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- EMDB-35241: Cryo-EM structure of TIR-APAZ/Ago-gRNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-35241
TitleCryo-EM structure of TIR-APAZ/Ago-gRNA complex
Map data
Sample
  • Complex: Cryo-EM structure of TIR-APAZ/Ago-gRNA complex
    • RNA: RNA (5'-R(P*GP*A)-3')
    • Protein or peptide: Piwi domain-containing protein
    • Protein or peptide: TIR domain-containing protein
Keywordsa protein complex / VIRAL PROTEIN-ANTIVIRAL PROTEIN complex
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: 3.7 Å
AuthorsZhang H / Li Z / Yu GM / Li XZ / Wang XS
Funding support1 items
OrganizationGrant numberCountry
Other government
CitationJournal: Cell Res / Year: 2023
Title: Structural insights into mechanisms of Argonaute protein-associated NADase activation in bacterial immunity.
Authors: Xiaoshen Wang / Xuzichao Li / Guimei Yu / Lingling Zhang / Chendi Zhang / Yong Wang / Fumeng Liao / Yanan Wen / Hang Yin / Xiang Liu / Yong Wei / Zhuang Li / Zengqin Deng / Heng Zhang /
Abstract: Nicotinamide adenine dinucleotide (NAD) is a central metabolite in cellular processes. Depletion of NAD has been demonstrated to be a prevalent theme in both prokaryotic and eukaryotic immune ...Nicotinamide adenine dinucleotide (NAD) is a central metabolite in cellular processes. Depletion of NAD has been demonstrated to be a prevalent theme in both prokaryotic and eukaryotic immune responses. Short prokaryotic Argonaute proteins (Agos) are associated with NADase domain-containing proteins (TIR-APAZ or SIR2-APAZ) encoded in the same operon. They confer immunity against mobile genetic elements, such as bacteriophages and plasmids, by inducing NAD depletion upon recognition of target nucleic acids. However, the molecular mechanisms underlying the activation of such prokaryotic NADase/Ago immune systems remain unknown. Here, we report multiple cryo-EM structures of NADase/Ago complexes from two distinct systems (TIR-APAZ/Ago and SIR2-APAZ/Ago). Target DNA binding triggers tetramerization of the TIR-APAZ/Ago complex by a cooperative self-assembly mechanism, while the heterodimeric SIR2-APAZ/Ago complex does not assemble into higher-order oligomers upon target DNA binding. However, the NADase activities of these two systems are unleashed via a similar closed-to-open transition of the catalytic pocket, albeit by different mechanisms. Furthermore, a functionally conserved sensor loop is employed to inspect the guide RNA-target DNA base pairing and facilitate the conformational rearrangement of Ago proteins required for the activation of these two systems. Overall, our study reveals the mechanistic diversity and similarity of Ago protein-associated NADase systems in prokaryotic immune response.
History
DepositionFeb 3, 2023-
Header (metadata) releaseJul 5, 2023-
Map releaseJul 5, 2023-
UpdateSep 13, 2023-
Current statusSep 13, 2023Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_35241.png

Downloads & links

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Map

FileDownload / File: emd_35241.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.85 Å
Density
Contour LevelBy AUTHOR: 4.0
Minimum - Maximum-21.058567 - 31.430243000000001
Average (Standard dev.)0.0031751336 (±0.9998776)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 170.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_35241_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_35241_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 : Cryo-EM structure of TIR-APAZ/Ago-gRNA complex

EntireName: Cryo-EM structure of TIR-APAZ/Ago-gRNA complex
Components
  • Complex: Cryo-EM structure of TIR-APAZ/Ago-gRNA complex
    • RNA: RNA (5'-R(P*GP*A)-3')
    • Protein or peptide: Piwi domain-containing protein
    • Protein or peptide: TIR domain-containing protein

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Supramolecule #1: Cryo-EM structure of TIR-APAZ/Ago-gRNA complex

SupramoleculeName: Cryo-EM structure of TIR-APAZ/Ago-gRNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Maribacter polysiphoniae (bacteria)

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Macromolecule #1: RNA (5'-R(P*GP*A)-3')

MacromoleculeName: RNA (5'-R(P*GP*A)-3') / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Maribacter polysiphoniae (bacteria)
Molecular weightTheoretical: 629.454 Da
SequenceString:
GA

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Macromolecule #2: Piwi domain-containing protein

MacromoleculeName: Piwi domain-containing protein / 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: TIR domain-containing protein

MacromoleculeName: TIR domain-containing protein / type: protein_or_peptide / ID: 3 / Number of copies: 1 / 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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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: 2.5 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / 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: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 200000
FSC plot (resolution estimation)

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