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- EMDB-40713: Monomeric MapSPARTA bound with guide RNA and target DNA hybrid -

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Basic information

Entry
Database: EMDB / ID: EMD-40713
TitleMonomeric MapSPARTA bound with guide RNA and target DNA hybrid
Map data
Sample
  • Complex: Complex of MapSPARTA binding with gRNA tDNA hybrid
    • Protein or peptide: TIR-APAZ
    • Protein or peptide: short pAgo
    • RNA: guide RNA
    • DNA: target DNA
  • Ligand: MAGNESIUM ION
KeywordsgRNA mediated DNA binding / Microbiology / Argonaute / microbic immune system / IMMUNE SYSTEM
Function / homology
Function and homology information


nucleic acid binding / signal transduction
Similarity search - Function
TIR domain / TIR domain profile. / Toll/interleukin-1 receptor homology (TIR) domain / Toll/interleukin-1 receptor homology (TIR) domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
Piwi domain-containing protein / TIR domain-containing protein
Similarity search - Component
Biological speciesMaribacter polysiphoniae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.28 Å
AuthorsShen ZF / Yang XY / Fu TM
Funding support United States, 1 items
OrganizationGrant numberCountry
Not funded United States
CitationJournal: Nature / Year: 2023
Title: Oligomerization-mediated activation of a short prokaryotic Argonaute.
Authors: Zhangfei Shen / Xiao-Yuan Yang / Shiyu Xia / Wei Huang / Derek J Taylor / Kotaro Nakanishi / Tian-Min Fu /
Abstract: Although eukaryotic and long prokaryotic Argonaute proteins (pAgos) cleave nucleic acids, some short pAgos lack nuclease activity and hydrolyse NAD(P) to induce bacterial cell death. Here we present ...Although eukaryotic and long prokaryotic Argonaute proteins (pAgos) cleave nucleic acids, some short pAgos lack nuclease activity and hydrolyse NAD(P) to induce bacterial cell death. Here we present a hierarchical activation pathway for SPARTA, a short pAgo consisting of an Argonaute (Ago) protein and TIR-APAZ, an associated protein. SPARTA progresses through distinct oligomeric forms, including a monomeric apo state, a monomeric RNA-DNA-bound state, two dimeric RNA-DNA-bound states and a tetrameric RNA-DNA-bound active state. These snapshots together identify oligomerization as a mechanistic principle of SPARTA activation. The RNA-DNA-binding channel of apo inactive SPARTA is occupied by an auto-inhibitory motif in TIR-APAZ. After the binding of RNA-DNA, SPARTA transitions from a monomer to a symmetric dimer and then an asymmetric dimer, in which two TIR domains interact through charge and shape complementarity. Next, two dimers assemble into a tetramer with a central TIR cluster responsible for hydrolysing NAD(P). In addition, we observe unique features of interactions between SPARTA and RNA-DNA, including competition between the DNA 3' end and the auto-inhibitory motif, interactions between the RNA G2 nucleotide and Ago, and splaying of the RNA-DNA duplex by two loops exclusive to short pAgos. Together, our findings provide a mechanistic basis for the activation of short pAgos, a large section of the Ago superfamily.
History
DepositionMay 4, 2023-
Header (metadata) releaseAug 23, 2023-
Map releaseAug 23, 2023-
UpdateSep 20, 2023-
Current statusSep 20, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40713.png

Downloads & links

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Map

FileDownload / File: emd_40713.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.95 Å
Density
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.6431066 - 1.2912396
Average (Standard dev.)-0.0019577655 (±0.029806077)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 243.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: sharped map

Fileemd_40713_additional_1.map
Annotationsharped map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_40713_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_40713_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 : Complex of MapSPARTA binding with gRNA tDNA hybrid

EntireName: Complex of MapSPARTA binding with gRNA tDNA hybrid
Components
  • Complex: Complex of MapSPARTA binding with gRNA tDNA hybrid
    • Protein or peptide: TIR-APAZ
    • Protein or peptide: short pAgo
    • RNA: guide RNA
    • DNA: target DNA
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Complex of MapSPARTA binding with gRNA tDNA hybrid

SupramoleculeName: Complex of MapSPARTA binding with gRNA tDNA hybrid / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Maribacter polysiphoniae (bacteria)

