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- EMDB-35592: Cryo-EM structure of the target ssDNA-bound SIR2-APAZ/Ago-gRNA qu... -

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Basic information

Entry
Database: EMDB / ID: EMD-35592
TitleCryo-EM structure of the target ssDNA-bound SIR2-APAZ/Ago-gRNA quaternary complex
Map data
Sample
  • Complex: Cryo-EM structure of the target ssDNA-bound SIR2-APAZ/Ago-gRNA quaternary complex
    • Protein or peptide: Sir2 superfamily protein
    • Protein or peptide: Piwi domain proteinPiwi
    • RNA: RNA (5'-R(P*AP*UP*AP*AP*UP*GP*GP*UP*UP*UP*CP*UP*UP*AP*GP*AP*CP*GP*UP*CP*GP*UP*UP*U)-3')
    • DNA: DNA (5'-D(P*AP*AP*CP*GP*AP*CP*GP*TP*CP*TP*AP*AP*GP*AP*AP*AP*CP*CP*AP*TP*TP*AP*A)-3')
  • Ligand: MAGNESIUM ION
Keywordsa protein / ANTIVIRAL PROTEIN
Function / homologySIR2-like domain / SIR2-like domain / DHS-like NAD/FAD-binding domain superfamily / Ribonuclease H superfamily / nucleic acid binding / Ribonuclease H-like superfamily / Piwi domain protein / Sir2 superfamily protein
Function and homology information
Biological speciesGeobacter sulfurreducens (strain ATCC 51573 / DSM 12127 / PCA) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 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
DepositionMar 8, 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_35592.png

Downloads & links

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Map

FileDownload / File: emd_35592.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.9066 Å
Density
Contour LevelBy AUTHOR: 0.35
Minimum - Maximum-1.9551997 - 3.2797503
Average (Standard dev.)0.0008064514 (±0.072284184)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 217.584 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_35592_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_35592_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 the target ssDNA-bound SIR2-APAZ/Ago-gRNA qu...

EntireName: Cryo-EM structure of the target ssDNA-bound SIR2-APAZ/Ago-gRNA quaternary complex
Components
  • Complex: Cryo-EM structure of the target ssDNA-bound SIR2-APAZ/Ago-gRNA quaternary complex
    • Protein or peptide: Sir2 superfamily protein
    • Protein or peptide: Piwi domain proteinPiwi
    • RNA: RNA (5'-R(P*AP*UP*AP*AP*UP*GP*GP*UP*UP*UP*CP*UP*UP*AP*GP*AP*CP*GP*UP*CP*GP*UP*UP*U)-3')
    • DNA: DNA (5'-D(P*AP*AP*CP*GP*AP*CP*GP*TP*CP*TP*AP*AP*GP*AP*AP*AP*CP*CP*AP*TP*TP*AP*A)-3')
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Cryo-EM structure of the target ssDNA-bound SIR2-APAZ/Ago-gRNA qu...

SupramoleculeName: Cryo-EM structure of the target ssDNA-bound SIR2-APAZ/Ago-gRNA quaternary complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Geobacter sulfurreducens (strain ATCC 51573 / DSM 12127 / PCA) (bacteria)

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Macromolecule #1: Sir2 superfamily protein

MacromoleculeName: Sir2 superfamily protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Geobacter sulfurreducens (strain ATCC 51573 / DSM 12127 / PCA) (bacteria)
Molecular weightTheoretical: 66.645922 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDVLTDNEFY QHYLQNSQHM MWFLGAGTSR SAGLPTASDI IWDLKHRYYC LHENQDYQKH DINNHAIKSK IQSYMDSKGF PLQWSPEEY SFYFELVFRD DYEAQRKYLL EALASRKVSL NIGHRVLAAL LEMNQTKVVF TTNFDDVIET AFSDISGKHL S VYHLEGSY ...String:
MDVLTDNEFY QHYLQNSQHM MWFLGAGTSR SAGLPTASDI IWDLKHRYYC LHENQDYQKH DINNHAIKSK IQSYMDSKGF PLQWSPEEY SFYFELVFRD DYEAQRKYLL EALASRKVSL NIGHRVLAAL LEMNQTKVVF TTNFDDVIET AFSDISGKHL S VYHLEGSY AALSALNTEA FPIYAKIHGD FRYQKIKNLT PDLQTNDREI HKCFLAAAIR FGLVVSGYSG RDENVMTMLR AA IDQNNAF PHGLYWTVPS ISKSEPAVQD LITYAQGKGV RAYLVETGTF DEMLSKIWRQ VKDKPAAIDA KVRTARVCPV SIP LPGPGK SFPALRTNAL PVVTQSIRCG VVTLASPITF SELKERISQK SPKALLTYTE KVLFLGGEPE IRKIFSNDEI NSIG QYYID EIAQSVAAST FLKSFVEEAI LTALLREKPI LHRVRHRTHY AVIPNASAKD DRFLDLRKAV GFKGDLGYIT GNVTN AKEL SWAEAVSIRL EERGGKLWIM LKPEIWIKPL DRREEATDFI RSRRRYRFNQ CSYQILDAWI KILFGSIGGG GTVNIS CFP DAEFKAEFEI GTRTAFSLGV GYGR

