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- PDB-9dhx: human Argonaute2 R315V/H316A - guide RNA in complex with a fully ... -

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Basic information

Entry
Database: PDB / ID: 9dhx
Titlehuman Argonaute2 R315V/H316A - guide RNA in complex with a fully complementary target
Components
  • Argonaute2 R315V/H316A
  • guide RNA
  • target RNA
KeywordsRNA BINDING PROTEIN / RNP / RNA / Argonaute2 / Ago2
Function / homology
Function and homology information


: / endoribonuclease activity, cleaving miRNA-paired mRNA / endoribonuclease activity, cleaving siRNA-paired mRNA / siRNA-mediated gene silencing by mRNA destabilization / Post-transcriptional silencing by small RNAs / Competing endogenous RNAs (ceRNAs) regulate PTEN translation / Regulation of CDH11 mRNA translation by microRNAs / Regulation of NPAS4 mRNA translation / Regulation of PTEN mRNA translation / miRNA-mediated gene silencing by mRNA destabilization ...: / endoribonuclease activity, cleaving miRNA-paired mRNA / endoribonuclease activity, cleaving siRNA-paired mRNA / siRNA-mediated gene silencing by mRNA destabilization / Post-transcriptional silencing by small RNAs / Competing endogenous RNAs (ceRNAs) regulate PTEN translation / Regulation of CDH11 mRNA translation by microRNAs / Regulation of NPAS4 mRNA translation / Regulation of PTEN mRNA translation / miRNA-mediated gene silencing by mRNA destabilization / negative regulation of amyloid precursor protein biosynthetic process / RNA stabilization / Small interfering RNA (siRNA) biogenesis / Regulation of CDH1 mRNA translation by microRNAs / positive regulation of trophoblast cell migration / Transcriptional Regulation by MECP2 / RISC-loading complex / miRNA metabolic process / mRNA cap binding / RISC complex assembly / regulatory ncRNA-mediated post-transcriptional gene silencing / miRNA-mediated gene silencing by inhibition of translation / miRNA processing / RNA 7-methylguanosine cap binding / pre-miRNA processing / regulation of synapse maturation / siRNA binding / mRNA 3'-UTR AU-rich region binding / M-decay: degradation of maternal mRNAs by maternally stored factors / siRNA processing / Regulation of MITF-M-dependent genes involved in apoptosis / RISC complex / TGFBR3 expression / regulatory ncRNA-mediated gene silencing / Regulation of RUNX1 Expression and Activity / P-body assembly / miRNA binding / MicroRNA (miRNA) biogenesis / positive regulation of nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / positive regulation of nuclear-transcribed mRNA poly(A) tail shortening / RNA polymerase II complex binding / Regulation of MECP2 expression and activity / core promoter sequence-specific DNA binding / NR1H3 & NR1H2 regulate gene expression linked to cholesterol transport and efflux / Nuclear events stimulated by ALK signaling in cancer / RNA endonuclease activity / translation initiation factor activity / negative regulation of translational initiation / post-embryonic development / TP53 Regulates Metabolic Genes / positive regulation of translation / Transcriptional regulation by small RNAs / P-body / MAPK6/MAPK4 signaling / Pre-NOTCH Transcription and Translation / positive regulation of angiogenesis / cytoplasmic ribonucleoprotein granule / double-stranded RNA binding / Ca2+ pathway / Estrogen-dependent gene expression / single-stranded RNA binding / postsynapse / translation / dendrite / glutamatergic synapse / positive regulation of transcription by RNA polymerase II / RNA binding / extracellular exosome / nucleoplasm / membrane / metal ion binding / nucleus / cytoplasm / cytosol
Similarity search - Function
Protein argonaute-2 / 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 ...Protein argonaute-2 / 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 profile. / Piwi domain / 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-2
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsSarkar, S. / Gebert, L.F.R. / MacRae, I.J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R35GM127090 United States
CitationJournal: Nat Struct Mol Biol / Year: 2026
Title: Catalytic activation of human Argonaute 2 requires RNA duplex deformation.
Authors: Sucharita Sarkar / Luca F R Gebert / Ian J MacRae /
Abstract: Small interfering RNAs (siRNAs) are an expanding class of RNA therapeutics, with seven drugs approved by the US Food and Drug Administration and many more in development. Rational design, however, ...Small interfering RNAs (siRNAs) are an expanding class of RNA therapeutics, with seven drugs approved by the US Food and Drug Administration and many more in development. Rational design, however, has been limited by incomplete understanding of how human Argonaute 2 (hAgo2) catalyzes target cleavage. Here we report high-resolution cryo-electron microscopy structures of hAgo2 bound to a target RNA in catalytic and noncatalytic conformations. The structures reveal that guide-target pairing alone is insufficient for slicing and catalysis requires deformation of the duplex through a coordinated network of RNA-protein interactions. Expansion of the central major groove positions the scissile phosphate, while compression toward the supplementary region docks the duplex into the hAgo2 cleft. A kink after guide nucleotide g6 disrupts seed-only pairing conformation and promotes the extended pairing required for catalysis. This rearrangement enables repositioning of K709 within the active site, while a pyrimidine at target position t10 optimally aligns R710 to accelerate cleavage. These findings provide a structural framework linking siRNA duplex geometry to catalytic efficiency and inform rational design of siRNAs with improved potency and specificity.
History
DepositionSep 4, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 4, 2026Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Argonaute2 R315V/H316A
B: guide RNA
C: target RNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)110,3834
Polymers110,3593
Non-polymers241
Water362
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Argonaute2 R315V/H316A / Argonaute2 / hAgo2 / Argonaute RISC catalytic component 2 / Eukaryotic translation initiation ...Argonaute2 / hAgo2 / Argonaute RISC catalytic component 2 / Eukaryotic translation initiation factor 2C 2 / eIF-2C 2 / eIF2C 2 / PAZ Piwi domain protein / PPD / Protein slicer


Mass: 97277.062 Da / Num. of mol.: 1 / Mutation: R315V, H316A, S387D, S824A, S828D, S831D, S834A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AGO2, EIF2C2 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q9UKV8, Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters
#2: RNA chain guide RNA


Mass: 6481.856 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: RNA chain target RNA


Mass: 6600.012 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
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O
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: human Argonaute-2 R315V/H316A - miR-122 in complex with a fully complementary target
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTRISTRIS1
2100 mMNaClNaCl1
30.5 mMTCEPTCEP1
SpecimenConc.: 1.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 190000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.2 sec. / Electron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 6 / Num. of real images: 15000
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.3particle selection
2EPUimage acquisition
4cryoSPARC4.3CTF correction
7UCSF ChimeraX1.7.1model fitting
11cryoSPARC4.3classification
12cryoSPARC4.33D reconstruction
19PHENIX1.20.1model refinement
20Coot0.9.8.1model refinement
21UCSF ChimeraX1.7.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3286293
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 264433 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6N4O

6n4o


Pdb chain-ID: A / Accession code: 6N4O / Source name: PDB / Type: experimental model
RefinementHighest resolution: 3.1 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0046624
ELECTRON MICROSCOPYf_angle_d0.5469162
ELECTRON MICROSCOPYf_dihedral_angle_d7.8691250
ELECTRON MICROSCOPYf_chiral_restr0.0421052
ELECTRON MICROSCOPYf_plane_restr0.0051027

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