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- PDB-9c0i: Structure of the DRT2 reverse transcriptase in complex with its n... -

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Basic information

Entry
Database: PDB / ID: 9c0i
TitleStructure of the DRT2 reverse transcriptase in complex with its non-coding RNA
Components
  • DNA primer
  • DRT2 ncRNA
  • Reverse transcriptase/maturase family protein
KeywordsTRANSFERASE/DNA/RNA / reverse transcriptase / rolling circle amplification / phage defense / TRANSFERASE-DNA-RNA complex
Function / homology
Function and homology information


RNA-directed DNA polymerase activity
Similarity search - Function
: / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
: / : / DNA / RNA / RNA (> 10) / RNA (> 100) / Reverse transcriptase/maturase family protein
Similarity search - Component
Biological speciesKlebsiella pneumoniae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.91 Å
AuthorsWilkinson, M.E. / Zhang, F.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Science / Year: 2024
Title: Phage-triggered reverse transcription assembles a toxic repetitive gene from a noncoding RNA.
Authors: Max E Wilkinson / David Li / Alex Gao / Rhiannon K Macrae / Feng Zhang /
Abstract: Reverse transcription has frequently been co-opted for cellular functions and in prokaryotes is associated with protection against viral infection, but the underlying mechanisms of defense are ...Reverse transcription has frequently been co-opted for cellular functions and in prokaryotes is associated with protection against viral infection, but the underlying mechanisms of defense are generally unknown. Here, we show that in the DRT2 defense system, the reverse transcriptase binds a neighboring pseudoknotted noncoding RNA. Upon bacteriophage infection, a template region of this RNA is reverse transcribed into an array of tandem repeats that reconstitute a promoter and open reading frame, allowing expression of a toxic repetitive protein and an abortive infection response. Biochemical reconstitution of this activity and cryo-electron microscopy provide a molecular basis for repeat synthesis. Gene synthesis from a noncoding RNA is a previously unknown mode of genetic regulation in prokaryotes.
History
DepositionMay 25, 2024Deposition site: RCSB / Processing site: RCSB
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Reverse transcriptase/maturase family protein
P: DNA primer
R: DRT2 ncRNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)142,3235
Polymers142,2593
Non-polymers632
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Reverse transcriptase/maturase family protein / antiviral reverse transcriptase Drt2


Mass: 49788.645 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: QIG75_19540 / Production host: Escherichia coli (E. coli) / References: UniProt: A0AA43TDM1
#2: DNA chain DNA primer


Mass: 1519.048 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Production host: Escherichia coli (E. coli)
#3: RNA chain DRT2 ncRNA


Mass: 90951.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Production host: Escherichia coli (E. coli) / References: GenBank: 979528538
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: K
Has ligand of interestN
Has protein modificationN

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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: DRT2 reverse transcriptase ribonucleoprotein complex / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.14 MDa / Experimental value: NO
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.9
SpecimenEmbedding 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: 2300 nm / Nominal defocus min: 900 nm
Image recordingElectron dose: 40.7 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 28211

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Processing

EM software
IDNameVersionCategory
8PHENIXmodel refinement
13RELION43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4628424
3D reconstructionResolution: 2.91 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 230001 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0058260
ELECTRON MICROSCOPYf_angle_d0.95112072
ELECTRON MICROSCOPYf_dihedral_angle_d14.292805
ELECTRON MICROSCOPYf_chiral_restr0.0491491
ELECTRON MICROSCOPYf_plane_restr0.008805

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