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- PDB-7xjg: Cryo-EM structure of E.coli retron-Ec86 in complex with its effec... -

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Basic information

Entry
Database: PDB / ID: 7xjg
TitleCryo-EM structure of E.coli retron-Ec86 in complex with its effector at 2.5 angstrom
Components
  • DNA (105-MER)
  • RNA (14-MER)
  • RNA (81-MER)
  • RNA-directed DNA polymerase from retron EC86
  • retron St85 family effector protein
KeywordsRNA BINDING PROTEIN/DNA/RNA / Reverse 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 (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
: / DNA / DNA (> 10) / DNA (> 100) / RNA / RNA (> 10) / Uncharacterized protein / Retron Ec86 reverse transcriptase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.51 Å
AuthorsWang, Y.J. / Guan, Z.Y. / Zou, T.T.
Funding support China, 2items
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, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 14, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 3, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA-directed DNA polymerase from retron EC86
C: retron St85 family effector protein
D: DNA (105-MER)
E: RNA (81-MER)
F: RNA (14-MER)
B: RNA-directed DNA polymerase from retron EC86
G: DNA (105-MER)
H: RNA (81-MER)
I: RNA (14-MER)
J: retron St85 family effector protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)272,55112
Polymers272,50210
Non-polymers492
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 4 molecules ABCJ

#1: Protein RNA-directed DNA polymerase from retron EC86 / EC86-RT / Reverse transcriptase


Mass: 37832.070 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P23070, RNA-directed DNA polymerase
#2: Protein retron St85 family effector protein


Mass: 35538.242 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: B / BL21-DE3 / Gene: ECBD_2767 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A140NAX8

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RNA chain , 2 types, 4 molecules EHFI

#4: RNA chain RNA (81-MER)


Mass: 25870.203 Da / Num. of mol.: 2
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
#5: RNA chain RNA (14-MER)


Mass: 4541.771 Da / Num. of mol.: 2
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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DNA chain / Non-polymers , 2 types, 4 molecules DG

#3: DNA chain DNA (105-MER)


Mass: 32468.803 Da / Num. of mol.: 2
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
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN

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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 (RT-msDNA-RNA) / Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenConc.: 0.4 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: 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: 1500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 53.68 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
EM softwareName: cryoSPARC / Version: 3.1 / Category: final Euler assignment
CTF correctionType: NONE
3D reconstructionResolution: 2.51 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 530544 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00513862
ELECTRON MICROSCOPYf_angle_d0.71219665
ELECTRON MICROSCOPYf_dihedral_angle_d22.2785762
ELECTRON MICROSCOPYf_chiral_restr0.0462319
ELECTRON MICROSCOPYf_plane_restr0.0051697

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