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- EMDB-49056: Structure of the Retron IA Complex without the HNH Nuclease -

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Basic information

Entry
Database: EMDB / ID: EMD-49056
TitleStructure of the Retron IA Complex without the HNH Nuclease
Map dataStructure of the Retron IA Complex without the HNH Nuclease
Sample
  • Complex: Retron IA surveillance complex with HNH nuclease bound in the "down" orientation
    • Protein or peptide: AAA family ATPase
    • Protein or peptide: RNA-directed DNA polymerase
    • DNA: Retron IA msDNA
    • RNA: Retron IA ncRNA
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
KeywordsRetron / IA / Immune / Transferase-DNA-RNA complex
Function / homology
Function and homology information


DNA synthesis involved in DNA repair / RNA-directed DNA polymerase activity / RNA-directed DNA polymerase / double-strand break repair / defense response to virus / RNA binding / ATP binding
Similarity search - Function
RNA-directed DNA polymerase (reverse transcriptase), msDNA / AAA domain, group 15 / AAA ATPase domain / : / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
RNA-directed DNA polymerase / AAA family ATPase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.99 Å
AuthorsBurman N / Thomas-George J / Wilkinson R / Wiedenheft B
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5F31GM153146-02 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R35GM134867 United States
CitationJournal: bioRxiv / Year: 2025
Title: Structural basis of antiphage defense by an ATPase-associated reverse transcriptase.
Authors: Jerrin Thomas George / Nathaniel Burman / Royce A Wilkinson / Senuri de Silva / Quynh McKelvey-Pham / Murat Buyukyoruk / Adelaide Dale / Hannah Landman / Ava Graham / Steven Z DeLuca / Blake Wiedenheft
Abstract: Reverse transcriptases (RTs) have well-established roles in the replication and spread of retroviruses and retrotransposons. However, recent evidence suggests that RTs have been conscripted by cells ...Reverse transcriptases (RTs) have well-established roles in the replication and spread of retroviruses and retrotransposons. However, recent evidence suggests that RTs have been conscripted by cells for diverse roles in antiviral defense. Here we determine structures of a type I-A retron, which explain how RNA, DNA, RT, HNH-nuclease and four molecules of an SMC-family ATPase assemble into a 364 kDa complex that provides phage defense. We show that phage-encoded nucleases trigger degradation of the retron-associated DNA, leading to disassembly of the retron and activation of the HNH nuclease. The HNH nuclease cleaves tRNA , stalling protein synthesis and arresting viral replication. Taken together, these data reveal diverse and paradoxical roles for RTs in the perpetuation and elimination of genetic parasites.
History
DepositionFeb 5, 2025-
Header (metadata) releaseApr 23, 2025-
Map releaseApr 23, 2025-
UpdateApr 23, 2025-
Current statusApr 23, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_49056.png

Downloads & links

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Map

FileDownload / File: emd_49056.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of the Retron IA Complex without the HNH Nuclease
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.91 Å/pix.
x 480 pix.
= 434.928 Å		0.91 Å/pix.
x 480 pix.
= 434.928 Å		0.91 Å/pix.
x 480 pix.
= 434.928 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9061 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015
Minimum - Maximum-0.10702835 - 0.28486577
Average (Standard dev.)-0.000009570875 (±0.0043392135)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 434.92798 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map B

Fileemd_49056_half_map_1.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_49056_half_map_2.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Retron IA surveillance complex with HNH nuclease bound in the "do...

EntireName: Retron IA surveillance complex with HNH nuclease bound in the "down" orientation
Components
  • Complex: Retron IA surveillance complex with HNH nuclease bound in the "down" orientation
    • Protein or peptide: AAA family ATPase
    • Protein or peptide: RNA-directed DNA polymerase
    • DNA: Retron IA msDNA
    • RNA: Retron IA ncRNA
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: Retron IA surveillance complex with HNH nuclease bound in the "do...

