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- PDB-8iby: Structure of R2 with 5'ORF -

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Basic information

Entry
Database: PDB / ID: 8iby
TitleStructure of R2 with 5'ORF
Components
  • 5'ORF RNA
  • Reverse transcriptase-like protein
KeywordsRNA BINDING PROTEIN/RNA / R2 complex / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


RNA-directed DNA polymerase activity / zinc ion binding
Similarity search - Function
Zinc finger C2H2 type domain profile. / Zinc finger C2H2 type domain signature. / Zinc finger C2H2-type / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA (> 100) / Reverse transcriptase-like protein
Similarity search - Component
Biological speciesBombyx mori (domestic silkworm)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.47 Å
AuthorsDeng, P. / Tan, S. / Wang, J. / Liu, J.J.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Cell / Year: 2023
Title: Structural RNA components supervise the sequential DNA cleavage in R2 retrotransposon.
Authors: Pujuan Deng / Shun-Qing Tan / Qi-Yu Yang / Liangzheng Fu / Yachao Wu / Han-Zhou Zhu / Lei Sun / Zhangbin Bao / Yi Lin / Qiangfeng Cliff Zhang / Haoyi Wang / Jia Wang / Jun-Jie Gogo Liu /
Abstract: Retroelements are the widespread jumping elements considered as major drivers for genome evolution, which can also be repurposed as gene-editing tools. Here, we determine the cryo-EM structures of ...Retroelements are the widespread jumping elements considered as major drivers for genome evolution, which can also be repurposed as gene-editing tools. Here, we determine the cryo-EM structures of eukaryotic R2 retrotransposon with ribosomal DNA target and regulatory RNAs. Combined with biochemical and sequencing analysis, we reveal two essential DNA regions, Drr and Dcr, required for recognition and cleavage. The association of 3' regulatory RNA with R2 protein accelerates the first-strand cleavage, blocks the second-strand cleavage, and initiates the reverse transcription starting from the 3'-tail. Removing 3' regulatory RNA by reverse transcription allows the association of 5' regulatory RNA and initiates the second-strand cleavage. Taken together, our work explains the DNA recognition and RNA supervised sequential retrotransposition mechanisms by R2 machinery, providing insights into the retrotransposon and application reprogramming.
History
DepositionFeb 10, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Sep 20, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
E: 5'ORF RNA
C: Reverse transcriptase-like protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)196,3713
Polymers196,3062
Non-polymers651
Water00
1


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

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Components

#1: RNA chain 5'ORF RNA


Mass: 72943.172 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bombyx mori (domestic silkworm) / Production host: Escherichia coli (E. coli)
#2: Protein Reverse transcriptase-like protein


Mass: 123362.430 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bombyx mori (domestic silkworm) / Production host: Escherichia coli (E. coli) / References: UniProt: V9H052
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: R2 complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.17728 MDa / Experimental value: NO
Source (natural)Organism: Bombyx mori (domestic silkworm)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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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 magnification: 64000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 1000 nm / Cs: 0.01 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
4cryoSPARC3.31CTF correction
7UCSF Chimera1.6model fitting
9PHENIX1.2model refinement
10RELION3.1initial Euler assignment
11cryoSPARC3.31final Euler assignment
12cryoSPARC3.31classification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.47 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 264298 / Symmetry type: POINT
Atomic model buildingProtocol: BACKBONE TRACE / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00311129
ELECTRON MICROSCOPYf_angle_d0.60916011
ELECTRON MICROSCOPYf_dihedral_angle_d17.8293258
ELECTRON MICROSCOPYf_chiral_restr0.0351942
ELECTRON MICROSCOPYf_plane_restr0.0041315

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