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- EMDB-25446: Cryo-EM structure of Arabidopsis Ago10-guide RNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-25446
TitleCryo-EM structure of Arabidopsis Ago10-guide RNA complex
Map data
Sample
  • Complex: AtAgo10-guide RNA complex
    • Protein or peptide: Protein argonaute 10
    • RNA: 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')
  • Ligand: MAGNESIUM ION
KeywordsmiRNA / siRNA / RNA silencing / Argonaute / plant / GENE REGULATION
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 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
Protein argonaute 10
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.26 Å
AuthorsXiao Y / MacRae IJ
Funding support United States, 1 items
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 18, 2021-
Header (metadata) releaseNov 23, 2022-
Map releaseNov 23, 2022-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25446.png

Downloads & links

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Map

FileDownload / File: emd_25446.map.gz / Format: CCP4 / Size: 134.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.57 Å/pix.
x 328 pix.
= 185.648 Å		0.57 Å/pix.
x 328 pix.
= 185.648 Å		0.57 Å/pix.
x 328 pix.
= 185.648 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.566 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.003
Minimum - Maximum-0.0022430215 - 0.012653796
Average (Standard dev.)0.000031453026 (±0.0006004572)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions328328328
Spacing328328328
CellA=B=C: 185.648 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Local resolution filtered map

Fileemd_25446_additional_1.map
AnnotationLocal resolution filtered map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_25446_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_25446_half_map_2.map
Projections & Slices
AxesZYX

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Sample components

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Entire : AtAgo10-guide RNA complex

EntireName: AtAgo10-guide RNA complex
Components
  • Complex: AtAgo10-guide RNA complex
    • Protein or peptide: Protein argonaute 10
    • RNA: 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')
  • Ligand: MAGNESIUM ION

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Supramolecule #1: AtAgo10-guide RNA complex

SupramoleculeName: AtAgo10-guide RNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: The complex of Arabidopsis Argonaute10 with a synthetic guide RNA
Source (natural)Organism: Arabidopsis thaliana (thale cress)

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Macromolecule #1: Protein argonaute 10

MacromoleculeName: Protein argonaute 10 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 110.981633 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MPIRQMKDSS ETHLVIKTQP LKHHNPKTVQ NGKIPPPSPS PVTVTTPATV TQSQASSPSP PSKNRSRRRN RGGRKSDQGD VCMRPSSRP RKPPPPSQTT SSAVSVATAG EIVAVNHQMQ MGVRKNSNFA PRPGFGTLGT KCIVKANHFL ADLPTKDLNQ Y DVTITPEV ...String:
MPIRQMKDSS ETHLVIKTQP LKHHNPKTVQ NGKIPPPSPS PVTVTTPATV TQSQASSPSP PSKNRSRRRN RGGRKSDQGD VCMRPSSRP RKPPPPSQTT SSAVSVATAG EIVAVNHQMQ MGVRKNSNFA PRPGFGTLGT KCIVKANHFL ADLPTKDLNQ Y DVTITPEV SSKSVNRAII AELVRLYKES DLGRRLPAYD GRKSLYTAGE LPFTWKEFSV KIVDEDDGII NGPKRERSYK VA IKFVARA NMHHLGEFLA GKRADCPQEA VQILDIVLRE LSVKRFCPVG RSFFSPDIKT PQRLGEGLES WCGFYQSIRP TQM GLSLNI DMASAAFIEP LPVIEFVAQL LGKDVLSKPL SDSDRVKIKK GLRGVKVEVT HRANVRRKYR VAGLTTQPTR ELMF PVDEN CTMKSVIEYF QEMYGFTIQH THLPCLQVGN QKKASYLPME ACKIVEGQRY TKRLNEKQIT ALLKVTCQRP RDREN DILR TVQHNAYDQD PYAKEFGMNI SEKLASVEAR ILPAPWLKYH ENGKEKDCLP QVGQWNMMNK KMINGMTVSR WACVNF SRS VQENVARGFC NELGQMCEVS GMEFNPEPVI PIYSARPDQV EKALKHVYHT SMNKTKGKEL ELLLAILPDN NGSLYGD LK RICETELGLI SQCCLTKHVF KISKQYLANV SLKINVKMGG RNTVLVDAIS CRIPLVSDIP TIIFGADVTH PENGEESS P SIAAVVASQD WPEVTKYAGL VCAQAHRQEL IQDLYKTWQD PVRGTVSGGM IRDLLISFRK ATGQKPLRII FYRAGVSEG QFYQVLLYEL DAIRKACASL EPNYQPPVTF IVVQKRHHTR LFANNHRDKN STDRSGNILP GTVVDTKICH PTEFDFYLCS HAGIQGTSR PAHYHVLWDE NNFTADGIQS LTNNLCYTYA RCTRSVSIVP PAYYAHLAAF RARFYLEPEI MQDNGSPGKK N TKTTTVGD VGVKPLPALK ENVKRVMFYC

UniProtKB: Protein argonaute 10

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

MacromoleculeName: 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')
type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.788021 KDa
SequenceString:
UGGAGUGUGA CAAUGGUGUU U

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / 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

Concentration0.3 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
10.0 mMC4H11NO3Tris
100.0 mMNaClNaCl
0.5 mMC9H15O6PTCEP
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 2656 / Average electron dose: 66.51 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.3 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 73000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 736209
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.26 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 53766
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1-cuda)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1-cuda)
FSC plot (resolution estimation)

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