SupramoleculeName: Retron IA surveillance complex with HNH nuclease bound in the "down" orientation
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Escherichia coli (E. coli) / Strain: FORC_082

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Macromolecule #1: AAA family ATPase

MacromoleculeName: AAA family ATPase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: FORC_082
Molecular weightTheoretical: 63.393953 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MWSHPQFEKI NKMNLETCYV DFLELESHVI NEDYLKESVE LQKLISTLNE SKFHLNKIGI HDFKRIRELQ ISLEDDLTVF VGDNGFGKS TILDAIAIVL SWLRSNIEKE SKPGTYIKSH EVNNSVDVEY ASIDANIKLK DFNTSILITK AKEGAYYSRN N ELLGVKKL ...String:
MWSHPQFEKI NKMNLETCYV DFLELESHVI NEDYLKESVE LQKLISTLNE SKFHLNKIGI HDFKRIRELQ ISLEDDLTVF VGDNGFGKS TILDAIAIVL SWLRSNIEKE SKPGTYIKSH EVNNSVDVEY ASIDANIKLK DFNTSILITK AKEGAYYSRN N ELLGVKKL ASIYRLVNKY VDNASLPLMA YYSIARSYIG GGVDRKRKNA KTKTVWSKFD VYDEIEFDRN DFTDFFQWLV FL HNRASQE KLSESQTTIN ALFSDIQSLK ATLTQLSAID NIDSTVIKGL ELSLKEKLNY MKSLQSGEHK FNNAVSLYDS VIN TILKFL PEFQWIKLVY GDDDYKIILK KGEVELDIQQ LSQGEKTIFT LVGDLARRLI LLNPNLSNPL LGYGIVLIDE IDLH LHPQW QQTIIERLTS TFPNVQFVIT THSPQVLSTV SSRSVRILQE VEVDGVNDLI VSHPDYQIKG VSNQDALLYG MRTDP IPST KENGWLEEYK KLVELNRYSS DEALLLREKV IKHFGLDHPL VQECDDLISV LEFKNKINQH FSGSKDVK

UniProtKB: AAA family ATPase

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Macromolecule #2: RNA-directed DNA polymerase

MacromoleculeName: RNA-directed DNA polymerase / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed DNA polymerase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 36.046191 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MQLTSKIISK FNYNRLAFQL LLNEAPKKYK VYYIPKRGAG FRVIAQPTKE LKNVQRFIVS LLQPKLPVHH KAMAYEYKKS IKDNALLHK DNNYILKMDF QNFFNKIKPD IFFSKLENTG LKLDSFDENT LRNLLFWRPG KKRSTTLILS VGAPSSPFIS N FVMYDFDK ...String:
MQLTSKIISK FNYNRLAFQL LLNEAPKKYK VYYIPKRGAG FRVIAQPTKE LKNVQRFIVS LLQPKLPVHH KAMAYEYKKS IKDNALLHK DNNYILKMDF QNFFNKIKPD IFFSKLENTG LKLDSFDENT LRNLLFWRPG KKRSTTLILS VGAPSSPFIS N FVMYDFDK SLDDWCRNNG ITYSRYADDI TFSTNIKDIL CRVPKVVKKM LSLHVPGLSI NESKTIFTSM AHNRHVTGVT LT PQGNLSI GRDRKRMLFA KIHKYSLGLL SSEEINKTKG MIAFANYLEG DFLLRLQKKY GCELITKFLM EGNK

UniProtKB: RNA-directed DNA polymerase

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Macromolecule #3: Retron IA msDNA

