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- EMDB-19792: Retron-Eco1 -1 turn mutant filament with ADP-ribosylated Effector... -

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Basic information

Entry
Database: EMDB / ID: EMD-19792
TitleRetron-Eco1 -1 turn mutant filament with ADP-ribosylated Effector (Consensus refinement)
Map dataRetron-Eco1 "-1 turn" mutant with ADPr. Consensus refinement.
Sample
  • Complex: Filamentous assembly of Retron-Eco1 "-1 turn" mutant in complex with ADPr, containing RT-msr-msDNA-Effector complex (Consensus refinement)
    • Protein or peptide: Retron-Eco1 Reverse Transcriptase
    • RNA: Retron-Eco1-msr
    • RNA: Retron-Eco1-A2
    • Protein or peptide: Retron-Eco1-Effector
    • DNA: Retron-Eco1-msDNA "-1 turn"
Keywordsretron / N-glycosidase / DNA-RNA-Protein complex / IMMUNE SYSTEM
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: 2.43 Å
AuthorsCarabias del Rey A / Montoya G / Pape T
Funding support Denmark, 2 items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF14CC0001 Denmark
LundbeckfondenR380-2021-1448 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
DepositionMar 5, 2024-
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_19792.png

Downloads & links

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Map

FileDownload / File: emd_19792.map.gz / Format: CCP4 / Size: 1.2 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRetron-Eco1 "-1 turn" mutant with ADPr. Consensus refinement.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 686 pix.
= 497.35 Å		0.73 Å/pix.
x 686 pix.
= 497.35 Å		0.73 Å/pix.
x 686 pix.
= 497.35 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.725 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.09
Minimum - Maximum-0.23190123 - 0.6124642
Average (Standard dev.)0.00000748691 (±0.0076458934)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions686686686
Spacing686686686
CellA=B=C: 497.35 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Retron-Eco1 "-1 turn" mutant with ADPr. Consensus refinement....

Fileemd_19792_half_map_1.map
AnnotationRetron-Eco1 "-1 turn" mutant with ADPr. Consensus refinement. Half map B.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Retron-Eco1 "-1 turn" mutant with ADPr. Consensus refinement....

Fileemd_19792_half_map_2.map
AnnotationRetron-Eco1 "-1 turn" mutant with ADPr. Consensus refinement. Half map A.
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 "-1 turn" mutant in complex w...

EntireName: Filamentous assembly of Retron-Eco1 "-1 turn" mutant in complex with ADPr, containing RT-msr-msDNA-Effector complex (Consensus refinement)
Components
  • Complex: Filamentous assembly of Retron-Eco1 "-1 turn" mutant in complex with ADPr, containing RT-msr-msDNA-Effector complex (Consensus refinement)
    • Protein or peptide: Retron-Eco1 Reverse Transcriptase
    • RNA: Retron-Eco1-msr
    • RNA: Retron-Eco1-A2
    • Protein or peptide: Retron-Eco1-Effector
    • DNA: Retron-Eco1-msDNA "-1 turn"

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Supramolecule #1: Filamentous assembly of Retron-Eco1 "-1 turn" mutant in complex w...

SupramoleculeName: Filamentous assembly of Retron-Eco1 "-1 turn" mutant in complex with ADPr, containing RT-msr-msDNA-Effector complex (Consensus refinement)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5 / Details: Filament formed by addition of NAD+
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-Eco1 Reverse Transcriptase

