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- EMDB-25482: Cryo-EM structure of Arabidopsis Ago10-guide-target RNA complex i... -

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Basic information

Entry
Database: EMDB / ID: EMD-25482
TitleCryo-EM structure of Arabidopsis Ago10-guide-target RNA complex in a bent duplex conformation
Map data
Sample
  • Complex: AtAgo10-guide-target 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')
    • RNA: RNA (5'-R(P*CP*CP*AP*UP*UP*GP*UP*CP*AP*CP*AP*CP*UP*CP*CP*AP*AP*A)-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 / 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
Protein argonaute 10
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.79 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 21, 2021-
Header (metadata) releaseNov 23, 2022-
Map releaseNov 23, 2022-
UpdateJul 5, 2023-
Current statusJul 5, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25482.png

Downloads & links

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Map

FileDownload / File: emd_25482.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.91 Å
Density
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.039292533 - 0.08276254
Average (Standard dev.)0.00007908888 (±0.003121295)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 182.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Local resolution filtered map

Fileemd_25482_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_25482_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_25482_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : AtAgo10-guide-target RNA complex

EntireName: AtAgo10-guide-target RNA complex
Components
  • Complex: AtAgo10-guide-target 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')
    • RNA: RNA (5'-R(P*CP*CP*AP*UP*UP*GP*UP*CP*AP*CP*AP*CP*UP*CP*CP*AP*AP*A)-3')
  • Ligand: MAGNESIUM ION

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Supramolecule #1: AtAgo10-guide-target RNA complex

SupramoleculeName: AtAgo10-guide-target RNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: The complex of Arabidopsis Argonaute10 with a synthetic guide RNA and a target pairing to 2-16nt of guide guide
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

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

MacromoleculeName: RNA (5'-R(P*CP*CP*AP*UP*UP*GP*UP*CP*AP*CP*AP*CP*UP*CP*CP*AP*AP*A)-3')
type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 5.636419 KDa
SequenceString:
CCAUUGUCAC ACUCCAAA

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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

Concentration0.3 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
10.0 mMC4H11NO3Tris
100.0 mMNaClSodium chlorideNaClSodium chloride
0.5 mMC9H15O6PTCEP
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.7 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 45000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 2049 / Average electron dose: 66.88 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1935081
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1-cuda)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1-cuda)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.79 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1-cuda) / Number images used: 28499
FSC plot (resolution estimation)

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