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- EMDB-17305: cryoEM structure of SPARTA complex dimer-3 -

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Basic information

Entry
Database: EMDB / ID: EMD-17305
TitlecryoEM structure of SPARTA complex dimer-3
Map datadimer3
Sample
  • Complex: a prokaryotic argonaute-TIR abortive immunity system
    • Protein or peptide: TIR domain-containing protein
    • Protein or peptide: Piwi domain-containing protein
  • RNA: RNA (18-MER)
  • DNA: DNA (16-MER)
KeywordsSPARTA / TIR / prokaryotic argonaute / DNA BINDING PROTEIN
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: 3.89 Å
AuthorsEkundayo B / Ni DC / Lu XH / Stahlberg H
Funding support Switzerland, 2 items
OrganizationGrant numberCountry
Swiss National Science FoundationCRSII5_177195 Switzerland
Swiss National Science FoundationIZLCZ0_206089 Switzerland
CitationJournal: Sci Adv / Year: 2023
Title: Activation mechanism of a short argonaute-TIR prokaryotic immune system.
Authors: Dongchun Ni / Xuhang Lu / Henning Stahlberg / Babatunde Ekundayo /
Abstract: Short prokaryotic argonaute (pAgo) and toll/interleukin-1 receptor/resistance protein (TIR)-analog of PAZ (APAZ) form a heterodimeric SPARTA complex that provides immunity to its prokaryotic host ...Short prokaryotic argonaute (pAgo) and toll/interleukin-1 receptor/resistance protein (TIR)-analog of PAZ (APAZ) form a heterodimeric SPARTA complex that provides immunity to its prokaryotic host through an abortive infection mechanism. Monomeric SPARTA senses foreign RNA/DNA duplexes to assemble an active tetramer resulting in cell death by nicotinamide adenine dinucleotide (oxidized form) (NAD) depletion via an unknown mechanism. We report nine structures of SPARTA in different functional states at a resolution range of 4.2 to 2.9 angstroms, revealing its activation mechanism. Inactive SPARTA monomers bind to RNA/DNA duplexes to form symmetric dimers mediated by the association of Ago subunits. The initiation of tetramer assembly induces flexibility of the TIR domains enabling a symmetry-breaking rotational movement of a TIR domain in the dimer units which facilitates the TIR oligomerization, resulting in the formation of the substrate binding pocket and the activation of the SPARTA complex's NADase activity. Our findings provide detailed structural and mechanistic insights into activating a short argonaute defense system.
History
DepositionMay 9, 2023-
Header (metadata) releaseAug 16, 2023-
Map releaseAug 16, 2023-
UpdateAug 23, 2023-
Current statusAug 23, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17305.png

Downloads & links

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Map

FileDownload / File: emd_17305.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationdimer3
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.21 Å/pix.
x 300 pix.
= 363. Å		1.21 Å/pix.
x 300 pix.
= 363. Å		1.21 Å/pix.
x 300 pix.
= 363. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.21 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0718
Minimum - Maximum-0.2913188 - 0.5847143
Average (Standard dev.)0.00049237336 (±0.01786989)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 363.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17305_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_17305_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_17305_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 : a prokaryotic argonaute-TIR abortive immunity system

EntireName: a prokaryotic argonaute-TIR abortive immunity system
Components
  • Complex: a prokaryotic argonaute-TIR abortive immunity system
    • Protein or peptide: TIR domain-containing protein
    • Protein or peptide: Piwi domain-containing protein
  • RNA: RNA (18-MER)
  • DNA: DNA (16-MER)

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Supramolecule #1: a prokaryotic argonaute-TIR abortive immunity system

SupramoleculeName: a prokaryotic argonaute-TIR abortive immunity system / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Maribacter polysiphoniae (bacteria)
Molecular weightTheoretical: 350 KDa

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Macromolecule #1: TIR domain-containing protein

MacromoleculeName: TIR domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Maribacter polysiphoniae (bacteria)
Molecular weightTheoretical: 53.270594 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRNKIFISHA TPDDNDFTRW LALKLIGLGY EVWCDILFLD KGVDFWSNIE KVIREDTCKF LLVSSSYSNQ REGVLKELAV AAKVKKQLK DDKFIIPLAI DEQLSYDDIN IDIVRLNAID FKMSWARGLK DILEAFEKQK VPKEVADASK SNLLYQQIFL H DKSVIEKE ...String:
MRNKIFISHA TPDDNDFTRW LALKLIGLGY EVWCDILFLD KGVDFWSNIE KVIREDTCKF LLVSSSYSNQ REGVLKELAV AAKVKKQLK DDKFIIPLAI DEQLSYDDIN IDIVRLNAID FKMSWARGLK DILEAFEKQK VPKEVADASK SNLLYQQIFL H DKSVIEKE EIYDSNWLSI LSFPEELRFH EYNWMLPKRF DVRELTFPAV RYKNYLCTFA WAYDFTYHLP KTETYHKSKT IR IPTEEIL SGSYDSNFIR NAECKRLIVQ LLNKAFELRM KDKEVQEYEM SNKTAYWLEK GKLEKDKFEK TMLVGKQKDK NWH FAISGA SKLYPFPVLM ISSHIFFTAD GKKLIDSSSV QHSSRRRQGK NWWNNTWRTK LLAFIKYLSD DDTSFYLEMG SEEK VFVSN EPVKFKGNVS YNIPEKNTLE EEAELSGFNQ GEDIEELEEL IENLEAE

UniProtKB: TIR domain-containing protein

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Macromolecule #2: Piwi domain-containing protein

MacromoleculeName: Piwi domain-containing protein / type: protein_or_peptide / ID: 2 / Number of copies: 2 / 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: RNA (18-MER)

MacromoleculeName: RNA (18-MER) / type: rna / ID: 3 / Number of copies: 2
Source (natural)Organism: Maribacter polysiphoniae (bacteria)
Molecular weightTheoretical: 5.466027 KDa
SequenceString:
UUUUUUUUUU UUUUUUUU

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Macromolecule #4: DNA (16-MER)

MacromoleculeName: DNA (16-MER) / type: dna / ID: 4 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Maribacter polysiphoniae (bacteria)
Molecular weightTheoretical: 4.966352 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(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

Concentration1 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.89 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 25123
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-8ozd:
cryoEM structure of SPARTA complex dimer-3

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