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- PDB-7swq: Cryo-EM structure of Arabidopsis Ago10-guide-target RNA complex i... -

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Basic information

Entry
Database: PDB / ID: 7swq
TitleCryo-EM structure of Arabidopsis Ago10-guide-target RNA complex in a bent duplex conformation
Components
  • Protein argonaute 10
  • RNA (5'-R(P*CP*CP*AP*UP*UP*GP*UP*CP*AP*CP*AP*CP*UP*CP*CP*AP*AP*A)-3')
  • RNA (5'-R(P*UP*GP*GP*AP*GP*UP*GP*UP*GP*AP*CP*AP*AP*UP*GP*GP*UP*GP*UP*UP*U)-3')
KeywordsGENE REGULATION / miRNA / siRNA / RNA silencing / Argonaute / plant
Function / homology
Function and homology information


regulation of shoot apical meristem development / primary shoot apical meristem specification / regulation of meristem structural organization / miRNA metabolic process / regulatory ncRNA-mediated gene silencing / miRNA binding / plastid / somatic stem cell population maintenance / regulation of translation / defense response to virus ...regulation of shoot apical meristem development / primary shoot apical meristem specification / regulation of meristem structural organization / miRNA metabolic process / regulatory ncRNA-mediated gene silencing / miRNA binding / plastid / somatic stem cell population maintenance / regulation of translation / defense response to virus / ribonucleoprotein complex / cytoplasm
Similarity search - Function
Protein argonaute, Mid domain / Argonaute-like, PIWI domain / Mid domain of argonaute / Argonaute linker 2 domain / Protein argonaute, N-terminal / N-terminal domain of argonaute / Argonaute linker 2 domain / DUF1785 / Argonaute, linker 1 domain / Argonaute linker 1 domain ...Protein argonaute, Mid domain / Argonaute-like, PIWI domain / Mid domain of argonaute / Argonaute linker 2 domain / Protein argonaute, N-terminal / N-terminal domain of argonaute / Argonaute linker 2 domain / DUF1785 / Argonaute, linker 1 domain / Argonaute linker 1 domain / Piwi domain / Piwi domain profile. / Piwi domain / Piwi / PAZ domain superfamily / PAZ / PAZ domain / PAZ domain / PAZ domain profile. / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / Protein argonaute 10
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.79 Å
AuthorsXiao, Y. / MacRae, I.J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM127090 United States
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Structural basis for RNA slicing by a plant Argonaute.
Authors: Yao Xiao / Shintaro Maeda / Takanori Otomo / Ian J MacRae /
Abstract: Argonaute (AGO) proteins use small RNAs to recognize transcripts targeted for silencing in plants and animals. Many AGOs cleave target RNAs using an endoribonuclease activity termed 'slicing'. ...Argonaute (AGO) proteins use small RNAs to recognize transcripts targeted for silencing in plants and animals. Many AGOs cleave target RNAs using an endoribonuclease activity termed 'slicing'. Slicing by DNA-guided prokaryotic AGOs has been studied in detail, but structural insights into RNA-guided slicing by eukaryotic AGOs are lacking. Here we present cryogenic electron microscopy structures of the Arabidopsis thaliana Argonaute10 (AtAgo10)-guide RNA complex with and without a target RNA representing a slicing substrate. The AtAgo10-guide-target complex adopts slicing-competent and slicing-incompetent conformations that are unlike known prokaryotic AGO structures. AtAgo10 slicing activity is licensed by docking target (t) nucleotides t9-t13 into a surface channel containing the AGO endoribonuclease active site. A β-hairpin in the L1 domain secures the t9-t13 segment and coordinates t9-t13 docking with extended guide-target pairing. Results show that prokaryotic and eukaryotic AGOs use distinct mechanisms for achieving target slicing and provide insights into small interfering RNA potency.
History
DepositionNov 21, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 23, 2022Provider: repository / Type: Initial release
Revision 1.1May 17, 2023Group: Database references / Source and taxonomy / Category: citation / citation_author / pdbx_entity_src_syn
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _pdbx_entity_src_syn.ncbi_taxonomy_id / _pdbx_entity_src_syn.organism_scientific
Revision 1.2Jul 5, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Protein argonaute 10
B: RNA (5'-R(P*UP*GP*GP*AP*GP*UP*GP*UP*GP*AP*CP*AP*AP*UP*GP*GP*UP*GP*UP*UP*U)-3')
D: RNA (5'-R(P*CP*CP*AP*UP*UP*GP*UP*CP*AP*CP*AP*CP*UP*CP*CP*AP*AP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)123,4304
Polymers123,4063
Non-polymers241
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Protein argonaute 10 / Protein PINHEAD / Protein ZWILLE


Mass: 110981.633 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: AGO10, PNH, ZLL, At5g43810, MQD19.17 / Variant: D795A / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9XGW1
#2: RNA chain RNA (5'-R(P*UP*GP*GP*AP*GP*UP*GP*UP*GP*AP*CP*AP*AP*UP*GP*GP*UP*GP*UP*UP*U)-3')


Mass: 6788.021 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: RNA chain RNA (5'-R(P*CP*CP*AP*UP*UP*GP*UP*CP*AP*CP*AP*CP*UP*CP*CP*AP*AP*A)-3')


Mass: 5636.419 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: AtAgo10-guide-target RNA complex / Type: COMPLEX
Details: The complex of Arabidopsis Argonaute10 with a synthetic guide RNA and a target pairing to 2-16nt of guide guide
Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTrisC4H11NO31
2100 mMNaClSodium chlorideNaClSodium chloride1
30.5 mMTCEPC9H15O6P1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 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 FIELDBright-field microscopy / Nominal magnification: 45000 X / Nominal defocus max: 1700 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 66.88 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 2049
Image scansMovie frames/image: 42

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Processing

EM software
IDNameVersionCategory
4GctfCTF correction
10RELION3.1-cudainitial Euler assignment
11RELION3.1-cudafinal Euler assignment
13RELION3.1-cuda3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1935081
3D reconstructionResolution: 3.79 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 28499 / Symmetry type: POINT

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