L ...Authors: Arturo Carabias / Sarah Camara-Wilpert / Mario Rodríguez Mestre / Blanca Lopéz-Méndez / Ivo A Hendriks / Ruiliang Zhao / Tillmann Pape / Anders Fuglsang / Sean Hoi-Ching Luk / Michael L Nielsen / Rafael Pinilla-Redondo / Guillermo Montoya / Abstract: Retrons are toxin-antitoxin systems protecting bacteria against bacteriophages via abortive infection. The Retron-Eco1 antitoxin is formed by a reverse transcriptase (RT) and a non-coding RNA (ncRNA) ...Retrons are toxin-antitoxin systems protecting bacteria against bacteriophages via abortive infection. The Retron-Eco1 antitoxin is formed by a reverse transcriptase (RT) and a non-coding RNA (ncRNA)/multi-copy single-stranded DNA (msDNA) hybrid that neutralizes an uncharacterized toxic effector. Yet, the molecular mechanisms underlying phage defense remain unknown. Here, we show that the N-glycosidase effector, which belongs to the STIR superfamily, hydrolyzes NAD during infection. Cryoelectron microscopy (cryo-EM) analysis shows that the msDNA stabilizes a filament that cages the effector in a low-activity state in which ADPr, a NAD hydrolysis product, is covalently linked to the catalytic E106 residue. Mutations shortening the msDNA induce filament disassembly and the effector's toxicity, underscoring the msDNA role in immunity. Furthermore, we discovered a phage-encoded Retron-Eco1 inhibitor (U56) that binds ADPr, highlighting the intricate interplay between retron systems and phage evolution. Our work outlines the structural basis of Retron-Eco1 defense, uncovering ADPr's pivotal role in immunity. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8qbl.cif.gz | 1 MB | Display | PDBx/mmCIF format |
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PDB format | pdb8qbl.ent.gz | 794.8 KB | Display | PDB format |
PDBx/mmJSON format | 8qbl.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8qbl_validation.pdf.gz | 1.6 MB | Display | wwPDB validaton report |
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Full document | 8qbl_full_validation.pdf.gz | 1.6 MB | Display | |
Data in XML | 8qbl_validation.xml.gz | 112.7 KB | Display | |
Data in CIF | 8qbl_validation.cif.gz | 180.4 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/qb/8qbl ftp://data.pdbj.org/pub/pdb/validation_reports/qb/8qbl | HTTPS FTP |
-Related structure data
Related structure data | 18314MC 8qbkC 8qbmC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Protein , 2 types, 11 molecules AIKPUZGEFHT
#1: Protein | Mass: 39984.391 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3) / Gene: ret Production host: Escherichia coli str. K-12 substr. MG1655 (bacteria) Strain (production host): Escherichia coli str. K-12 substr. MG1655 (DE3) References: UniProt: P23070 #4: Protein | Mass: 35480.207 Da / Num. of mol.: 5 / Mutation: E106A Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3) / Gene: LM2_00875 Production host: Escherichia coli str. K-12 substr. MG1655 (bacteria) Strain (production host): Escherichia coli str. K-12 substr. MG1655 (DE3) References: UniProt: P0DV88 |
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-RNA chain , 2 types, 12 molecules CMOWbRDNSXYc
#2: RNA chain | Mass: 26199.410 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3) Production host: Escherichia coli str. K-12 substr. MG1655 (bacteria) Strain (production host): Escherichia coli str. K-12 substr. MG1655 (DE3) References: GenBank: 296142109 #3: RNA chain | Mass: 4541.771 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3) Production host: Escherichia coli str. K-12 substr. MG1655 (bacteria) Strain (production host): Escherichia coli str. K-12 substr. MG1655 (DE3) |
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-DNA chain , 1 types, 6 molecules LBJQVa
#5: DNA chain | Mass: 26320.875 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3) Production host: Escherichia coli str. K-12 substr. MG1655 (bacteria) Strain (production host): Escherichia coli str. K-12 substr. MG1655 (DE3) References: GenBank: 296142109 |
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-Non-polymers , 2 types, 9 molecules
#6: Chemical | ChemComp-MG / #7: Chemical | ChemComp-NAD / |
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-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Filamentous assembly of Retron-Eco1-E106A mutant containing RT-msr-msDNA-Effector complex (2 segments) Type: COMPLEX Details: Filament as purified from the bacteria after overexpression. No ligand added. Entity ID: #1-#5 / Source: RECOMBINANT | ||||||||||||||||||||
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Molecular weight | Value: 21 kDa/nm / Experimental value: NO | ||||||||||||||||||||
Source (natural) | Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: Escherichia coli BL21(DE3) | ||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli str. K-12 substr. MG1655 (bacteria) Strain: Escherichia coli str. K-12 substr. MG1655 (DE3) | ||||||||||||||||||||
Buffer solution | pH: 7.5 Details: 20 mM HEPES (pH 7.5),200 mM KCl, 5 mM MgCl2, 1mM TCEP | ||||||||||||||||||||
Buffer component |
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Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: The complex was prepared at a concentration corresponding to A260nm = ~9 | ||||||||||||||||||||
Specimen support | Details: 10 mAmp / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 | ||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Blotting time 3 seconds |
-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: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 1900 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN |
Image recording | Electron dose: 41 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 7250 |
Image scans | Width: 4096 / Height: 4096 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1474697 | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 358067 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: REAL / Details: in ChimeraX | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | Accession code: P23070 / Source name: AlphaFold / Type: in silico model | ||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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