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- EMDB-36114: SPARTA dimer bound with guide-target -

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Basic information

Entry
Database: EMDB / ID: EMD-36114
TitleSPARTA dimer bound with guide-target
Map data
Sample
  • Complex: Short ago complexed with TIR-APAZ
    • Protein or peptide: Piwi domain-containing protein
    • Protein or peptide: TIR domain-containing protein
    • RNA: RNA (5'-R(P*UP*GP*AP*CP*GP*GP*CP*UP*CP*UP*AP*AP*UP*CP*UP*AP*UP*UP*AP*GP*U)-3')
    • DNA: DNA (25-MER)
  • Ligand: MAGNESIUM ION
KeywordsSPARTA / Ago / Tir / DNA BINDING PROTEIN/RNA/DNA / DNA BINDING PROTEIN-RNA-DNA complex
Function / homologyTIR domain / Toll/interleukin-1 receptor homology (TIR) domain / Toll/interleukin-1 receptor homology (TIR) domain superfamily / Ribonuclease H superfamily / nucleic acid binding / Ribonuclease H-like superfamily / signal transduction / Piwi domain-containing protein / TIR domain-containing protein
Function and homology information
Biological speciesThermoflavifilum thermophilum (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsLi ZX / Guo LJ / Huang PP / Xiao YB / Chen MR
Funding support China, 5 items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)Nos.2018YFA0902000 China
Ministry of Science and Technology (MoST, China)ZX-2021ZD0203404 China
National Natural Science Foundation of China (NSFC)Nos. 32271330 China
National Natural Science Foundation of China (NSFC)Nos. 32000889 China
National Natural Science Foundation of China (NSFC)31970547 China
CitationJournal: Nat Chem Biol / Year: 2024
Title: Auto-inhibition and activation of a short Argonaute-associated TIR-APAZ defense system.
Authors: Lijie Guo / Pingping Huang / Zhaoxing Li / Young-Cheul Shin / Purui Yan / Meiling Lu / Meirong Chen / Yibei Xiao /
Abstract: Short prokaryotic Ago accounts for most prokaryotic Argonaute proteins (pAgos) and is involved in defending bacteria against invading nucleic acids. Short pAgo associated with TIR-APAZ (SPARTA) has ...Short prokaryotic Ago accounts for most prokaryotic Argonaute proteins (pAgos) and is involved in defending bacteria against invading nucleic acids. Short pAgo associated with TIR-APAZ (SPARTA) has been shown to oligomerize and deplete NAD upon guide-mediated target DNA recognition. However, the molecular basis of SPARTA inhibition and activation remains unknown. In this study, we determined the cryogenic electron microscopy structures of Crenotalea thermophila SPARTA in its inhibited, transient and activated states. The SPARTA monomer is auto-inhibited by its acidic tail, which occupies the guide-target binding channel. Guide-mediated target binding expels this acidic tail and triggers substantial conformational changes to expose the Ago-Ago dimerization interface. As a result, SPARTA assembles into an active tetramer, where the four TIR domains are rearranged and packed to form NADase active sites. Together with biochemical evidence, our results provide a panoramic vision explaining SPARTA auto-inhibition and activation and expand understanding of pAgo-mediated bacterial defense systems.
History
DepositionMay 4, 2023-
Header (metadata) releaseMar 6, 2024-
Map releaseMar 6, 2024-
UpdateApr 10, 2024-
Current statusApr 10, 2024Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_36114.png

Downloads & links

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Map

FileDownload / File: emd_36114.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 0.09
Minimum - Maximum-0.34425285 - 0.7670157
Average (Standard dev.)-0.00021447834 (±0.015891992)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 403.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_36114_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_36114_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 : Short ago complexed with TIR-APAZ

EntireName: Short ago complexed with TIR-APAZ
Components
  • Complex: Short ago complexed with TIR-APAZ
    • Protein or peptide: Piwi domain-containing protein
    • Protein or peptide: TIR domain-containing protein
    • RNA: RNA (5'-R(P*UP*GP*AP*CP*GP*GP*CP*UP*CP*UP*AP*AP*UP*CP*UP*AP*UP*UP*AP*GP*U)-3')
    • DNA: DNA (25-MER)
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Short ago complexed with TIR-APAZ

SupramoleculeName: Short ago complexed with TIR-APAZ / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Thermoflavifilum thermophilum (bacteria)

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Macromolecule #1: Piwi domain-containing protein

