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- EMDB-45086: Structure of the elongating DRT2 reverse transcriptase in complex... -

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Basic information

Entry
Database: EMDB / ID: EMD-45086
TitleStructure of the elongating DRT2 reverse transcriptase in complex with its non-coding RNA and dNTPs
Map dataSharpened map of the elongating UG2 reverse transcriptase ribonucleoprotein complex
Sample
  • Complex: UG2 reverse transcriptase ribonucleoprotein complex, elongating state
    • Protein or peptide: Reverse transcriptase/maturase family protein
    • DNA: ccDNA
    • RNA: DRT2 ncRNA
  • Ligand: MAGNESIUM ION
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE
  • Ligand: POTASSIUM ION
Keywordsreverse transcriptase / rolling circle amplification / phage defense / TRANSFERASE-DNA-RNA complex
Function / homology: / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / Reverse transcriptase (RNA-dependent DNA polymerase) / RNA-directed DNA polymerase activity / DNA/RNA polymerase superfamily / Reverse transcriptase/maturase family protein
Function and homology information
Biological speciesKlebsiella pneumoniae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.17 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-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45086.png

Downloads & links

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Map

FileDownload / File: emd_45086.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map of the elongating UG2 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-0.08474546 - 0.13420972
Average (Standard dev.)-0.00014777922 (±0.0037313404)
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: UG2 RNP with dNTPs refinement, half-map 1

Fileemd_45086_half_map_1.map
AnnotationUG2 RNP with dNTPs refinement, half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: UG2 RNP with dNTPs refinement, half-map 2

Fileemd_45086_half_map_2.map
AnnotationUG2 RNP with dNTPs 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 : UG2 reverse transcriptase ribonucleoprotein complex, elongating state

EntireName: UG2 reverse transcriptase ribonucleoprotein complex, elongating state
Components
  • Complex: UG2 reverse transcriptase ribonucleoprotein complex, elongating state
    • Protein or peptide: Reverse transcriptase/maturase family protein
    • DNA: ccDNA
    • RNA: DRT2 ncRNA
  • Ligand: MAGNESIUM ION
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE
  • Ligand: POTASSIUM ION

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Supramolecule #1: UG2 reverse transcriptase ribonucleoprotein complex, elongating state

SupramoleculeName: UG2 reverse transcriptase ribonucleoprotein complex, elongating state
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: ccDNA

MacromoleculeName: ccDNA / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 2.745821 KDa
SequenceString:
(DT)(DA)(DT)(DG)(DC)(DT)(DG)(DT)(DA)

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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

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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: THYMIDINE-5'-TRIPHOSPHATE

MacromoleculeName: THYMIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: TTP
Molecular weightTheoretical: 482.168 Da
Chemical component information

ChemComp-TTP:
THYMIDINE-5'-TRIPHOSPHATE

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Macromolecule #6: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 6 / 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: 1 / Number real images: 18453 / Average electron dose: 40.3 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3313207
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.17 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 136601
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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