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

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Basic information

Entry
Database: PDB / ID: 9n6c
TitleStructure of the Retron IA Complex without the HNH Nuclease
Components
  • AAA family ATPase
  • RNA-directed DNA polymerase
  • Retron IA msDNA
  • Retron IA ncRNA
KeywordsTransferase/DNA/RNA / Retron / 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
ADENOSINE-5'-TRIPHOSPHATE / : / DNA / DNA (> 10) / DNA (> 100) / RNA / RNA (> 10) / RNA (> 100) / RNA-directed DNA polymerase / AAA family ATPase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.99 Å
AuthorsBurman, N. / Thomas-George, J. / Wilkinson, R. / Wiedenheft, B.
Funding support United States, 2items
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, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 23, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: AAA family ATPase
B: AAA family ATPase
C: AAA family ATPase
D: AAA family ATPase
E: RNA-directed DNA polymerase
H: Retron IA msDNA
I: Retron IA ncRNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)393,05410
Polymers391,5337
Non-polymers1,5223
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
AAA family ATPase


Mass: 63393.953 Da / Num. of mol.: 4 / Mutation: N-terminal MWSHPQFEK, del native fMet
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: FORC_082 / Gene: QY721_001703 / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): AI / References: UniProt: A0AAD2V6K7
#2: Protein RNA-directed DNA polymerase


Mass: 36046.191 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: QY721_001704 / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): AI
References: UniProt: A0AAD2V6H6, RNA-directed DNA polymerase
#3: DNA chain Retron IA msDNA


Mass: 49825.891 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: FORC_082 / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): AI / References: GenBank: 1566436237
#4: RNA chain Retron IA ncRNA


Mass: 52084.648 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: FORC_082 / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): AI / References: GenBank: 1566436237
#5: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
Has ligand of interestY
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: Retron IA surveillance complex with HNH nuclease bound in the "down" orientation
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: FORC_082
Source (recombinant)Organism: Escherichia coli BL21 (bacteria) / Strain: AI
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 45000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 56.69 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 13695

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCv4.6.2particle selectioncryoSPARCs template picker was used to automatically pick particles
4cryoSPARCv4.6.2CTF correctioncryoSPARC's patch CTF estimation was used to estimate defocus on-the-fly. Per Particle CTF correction was enabled during final refinements.
7UCSF ChimeraX1.9model fittingChimeraX's fit in map command was used to fit individual chains predicted in Alphafold to the density
9cryoSPARCv4.6.2initial Euler assignmentMulti-Class Ab Initio reconstruction was used to obtain initial angle estimates for selected particles
10cryoSPARCv4.6.2final Euler assignmentNon-Uniform Refinement was used to assign final angles for selected particles
11cryoSPARCv4.6.2classificationFocused 3-D classification using masks generated from the consensus volume was used to sort particles
12cryoSPARCv4.6.2classificationA final round of 2-D classification was applied to remove poorly aligned particles prior to reconstruction
13cryoSPARCv4.6.23D reconstructionNon-Uniform refinement with minimize over per particle scale, optimize per particle defocus, optimize per exposure group CTF parames enabled
14ISOLDE1.6.0model refinementISOLDE was used to improve the fit of rigid body docked structures into the density
15PHENIX1.21.2-5419model refinementRealspace Refinement was used to obtain final models for deposition after manual editing
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5915380
Details: Particles picked using a 20 Angstrom lowpass filtered template volume that was produced de novo using blob picked particles
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 513948 / Algorithm: FOURIER SPACE
Details: Reported resolution from cryoSPARC's GSFSC estimation
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: Alphafold 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 ...Details: Alphafold 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.
Atomic model buildingDetails: The structure of each subunit was predicted individually in Alphafold before rigid body fitting in ChimeraX
Source name: AlphaFold / Type: in silico model

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