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- EMDB-31827: Cryo-EM structure of E.coli retron-Ec86 (RT-msDNA-RNA) at 3.2 angstrom -

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Basic information

Entry
Database: EMDB / ID: EMD-31827
TitleCryo-EM structure of E.coli retron-Ec86 (RT-msDNA-RNA) at 3.2 angstrom
Map data
Sample
  • Complex: retron-Ec86 (RT-msDNA-RNA)
    • Protein or peptide: RNA-directed DNA polymerase from retron EC86Reverse transcriptase
    • DNA: DNA (105-MER)
    • RNA: RNA (81-MER)
    • RNA: RNA (5'-R(P*CP*GP*UP*AP*AP*GP*GP*G)-3')
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 reverse transcriptase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.12 Å
AuthorsWang YJ / Guan ZY / Zou TT
Funding support China, 2 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)2018YFA0507700 China
National Natural Science Foundation of China (NSFC)32070174 China
CitationJournal: Nat Microbiol / Year: 2022
Title: Cryo-EM structures of Escherichia coli Ec86 retron complexes reveal architecture and defence mechanism.
Authors: Yanjing Wang / Zeyuan Guan / Chen Wang / Yangfan Nie / Yibei Chen / Zhaoyang Qian / Yongqing Cui / Han Xu / Qiang Wang / Fen Zhao / Delin Zhang / Pan Tao / Ming Sun / Ping Yin / Shuangxia ...Authors: Yanjing Wang / Zeyuan Guan / Chen Wang / Yangfan Nie / Yibei Chen / Zhaoyang Qian / Yongqing Cui / Han Xu / Qiang Wang / Fen Zhao / Delin Zhang / Pan Tao / Ming Sun / Ping Yin / Shuangxia Jin / Shan Wu / Tingting Zou /
Abstract: First discovered in the 1980s, retrons are bacterial genetic elements consisting of a reverse transcriptase and a non-coding RNA (ncRNA). Retrons mediate antiphage defence in bacteria but their ...First discovered in the 1980s, retrons are bacterial genetic elements consisting of a reverse transcriptase and a non-coding RNA (ncRNA). Retrons mediate antiphage defence in bacteria but their structure and defence mechanisms are unknown. Here, we investigate the Escherichia coli Ec86 retron and use cryo-electron microscopy to determine the structures of the Ec86 (3.1 Å) and cognate effector-bound Ec86 (2.5 Å) complexes. The Ec86 reverse transcriptase exhibits a characteristic right-hand-like fold consisting of finger, palm and thumb subdomains. Ec86 reverse transcriptase reverse-transcribes part of the ncRNA into satellite, multicopy single-stranded DNA (msDNA, a DNA-RNA hybrid) that we show wraps around the reverse transcriptase electropositive surface. In msDNA, both inverted repeats are present and the 3' sides of the DNA/RNA chains are close to the reverse transcriptase active site. The Ec86 effector adopts a two-lobe fold and directly binds reverse transcriptase and msDNA. These findings offer insights into the structure-function relationship of the retron-effector unit and provide a structural basis for the optimization of retron-based genome editing systems.
History
DepositionAug 26, 2021-
Header (metadata) releaseAug 31, 2022-
Map releaseAug 31, 2022-
UpdateSep 14, 2022-
Current statusSep 14, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_31827.png

Downloads & links

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Map

FileDownload / File: emd_31827.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.85 Å
Density
Contour LevelBy AUTHOR: 0.35
Minimum - Maximum-1.9879236 - 2.491176
Average (Standard dev.)0.002994435 (±0.059956674)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 238.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : retron-Ec86 (RT-msDNA-RNA)

EntireName: retron-Ec86 (RT-msDNA-RNA)
Components
  • Complex: retron-Ec86 (RT-msDNA-RNA)
    • Protein or peptide: RNA-directed DNA polymerase from retron EC86Reverse transcriptase
    • DNA: DNA (105-MER)
    • RNA: RNA (81-MER)
    • RNA: RNA (5'-R(P*CP*GP*UP*AP*AP*GP*GP*G)-3')

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Supramolecule #1: retron-Ec86 (RT-msDNA-RNA)

SupramoleculeName: retron-Ec86 (RT-msDNA-RNA) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #1: RNA-directed DNA polymerase from retron EC86

MacromoleculeName: RNA-directed DNA polymerase from retron EC86 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: RNA-directed DNA polymerase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 36.484641 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

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Macromolecule #2: DNA (105-MER)

MacromoleculeName: DNA (105-MER) / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 32.468803 KDa
SequenceString: (DG)(DA)(DA)(DA)(DG)(DT)(DT)(DG)(DC)(DG) (DC)(DA)(DC)(DC)(DC)(DT)(DT)(DA)(DC)(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) ...String:
(DG)(DA)(DA)(DA)(DG)(DT)(DT)(DG)(DC)(DG) (DC)(DA)(DC)(DC)(DC)(DT)(DT)(DA)(DC)(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)(DA)

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Macromolecule #3: RNA (81-MER)

MacromoleculeName: RNA (81-MER) / type: rna / ID: 3 / Number of copies: 2
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 25.870203 KDa
SequenceString:
UGCGCACCCU UAGCGAGAGG UUUAUCAUUA AGGUCAACCU CUGGAUGUUG UUUCGGCAUC CUGCAUUGAA UCUGAGUUAC U

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Macromolecule #4: RNA (5'-R(P*CP*GP*UP*AP*AP*GP*GP*G)-3')

MacromoleculeName: RNA (5'-R(P*CP*GP*UP*AP*AP*GP*GP*G)-3') / type: rna / ID: 4 / Number of copies: 2
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 4.541771 KDa
SequenceString:
CGUAAGGGUG CGCA

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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

Concentration0.4 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 53.68 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.12 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 144586

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