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- PDB-8ubc: Diversity-generating retroelement (DGR) ribonucleoprotein reverse... -

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Basic information

Entry
Database: PDB / ID: 8ubc
TitleDiversity-generating retroelement (DGR) ribonucleoprotein reverse transcriptase - Resting State 1b
Components
  • Avd
  • Diversity-generating retroelement (DGR) RNA Sp
  • Diversity-generating retroelement (DGR) RNA TR
  • Reverse transcriptase
KeywordsRNA BINDING PROTEIN/RNA / Reverse transcriptase / Ribonucleoprotein / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


5,6,7,8-tetrahydromethanopterin hydro-lyase / carbon-nitrogen lyase activity / formaldehyde catabolic process / carbohydrate biosynthetic process / RNA-directed DNA polymerase activity / one-carbon metabolic process / cytoplasm
Similarity search - Function
: / bAvd-like / Formaldehyde-activating enzyme / Formaldehyde-activating enzyme superfamily / Formaldehyde-activating enzyme (Fae) / 23S rRNA-intervening sequence superfamily / : / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. ...: / bAvd-like / Formaldehyde-activating enzyme / Formaldehyde-activating enzyme superfamily / Formaldehyde-activating enzyme (Fae) / 23S rRNA-intervening sequence superfamily / : / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / Ribosomal protein S5 domain 2-type fold / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
: / RNA / RNA (> 10) / RNA (> 100) / Bbp7 / Reverse transcriptase / 5,6,7,8-tetrahydromethanopterin hydro-lyase
Similarity search - Component
Biological speciesBordetella phage BPP-1 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.29 Å
AuthorsBiswas, T. / Handa, S. / Ghosh, P.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R01GM132720-03 United States
CitationJournal: Nature / Year: 2025
Title: RNA control of reverse transcription in a diversity-generating retroelement.
Authors: Sumit Handa / Tapan Biswas / Jeet Chakraborty / Gourisankar Ghosh / Blair G Paul / Partho Ghosh /
Abstract: Diversity-generating retroelements (DGRs) create massive protein sequence variation (up to 10) in ecologically diverse microorganisms. A recent survey identified around 31,000 DGRs from more than ...Diversity-generating retroelements (DGRs) create massive protein sequence variation (up to 10) in ecologically diverse microorganisms. A recent survey identified around 31,000 DGRs from more than 1,500 bacterial and archaeal genera, constituting more than 90 environment types. DGRs are especially enriched in the human gut microbiome and nano-sized microorganisms that seem to comprise most microbial life and maintain DGRs despite reduced genomes. DGRs are also implicated in the emergence of multicellularity. Variation occurs during reverse transcription of a protein-encoding RNA template coupled to misincorporation at adenosines. In the prototypical Bordetella bacteriophage DGR, the template must be surrounded by upstream and downstream RNA segments for complementary DNA synthesis to be carried out by a complex of the DGR reverse transcriptase bRT and associated protein Avd. The function of the surrounding RNA was unknown. Here we show through cryogenic electron microscopy that this RNA envelops bRT and lies over the barrel-shaped Avd, forming an intimate ribonucleoprotein. An abundance of essential interactions in the ribonucleoprotein precisely position an RNA homoduplex in the bRT active site for initiation of reverse transcription. Our results explain how the surrounding RNA primes complementary DNA synthesis, promotes processivity, terminates polymerization and strictly limits mutagenesis to specific proteins through mechanisms that are probably conserved in DGRs belonging to distant taxa.
History
DepositionSep 22, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 23, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 15, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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.title / _citation.year / _em_admin.last_update
Revision 1.2Jan 22, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.3Mar 12, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Reverse transcriptase
B: Avd
C: Avd
D: Avd
E: Avd
F: Avd
H: Diversity-generating retroelement (DGR) RNA TR
I: Diversity-generating retroelement (DGR) RNA Sp


Theoretical massNumber of molelcules
Total (without water)253,2938
Polymers253,2938
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, EM map and derived model, gel filtration, Superdex 200
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Reverse transcriptase


Mass: 38099.836 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bordetella phage BPP-1 (virus) / Gene: brt / Production host: Escherichia coli (E. coli) / References: UniProt: Q775D8
#2: Protein
Avd / Formaldehyde-activating enzyme / Fae


Mass: 31753.635 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bordetella phage BPP-1 (virus) / Gene: bbp7, fae, MexAM1_META1p1766 / Production host: Escherichia coli (E. coli)
References: UniProt: Q775D7, UniProt: Q9FA38, 5,6,7,8-tetrahydromethanopterin hydro-lyase
#3: RNA chain Diversity-generating retroelement (DGR) RNA TR / DGR RNA TR


Mass: 11383.714 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Bordetella phage BPP-1 (virus)
#4: RNA chain Diversity-generating retroelement (DGR) RNA Sp / DGR RNA Sp


Mass: 45041.664 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Bordetella phage BPP-1 (virus) / References: GenBank: 41179361
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: Diversity-generating retroelement (DGR) ribonucleoprotein reverse transcriptase with dCpCpp
Type: COMPLEX
Details: Avd protein contains Methylobacterium extorquens AM1 Fae protein at its C-terminus
Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.173 MDa / Experimental value: NO
Source (natural)Organism: Bordetella phage BPP-1 (virus)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
175 mMPottasium ChlorideKCl1
230 mMAmmonium sulfate(NH4)2SO41
32 mMDithiothreitolDTT1
43 mMMagnesium ChlorideMgCl21
550 mMTrizma baseTris1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 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 defocus max: 2000 nm / Nominal defocus min: 400 nm
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 6763

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Processing

EM software
IDNameVersionCategory
1RELIONparticle selection
2cryoSPARCparticle selection
3Topazparticle selection
4Gautomatchparticle selection
5SerialEMimage acquisition
7GctfCTF correction
8CTFFINDCTF correction
11Cootmodel fitting
15cryoSPARCclassification
16cryoSPARC3.1.13D reconstruction
17CCP4 packagemodel refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.29 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 109161 / Symmetry type: POINT

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