MacromoleculeName: Retron IA msDNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli) / Strain: FORC_082
Molecular weightTheoretical: 49.825891 KDa
SequenceString: (DC)(DT)(DC)(DT)(DT)(DC)(DA)(DC)(DT)(DA) (DA)(DA)(DG)(DA)(DC)(DA)(DG)(DC)(DG)(DA) (DA)(DA)(DG)(DA)(DC)(DA)(DC)(DA)(DG) (DA)(DT)(DT)(DT)(DC)(DT)(DC)(DC)(DT)(DT) (DC) (DG)(DC)(DA)(DT)(DA)(DT) ...String:
(DC)(DT)(DC)(DT)(DT)(DC)(DA)(DC)(DT)(DA) (DA)(DA)(DG)(DA)(DC)(DA)(DG)(DC)(DG)(DA) (DA)(DA)(DG)(DA)(DC)(DA)(DC)(DA)(DG) (DA)(DT)(DT)(DT)(DC)(DT)(DC)(DC)(DT)(DT) (DC) (DG)(DC)(DA)(DT)(DA)(DT)(DC)(DT) (DG)(DC)(DC)(DC)(DC)(DG)(DG)(DG)(DC)(DA) (DG)(DG) (DG)(DA)(DT)(DG)(DC)(DG)(DA) (DA)(DG)(DG)(DA)(DG)(DA)(DA)(DA)(DT)(DC) (DT)(DG)(DT) (DG)(DT)(DC)(DT)(DT)(DT) (DC)(DG)(DC)(DA)(DA)(DC)(DC)(DC)(DT)(DA) (DA)(DA)(DC)(DC) (DG)(DG)(DA)(DC)(DA) (DA)(DT)(DA)(DA)(DA)(DT)(DT)(DT)(DA)(DA) (DG)(DC)(DC)(DT)(DG) (DG)(DC)(DT)(DA) (DA)(DT)(DC)(DA)(DC)(DC)(DC)(DG)(DG)(DC) (DT)(DG)(DG)(DA)(DA)(DC) (DC)(DA)(DA) (DT)(DG)(DT)(DT)(DC)(DC)(DT)(DA)(DC)(DA) (DC)(DT)(DA)(DA)(DA)(DG)(DA) (DG)(DC)

GENBANK: GENBANK: CP026641.1

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Macromolecule #4: Retron IA ncRNA

MacromoleculeName: Retron IA ncRNA / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli) / Strain: FORC_082
Molecular weightTheoretical: 52.084648 KDa
SequenceString:
GCUCUUUAGU GUAGGAACAU UGGUUCCAGC CGGGUGAUUA GCCAGGCUUA AAUUUAUUGU CCGGUUUAGG GUUGCGAAAG ACACAGAUU UCUCCUUCGC AUCCCUGCCC GGGGCAGAUA UGCGAAGGAG AAAUCUGUGU CUUUCGCUGU CUUUAGUGAA G AG

GENBANK: GENBANK: CP026641.1

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Macromolecule #5: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 3 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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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: 7.5
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 13695 / Average electron dose: 56.69 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 45000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5915380
Details: Particles picked using a 20 Angstrom lowpass filtered template volume that was produced de novo using blob picked particles
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.6.2)
Software - details: Non-Uniform refinement with minimize over per particle scale, optimize per particle defocus, optimize per exposure group CTF parames enabled
Details: Reported resolution from cryoSPARC's GSFSC estimation
Number images used: 513948
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.6.2)
Software - details: Multi-Class Ab Initio reconstruction was used to obtain initial angle estimates for selected particles
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.6.2)
Software - details: Non-Uniform Refinement was used to assign final angles for selected particles
Final 3D classificationNumber classes: 3 / Avg.num./class: 414987
Software:
Namedetails
cryoSPARC (ver. v4.6.2)Focused 3-D classification using masks generated from the consensus volume was used to sort particles
cryoSPARC (ver. v4.6.2)A final round of 2-D classification was applied to remove poorly aligned particles prior to reconstruction

Details: Focused 3-D Classification was used to parse particle stacks based on HNH occupancy and orientation
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Details: The structure of each subunit was predicted individually in Alphafold before rigid body fitting in ChimeraX
SoftwareName: UCSF ChimeraX (ver. 1.9)
Details: ChimeraX's fit in map command was used to fit individual chains predicted in Alphafold to the density
DetailsAlphafold predicted structures of each individual subunit were first rigid body fit in ChimeraX using the fit in map command before initial relaxation using ISOLDE and final refinement in PHENIX RealSpaceRefinement. COOT was used for manual editing.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-9n6c:
Structure of the Retron IA Complex without the HNH Nuclease

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