UniProtKB: Sir2 superfamily protein

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Macromolecule #2: Piwi domain protein

MacromoleculeName: Piwi domain protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Geobacter sulfurreducens (strain ATCC 51573 / DSM 12127 / PCA) (bacteria)
Molecular weightTheoretical: 53.325566 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MADNLSQLAA HSTIPEPLLL FKDNRTDTHP LRGLSQYGPY SACFNLPGQV RLAYLAPTEH MRKLDAIVRE LQNPATPKEA TNYYVEYGG FEKVFKVPLV MPQEHLRCLA LDECHGVAAN GNGLALADKI VQSMSGLFRQ KHAFDVLLVY LPASWKKCFE Y DGFDLHDR ...String:
MADNLSQLAA HSTIPEPLLL FKDNRTDTHP LRGLSQYGPY SACFNLPGQV RLAYLAPTEH MRKLDAIVRE LQNPATPKEA TNYYVEYGG FEKVFKVPLV MPQEHLRCLA LDECHGVAAN GNGLALADKI VQSMSGLFRQ KHAFDVLLVY LPASWKKCFE Y DGFDLHDR IKAKVAPLNL PIQIINDTAL TRQCRANVMW GVSVALYAKA GGIPWKLADW DKDEAYIGLS YAIKKNAEGQ EY TTCCSQV FDPDGTGFEF VAYDTREFIT DRKGNPYLSY QEMQSVLSKS LHLYQSSHNG RMPRKIFIHK TTHFTEDEIQ GAF DSFSSS TEIELVQIIQ STNWYGLKVD GKKGDKPVAP ASYPVDRGLY QPLTESECLL WTQGSVMGVN QQNPGQPVFK EAAL TPLPN PIMLRRFSGN GGWHATCSSI LALTKVDWNN NTLYKKLPVT LVYSQVFADV VKQTPEIVNE IYDYRFFM

UniProtKB: Piwi domain protein

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

MacromoleculeName: RNA (5'-R(P*AP*UP*AP*AP*UP*GP*GP*UP*UP*UP*CP*UP*UP*AP*GP*AP*CP*GP*UP*CP*GP*UP*UP*U)-3')
type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Geobacter sulfurreducens (strain ATCC 51573 / DSM 12127 / PCA) (bacteria)
Molecular weightTheoretical: 7.610471 KDa
SequenceString:
AUAAUGGUUU CUUAGACGUC GUUU

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Macromolecule #4: DNA (5'-D(P*AP*AP*CP*GP*AP*CP*GP*TP*CP*TP*AP*AP*GP*AP*AP*AP*CP*CP...

MacromoleculeName: DNA (5'-D(P*AP*AP*CP*GP*AP*CP*GP*TP*CP*TP*AP*AP*GP*AP*AP*AP*CP*CP*AP*TP*TP*AP*A)-3')
type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Geobacter sulfurreducens (strain ATCC 51573 / DSM 12127 / PCA) (bacteria)
Molecular weightTheoretical: 7.050615 KDa
SequenceString:
(DA)(DA)(DC)(DG)(DA)(DC)(DG)(DT)(DC)(DT) (DA)(DA)(DG)(DA)(DA)(DA)(DC)(DC)(DA)(DT) (DT)(DA)(DA)

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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
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: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 380948
FSC plot (resolution estimation)

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