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Macromolecule #1: TIR-APAZ

MacromoleculeName: TIR-APAZ / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Maribacter polysiphoniae (bacteria)
Molecular weightTheoretical: 53.139398 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: RNKIFISHAT PDDNDFTRWL ALKLIGLGYE VWCDILFLDK GVDFWSNIEK VIREDTCKFL LVSSSYSNQR EGVLKELAVA AKVKKQLKD DKFIIPLAID EQLSYDDINI DIVRLNAIDF KMSWARGLKD ILEAFEKQKV PKEVADASKS NLLYQQIFLH D KSVIEKEE ...String:
RNKIFISHAT PDDNDFTRWL ALKLIGLGYE VWCDILFLDK GVDFWSNIEK VIREDTCKFL LVSSSYSNQR EGVLKELAVA AKVKKQLKD DKFIIPLAID EQLSYDDINI DIVRLNAIDF KMSWARGLKD ILEAFEKQKV PKEVADASKS NLLYQQIFLH D KSVIEKEE IYDSNWLSIL SFPEELRFHE YNWMLPKRFD VRELTFPAVR YKNYLCTFAW AYDFTYHLPK TETYHKSKTI RI PTEEILS GSYDSNFIRN AECKRLIVQL LNKAFELRMK DKEVQEYEMS NKTAYWLEKG KLEKDKFEKT MLVGKQKDKN WHF AISGAS KLYPFPVLMI SSHIFFTADG KKLIDSSSVQ HSSRRRQGKN WWNNTWRTKL LAFIKYLSDD DTSFYLEMGS EEKV FVSNE PVKFKGNVSY NIPEKNTLEE EAELSGFNQG EDIEELEELI ENLEAE

UniProtKB: TIR domain-containing protein

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Macromolecule #2: short pAgo

MacromoleculeName: short pAgo / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Maribacter polysiphoniae (bacteria)
Molecular weightTheoretical: 58.09141 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKELIYIEEP KILFAHGQKC TDARDGLALF GPLNNLYGIK SGVIGTKQGL KIFRDYLDHI QKPIYNSNSI TRPMFPGFEA VFDCKWEST GITFKEVTNE DIGKFLYNSS THKRTYDLVS LFIDKIISAN KNEDENVDVW FVIVPDEIYK YCRPNSVLPK E MVQTKALM ...String:
MKELIYIEEP KILFAHGQKC TDARDGLALF GPLNNLYGIK SGVIGTKQGL KIFRDYLDHI QKPIYNSNSI TRPMFPGFEA VFDCKWEST GITFKEVTNE DIGKFLYNSS THKRTYDLVS LFIDKIISAN KNEDENVDVW FVIVPDEIYK YCRPNSVLPK E MVQTKALM SKSKAKSFRY EPSLFPDINI ELKEQEKEAE TYNYDAQFHD QFKARLLKHT IPTQIFREST LAWRDFKNAF GL PIRDFSK IEGHLAWTIS TAAFYKAGGK PWKLSDVRNG VCYLGLVYKK VEKSKNPRNA CCAAQMFLDN GDGTVFKGEV GPW YNPKNG QYHLEPKEAK ALLSQSLQSY KEQIGEYPKE VFIHAKTRFN HQEWDAFLEV TPKETNLVGV TISKTKPLKL YKTE GDYTI LRGNAYVVNE RSAFLWTVGY VPKIQTALSM EVPNPLFIEI NKGEADIKQV LKDILSLTKL NYNACIFADG EPVTL RFAD KIGEILTAST DIKTPPLAFK YYI

UniProtKB: Piwi domain-containing protein

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Macromolecule #3: guide RNA

MacromoleculeName: guide RNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Maribacter polysiphoniae (bacteria)
Molecular weightTheoretical: 6.651949 KDa
SequenceString:
UGACGGCUCU AAUCUAUUAG U

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Macromolecule #4: target DNA

MacromoleculeName: target DNA / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Maribacter polysiphoniae (bacteria)
Molecular weightTheoretical: 7.675 KDa
SequenceString:
(DC)(DA)(DA)(DC)(DT)(DA)(DA)(DT)(DA)(DG) (DA)(DT)(DT)(DA)(DG)(DA)(DG)(DC)(DC)(DG) (DT)(DC)(DA)(DA)(DT)

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / 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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: RANDOM ASSIGNMENT
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 163800
FSC plot (resolution estimation)

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