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- PDB-8gh6: Bombyx mori R2 retrotransposon initiating target-primed reverse t... -

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Basic information

Entry
Database: PDB / ID: 8gh6
TitleBombyx mori R2 retrotransposon initiating target-primed reverse transcription
Components
  • (28S DNA bottom strand, ...) x 2
  • 28S DNA top strand
  • R2Bm 3'UTR RNA
  • Reverse transcriptase-like protein
KeywordsRNA BINDING PROTEIN/RNA/DNA / retrotransposon / LINE / reverse transcriptase / endonuclease / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA-DNA complex
Function / homology
Function and homology information


RNA-directed DNA polymerase activity
Similarity search - Function
Zinc finger C2H2 type domain profile. / Zinc finger C2H2 type domain signature. / Zinc finger C2H2-type / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
THYMIDINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / 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: 3.08 Å
AuthorsWilkinson, M.E. / Zhang, F.
Funding support United States, 2items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)R01HG009761 United States
CitationJournal: Science / Year: 2023
Title: Structure of the R2 non-LTR retrotransposon initiating target-primed reverse transcription.
Authors: Max E Wilkinson / Chris J Frangieh / Rhiannon K Macrae / Feng Zhang /
Abstract: Non-long terminal repeat (non-LTR) retrotransposons, or long interspersed nuclear elements (LINEs), are an abundant class of eukaryotic transposons that insert into genomes by target-primed reverse ...Non-long terminal repeat (non-LTR) retrotransposons, or long interspersed nuclear elements (LINEs), are an abundant class of eukaryotic transposons that insert into genomes by target-primed reverse transcription (TPRT). During TPRT, a target DNA sequence is nicked and primes reverse transcription of the retrotransposon RNA. Here, we report the cryo-electron microscopy structure of the R2 non-LTR retrotransposon initiating TPRT at its ribosomal DNA target. The target DNA sequence is unwound at the insertion site and recognized by an upstream motif. An extension of the reverse transcriptase (RT) domain recognizes the retrotransposon RNA and guides the 3' end into the RT active site to template reverse transcription. We used Cas9 to retarget R2 in vitro to non-native sequences, suggesting future use as a reprogrammable RNA-based gene-insertion tool.
History
DepositionMar 9, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 5, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 12, 2023Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.year
Revision 1.2Apr 19, 2023Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.3May 3, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.4Aug 16, 2023Group: Data collection / Other
Category: chem_comp_atom / chem_comp_bond / pdbx_database_status
Item: _pdbx_database_status.pdb_format_compatible

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Reverse transcriptase-like protein
B: 28S DNA bottom strand, 3' side
P: 28S DNA bottom strand, 5' side (priming strand)
R: R2Bm 3'UTR RNA
T: 28S DNA top strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)255,8129
Polymers255,1755
Non-polymers6374
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
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: 21507.758 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Bombyx mori (domestic silkworm)
#3: DNA chain 28S DNA bottom strand, 5' side (priming strand)


Mass: 7439.812 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Bombyx mori (domestic silkworm)

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

#1: Protein Reverse transcriptase-like protein


Mass: 123358.352 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
#4: RNA chain R2Bm 3'UTR RNA


Mass: 81213.930 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Bombyx mori (domestic silkworm)
#5: DNA chain 28S DNA top strand


Mass: 21654.875 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Bombyx mori (domestic silkworm)

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Non-polymers , 3 types, 4 molecules

#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-TTP / THYMIDINE-5'-TRIPHOSPHATE


Mass: 482.168 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N2O14P3 / Feature type: SUBJECT OF INVESTIGATION

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Details

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: Bombyx mori R2 retrotransposon initiating target-primed reverse transcription
Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.23 MDa / Experimental value: NO
Source (natural)Organism: Bombyx mori (domestic silkworm)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.9
Details: 20 mM HEPES-KOH pH 7.9, 500 mM potassium acetate, 5 mM magnesium acetate, 1 mM TCEP
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: A260 = 3
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
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: 2500 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: 0.69 sec. / Electron dose: 42.2 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 16551
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategory
1Topazparticle selection
2EPUimage acquisition
4CTFFIND4.1CTF correction
7ISOLDEmodel fitting
8Cootmodel fitting
11RELION4final Euler assignment
12RELION4classification
13RELION43D reconstruction
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1085471
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.08 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 39616 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 103.35 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005711366
ELECTRON MICROSCOPYf_angle_d1.081816139
ELECTRON MICROSCOPYf_chiral_restr0.05751814
ELECTRON MICROSCOPYf_plane_restr0.01021449
ELECTRON MICROSCOPYf_dihedral_angle_d24.63592851

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