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- PDB-9jm0: retron Ec86-effector fiber -

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Basic information

Entry
Database: PDB / ID: 9jm0
Titleretron Ec86-effector fiber
Components
  • DNA (85-MER)
  • RNA (5'-R(P*CP*GP*UP*AP*AP*GP*GP*GP*UP*GP*CP*GP*CP*A)-3')
  • RNA (82-MER)
  • Retron Ec86 putative ribosyltransferase/DNA-binding protein
  • Retron Ec86 reverse transcriptase
KeywordsRNA BINDING PROTEIN/DNA/RNA / Reverse transcriptase / RNA BINDING PROTEIN-DNA-RNA complex
Function / homology
Function and homology information


RNA-directed DNA polymerase / telomerase activity / defense response to virus / RNA binding / metal ion binding
Similarity search - Function
: / RNA-directed DNA polymerase (reverse transcriptase), msDNA / : / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Chem-AR6 / NICOTINAMIDE / : / DNA / DNA (> 10) / RNA / RNA (> 10) / Retron Ec86 putative ribosyltransferase/DNA-binding protein / Retron Ec86 reverse transcriptase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsWang, Y.J. / Guan, Z.Y. / Wang, C. / Zou, T.T.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Cell Rep / Year: 2024
Title: DNA methylation activates retron Ec86 filaments for antiphage defense.
Authors: Yanjing Wang / Chen Wang / Zeyuan Guan / Jie Cao / Jia Xu / Shuangshuang Wang / Yongqing Cui / Qiang Wang / Yibei Chen / Yongqi Yin / Delin Zhang / Hongbo Liu / Ming Sun / Shuangxia Jin / ...Authors: Yanjing Wang / Chen Wang / Zeyuan Guan / Jie Cao / Jia Xu / Shuangshuang Wang / Yongqing Cui / Qiang Wang / Yibei Chen / Yongqi Yin / Delin Zhang / Hongbo Liu / Ming Sun / Shuangxia Jin / Pan Tao / Tingting Zou /
Abstract: Retrons are a class of multigene antiphage defense systems typically consisting of a retron reverse transcriptase, a non-coding RNA, and a cognate effector. Although triggers for several retron ...Retrons are a class of multigene antiphage defense systems typically consisting of a retron reverse transcriptase, a non-coding RNA, and a cognate effector. Although triggers for several retron systems have been discovered recently, the complete mechanism by which these systems detect invading phages and mediate defense remains unclear. Here, we focus on the retron Ec86 defense system, elucidating its modes of activation and mechanisms of action. We identified a phage-encoded DNA cytosine methyltransferase (Dcm) as a trigger of the Ec86 system and demonstrated that Ec86 is activated upon multicopy single-stranded DNA (msDNA) methylation. We further elucidated the structure of a tripartite retron Ec86-effector filament assembly that is primed for activation by Dcm and capable of hydrolyzing nicotinamide adenine dinucleotide (NAD). These findings provide insights into the retron Ec86 defense mechanism and underscore an emerging theme of antiphage defense through supramolecular complex assemblies.
History
DepositionSep 20, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Dec 18, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Retron Ec86 reverse transcriptase
C: Retron Ec86 putative ribosyltransferase/DNA-binding protein
D: DNA (85-MER)
E: RNA (82-MER)
F: RNA (5'-R(P*CP*GP*UP*AP*AP*GP*GP*GP*UP*GP*CP*GP*CP*A)-3')
B: Retron Ec86 reverse transcriptase
G: DNA (85-MER)
H: RNA (82-MER)
I: RNA (5'-R(P*CP*GP*UP*AP*AP*GP*GP*GP*UP*GP*CP*GP*CP*A)-3')
J: Retron Ec86 putative ribosyltransferase/DNA-binding protein
K: Retron Ec86 reverse transcriptase
L: Retron Ec86 putative ribosyltransferase/DNA-binding protein
M: DNA (85-MER)
N: RNA (82-MER)
O: RNA (5'-R(P*CP*GP*UP*AP*AP*GP*GP*GP*UP*GP*CP*GP*CP*A)-3')
P: Retron Ec86 reverse transcriptase
Q: DNA (85-MER)
R: RNA (82-MER)
S: RNA (5'-R(P*CP*GP*UP*AP*AP*GP*GP*GP*UP*GP*CP*GP*CP*A)-3')
T: Retron Ec86 putative ribosyltransferase/DNA-binding protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)522,41122
Polymers521,72920
Non-polymers6812
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 8 molecules ABKPCJLT

#1: Protein
Retron Ec86 reverse transcriptase / Ec86-RT / ORF320 / Reverse transcriptase / RT


Mass: 37832.070 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: ret, LM2_00877 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P23070, RNA-directed DNA polymerase
#2: Protein
Retron Ec86 putative ribosyltransferase/DNA-binding protein / ORF223


Mass: 35538.242 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: LM2_00875 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0DV88

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DNA chain , 1 types, 4 molecules DGMQ

#3: DNA chain
DNA (85-MER)


Mass: 26320.875 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: GenBank: 341167

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RNA chain , 2 types, 8 molecules EHNRFIOS

#4: RNA chain
RNA (82-MER)


Mass: 26199.410 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: msrRNA / Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: GenBank: 341167
#5: RNA chain
RNA (5'-R(P*CP*GP*UP*AP*AP*GP*GP*GP*UP*GP*CP*GP*CP*A)-3')


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

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Non-polymers , 2 types, 2 molecules

#6: Chemical ChemComp-NCA / NICOTINAMIDE


Mass: 122.125 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H6N2O / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM
#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.: 1 / Source method: obtained synthetically / Formula: C15H23N5O14P2 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: retron Ec86-effector fiber / Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI SPIRIT
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 270375 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00429609
ELECTRON MICROSCOPYf_angle_d0.62942241
ELECTRON MICROSCOPYf_dihedral_angle_d27.1729100
ELECTRON MICROSCOPYf_chiral_restr0.0394965
ELECTRON MICROSCOPYf_plane_restr0.0043477

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