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- PDB-9dou: Taeniopygia guttata R2 retrotransposon (R2Tg) initiating target-p... -

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Basic information

Entry
Database: PDB / ID: 9dou
TitleTaeniopygia guttata R2 retrotransposon (R2Tg) initiating target-primed reverse transcription
Components
  • (28S DNA bottom strand, ...) x 2
  • 28S DNA top strand
  • R2Tg 3'UTR RNA
  • R2Tg retrotransposon ORF
KeywordsRNA BINDING PROTEIN/RNA/DNA / retrotransposon / LINE / reverse transcriptase / endonuclease / RNA BINDING PROTEIN-RNA-DNA complex
Function / homologyTHYMIDINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA (> 100) / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesTaeniopygia guttata (zebra finch)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsWilkinson, M.E. / Edmonds, K.H.K. / Zhang, F.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: To Be Published
Title: Structure and biochemistry-guided engineering of an all-RNA system for DNA insertion with R2 retrotransposons
Authors: Edmonds, K.H.K. / Wilkinson, M.E. / Strebinger, D. / Chen, H. / Lash, B. / Zhu, S. / Liu, D. / Zilberzwige-Tal, S. / Ladha, A. / Walsh, M.L. / Frangieh, C.J. / Macrae, R. / Wang, X. / Zhang, F.
History
DepositionSep 19, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 21, 2025Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: R2Tg retrotransposon ORF
B: 28S DNA bottom strand, 3' side
P: 28S DNA bottom strand, 5' side (priming strand)
R: R2Tg 3'UTR RNA
T: 28S DNA top strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)398,92811
Polymers398,1595
Non-polymers7686
Water00
1


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

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Components

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28S DNA bottom strand, ... , 2 types, 2 molecules BP

#2: DNA chain 28S DNA bottom strand, 3' side


Mass: 65051.477 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Taeniopygia guttata (zebra finch)
#3: DNA chain 28S DNA bottom strand, 5' side (priming strand)


Mass: 3997.607 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Taeniopygia guttata (zebra finch)

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Protein / RNA chain / DNA chain , 3 types, 3 molecules ART

#1: Protein R2Tg retrotransposon ORF


Mass: 157228.484 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Taeniopygia guttata (zebra finch) / Production host: Saccharomyces cerevisiae (brewer's yeast)
#4: RNA chain R2Tg 3'UTR RNA


Mass: 106656.273 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Taeniopygia guttata (zebra finch)
#5: DNA chain 28S DNA top strand


Mass: 65225.641 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Taeniopygia guttata (zebra finch)

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Non-polymers , 3 types, 6 molecules

#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#7: Chemical ChemComp-TTP / THYMIDINE-5'-TRIPHOSPHATE


Mass: 482.168 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N2O14P3
#8: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn

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Details

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: Taeniopygia guttata R2 retrotransposon initiating target-primed reverse transcription
Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.38 MDa / Experimental value: NO
Source (natural)Organism: Taeniopygia guttata (zebra finch)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.9
Details: 20 mM HEPES-KOH pH 7.9, 400 mM potassium acetate, 5 mM magnesium acetate, 0.1 mM dTTP
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: A260 = 17
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 285 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: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.02 sec. / Electron dose: 59.6 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10627
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
2EPUimage acquisition
4CTFFIND4.1CTF correction
7ISOLDEmodel fitting
8Cootmodel fitting
10PHENIXmodel refinement
12RELION5final Euler assignment
13RELION5classification
14RELION53D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 323700
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30612 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00611015
ELECTRON MICROSCOPYf_angle_d1.0915539
ELECTRON MICROSCOPYf_dihedral_angle_d23.4212831
ELECTRON MICROSCOPYf_chiral_restr0.0571785
ELECTRON MICROSCOPYf_plane_restr0.0091447

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