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Yorodumi- EMDB-18314: Retron-Eco1 filament with inactive effector (E106A, 2 segments) -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-18314 | |||||||||
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Title | Retron-Eco1 filament with inactive effector (E106A, 2 segments) | |||||||||
Map data | Retron-Eco1-E106A filament (Local) SHARPENED MAP | |||||||||
Sample |
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Keywords | retron / 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 | |||||||||
Biological species | Escherichia coli BL21(DE3) (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.66 Å | |||||||||
Authors | Carabias del Rey A / Montoya G | |||||||||
Funding support | Denmark, 1 items
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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
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_18314.map.gz | 727.4 MB | EMDB map data format | |
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Header (meta data) | emd-18314-v30.xml emd-18314.xml | 26.1 KB 26.1 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_18314_fsc.xml | 19.8 KB | Display | FSC data file |
Images | emd_18314.png | 79.3 KB | ||
Filedesc metadata | emd-18314.cif.gz | 7.5 KB | ||
Others | emd_18314_additional_1.map.gz emd_18314_half_map_1.map.gz emd_18314_half_map_2.map.gz | 413.2 MB 765.8 MB 765.8 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-18314 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-18314 | HTTPS FTP |
-Validation report
Summary document | emd_18314_validation.pdf.gz | 801 KB | Display | EMDB validaton report |
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Full document | emd_18314_full_validation.pdf.gz | 800.6 KB | Display | |
Data in XML | emd_18314_validation.xml.gz | 27.8 KB | Display | |
Data in CIF | emd_18314_validation.cif.gz | 36.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-18314 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-18314 | HTTPS FTP |
-Related structure data
Related structure data | 8qblMC 8qbkC 8qbmC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_18314.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | Retron-Eco1-E106A filament (Local) SHARPENED MAP | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.832 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: Retron-Eco1-E106A filament (Local) SHARPENED MAP
File | emd_18314_additional_1.map | ||||||||||||
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Annotation | Retron-Eco1-E106A filament (Local) SHARPENED MAP | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Retron-Eco1-E106A filament (Local) Half Map A
File | emd_18314_half_map_1.map | ||||||||||||
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Annotation | Retron-Eco1-E106A filament (Local) Half Map A | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Retron-Eco1-E106A filament (Local) Half Map B
File | emd_18314_half_map_2.map | ||||||||||||
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Annotation | Retron-Eco1-E106A filament (Local) Half Map B | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Filamentous assembly of Retron-Eco1-E106A mutant containing RT-ms...
Entire | Name: Filamentous assembly of Retron-Eco1-E106A mutant containing RT-msr-msDNA-Effector complex (2 segments) |
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Components |
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-Supramolecule #1: Filamentous assembly of Retron-Eco1-E106A mutant containing RT-ms...
Supramolecule | Name: 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. |
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Source (natural) | Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: Escherichia coli BL21(DE3) |
Molecular weight | Theoretical: 21 kDa/nm |
-Macromolecule #1: Retron Ec86 reverse transcriptase
Macromolecule | Name: Retron Ec86 reverse transcriptase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO |
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Source (natural) | Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3) |
Molecular weight | Theoretical: 39.984391 KDa |
Recombinant expression | Organism: Escherichia coli str. K-12 substr. MG1655 (bacteria) |
Sequence | String: 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 |
-Macromolecule #4: Retron Ec86 putative ribosyltransferase/DNA-binding protein
Macromolecule | Name: Retron Ec86 putative ribosyltransferase/DNA-binding protein type: protein_or_peptide / ID: 4 / Number of copies: 5 / Enantiomer: LEVO |
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Source (natural) | Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3) |
Molecular weight | Theoretical: 35.480207 KDa |
Recombinant expression | Organism: Escherichia coli str. K-12 substr. MG1655 (bacteria) |
Sequence | String: 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 |
-Macromolecule #2: Retron-Eco1-msr
Macromolecule | Name: Retron-Eco1-msr / type: rna / ID: 2 / Number of copies: 6 |
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Source (natural) | Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3) |
Molecular weight | Theoretical: 26.19941 KDa |
Sequence | String: AUGCGCACCC UUAGCGAGAG GUUUAUCAUU AAGGUCAACC UCUGGAUGUU GUUUCGGCAU CCUGCAUUGA AUCUGAGUUA CU GENBANK: GENBANK: CP001509.3 |
-Macromolecule #3: Retron-Eco1-A2
Macromolecule | Name: Retron-Eco1-A2 / type: rna / ID: 3 / Number of copies: 6 |
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Source (natural) | Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3) |
Molecular weight | Theoretical: 4.541771 KDa |
Sequence | String: CGUAAGGGUG CGCA |
-Macromolecule #5: Retron-Eco1 msDNA
Macromolecule | Name: Retron-Eco1 msDNA / type: dna / ID: 5 / Number of copies: 6 / Classification: DNA |
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Source (natural) | Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3) |
Molecular weight | Theoretical: 26.320875 KDa |
Sequence | 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) ...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 |
-Macromolecule #6: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 5 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #7: NICOTINAMIDE-ADENINE-DINUCLEOTIDE
Macromolecule | Name: NICOTINAMIDE-ADENINE-DINUCLEOTIDE / type: ligand / ID: 7 / Number of copies: 4 / Formula: NAD |
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Molecular weight | Theoretical: 663.425 Da |
Chemical component information | ChemComp-NAD: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | filament |
-Sample preparation
Buffer | pH: 7.5 Component:
Details: 20 mM HEPES (pH 7.5),200 mM KCl, 5 mM MgCl2, 1mM TCEP | ||||||||||
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Grid | Model: 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 | ||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time 3 seconds. | ||||||||||
Details | The complex was prepared at a concentration corresponding to A260nm = ~9 |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film 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 beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.9000000000000001 µm / Nominal defocus min: 0.6 µm |
Sample stage | Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |