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- EMDB-36986: Cryo-EM structure of Crt-SPARTA-gRNA-tDNA dimer (conformation-1) -

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Basic information

Entry
Database: EMDB / ID: EMD-36986
TitleCryo-EM structure of Crt-SPARTA-gRNA-tDNA dimer (conformation-1)
Map data
Sample
  • Complex: The dimer of prokaryotic Argonaute System with DNA and RNA
    • Protein or peptide: Piwi domain-containing protein
    • Protein or peptide: TIR domain-containing protein
    • DNA: DNA (45-mer)
    • RNA: RNA (5'-R(P*UP*GP*AP*GP*GP*UP*AP*GP*UP*AP*GP*GP*UP*UP*GP*UP*AP*UP*AP*GP*U)-3')
KeywordsAgo / DNA/RNA / Dimer / DNA BINDING PROTEIN / CELL INVASION / DNA-RNA-CELL INVASION 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) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.49 Å
AuthorsGao X / Shang K / Zhu K / Wang L / Mu Z / Fu X / Yu X / Qin B / Zhu H / Ding W / Cui S
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nature / Year: 2024
Title: Nucleic-acid-triggered NADase activation of a short prokaryotic Argonaute.
Authors: Xiaopan Gao / Kun Shang / Kaixiang Zhu / Linyue Wang / Zhixia Mu / Xingke Fu / Xia Yu / Bo Qin / Hongtao Zhu / Wei Ding / Sheng Cui /
Abstract: Argonaute (Ago) proteins mediate RNA- or DNA-guided inhibition of nucleic acids. Although the mechanisms used by eukaryotic Ago proteins and long prokaryotic Ago proteins (pAgos) are known, that used ...Argonaute (Ago) proteins mediate RNA- or DNA-guided inhibition of nucleic acids. Although the mechanisms used by eukaryotic Ago proteins and long prokaryotic Ago proteins (pAgos) are known, that used by short pAgos remains elusive. Here we determined the cryo-electron microscopy structures of a short pAgo and the associated TIR-APAZ proteins (SPARTA) from Crenotalea thermophila (Crt): a free-state Crt-SPARTA; a guide RNA-target DNA-loaded Crt-SPARTA; two Crt-SPARTA dimers with distinct TIR organization; and a Crt-SPARTA tetramer. These structures reveal that Crt-SPARTA is composed of a bilobal-fold Ago lobe that connects with a TIR lobe. Whereas the Crt-Ago contains a MID and a PIWI domain, Crt-TIR-APAZ has a TIR domain, an N-like domain, a linker domain and a trigger domain. The bound RNA-DNA duplex adopts a B-form conformation that is recognized by base-specific contacts. Nucleic acid binding causes conformational changes because the trigger domain acts as a 'roadblock' that prevents the guide RNA 5' ends and the target DNA 3' ends from reaching their canonical pockets; this disorders the MID domain and promotes Crt-SPARTA dimerization. Two RNA-DNA-loaded Crt-SPARTA dimers form a tetramer through their TIR domains. Four Crt-TIR domains assemble into two parallel head-to-tail-organized TIR dimers, indicating an NADase-active conformation, which is supported by our mutagenesis study. Our results reveal the structural basis of short-pAgo-mediated defence against invading nucleic acids, and provide insights for optimizing the detection of SPARTA-based programmable DNA sequences.
History
DepositionAug 1, 2023-
Header (metadata) releaseOct 18, 2023-
Map releaseOct 18, 2023-
UpdateFeb 7, 2024-
Current statusFeb 7, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36986.png

Downloads & links

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Map

FileDownload / File: emd_36986.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.04 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.28218296 - 0.8888461
Average (Standard dev.)0.0006772044 (±0.0392465)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 266.24 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_36986_msk_1.map
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Additional map: #1

Fileemd_36986_additional_1.map
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Half map: #2

Fileemd_36986_half_map_1.map
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Half map: #1

Fileemd_36986_half_map_2.map
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Sample components

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Entire : The dimer of prokaryotic Argonaute System with DNA and RNA

EntireName: The dimer of prokaryotic Argonaute System with DNA and RNA
Components
  • Complex: The dimer of prokaryotic Argonaute System with DNA and RNA
    • Protein or peptide: Piwi domain-containing protein
    • Protein or peptide: TIR domain-containing protein
    • DNA: DNA (45-mer)
    • RNA: RNA (5'-R(P*UP*GP*AP*GP*GP*UP*AP*GP*UP*AP*GP*GP*UP*UP*GP*UP*AP*UP*AP*GP*U)-3')

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Supramolecule #1: The dimer of prokaryotic Argonaute System with DNA and RNA

SupramoleculeName: The dimer of prokaryotic Argonaute System with DNA and RNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #3-#4, #1-#2
Source (natural)Organism: Thermoflavifilum thermophilum (bacteria)

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Macromolecule #1: DNA (45-mer)

MacromoleculeName: DNA (45-mer) / type: dna / ID: 1 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.721869 KDa
SequenceString:
(DA)(DA)(DA)(DC)(DG)(DA)(DC)(DG)(DG)(DC) (DC)(DA)(DG)(DT)(DG)(DC)(DC)(DA)(DA)(DG) (DC)(DA)(DA)(DA)(DC)(DT)(DA)(DT)(DA) (DC)(DA)(DA)(DC)(DC)(DT)(DA)(DC)(DT)(DA) (DC) (DC)(DT)(DC)(DA)(DT)

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Macromolecule #2: RNA (5'-R(P*UP*GP*AP*GP*GP*UP*AP*GP*UP*AP*GP*GP*UP*UP*GP*UP*AP*UP...

MacromoleculeName: RNA (5'-R(P*UP*GP*AP*GP*GP*UP*AP*GP*UP*AP*GP*GP*UP*UP*GP*UP*AP*UP*AP*GP*U)-3')
type: rna / ID: 2 / Number of copies: 2
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.812045 KDa
SequenceString:
UGAGGUAGUA GGUUGUAUAG U

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Macromolecule #3: Piwi domain-containing protein

MacromoleculeName: Piwi domain-containing protein / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Thermoflavifilum thermophilum (bacteria)
Molecular weightTheoretical: 58.304848 KDa
Recombinant expressionOrganism: Expression vector pET-mod (others)
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 #4: TIR domain-containing protein

MacromoleculeName: TIR domain-containing protein / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Thermoflavifilum thermophilum (bacteria)
Molecular weightTheoretical: 53.256734 KDa
Recombinant expressionOrganism: Expression vector pET-mod (others)
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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.5 µm / Calibrated defocus min: 1.8 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 66.0 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: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.49 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 614651

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Correlation coefficient
Output model

PDB-8k9g:
Cryo-EM structure of Crt-SPARTA-gRNA-tDNA dimer (conformation-1)

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