MacromoleculeName: Retron-Eco1 Reverse Transcriptase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21
Recombinant expressionOrganism: Escherichia coli str. K-12 substr. MG1655 (bacteria)
SequenceString: MKSAEYLNTF RLRNLGLPVM NNLHDMSKAT RISVETLRLL IYTADFRYRI YTVEKKGPEK RMRTIYQPSR ELKALQGWVL RNILDKLSSS PFSIGFEKHQ SILNNATPHI GANFILNIDL EDFFPSLTAN KVFGVFHSLG YNRLISSVLT KICCYKNLLP QGAPSSPKLA ...String:
MKSAEYLNTF RLRNLGLPVM NNLHDMSKAT RISVETLRLL IYTADFRYRI YTVEKKGPEK RMRTIYQPSR ELKALQGWVL RNILDKLSSS PFSIGFEKHQ SILNNATPHI GANFILNIDL EDFFPSLTAN KVFGVFHSLG YNRLISSVLT KICCYKNLLP QGAPSSPKLA NLICSKLDYR IQGYAGSRGL IYTRYADDLT LSAQSMKKVV KARDFLFSII PSEGLVINSK KTCISGPRSQ RKVTGLVISQ EKVGIGREKY KEIRAKIHHI FCGKSSEIEH VRGWLSFILS VDSKSHRRLI TYISKLEKKY GKNPLNKAKT GSEFELENLY FQGELRRQAS ALEHHHHHH

UniProtKB: Retron Ec86 reverse transcriptase

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Macromolecule #4: Retron-Eco1-Effector

MacromoleculeName: Retron-Eco1-Effector / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
Recombinant expressionOrganism: Escherichia coli str. K-12 substr. MG1655 (bacteria)
SequenceString: MNKKFTDEQQ QQLIGHLTKK GFYRGANIKI TIFLCGGDVA NHQSWRHQLS QFLAKFSDVD IFYPEDLFDD LLAGQGQHSL LSLENILAEA VDVIILFPES PGSFTELGAF SNNENLRRKL ICIQDAKFKS KRSFINYGPV RLLRKFNSKS VLRCSSNELK EMCDSSIDVA ...String:
MNKKFTDEQQ QQLIGHLTKK GFYRGANIKI TIFLCGGDVA NHQSWRHQLS QFLAKFSDVD IFYPEDLFDD LLAGQGQHSL LSLENILAEA VDVIILFPES PGSFTELGAF SNNENLRRKL ICIQDAKFKS KRSFINYGPV RLLRKFNSKS VLRCSSNELK EMCDSSIDVA RKLRLYKKLM ASIKKVRKEN KVSKDIGNIL YAERFLLPCI YLLDSVNYRT LCELAFKAIK QDDVLSKIIV RSVVSRLINE RKILQMTDGY QVTALGASYV RSVFDRKTLD RLRLEIMNFE NRRKSTFNYD KIPYAHP

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
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
SequenceString:
AUGCGCACCC UUAGCGAGAG GUUUAUCAUU AAGGUCAACC UCUGGAUGUU GUUUCGGCAU CCUGCAUUGA AUCUGAGUUA CU

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Macromolecule #3: Retron-Eco1-A2

MacromoleculeName: Retron-Eco1-A2 / type: rna / ID: 3
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
SequenceString:
CGUAAGGGUG CGCA

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Macromolecule #5: Retron-Eco1-msDNA "-1 turn"

MacromoleculeName: Retron-Eco1-msDNA "-1 turn" / type: dna / ID: 5 / Classification: DNA
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
SequenceString:
gtcagaaaaa acgggtttcc tggttggctc AGGCgttcca acaaggaaaa cagacagtaa ctcag

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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 / Pretreatment - Pressure: 0.03 kPa / Details: 10 mAmps
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 = ~12

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 100.0 K / Max: 100.0 K
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 5 eV
Details: The slit was recentered every 6 hours during data collection
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 26559 / Average exposure time: 2.6 sec. / Average electron dose: 50.0 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: 2.0 µm / Nominal defocus min: 0.4 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4771292
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.43 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.4.1) / Number images used: 1003701
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.4.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.4.1)
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. v4.4.1) / Software - details: Ab inition reconstruction
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8qbm


Chain - Source name: PDB / Chain - Initial model type: experimental model
Detailsin ChimeraX
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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