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- EMDB-18314: Retron-Eco1 filament with inactive effector (E106A, 2 segments) -

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Basic information

Entry
Database: EMDB / ID: EMD-18314
TitleRetron-Eco1 filament with inactive effector (E106A, 2 segments)
Map dataRetron-Eco1-E106A filament (Local) SHARPENED MAP
Sample
  • Complex: Filamentous assembly of Retron-Eco1-E106A mutant containing RT-msr-msDNA-Effector complex (2 segments)
    • Protein or peptide: Retron Ec86 reverse transcriptase
    • RNA: Retron-Eco1-msr
    • RNA: Retron-Eco1-A2
    • Protein or peptide: Retron Ec86 putative ribosyltransferase/DNA-binding protein
    • DNA: Retron-Eco1 msDNA
  • Ligand: MAGNESIUM ION
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE
Keywordsretron / IMMUNE SYSTEM / 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
: / RNA-directed DNA polymerase (reverse transcriptase), msDNA / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Retron Ec86 putative ribosyltransferase/DNA-binding protein / Retron Ec86 reverse transcriptase
Similarity search - Component
Biological speciesEscherichia coli BL21(DE3) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.66 Å
AuthorsCarabias del Rey A / Montoya G
Funding support Denmark, 1 items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF14CC0001 Denmark
CitationJournal: 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
DepositionAug 24, 2023-
Header (metadata) releaseJun 5, 2024-
Map releaseJun 5, 2024-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18314.png

Downloads & links

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Map

FileDownload / File: emd_18314.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRetron-Eco1-E106A filament (Local) SHARPENED MAP
Voxel sizeX=Y=Z: 0.832 Å
Density
Contour LevelBy AUTHOR: 7.0
Minimum - Maximum-60.027614999999997 - 85.155540000000002
Average (Standard dev.)0.000000000002161 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 499.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Retron-Eco1-E106A filament (Local) SHARPENED MAP

Fileemd_18314_additional_1.map
AnnotationRetron-Eco1-E106A filament (Local) SHARPENED MAP
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Retron-Eco1-E106A filament (Local) Half Map A

Fileemd_18314_half_map_1.map
AnnotationRetron-Eco1-E106A filament (Local) Half Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Retron-Eco1-E106A filament (Local) Half Map B

Fileemd_18314_half_map_2.map
AnnotationRetron-Eco1-E106A filament (Local) Half Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Filamentous assembly of Retron-Eco1-E106A mutant containing RT-ms...

EntireName: Filamentous assembly of Retron-Eco1-E106A mutant containing RT-msr-msDNA-Effector complex (2 segments)
Components
  • Complex: Filamentous assembly of Retron-Eco1-E106A mutant containing RT-msr-msDNA-Effector complex (2 segments)
    • Protein or peptide: Retron Ec86 reverse transcriptase
    • RNA: Retron-Eco1-msr
    • RNA: Retron-Eco1-A2
    • Protein or peptide: Retron Ec86 putative ribosyltransferase/DNA-binding protein
    • DNA: Retron-Eco1 msDNA
  • Ligand: MAGNESIUM ION
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

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Supramolecule #1: Filamentous assembly of Retron-Eco1-E106A mutant containing RT-ms...

SupramoleculeName: Filamentous assembly of Retron-Eco1-E106A mutant containing RT-msr-msDNA-Effector complex (2 segments)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Details: Filament as purified from the bacteria after overexpression. No ligand added.
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: Escherichia coli BL21(DE3)
Molecular weightTheoretical: 21 kDa/nm

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Macromolecule #1: Retron Ec86 reverse transcriptase

MacromoleculeName: Retron Ec86 reverse transcriptase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
Molecular weightTheoretical: 39.984391 KDa
Recombinant expressionOrganism: Escherichia coli str. K-12 substr. MG1655 (bacteria)
SequenceString: MKSAEYLNTF RLRNLGLPVM NNLHDMSKAT RISVETLRLL IYTADFRYRI YTVEKKGPEK RMRTIYQPSR ELKALQGWVL RNILDKLSS SPFSIGFEKH QSILNNATPH IGANFILNID LEDFFPSLTA NKVFGVFHSL GYNRLISSVL TKICCYKNLL P QGAPSSPK ...String:
MKSAEYLNTF RLRNLGLPVM NNLHDMSKAT RISVETLRLL IYTADFRYRI YTVEKKGPEK RMRTIYQPSR ELKALQGWVL RNILDKLSS SPFSIGFEKH QSILNNATPH IGANFILNID LEDFFPSLTA NKVFGVFHSL GYNRLISSVL TKICCYKNLL P QGAPSSPK LANLICSKLD YRIQGYAGSR GLIYTRYADD LTLSAQSMKK VVKARDFLFS IIPSEGLVIN SKKTCISGPR SQ RKVTGLV ISQEKVGIGR EKYKEIRAKI HHIFCGKSSE IEHVRGWLSF ILSVDSKSHR RLITYISKLE KKYGKNPLNK AKT GSEFEL ENLYFQGELR RQASALEHHH HHH

UniProtKB: Retron Ec86 reverse transcriptase

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Macromolecule #4: Retron Ec86 putative ribosyltransferase/DNA-binding protein

