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- EMDB-36070: SPARTA monomer bound with guide-target, state 2 -

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Basic information

Entry
Database: EMDB / ID: EMD-36070
TitleSPARTA monomer bound with guide-target, state 2
Map data
Sample
  • Complex: Short ago complexed with TIR-APAZ
    • 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)
    • Protein or peptide: Piwi domain-containing protein
    • Protein or peptide: TIR domain-containing protein
  • Ligand: MAGNESIUM ION
KeywordsSPARTA / Ago / Tir / DNA BINDING PROTEIN/DNA/RNA / DNA BINDING PROTEIN-DNA-RNA 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 1, 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_36070.png

Downloads & links

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Map

FileDownload / File: emd_36070.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 256 pix.
= 268.8 Å		1.05 Å/pix.
x 256 pix.
= 268.8 Å		1.05 Å/pix.
x 256 pix.
= 268.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.06
Minimum - Maximum-0.3645371 - 0.71136135
Average (Standard dev.)-0.000052798692 (±0.01575059)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 268.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_36070_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_36070_half_map_2.map
Projections & Slices
AxesZYX

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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
    • 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)
    • Protein or peptide: Piwi domain-containing protein
    • Protein or peptide: TIR domain-containing protein
  • 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: #3-#4, #1-#2
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: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermoflavifilum thermophilum (bacteria)
Molecular weightTheoretical: 61.857793 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSH MASMTGGQQM GRGSMKELIY IEEPSILFAH GQKCTDPRDG LALFGPLNQI YGIKSGVVGT QKGLQIFKS YLDKIQKPIY NHNNITRPMF PGFEAVFGCK WESQNIVFKE ITDEEIRRYL FNASTHKRTY DLVTLFNDKI I TANKNDEE ...String:
MGSSHHHHHH SSGLVPRGSH MASMTGGQQM GRGSMKELIY IEEPSILFAH GQKCTDPRDG LALFGPLNQI YGIKSGVVGT QKGLQIFKS YLDKIQKPIY NHNNITRPMF PGFEAVFGCK WESQNIVFKE ITDEEIRRYL FNASTHKRTY DLVTLFNDKI I TANKNDEE RVDVWFVIVP EEIYKYCRPN SVLPNELVQT KSLISKSKAK SFRYTPTLFE EFNKKLKEVE KEAKTYNYDA QF HDQLKAR LLEHTIPTQI LRESTLAWRD FKNTFGAPIR DFSKIEGHLA WTISTAAYYK AGGKPWKLGD IRPGVCYLGL VYK KIEKSK NPQNACCAAQ MFLDNGDGTV FKGEVGPWYN PEKGEYHLKP KEAKALLTQA LESYKEQNKS YPKEVFIHAR TRFN DEEWN AFNEVTPKNT NLVGVTITKS KPLKLYKTEG AFPIMRGNAY IVDEKKAFLW TLGFVPKLQS TLSMEVPNPI FIEIN KGEA EIQQVLKDIL ALTKLNYNAC IYADGEPVTL RFANKIGEIL TASTEIKTPP LAFKYYI

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: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermoflavifilum thermophilum (bacteria)
Molecular weightTheoretical: 56.809668 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSH MASMTGGQQM GRGSMRNKIF ISHATPEDDD FTRWLSLKLI GLGYEVWCDI LFLDKGVDFW STIEKEIRE NTCKFLIVSS TAGNKREGVL KELAVATKVK KHLQDDMFII PLAIDENLSY DDINIEIVRL NAIDFKKSWA K GLQDLLDA ...String:
MGSSHHHHHH SSGLVPRGSH MASMTGGQQM GRGSMRNKIF ISHATPEDDD FTRWLSLKLI GLGYEVWCDI LFLDKGVDFW STIEKEIRE NTCKFLIVSS TAGNKREGVL KELAVATKVK KHLQDDMFII PLAIDENLSY DDINIEIVRL NAIDFKKSWA K GLQDLLDA FEKQNVPKKP PDHSKSNLLY QQIFLHDKQA IEKEETYDSN WFPIISFPNE LRFHRYDWRL PKQFDVRTLA FP AIRYKEY LCTFAWEYDF IHQLPKTETY NGQESIRIST SDILSGRYDT DFIRNYECQR LIVQLINKAF ELRMKDKNVR EYQ MSKTFA YWIEKGKLEK DKFEKIKLVG KQKNKYWHFG ISAAGKLYPS PVLMVSSHII FTMDGINLIK SKSIQHSSRR KQGK NWWND KWREKLLAFI RFLSDDQNAI YLNVGSEEKI LISNKPLKFF GKMSYVTPSE VTLEEESVLA DINNFEEDTE DLDEL EDIE

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: 1
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: 1 / 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: 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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 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: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 117819
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: RANDOM ASSIGNMENT

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-8j8h:
SPARTA monomer bound with guide-target, state 2

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