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

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Basic information

Entry
Database: PDB / ID: 7swf
TitleCryo-EM structure of Arabidopsis Ago10-guide-target RNA complex in a central 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*A)-3')
  • RNA (5'-R(P*UP*GP*GP*AP*GP*UP*GP*UP*GP*AP*CP*AP*AP*UP*GP*GP*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 / Mid domain of argonaute / Argonaute linker 2 domain / Protein argonaute, N-terminal / Argonaute-like, PIWI domain / N-terminal domain of argonaute / Argonaute linker 2 domain / DUF1785 / Argonaute, linker 1 domain / Argonaute linker 1 domain ...Protein argonaute, Mid domain / Mid domain of argonaute / Argonaute linker 2 domain / Protein argonaute, N-terminal / Argonaute-like, PIWI domain / 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 profile. / PAZ domain / 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)
unidentified (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: Nucleic Acids Res / Year: 2022
Title: The molecular mechanism of microRNA duplex selectivity of Arabidopsis ARGONAUTE10.
Authors: Yao Xiao / Ian J MacRae /
Abstract: Small RNAs (sRNAs), including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are essential gene regulators for plant and animal development. The loading of sRNA duplexes into the proper ...Small RNAs (sRNAs), including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are essential gene regulators for plant and animal development. The loading of sRNA duplexes into the proper ARGONAUTE (AGO) protein is a key step to forming a functional silencing complex. In Arabidopsis thaliana, the specific loading of miR166/165 into AGO10 (AtAGO10) is critical for the maintenance of the shoot apical meristem, the source of all shoot organs, but the mechanism by which AtAGO10 distinguishes miR166/165 from other cellular miRNAs is not known. Here, we show purified AtAGO10 alone lacks loading selectivity towards miR166/165 duplexes. However, phosphate and HSP chaperone systems reshape the selectivity of AtAGO10 to its physiological substrates. A loop in the AtAGO10 central cleft is essential for recognizing specific mismatches opposite the guide strand 3' region in miR166/165 duplexes. Replacing this loop with the equivalent loop from Homo sapiens AGO2 (HsAGO2) changes AtAGO10 miRNA loading behavior such that 3' region mismatches are ignored and mismatches opposite the guide 5' end instead drive loading, as in HsAGO2. Thus, this study uncovers the molecular mechanism underlying the miR166/165 selectivity of AtAGO10, essential for plant development, and provides new insights into how miRNA duplex structures are recognized for sRNA sorting.
History
DepositionNov 19, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 3, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 5, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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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*U)-3')
D: RNA (5'-R(P*CP*CP*AP*UP*UP*GP*UP*CP*AP*CP*AP*CP*UP*CP*CP*AP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)123,4304
Polymers123,4063
Non-polymers241
Water00
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*U)-3')


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


Mass: 5636.419 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) unidentified (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 mMNaClNaCl1
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 FIELD / 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

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
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
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.79 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 38833 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.017083
ELECTRON MICROSCOPYf_angle_d0.8759744
ELECTRON MICROSCOPYf_dihedral_angle_d17.3361244
ELECTRON MICROSCOPYf_chiral_restr0.0491116
ELECTRON MICROSCOPYf_plane_restr0.0061132

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