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- EMDB-62134: Cryo-EM Structure of hAGO2D669A-siRNA-target (19-nt) -

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Basic information

Entry
Database: EMDB / ID: EMD-62134
TitleCryo-EM Structure of hAGO2D669A-siRNA-target (19-nt)
Map data
Sample
  • Complex: Complex of hAGO2D669A-siRNA-target (19-nt)
    • Protein or peptide: Protein argonaute-2
    • RNA: RNA (5'-R(P*UP*AP*CP*AP*AP*GP*AP*GP*CP*CP*UP*UP*UP*CP*UP*GP*UP*UP*G)-3')
    • RNA: RNA (5'-R(P*CP*AP*AP*CP*AP*GP*AP*AP*AP*GP*GP*CP*UP*CP*UP*UP*GP*UP*U)-3')
  • Ligand: MAGNESIUM ION
KeywordsArgonaute protein / siRNA / RNA BINDING PROTEIN/RNA / RNA BINDING PROTEIN
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 / miRNA-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 ...: / endoribonuclease activity, cleaving miRNA-paired mRNA / endoribonuclease activity, cleaving siRNA-paired mRNA / siRNA-mediated gene silencing by mRNA destabilization / miRNA-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 / negative regulation of amyloid precursor protein biosynthetic process / Small interfering RNA (siRNA) biogenesis / positive regulation of trophoblast cell migration / Transcriptional Regulation by MECP2 / miRNA metabolic process / RISC-loading complex / mRNA cap binding / regulatory ncRNA-mediated post-transcriptional gene silencing / RISC complex assembly / miRNA processing / miRNA-mediated gene silencing by inhibition of translation / pre-miRNA processing / RNA 7-methylguanosine cap binding / siRNA processing / regulation of synapse maturation / siRNA binding / Regulation of MITF-M-dependent genes involved in apoptosis / mRNA 3'-UTR AU-rich region binding / M-decay: degradation of maternal mRNAs by maternally stored factors / RISC complex / regulatory ncRNA-mediated gene silencing / TGFBR3 expression / Regulation of RUNX1 Expression and Activity / P-body assembly / miRNA binding / positive regulation of nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / MicroRNA (miRNA) biogenesis / 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 / Nuclear events stimulated by ALK signaling in cancer / NR1H3 & NR1H2 regulate gene expression linked to cholesterol transport and efflux / negative regulation of translational initiation / translation initiation factor activity / RNA endonuclease activity / positive regulation of translation / post-embryonic development / TP53 Regulates Metabolic Genes / Transcriptional regulation by small RNAs / P-body / MAPK6/MAPK4 signaling / Pre-NOTCH Transcription and Translation / cytoplasmic ribonucleoprotein granule / positive regulation of angiogenesis / 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 / metal ion binding / nucleus / membrane / cytosol / cytoplasm
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
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsLi ZZ / Xu QK / Wu JP / Shen EZ
Funding support1 items
OrganizationGrant numberCountry
Other private
CitationJournal: Cell Res / Year: 2025
Title: Mechanistic insights into RNA cleavage by human Argonaute2-siRNA complex.
Authors: Zhenzhen Li / Qikui Xu / Yan Zhang / Jing Zhong / Tianxiang Zhang / Junchao Xue / Shuxian Liu / Haishan Gao / Z Z Zhao Zhang / Jianping Wu / En-Zhi Shen /
Abstract: In animals, AGO-clade Argonaute proteins utilize small interfering RNAs (siRNAs) as guides to recognize target with complete complementarity, resulting in target RNA cleavage that is a critical step ...In animals, AGO-clade Argonaute proteins utilize small interfering RNAs (siRNAs) as guides to recognize target with complete complementarity, resulting in target RNA cleavage that is a critical step for target silencing. These proteins feature a constricted nucleic acid-binding channel that limits base pairing between the guide and target beyond the seed region. How the AGO-siRNA complexes overcome this structural limitation and achieve efficient target cleavage remains unclear. We performed cryo-electron microscopy of human AGO-siRNA complexes bound to target RNAs of increasing lengths to examine the conformational changes associated with target recognition and cleavage. Initially, conformational transition propagates from the opening of the PAZ domain and extends through a repositioning of the PIWI-L1-N domain toward the binding channel, facilitating the capture of siRNA-target duplex. Subsequent extension of base pairing drives the downward movement of the PIWI-L1-N domain to enable catalytic activation. Finally, further base pairing toward the 3' end of siRNA destabilizes the PAZ-N domain, resulting in a "uni-lobed" architecture, which might facilitate the multi-turnover action of the AGO-siRNA enzyme complex. In contrast to PIWI-clade Argonautes, the "uni-lobed" structure of the AGO complex makes multiple contacts with the target in the central region of the siRNA-target duplex, positioning it within the catalytic site. Our findings shed light on the stepwise mechanisms by which the AGO-siRNA complex executes target RNA cleavage and offer insights into the distinct operational modalities of AGO and PIWI proteins in achieving such cleavage.
History
DepositionOct 22, 2024-
Header (metadata) releaseJun 25, 2025-
Map releaseJun 25, 2025-
UpdateJun 25, 2025-
Current statusJun 25, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_62134.png

