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- EMDB-25472: Cryo-EM structure of Arabidopsis Ago10-guide-target RNA complex i... -

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Basic information

Entry
Database: EMDB / ID: EMD-25472
TitleCryo-EM structure of Arabidopsis Ago10-guide-target RNA complex in a central 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*U)-3')
    • RNA: RNA (5'-R(P*CP*CP*AP*UP*UP*GP*UP*CP*AP*CP*AP*CP*UP*CP*CP*AP*A)-3')
  • Ligand: MAGNESIUM ION
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) / unidentified (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: 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, 2021-
Header (metadata) releaseAug 3, 2022-
Map releaseAug 3, 2022-
UpdateOct 5, 2022-
Current statusOct 5, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25472.png

Downloads & links

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Map

FileDownload / File: emd_25472.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: 0.008
Minimum - Maximum-0.03761009 - 0.06994262
Average (Standard dev.)7.741307e-05 (±0.002742027)
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: #1

Fileemd_25472_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_25472_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_25472_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*U)-3')
    • RNA: RNA (5'-R(P*CP*CP*AP*UP*UP*GP*UP*CP*AP*CP*AP*CP*UP*CP*CP*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)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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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*U)-3')

MacromoleculeName: RNA (5'-R(P*UP*GP*GP*AP*GP*UP*GP*UP*GP*AP*CP*AP*AP*UP*GP*GP*U)-3')
type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: unidentified (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*A)-3')

MacromoleculeName: RNA (5'-R(P*CP*CP*AP*UP*UP*GP*UP*CP*AP*CP*AP*CP*UP*CP*CP*AP*A)-3')
type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: unidentified (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
CTF correctionSoftware - Name: Gctf
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 reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.79 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1-cuda) / Number images used: 38833
FSC plot (resolution estimation)

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