MacromoleculeName: Retron Ec86 putative ribosyltransferase/DNA-binding protein
type: protein_or_peptide / ID: 4 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
Molecular weightTheoretical: 35.480207 KDa
Recombinant expressionOrganism: Escherichia coli str. K-12 substr. MG1655 (bacteria)
SequenceString: MNKKFTDEQQ QQLIGHLTKK GFYRGANIKI TIFLCGGDVA NHQSWRHQLS QFLAKFSDVD IFYPEDLFDD LLAGQGQHSL LSLENILAE AVDVIILFPE SPGSFTALGA FSNNENLRRK LICIQDAKFK SKRSFINYGP VRLLRKFNSK SVLRCSSNEL K EMCDSSID ...String:
MNKKFTDEQQ QQLIGHLTKK GFYRGANIKI TIFLCGGDVA NHQSWRHQLS QFLAKFSDVD IFYPEDLFDD LLAGQGQHSL LSLENILAE AVDVIILFPE SPGSFTALGA FSNNENLRRK LICIQDAKFK SKRSFINYGP VRLLRKFNSK SVLRCSSNEL K EMCDSSID VARKLRLYKK LMASIKKVRK ENKVSKDIGN ILYAERFLLP CIYLLDSVNY RTLCELAFKA IKQDDVLSKI IV RSVVSRL INERKILQMT DGYQVTALGA SYVRSVFDRK TLDRLRLEIM NFENRRKSTF NYDKIPYAHP

UniProtKB: Retron Ec86 putative ribosyltransferase/DNA-binding protein

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Macromolecule #2: Retron-Eco1-msr

MacromoleculeName: Retron-Eco1-msr / type: rna / ID: 2 / Number of copies: 6
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
Molecular weightTheoretical: 26.19941 KDa
SequenceString:
AUGCGCACCC UUAGCGAGAG GUUUAUCAUU AAGGUCAACC UCUGGAUGUU GUUUCGGCAU CCUGCAUUGA AUCUGAGUUA CU

GENBANK: GENBANK: CP001509.3

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Macromolecule #3: Retron-Eco1-A2

MacromoleculeName: Retron-Eco1-A2 / type: rna / ID: 3 / Number of copies: 6
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
Molecular weightTheoretical: 4.541771 KDa
SequenceString:
CGUAAGGGUG CGCA

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Macromolecule #5: Retron-Eco1 msDNA

MacromoleculeName: Retron-Eco1 msDNA / type: dna / ID: 5 / Number of copies: 6 / Classification: DNA
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
Molecular weightTheoretical: 26.320875 KDa
SequenceString: (DG)(DT)(DC)(DA)(DG)(DA)(DA)(DA)(DA)(DA) (DA)(DC)(DG)(DG)(DG)(DT)(DT)(DT)(DC)(DC) (DT)(DG)(DG)(DT)(DT)(DG)(DG)(DC)(DT) (DC)(DG)(DG)(DA)(DG)(DA)(DG)(DC)(DA)(DT) (DC) (DA)(DG)(DG)(DC)(DG)(DA) ...String:
(DG)(DT)(DC)(DA)(DG)(DA)(DA)(DA)(DA)(DA) (DA)(DC)(DG)(DG)(DG)(DT)(DT)(DT)(DC)(DC) (DT)(DG)(DG)(DT)(DT)(DG)(DG)(DC)(DT) (DC)(DG)(DG)(DA)(DG)(DA)(DG)(DC)(DA)(DT) (DC) (DA)(DG)(DG)(DC)(DG)(DA)(DT)(DG) (DC)(DT)(DC)(DT)(DC)(DC)(DG)(DT)(DT)(DC) (DC)(DA) (DA)(DC)(DA)(DA)(DG)(DG)(DA) (DA)(DA)(DA)(DC)(DA)(DG)(DA)(DC)(DA)(DG) (DT)(DA)(DA) (DC)(DT)(DC)(DA)(DG)

GENBANK: GENBANK: CP001509.3

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 5 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

MacromoleculeName: NICOTINAMIDE-ADENINE-DINUCLEOTIDE / type: ligand / ID: 7 / Number of copies: 4 / Formula: NAD
Molecular weightTheoretical: 663.425 Da
Chemical component information

ChemComp-NAD:
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / NAD*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.5
Component:
ConcentrationName
20.0 mMHepes
200.0 mMKCl
1.0 mMTCEP
5.0 mMMgCl2

Details: 20 mM HEPES (pH 7.5),200 mM KCl, 5 mM MgCl2, 1mM TCEP
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Details: 10 mAmp
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time 3 seconds.
DetailsThe complex was prepared at a concentration corresponding to A260nm = ~9

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number real images: 7250 / Average electron dose: 41.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.9000000000000001 µm / Nominal defocus min: 0.6 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1474697
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.66 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.2.1) / Number images used: 358067
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.2.1)
Final 3D classificationNumber classes: 2 / Avg.num./class: 300000 / Software - Name: cryoSPARC (ver. v4.2.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3070


Chain - Source name: AlphaFold / Chain - Initial model type: in silico model
Detailsin ChimeraX
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8qbl:
Retron-Eco1 filament with inactive effector (E106A, 2 segments)

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