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- PDB-8r3z: Cryo-EM structure of the Asgard archaeal Argonaute HrAgo1 bound t... -

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Basic information

Entry
Database: PDB / ID: 8r3z
TitleCryo-EM structure of the Asgard archaeal Argonaute HrAgo1 bound to a guide RNA
Components
  • HrAgo1
  • RNA (5'-R(P*UP*GP*AP*GP*GP*U*(MG))-3')
KeywordsRNA BINDING PROTEIN / Argonaute / RNA
Function / homologyRNA / RNA (> 10)
Function and homology information
Biological speciesCandidatus Harpocratesius repetitus (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsFinocchio, G. / Swarts, D. / Jinek, M.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)European Union
CitationJournal: Nat Commun / Year: 2024
Title: RNA-guided RNA silencing by an Asgard archaeal Argonaute.
Authors: Carolien Bastiaanssen / Pilar Bobadilla Ugarte / Kijun Kim / Giada Finocchio / Yanlei Feng / Todd A Anzelon / Stephan Köstlbacher / Daniel Tamarit / Thijs J G Ettema / Martin Jinek / Ian J ...Authors: Carolien Bastiaanssen / Pilar Bobadilla Ugarte / Kijun Kim / Giada Finocchio / Yanlei Feng / Todd A Anzelon / Stephan Köstlbacher / Daniel Tamarit / Thijs J G Ettema / Martin Jinek / Ian J MacRae / Chirlmin Joo / Daan C Swarts / Fabai Wu /
Abstract: Argonaute proteins are the central effectors of RNA-guided RNA silencing pathways in eukaryotes, playing crucial roles in gene repression and defense against viruses and transposons. Eukaryotic ...Argonaute proteins are the central effectors of RNA-guided RNA silencing pathways in eukaryotes, playing crucial roles in gene repression and defense against viruses and transposons. Eukaryotic Argonautes are subdivided into two clades: AGOs generally facilitate miRNA- or siRNA-mediated silencing, while PIWIs generally facilitate piRNA-mediated silencing. It is currently unclear when and how Argonaute-based RNA silencing mechanisms arose and diverged during the emergence and early evolution of eukaryotes. Here, we show that in Asgard archaea, the closest prokaryotic relatives of eukaryotes, an evolutionary expansion of Argonaute proteins took place. In particular, a deep-branching PIWI protein (HrAgo1) encoded by the genome of the Lokiarchaeon 'Candidatus Harpocratesius repetitus' shares a common origin with eukaryotic PIWI proteins. Contrasting known prokaryotic Argonautes that use single-stranded DNA as guides and/or targets, HrAgo1 mediates RNA-guided RNA cleavage, and facilitates gene silencing when expressed in human cells and supplied with miRNA precursors. A cryo-EM structure of HrAgo1, combined with quantitative single-molecule experiments, reveals that the protein displays structural features and target-binding modes that are a mix of those of eukaryotic AGO and PIWI proteins. Thus, this deep-branching archaeal PIWI may have retained an ancestral molecular architecture that preceded the functional and mechanistic divergence of eukaryotic AGOs and PIWIs.
History
DepositionNov 10, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 19, 2024Provider: repository / Type: Initial release
Revision 1.0Jun 19, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Revision 1.1Jul 10, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update
Revision 1.2Jul 9, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details
Item: _em_admin.last_update / _em_software.name / _pdbx_entry_details.has_protein_modification
Revision 1.1Jul 9, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: HrAgo1
B: RNA (5'-R(P*UP*GP*AP*GP*GP*U*(MG))-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)102,2063
Polymers102,1822
Non-polymers241
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 HrAgo1


Mass: 95369.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Candidatus Harpocratesius repetitus (archaea)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#2: RNA chain RNA (5'-R(P*UP*GP*AP*GP*GP*U*(MG))-3')


Mass: 6812.045 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Candidatus Harpocratesius repetitus (archaea)
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN
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: Asgard archaeal Argonaute HrAgo1 in complex with the 5'-end of a guide RNA
Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Candidatus Harpocratesius repetitus (archaea)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 56.81 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 283659 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036541
ELECTRON MICROSCOPYf_angle_d0.6278862
ELECTRON MICROSCOPYf_dihedral_angle_d5.995890
ELECTRON MICROSCOPYf_chiral_restr0.045986
ELECTRON MICROSCOPYf_plane_restr0.0051098

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