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- EMDB-36385: Cryo-EM structure of the prokaryotic SPARSA system complex -

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Basic information

Entry
Database: EMDB / ID: EMD-36385
TitleCryo-EM structure of the prokaryotic SPARSA system complex
Map data
Sample
  • Complex: the Sir2/Ago/Guide RNA/recognition DNA complex
    • Complex: Sir2
      • Protein or peptide: Sir2 superfamily protein
    • Complex: Ago
      • Protein or peptide: Piwi domain protein
    • Complex: DNA/guide RNA duplex
      • DNA: DNA (5'-D(P*AP*CP*GP*AP*CP*GP*TP*CP*TP*AP*AP*GP*AP*AP*AP*CP*CP*AP*TP*TP*AP*T)-3')
      • 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*U)-3')
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE
Keywordsthe SPARSA antiviral system / NADase / argonaute protein / ANTIVIRAL PROTEIN / ANTIVIRAL PROTEIN-DNA-RNA complex
Function / homologySIR2-like domain / SIR2-like domain / DHS-like NAD/FAD-binding domain superfamily / nucleic acid binding / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Piwi domain protein / Sir2 superfamily protein
Function and homology information
Biological speciesGeobacter sulfurreducens PCA (bacteria) / Geobacter sulfurreducens (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsXu X / Zhen X / Long F
Funding support China, 3 items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2021YFA0909500 China
Ministry of Science and Technology (MoST, China)2018YFA0900400 China
Ministry of Education (MoE, China)2042019kf0185 China
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis of antiphage immunity generated by a prokaryotic Argonaute-associated SPARSA system.
Authors: Xiangkai Zhen / Xiaolong Xu / Le Ye / Song Xie / Zhijie Huang / Sheng Yang / Yanhui Wang / Jinyu Li / Feng Long / Songying Ouyang /
Abstract: Argonaute (Ago) proteins are ubiquitous across all kingdoms of life. Eukaryotic Agos (eAgos) use small RNAs to recognize transcripts for RNA silencing in eukaryotes. In contrast, the functions of ...Argonaute (Ago) proteins are ubiquitous across all kingdoms of life. Eukaryotic Agos (eAgos) use small RNAs to recognize transcripts for RNA silencing in eukaryotes. In contrast, the functions of prokaryotic counterparts (pAgo) are less well known. Recently, short pAgos in conjunction with the associated TIR or Sir2 (SPARTA or SPARSA) were found to serve as antiviral systems to combat phage infections. Herein, we present the cryo-EM structures of nicotinamide adenine dinucleotide (NAD)-bound SPARSA with and without nucleic acids at resolutions of 3.1 Å and 3.6 Å, respectively. Our results reveal that the APAZ (Analogue of PAZ) domain and the short pAgo form a featured architecture similar to the long pAgo to accommodate nucleic acids. We further identified the key residues for NAD binding and elucidated the structural basis for guide RNA and target DNA recognition. Using structural comparisons, molecular dynamics simulations, and biochemical experiments, we proposed a putative mechanism for NAD hydrolysis in which an H186 loop mediates nucleophilic attack by catalytic water molecules. Overall, our study provides mechanistic insight into the antiphage role of the SPARSA system.
History
DepositionJun 2, 2023-
Header (metadata) releaseJan 24, 2024-
Map releaseJan 24, 2024-
UpdateJan 24, 2024-
Current statusJan 24, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36385.png

Downloads & links

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Map

FileDownload / File: emd_36385.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.84 Å
Density
Contour LevelBy AUTHOR: 0.16
Minimum - Maximum-0.8175249 - 1.4406409
Average (Standard dev.)0.0015977646 (±0.033030067)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 215.04 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_36385_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_36385_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_36385_half_map_2.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : the Sir2/Ago/Guide RNA/recognition DNA complex

EntireName: the Sir2/Ago/Guide RNA/recognition DNA complex
Components
  • Complex: the Sir2/Ago/Guide RNA/recognition DNA complex
    • Complex: Sir2
      • Protein or peptide: Sir2 superfamily protein
    • Complex: Ago
      • Protein or peptide: Piwi domain protein
    • Complex: DNA/guide RNA duplex
      • DNA: DNA (5'-D(P*AP*CP*GP*AP*CP*GP*TP*CP*TP*AP*AP*GP*AP*AP*AP*CP*CP*AP*TP*TP*AP*T)-3')
      • 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*U)-3')
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

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Supramolecule #1: the Sir2/Ago/Guide RNA/recognition DNA complex

SupramoleculeName: the Sir2/Ago/Guide RNA/recognition DNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Geobacter sulfurreducens PCA (bacteria)

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Supramolecule #2: Sir2

SupramoleculeName: Sir2 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Geobacter sulfurreducens PCA (bacteria)

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Supramolecule #3: Ago

SupramoleculeName: Ago / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2

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Supramolecule #4: DNA/guide RNA duplex

SupramoleculeName: DNA/guide RNA duplex / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3-#4

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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 (bacteria)
Molecular weightTheoretical: 66.488727 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 GYG

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

MacromoleculeName: DNA (5'-D(P*AP*CP*GP*AP*CP*GP*TP*CP*TP*AP*AP*GP*AP*AP*AP*CP*CP*AP*TP*TP*AP*T)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.728394 KDa
SequenceString:
(DA)(DC)(DG)(DA)(DC)(DG)(DT)(DC)(DT)(DA) (DA)(DG)(DA)(DA)(DA)(DC)(DC)(DA)(DT)(DT) (DA)(DT)

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Macromolecule #4: 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*U)-3')
type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.998139 KDa
SequenceString:
AUAAUGGUUU CUUAGACGUC GU

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Macromolecule #5: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

MacromoleculeName: NICOTINAMIDE-ADENINE-DINUCLEOTIDE / type: ligand / ID: 5 / Number of copies: 1 / Formula: NAD
Molecular weightTheoretical: 663.425 Da
Chemical component information

ChemComp-NAD:
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / NAD*YM

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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

Concentration2.5 mg/mL
BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 50 sec.
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
SoftwareName: EPU (ver. FEI)
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / 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 / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 100134
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Output model

PDB-8jl0:
Cryo-EM structure of the prokaryotic SPARSA system complex

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