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

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Basic information

Entry
Database: PDB / ID: 8ubd
TitleDiversity-generating retroelement (DGR) ribonucleoprotein reverse transcriptase - Pre-active State 2
Components
  • Avd
  • Diversity-generating retroelement (DGR) RNA Sp
  • Diversity-generating retroelement (DGR) RNA TR
  • Diversity-generating retroelement (DGR) RNA avd
  • 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)
Methylorubrum extorquens (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.05 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
G: Diversity-generating retroelement (DGR) RNA avd
H: Diversity-generating retroelement (DGR) RNA TR
I: Diversity-generating retroelement (DGR) RNA Sp


Theoretical massNumber of molelcules
Total (without water)259,5699
Polymers259,5699
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


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. exp.) Methylorubrum extorquens (bacteria)
Gene: bbp7, fae / Strain: ATCC 14718 / DSM 1338 / JCM 2805 / NCIMB 9133 / AM1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q775D7, UniProt: Q9FA38
#3: RNA chain Diversity-generating retroelement (DGR) RNA avd


Mass: 6259.826 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Bordetella phage BPP-1 (virus)
#4: RNA chain Diversity-generating retroelement (DGR) RNA TR


Mass: 11399.714 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Bordetella phage BPP-1 (virus)
#5: 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 without dNTP
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
SpecimenEmbedding 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: 3397

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
7Cootmodel fitting
11cryoSPARCclassification
12cryoSPARC3.1.13D reconstruction
13CCP4 packagemodel refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 202034 / Symmetry type: POINT
RefinementResolution: 3.05→3.05 Å / Cor.coef. Fo:Fc: 0.938 / SU B: 13.656 / SU ML: 0.245 / ESU R: 0.506
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.34749 --
obs0.34749 87492 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 218.142 Å2
Refinement stepCycle: 1 / Total: 10244
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0110.01110773
ELECTRON MICROSCOPYr_bond_other_d00.0168316
ELECTRON MICROSCOPYr_angle_refined_deg1.9911.71415279
ELECTRON MICROSCOPYr_angle_other_deg0.5791.57919393
ELECTRON MICROSCOPYr_dihedral_angle_1_deg7.2775883
ELECTRON MICROSCOPYr_dihedral_angle_2_deg5.815565
ELECTRON MICROSCOPYr_dihedral_angle_3_deg25.11101259
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.1220.21814
ELECTRON MICROSCOPYr_gen_planes_refined0.0080.029986
ELECTRON MICROSCOPYr_gen_planes_other0.0020.022094
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it21.31316.9763553
ELECTRON MICROSCOPYr_mcbond_other21.31316.9763553
ELECTRON MICROSCOPYr_mcangle_it30.09825.4284429
ELECTRON MICROSCOPYr_mcangle_other30.09425.4374430
ELECTRON MICROSCOPYr_scbond_it19.63127.8487220
ELECTRON MICROSCOPYr_scbond_other19.6327.857221
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other29.50641.86110851
ELECTRON MICROSCOPYr_long_range_B_refined47.22752322
ELECTRON MICROSCOPYr_long_range_B_other47.22652323
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.1→3.181 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.435 6465 -
obs--100 %

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