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- EMDB-45085: Structure of the DRT2 reverse transcriptase in complex with its n... -

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Basic information

Entry
Database: EMDB / ID: EMD-45085
TitleStructure of the DRT2 reverse transcriptase in complex with its non-coding RNA
Map dataSharpened map of DRT2 reverse transcriptase ribonucleoprotein complex
Sample
  • Complex: DRT2 reverse transcriptase ribonucleoprotein complex
    • Protein or peptide: Reverse transcriptase/maturase family protein
    • DNA: DNA primer
    • RNA: DRT2 ncRNA
  • Ligand: MAGNESIUM ION
  • Ligand: POTASSIUM ION
Keywordsreverse transcriptase / rolling circle amplification / phage defense / TRANSFERASE-DNA-RNA complex
Function / homology: / RNA-directed DNA polymerase activity / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily / Reverse transcriptase/maturase family protein
Function and homology information
Biological speciesKlebsiella pneumoniae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.91 Å
AuthorsWilkinson ME / Zhang F
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Science / Year: 2024
Title: Phage-triggered reverse transcription assembles a toxic repetitive gene from a noncoding RNA.
Authors: Max E Wilkinson / David Li / Alex Gao / Rhiannon K Macrae / Feng Zhang /
Abstract: Reverse transcription has frequently been co-opted for cellular functions and in prokaryotes is associated with protection against viral infection, but the underlying mechanisms of defense are ...Reverse transcription has frequently been co-opted for cellular functions and in prokaryotes is associated with protection against viral infection, but the underlying mechanisms of defense are generally unknown. Here, we show that in the DRT2 defense system, the reverse transcriptase binds a neighboring pseudoknotted noncoding RNA. Upon bacteriophage infection, a template region of this RNA is reverse transcribed into an array of tandem repeats that reconstitute a promoter and open reading frame, allowing expression of a toxic repetitive protein and an abortive infection response. Biochemical reconstitution of this activity and cryo-electron microscopy provide a molecular basis for repeat synthesis. Gene synthesis from a noncoding RNA is a previously unknown mode of genetic regulation in prokaryotes.
History
DepositionMay 25, 2024-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateMay 21, 2025-
Current statusMay 21, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45085.png

Downloads & links

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Map

FileDownload / File: emd_45085.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map of DRT2 reverse transcriptase ribonucleoprotein complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.88 Å/pix.
x 240 pix.
= 212.16 Å		0.88 Å/pix.
x 240 pix.
= 212.16 Å		0.88 Å/pix.
x 240 pix.
= 212.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.884 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.06136795 - 0.15288427
Average (Standard dev.)0.00006283072 (±0.0037967784)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 212.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: DRT2 RNP refinement, half-map 1

Fileemd_45085_half_map_1.map
AnnotationDRT2 RNP refinement, half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: DRT2 RNP refinement, half-map 2

Fileemd_45085_half_map_2.map
AnnotationDRT2 RNP refinement, half-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : DRT2 reverse transcriptase ribonucleoprotein complex

EntireName: DRT2 reverse transcriptase ribonucleoprotein complex
Components
  • Complex: DRT2 reverse transcriptase ribonucleoprotein complex
    • Protein or peptide: Reverse transcriptase/maturase family protein
    • DNA: DNA primer
    • RNA: DRT2 ncRNA
  • Ligand: MAGNESIUM ION
  • Ligand: POTASSIUM ION

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Supramolecule #1: DRT2 reverse transcriptase ribonucleoprotein complex

SupramoleculeName: DRT2 reverse transcriptase ribonucleoprotein complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 140 KDa

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Macromolecule #1: Reverse transcriptase/maturase family protein

MacromoleculeName: Reverse transcriptase/maturase family protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 49.788645 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNNDDYPWFR KRGYLHFDEP VSLKKAVKYV SSPEKIIKHS FLPFLSFEVK SFKIKKDKST KQLSKTEKLR PIAYSSHLDS HIYAFYAEY LTGHYELLIQ ENNLHENILA FRSLNKSNIE FAKRAFDTIT EMGECSAVAL DLSGFFDNLD HQILKHQWCK V IGTEALPQ ...String:
MNNDDYPWFR KRGYLHFDEP VSLKKAVKYV SSPEKIIKHS FLPFLSFEVK SFKIKKDKST KQLSKTEKLR PIAYSSHLDS HIYAFYAEY LTGHYELLIQ ENNLHENILA FRSLNKSNIE FAKRAFDTIT EMGECSAVAL DLSGFFDNLD HQILKHQWCK V IGTEALPQ DHFAIYKSIT RYSKVDKNRA YEILGISKNN PKYNRRKICT PVDFRNKIRK NGLIIVNNSQ KGIPQGSPIS AL LSNIYML DFDIEMRDYA QERGGHYYRY CDDMLFIVPT KYNKTLAGDV AQRIKHLKVE LNTKKTEIRD FIYKDSTLVA NMP LQYLGF IFDGSNILLR SSSLARYSER MKRGVRLAKA TMDSKNRIRE NKGEALKALF KKKLYARYSH IGRRNFLTYG YRAA KIMNS KAIKRQLKPL QKRLENEILK

UniProtKB: Reverse transcriptase/maturase family protein

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Macromolecule #2: DNA primer

MacromoleculeName: DNA primer / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 1.519048 KDa
SequenceString:
(DG)(DA)(DT)(DA)(DT)

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Macromolecule #3: DRT2 ncRNA

MacromoleculeName: DRT2 ncRNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 90.951438 KDa
SequenceString: GGCCCUAAAC AAAGGUUUAG GGGUAUUGUA CAGGUUGUCA AGCCUCCCAC AGGUCUUGGU GAAACCAAUC ACUGUGACGA CGGUAAGCA ACACUUGGAU GAUAUUCAUA AUUGACUCCA CGCUACUGAU UACAUUAUAC AGCAUAUCUA ACAUUUGCGG C GAGGUUCA ...String:
GGCCCUAAAC AAAGGUUUAG GGGUAUUGUA CAGGUUGUCA AGCCUCCCAC AGGUCUUGGU GAAACCAAUC ACUGUGACGA CGGUAAGCA ACACUUGGAU GAUAUUCAUA AUUGACUCCA CGCUACUGAU UACAUUAUAC AGCAUAUCUA ACAUUUGCGG C GAGGUUCA CAAUUUGUAU UUAGGUACUG AUUGUGGAUG AGAAGGUUGG AGAAAGACCA CUUGGUUAAG CCGGAGGAUG UG UCCUAGA AUUGUCGCUA UUCUGUCAUC CUCCGGUUUU GCUAAU

GENBANK: GENBANK: CP014010.1

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: K
Molecular weightTheoretical: 39.098 Da

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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

BufferpH: 7.9
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Number real images: 28211 / Average electron dose: 40.7 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.3000000000000003 µm / Nominal defocus min: 0.9 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4628424
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.91 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 230001
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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