Downloads & links

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Map

FileDownload / File: emd_62134.map.gz / Format: CCP4 / Size: 13.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.14 Å/pix.
x 154 pix.
= 175.56 Å		1.14 Å/pix.
x 154 pix.
= 175.56 Å		1.14 Å/pix.
x 154 pix.
= 175.56 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.14 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.248
Minimum - Maximum-1.1720711 - 1.8565143
Average (Standard dev.)-0.00013257875 (±0.041119643)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions154154154
Spacing154154154
CellA=B=C: 175.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_62134_half_map_1.map
Projections & Slices
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Slices (1/2)
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Half map: #1

Fileemd_62134_half_map_2.map
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Sample components

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Entire : Complex of hAGO2D669A-siRNA-target (19-nt)

EntireName: Complex of hAGO2D669A-siRNA-target (19-nt)
Components
  • Complex: Complex of hAGO2D669A-siRNA-target (19-nt)
    • Protein or peptide: Protein argonaute-2
    • RNA: RNA (5'-R(P*UP*AP*CP*AP*AP*GP*AP*GP*CP*CP*UP*UP*UP*CP*UP*GP*UP*UP*G)-3')
    • RNA: RNA (5'-R(P*CP*AP*AP*CP*AP*GP*AP*AP*AP*GP*GP*CP*UP*CP*UP*UP*GP*UP*U)-3')
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Complex of hAGO2D669A-siRNA-target (19-nt)

SupramoleculeName: Complex of hAGO2D669A-siRNA-target (19-nt) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Protein argonaute-2

MacromoleculeName: Protein argonaute-2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 97.306164 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MYSGAGPALA PPAPPPPIQG YAFKPPPRPD FGTSGRTIKL QANFFEMDIP KIDIYHYELD IKPEKCPRRV NREIVEHMVQ HFKTQIFGD RKPVFDGRKN LYTAMPLPIG RDKVELEVTL PGEGKDRIFK VSIKWVSCVS LQALHDALSG RLPSVPFETI Q ALDVVMRH ...String:
MYSGAGPALA PPAPPPPIQG YAFKPPPRPD FGTSGRTIKL QANFFEMDIP KIDIYHYELD IKPEKCPRRV NREIVEHMVQ HFKTQIFGD RKPVFDGRKN LYTAMPLPIG RDKVELEVTL PGEGKDRIFK VSIKWVSCVS LQALHDALSG RLPSVPFETI Q ALDVVMRH LPSMRYTPVG RSFFTASEGC SNPLGGGREV WFGFHQSVRP SLWKMMLNID VSATAFYKAQ PVIEFVCEVL DF KSIEEQQ KPLTDSQRVK FTKEIKGLKV EITHCGQMKR KYRVCNVTRR PASHQTFPLQ QESGQTVECT VAQYFKDRHK LVL RYPHLP CLQVGQEQKH TYLPLEVCNI VAGQRCIKKL TDNQTSTMIR ATARSAPDRQ EEISKLMRSA SFNTDPYVRE FGIM VKDEM TDVTGRVLQP PSILYGGRNK AIATPVQGVW DMRNKQFHTG IEIKVWAIAC FAPQRQCTEV HLKSFTEQLR KISRD AGMP IQGQPCFCKY AQGADSVEPM FRHLKNTYAG LQLVVVILPG KTPVYAEVKR VGDTVLGMAT QCVQMKNVQR TTPQTL SNL CLKINVKLGG VNNILLPQGR PPVFQQPVIF LGADVTHPPA GDGKKPSIAA VVGSMDAHPN RYCATVRVQQ HRQEIIQ DL AAMVRELLIQ FYKSTRFKPT RIIFYRAGVS EGQFQQVLHH ELLAIREACI KLEKDYQPGI TFIVVQKRHH TRLFCTDK N ERVGKSGNIP AGTTVDTKIT HPTEFDFYLC SHAGIQGTSR PSHYHVLWDD NRFSSDELQI LTYQLCHTYV RCTRSVSIP APAYYAHLVA FRARYHLVDK EHDSAEGSHT SGQSNGRDHQ ALAKAVQVHQ DTLRTMYFA

UniProtKB: Protein argonaute-2

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Macromolecule #2: RNA (5'-R(P*UP*AP*CP*AP*AP*GP*AP*GP*CP*CP*UP*UP*UP*CP*UP*GP*UP*UP...

MacromoleculeName: RNA (5'-R(P*UP*AP*CP*AP*AP*GP*AP*GP*CP*CP*UP*UP*UP*CP*UP*GP*UP*UP*G)-3')
type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 6.016577 KDa
SequenceString:
UACAAGAGCC UUUCUGUUG

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Macromolecule #3: RNA (5'-R(P*CP*AP*AP*CP*AP*GP*AP*AP*AP*GP*GP*CP*UP*CP*UP*UP*GP*UP...

MacromoleculeName: RNA (5'-R(P*CP*AP*AP*CP*AP*GP*AP*AP*AP*GP*GP*CP*UP*CP*UP*UP*GP*UP*U)-3')
type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 6.062657 KDa
SequenceString:
CAACAGAAAG GCUCUUGUU

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 51296
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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