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- EMDB-42078: Diversity-generating retroelement (DGR) ribonucleoprotein reverse... -

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Basic information

Entry
Database: EMDB / ID: EMD-42078
TitleDiversity-generating retroelement (DGR) ribonucleoprotein reverse transcriptase - Pre-active State 1a
Map datamap
Sample
  • Complex: Diversity-generating retroelement (DGR) ribonucleoprotein with dCTP, dATP, and ddGTP
    • Protein or peptide: Reverse transcriptase
    • Protein or peptide: Avd
    • RNA: Diversity-generating retroelement (DGR) RNA avd
    • RNA: Diversity-generating retroelement (DGR) RNA TR
    • RNA: Diversity-generating retroelement (DGR) RNA Sp
KeywordsReverse 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 / one-carbon metabolic process / RNA-directed DNA polymerase activity / 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
Bbp7 / Reverse transcriptase / 5,6,7,8-tetrahydromethanopterin hydro-lyase
Similarity search - Component
Biological speciesBordetella phage BPP-1 (virus) / Methylorubrum extorquens (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.28 Å
AuthorsBiswas T / Handa S / Ghosh P
Funding support United States, 1 items
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, 2023-
Header (metadata) releaseOct 23, 2024-
Map releaseOct 23, 2024-
UpdateMar 12, 2025-
Current statusMar 12, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42078.png

Downloads & links

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Map

FileDownload / File: emd_42078.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmap
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1 Å/pix.
x 320 pix.
= 320. Å		1 Å/pix.
x 320 pix.
= 320. Å		1 Å/pix.
x 320 pix.
= 320. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.176
Minimum - Maximum-1.0176659 - 1.577191
Average (Standard dev.)-0.00025354055 (±0.02594582)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 320.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_42078_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap A

Fileemd_42078_half_map_1.map
Annotationhalfmap_A
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap B

Fileemd_42078_half_map_2.map
Annotationhalfmap_B
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Diversity-generating retroelement (DGR) ribonucleoprotein with dC...

EntireName: Diversity-generating retroelement (DGR) ribonucleoprotein with dCTP, dATP, and ddGTP
Components
  • Complex: Diversity-generating retroelement (DGR) ribonucleoprotein with dCTP, dATP, and ddGTP
    • Protein or peptide: Reverse transcriptase
    • Protein or peptide: Avd
    • RNA: Diversity-generating retroelement (DGR) RNA avd
    • RNA: Diversity-generating retroelement (DGR) RNA TR
    • RNA: Diversity-generating retroelement (DGR) RNA Sp

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Supramolecule #1: Diversity-generating retroelement (DGR) ribonucleoprotein with dC...

SupramoleculeName: Diversity-generating retroelement (DGR) ribonucleoprotein with dCTP, dATP, and ddGTP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Avd protein contains Methylobacterium extorquens AM1 Fae protein at its C-terminus
Source (natural)Organism: Bordetella phage BPP-1 (virus)
Molecular weightTheoretical: 173 KDa

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Macromolecule #1: Reverse transcriptase

MacromoleculeName: Reverse transcriptase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bordetella phage BPP-1 (virus)
Molecular weightTheoretical: 38.099836 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGKRHRNLID QITTWENLLD AYRKTSHGKR RTWGYLEFKE YDLANLLALQ AELKAGNYER GPYREFLVYE PKPRLISALE FKDRLVQHA LCNIVAPIFE AGLLPYTYAC RPDKGTHAGV CHVQAELRRT RATHFLKSDF SKFFPSIDRA ALYAMIDKKI H CAATRRLL ...String:
MGKRHRNLID QITTWENLLD AYRKTSHGKR RTWGYLEFKE YDLANLLALQ AELKAGNYER GPYREFLVYE PKPRLISALE FKDRLVQHA LCNIVAPIFE AGLLPYTYAC RPDKGTHAGV CHVQAELRRT RATHFLKSDF SKFFPSIDRA ALYAMIDKKI H CAATRRLL RVVLPDEGVG IPIGSLTSQL FANVYGGAVD RLLHDELKQR HWARYMDDIV VLGDDPEELR AVFYRLRDFA SE RLGLKIS HWQVAPVSRG INFLGYRIWP THKLLRKSSV KRAKRKVANF IKHGEDESLQ RFLASWSGHA QWADTHNLFT WME EQYGIA CH

UniProtKB: Reverse transcriptase

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Macromolecule #2: Avd

MacromoleculeName: Avd / type: protein_or_peptide / ID: 2 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Methylorubrum extorquens (bacteria) / Strain: ATCC 14718 / DSM 1338 / JCM 2805 / NCIMB 9133 / AM1
Molecular weightTheoretical: 31.753635 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEPIEEATKC YDQMLIVERY ERVISYLYPI AQSIPRKHGV AREMFLKCLL GQVELFIVAG KSNQVSKLYA ADAGLAMLRF WLRFLAGIQ KPHAMTPHQV ETAQVLIAEV GRILGSWIAR VNRKGTKVQV GEALVGDGNE VAHIDLIIGP RGSPAETAFC N GLVNNKHG ...String:
MEPIEEATKC YDQMLIVERY ERVISYLYPI AQSIPRKHGV AREMFLKCLL GQVELFIVAG KSNQVSKLYA ADAGLAMLRF WLRFLAGIQ KPHAMTPHQV ETAQVLIAEV GRILGSWIAR VNRKGTKVQV GEALVGDGNE VAHIDLIIGP RGSPAETAFC N GLVNNKHG FTSLLAVIAP NLPCKPNTLM FNKVTINDAR QAVQMFGPAQ HGVAMAVQDA VAEGIIPADE ADDLYVLVGV FI HWEAADD AKIQKYNYEA TKLSIQRAVN GEPKASVVTE QRKSATHPFA ANA

UniProtKB: Bbp7, 5,6,7,8-tetrahydromethanopterin hydro-lyase

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Macromolecule #3: Diversity-generating retroelement (DGR) RNA avd

MacromoleculeName: Diversity-generating retroelement (DGR) RNA avd / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Bordetella phage BPP-1 (virus)
Molecular weightTheoretical: 6.259826 KDa
SequenceString:
GGGGCAGGCU GGGAAAUAA

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Macromolecule #4: Diversity-generating retroelement (DGR) RNA TR

MacromoleculeName: Diversity-generating retroelement (DGR) RNA TR / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Bordetella phage BPP-1 (virus)
Molecular weightTheoretical: 11.422754 KDa
SequenceString:
CGCUGCUGCG CGGCGACUGU GCCCAUCACC UUCUUG

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Macromolecule #5: Diversity-generating retroelement (DGR) RNA Sp

MacromoleculeName: Diversity-generating retroelement (DGR) RNA Sp / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Bordetella phage BPP-1 (virus)
Molecular weightTheoretical: 45.041664 KDa
SequenceString:
CAUGGCUCUG CCAACGCUAC GGCUUGGCGG GCUGGCCUUU CCUCAAUAGG UGGUCAGCCG GUUCUGUCCU GCUUCGGCGA ACACGUUAC ACGGUUCGGC AAAACGUCGA UUACUGAAAA UGGAAAGGCG GGGCCGACUU C

GENBANK: GENBANK: NC_005357.1

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
75.0 mMKClPottasium Chloride
30.0 mM(NH4)2SO4Ammonium sulfate
2.0 mMDTTDithiothreitol
3.0 mMMgCl2Magnesium Chloride
50.0 mMTrisTrizma base
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 3559 / Average electron dose: 55.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.4 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1.1) / Number images used: 343150
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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