MacromoleculeName: Piwi domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Thermoflavifilum thermophilum (bacteria)
Molecular weightTheoretical: 58.304848 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MKELIYIEEP SILFAHGQKC TDPRDGLALF GPLNQIYGIK SGVVGTQKGL QIFKSYLDKI QKPIYNHNNI TRPMFPGFEA VFGCKWESQ NIVFKEITDE EIRRYLFNAS THKRTYDLVT LFNDKIITAN KNDEERVDVW FVIVPEEIYK YCRPNSVLPN E LVQTKSLI ...String:
MKELIYIEEP SILFAHGQKC TDPRDGLALF GPLNQIYGIK SGVVGTQKGL QIFKSYLDKI QKPIYNHNNI TRPMFPGFEA VFGCKWESQ NIVFKEITDE EIRRYLFNAS THKRTYDLVT LFNDKIITAN KNDEERVDVW FVIVPEEIYK YCRPNSVLPN E LVQTKSLI SKSKAKSFRY TPTLFEEFNK KLKEVEKEAK TYNYDAQFHD QLKARLLEHT IPTQILREST LAWRDFKNTF GA PIRDFSK IEGHLAWTIS TAAYYKAGGK PWKLGDIRPG VCYLGLVYKK IEKSKNPQNA CCAAQMFLDN GDGTVFKGEV GPW YNPEKG EYHLKPKEAK ALLTQALESY KEQNKSYPKE VFIHARTRFN DEEWNAFNEV TPKNTNLVGV TITKSKPLKL YKTE GAFPI MRGNAYIVDE KKAFLWTLGF VPKLQSTLSM EVPNPIFIEI NKGEAEIQQV LKDILALTKL NYNACIYADG EPVTL RFAN KIGEILTAST EIKTPPLAFK YYI

UniProtKB: Piwi domain-containing protein

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Macromolecule #2: TIR domain-containing protein

MacromoleculeName: TIR domain-containing protein / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Thermoflavifilum thermophilum (bacteria)
Molecular weightTheoretical: 53.256734 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MRNKIFISHA TPEDDDFTRW LSLKLIGLGY EVWCDILFLD KGVDFWSTIE KEIRENTCKF LIVSSTAGNK REGVLKELAV ATKVKKHLQ DDMFIIPLAI DENLSYDDIN IEIVRLNAID FKKSWAKGLQ DLLDAFEKQN VPKKPPDHSK SNLLYQQIFL H DKQAIEKE ...String:
MRNKIFISHA TPEDDDFTRW LSLKLIGLGY EVWCDILFLD KGVDFWSTIE KEIRENTCKF LIVSSTAGNK REGVLKELAV ATKVKKHLQ DDMFIIPLAI DENLSYDDIN IEIVRLNAID FKKSWAKGLQ DLLDAFEKQN VPKKPPDHSK SNLLYQQIFL H DKQAIEKE ETYDSNWFPI ISFPNELRFH RYDWRLPKQF DVRTLAFPAI RYKEYLCTFA WEYDFIHQLP KTETYNGQES IR ISTSDIL SGRYDTDFIR NYECQRLIVQ LINKAFELRM KDKNVREYQM SKTFAYWIEK GKLEKDKFEK IKLVGKQKNK YWH FGISAA GKLYPSPVLM VSSHIIFTMD GINLIKSKSI QHSSRRKQGK NWWNDKWREK LLAFIRFLSD DQNAIYLNVG SEEK ILISN KPLKFFGKMS YVTPSEVTLE EESVLADINN FEEDTEDLDE LEDIE

UniProtKB: TIR domain-containing protein

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

MacromoleculeName: RNA (5'-R(P*UP*GP*AP*CP*GP*GP*CP*UP*CP*UP*AP*AP*UP*CP*UP*AP*UP*UP*AP*GP*U)-3')
type: rna / ID: 3 / Number of copies: 2
Source (natural)Organism: Thermoflavifilum thermophilum (bacteria)
Molecular weightTheoretical: 6.651949 KDa
SequenceString:
UGACGGCUCU AAUCUAUUAG U

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Macromolecule #4: DNA (25-MER)

MacromoleculeName: DNA (25-MER) / type: dna / ID: 4 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Thermoflavifilum thermophilum (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: 2 / 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: 7.5
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: 2.4 µm / Nominal defocus min: 1.0 µm
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 / Average electron dose: 40.0 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: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 147489
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-8j9p:
SPARTA dimer bound with guide-target

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