[English] 日本語
Yorodumi
- PDB-8qbk: Retron-Eco1 filament with ADP-ribosylated Effector (local map wit... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 8qbk
TitleRetron-Eco1 filament with ADP-ribosylated Effector (local map with 1 segment)
Components
  • Retron Ec86 putative ribosyltransferase/DNA-binding protein
  • Retron Ec86 reverse transcriptase
  • Retron-Eco1 msDNA
  • Retron-Eco1-A2
  • Retron-Eco1-msr
KeywordsIMMUNE 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
: / 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
Chem-AR6 / : / DNA / DNA (> 10) / RNA / RNA (> 10) / Retron Ec86 putative ribosyltransferase/DNA-binding protein / Retron Ec86 reverse transcriptase
Similarity search - Component
Biological speciesEscherichia coli BL21 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.99 Å
AuthorsCarabias del Rey, A. / Montoya, G.
Funding support Denmark, 1items
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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 5, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

-
Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
F: Retron Ec86 putative ribosyltransferase/DNA-binding protein
G: Retron Ec86 putative ribosyltransferase/DNA-binding protein
A: Retron Ec86 reverse transcriptase
B: Retron-Eco1 msDNA
C: Retron-Eco1-msr
D: Retron-Eco1-A2
K: Retron Ec86 reverse transcriptase
L: Retron-Eco1 msDNA
M: Retron-Eco1-msr
N: Retron-Eco1-A2
P: Retron Ec86 reverse transcriptase
Q: Retron-Eco1 msDNA
R: Retron-Eco1-msr
S: Retron-Eco1-A2
T: Retron Ec86 putative ribosyltransferase/DNA-binding protein
U: Retron Ec86 reverse transcriptase
V: Retron-Eco1 msDNA
W: Retron-Eco1-msr
X: Retron-Eco1-A2
E: Retron Ec86 putative ribosyltransferase/DNA-binding protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)531,55526
Polymers530,33920
Non-polymers1,2166
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, Negative staining and Size exclussion Chromatography data
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

-
Components

-
Protein , 2 types, 8 molecules FGTEAKPU

#1: Protein
Retron Ec86 putative ribosyltransferase/DNA-binding protein / ORF223


Mass: 35538.242 Da / Num. of mol.: 4 / Mutation: ADP-ribosylated E106
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3) / Gene: LM2_00875 / Variant: Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): Escherichia coli / References: UniProt: P0DV88
#2: Protein
Retron Ec86 reverse transcriptase


Mass: 39984.391 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3) / Gene: ret / Variant: Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): Escherichia coli / References: UniProt: P23070

-
DNA chain , 1 types, 4 molecules BLQV

#3: DNA chain
Retron-Eco1 msDNA


Mass: 26320.875 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3) / Variant: Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): Escherichia coli / References: GenBank: 296142109

-
RNA chain , 2 types, 8 molecules CMRWDNSX

#4: RNA chain
Retron-Eco1-msr


Mass: 26199.410 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3) / Variant: Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): Escherichia coli / References: GenBank: 296142109
#5: RNA chain
Retron-Eco1-A2


Mass: 4541.771 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3) / Variant: Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): Escherichia coli

-
Non-polymers , 2 types, 6 molecules

#6: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#7: Chemical ChemComp-AR6 / [(2R,3S,4R,5R)-5-(6-AMINOPURIN-9-YL)-3,4-DIHYDROXY-OXOLAN-2-YL]METHYL [HYDROXY-[[(2R,3S,4R,5S)-3,4,5-TRIHYDROXYOXOLAN-2-YL]METHOXY]PHOSPHORYL] HYDROGEN PHOSPHATE / Adenosine-5-Diphosphoribose


Mass: 559.316 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C15H23N5O14P2 / Feature type: SUBJECT OF INVESTIGATION

-
Details

Has ligand of interestY

-
Experimental details

-
Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

-
Sample preparation

ComponentName: Filamentous assembly of Retron-Eco1 in complex with ADPr, containing RT-msr-msDNA-Effector complex (1 segment)
Type: COMPLEX / Details: Filament formed by addition of NAD+ / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightValue: 21 kDa/nm / Experimental value: NO
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: Escherichia coli BL21(DE3)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: Escherichia coli BL21(DE3)
Buffer solutionpH: 7.5
Details: 20 mM HEPES (pH 7.5),200 mM KCl, 5 mM MgCl2, 1mM TCEP
Buffer component
IDConc.NameBuffer-ID
120 mMHepes1
2200 mMKCl1
31 mMTCEP1
45 mMMgCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The complex was prepared at a concentration corresponding to A260nm = ~9
Specimen supportDetails: 10mAmps / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: 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
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Image recordingElectron dose: 39 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 7226
Image scansWidth: 4096 / Height: 4096

-
Processing

EM software
IDNameVersionCategory
1Topazparticle selection
2EPUimage acquisition
4cryoSPARCv4.2.1CTF correction
7UCSF ChimeraX1.6.1model fitting
9cryoSPARCv4.2.1initial Euler assignment
10cryoSPARCv4.2.1final Euler assignment
11cryoSPARCv4.2.1classification
12cryoSPARCv4.2.13D reconstruction
13PHENIX1.20-4487model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2432349
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 340412 / Algorithm: FOURIER SPACE / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Details: in ChimeraX
Atomic model buildingAccession code: P23070 / Source name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00328745
ELECTRON MICROSCOPYf_angle_d0.50541391
ELECTRON MICROSCOPYf_dihedral_angle_d21.5838203
ELECTRON MICROSCOPYf_chiral_restr0.0355024
ELECTRON MICROSCOPYf_plane_restr0.0043315

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbjlvh1.pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more