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- EMDB-33226: Cryo-EM structure of E.coli retron-Ec86 in complex with its effec... -

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Basic information

Entry
Database: EMDB / ID: EMD-33226
TitleCryo-EM structure of E.coli retron-Ec86 in complex with its effector at 2.5 angstrom
Map data
Sample
  • Complex: retron-Ec86 (RT-msDNA-RNA)
    • Protein or peptide: RNA-directed DNA polymerase from retron EC86
    • Protein or peptide: retron St85 family effector protein
    • DNA: DNA (105-MER)
    • RNA: RNA (81-MER)
    • RNA: RNA (14-MER)
  • Ligand: MAGNESIUM ION
KeywordsReverse transcriptase / RNA BINDING PROTEIN / RNA BINDING PROTEIN-DNA-RNA 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 domain / Reverse transcriptase (RT) catalytic domain profile. / Reverse transcriptase (RNA-dependent DNA polymerase) / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
UDP-3-O-(3-hydroxymyristoyl)glucosamine N-acyltransferase / Retron Ec86 reverse transcriptase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.51 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
DepositionApr 17, 2022-
Header (metadata) releaseSep 14, 2022-
Map releaseSep 14, 2022-
UpdateJul 3, 2024-
Current statusJul 3, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33226.png

Downloads & links

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Map

FileDownload / File: emd_33226.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 320 pix.
= 272. Å		0.85 Å/pix.
x 320 pix.
= 272. Å		0.85 Å/pix.
x 320 pix.
= 272. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.0017715769 - 2.0274458
Average (Standard dev.)0.0009138451 (±0.019660594)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 272.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_33226_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_33226_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 (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 EC86
    • Protein or peptide: retron St85 family effector protein
    • DNA: DNA (105-MER)
    • RNA: RNA (81-MER)
    • RNA: RNA (14-MER)
  • Ligand: MAGNESIUM ION

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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: #1-#5
Source (natural)Organism: Escherichia coli (E. coli)

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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: 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 St85 family effector protein

MacromoleculeName: retron St85 family effector protein / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: B / BL21-DE3
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: UDP-3-O-(3-hydroxymyristoyl)glucosamine N-acyltransferase

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Macromolecule #3: DNA (105-MER)

MacromoleculeName: DNA (105-MER) / type: dna / ID: 3 / 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)

GENBANK: GENBANK: M24408.1

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Macromolecule #4: RNA (81-MER)

MacromoleculeName: RNA (81-MER) / type: rna / ID: 4 / 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

GENBANK: GENBANK: M24408.1

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Macromolecule #5: RNA (14-MER)

MacromoleculeName: RNA (14-MER) / type: rna / ID: 5 / Number of copies: 2
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 4.541771 KDa
SequenceString:
CGUAAGGGUG CGCA

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 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

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

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 53.68 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

31827

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.51 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 530544
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1)

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