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- EMDB-61595: retron Ec86-effector fiber -

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Basic information

Entry
Database: EMDB / ID: EMD-61595
Titleretron Ec86-effector fiber
Map data
Sample
  • Complex: retron Ec86-effector fiber
    • Protein or peptide: Retron Ec86 reverse transcriptase
    • Protein or peptide: Retron Ec86 putative ribosyltransferase/DNA-binding protein
    • DNA: DNA (85-MER)
    • RNA: RNA (82-MER)
    • RNA: RNA (5'-R(P*CP*GP*UP*AP*AP*GP*GP*GP*UP*GP*CP*GP*CP*A)-3')
  • Ligand: NICOTINAMIDE
  • Ligand: [(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
KeywordsReverse transcriptase / RNA BINDING PROTEIN/DNA/RNA / 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
Retron Ec86 putative ribosyltransferase/DNA-binding protein / Retron Ec86 reverse transcriptase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsWang YJ / Guan ZY / Wang C / Zou TT
Funding support China, 1 items
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, 2024-
Header (metadata) releaseDec 18, 2024-
Map releaseDec 18, 2024-
UpdateDec 18, 2024-
Current statusDec 18, 2024Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_61595.png

Downloads & links

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Map

FileDownload / File: emd_61595.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 280 pix.
= 299.6 Å		1.07 Å/pix.
x 280 pix.
= 299.6 Å		1.07 Å/pix.
x 280 pix.
= 299.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-2.191449 - 4.1874213
Average (Standard dev.)0.0019668809 (±0.10469317)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 299.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_61595_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_61595_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : retron Ec86-effector fiber

EntireName: retron Ec86-effector fiber
Components
  • Complex: retron Ec86-effector fiber
    • Protein or peptide: Retron Ec86 reverse transcriptase
    • Protein or peptide: Retron Ec86 putative ribosyltransferase/DNA-binding protein
    • DNA: DNA (85-MER)
    • RNA: RNA (82-MER)
    • RNA: RNA (5'-R(P*CP*GP*UP*AP*AP*GP*GP*GP*UP*GP*CP*GP*CP*A)-3')
  • Ligand: NICOTINAMIDE
  • Ligand: [(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

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Supramolecule #1: retron Ec86-effector fiber

SupramoleculeName: retron Ec86-effector fiber / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Retron Ec86 reverse transcriptase

MacromoleculeName: Retron Ec86 reverse transcriptase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: RNA-directed DNA polymerase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 37.83207 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (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 LEHHHH HHHH

UniProtKB: Retron Ec86 reverse transcriptase

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Macromolecule #2: Retron Ec86 putative ribosyltransferase/DNA-binding protein

MacromoleculeName: Retron Ec86 putative ribosyltransferase/DNA-binding protein
type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 35.538242 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNKKFTDEQQ QQLIGHLTKK GFYRGANIKI TIFLCGGDVA NHQSWRHQLS QFLAKFSDVD IFYPEDLFDD LLAGQGQHSL LSLENILAE AVDVIILFPE SPGSFTELGA FSNNENLRRK LICIQDAKFK SKRSFINYGP VRLLRKFNSK SVLRCSSNEL K EMCDSSID ...String:
MNKKFTDEQQ QQLIGHLTKK GFYRGANIKI TIFLCGGDVA NHQSWRHQLS QFLAKFSDVD IFYPEDLFDD LLAGQGQHSL LSLENILAE AVDVIILFPE SPGSFTELGA 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 #3: DNA (85-MER)

MacromoleculeName: DNA (85-MER) / type: dna / ID: 3 / Number of copies: 4 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
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: M24408.1

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Macromolecule #4: RNA (82-MER)

MacromoleculeName: RNA (82-MER) / type: rna / ID: 4 / Details: msrRNA / Number of copies: 4
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 26.19941 KDa
SequenceString:
AUGCGCACCC UUAGCGAGAG GUUUAUCAUU AAGGUCAACC UCUGGAUGUU GUUUCGGCAU CCUGCAUUGA AUCUGAGUUA CU

GENBANK: GENBANK: M24408.1

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Macromolecule #5: RNA (5'-R(P*CP*GP*UP*AP*AP*GP*GP*GP*UP*GP*CP*GP*CP*A)-3')

MacromoleculeName: RNA (5'-R(P*CP*GP*UP*AP*AP*GP*GP*GP*UP*GP*CP*GP*CP*A)-3')
type: rna / ID: 5 / Number of copies: 4
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 4.541771 KDa
SequenceString:
CGUAAGGGUG CGCA

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Macromolecule #6: NICOTINAMIDE

MacromoleculeName: NICOTINAMIDE / type: ligand / ID: 6 / Number of copies: 1 / Formula: NCA
Molecular weightTheoretical: 122.125 Da
Chemical component information

ChemComp-NCA:
NICOTINAMIDE / medication*YM

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Macromolecule #7: [(2R,3S,4R,5R)-5-(6-AMINOPURIN-9-YL)-3,4-DIHYDROXY-OXOLAN-2-YL]ME...

MacromoleculeName: [(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
type: ligand / ID: 7 / Number of copies: 1 / Formula: AR6
Molecular weightTheoretical: 559.316 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / 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 / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 270375
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: PROJECTION MATCHING

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