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

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Basic information

Entry
Database: PDB / ID: 8jl0
TitleCryo-EM structure of the prokaryotic SPARSA system complex
Components
  • 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')
  • Piwi domain proteinPiwi
  • 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')
  • Sir2 superfamily protein
KeywordsANTIVIRAL PROTEIN/DNA/RNA / the SPARSA antiviral system / NADase / argonaute protein / ANTIVIRAL PROTEIN / ANTIVIRAL PROTEIN-DNA-RNA complex
Function / homology
Function and homology information


nucleic acid binding
Similarity search - Function
SIR2-like domain / SIR2-like domain / DHS-like NAD/FAD-binding domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / DNA / DNA (> 10) / RNA / RNA (> 10) / Piwi domain protein / Sir2 superfamily protein
Similarity search - Component
Biological speciesGeobacter sulfurreducens (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsXu, X. / Zhen, X. / Long, F.
Funding support China, 3items
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, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 24, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Sir2 superfamily protein
B: Piwi domain protein
D: 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')
C: 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')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)134,2045
Polymers133,5414
Non-polymers6631
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Sir2 superfamily protein


Mass: 66488.727 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacter sulfurreducens (bacteria) / Gene: GSU1360 / Production host: Escherichia coli (E. coli) / References: UniProt: Q74DF6
#2: Protein Piwi domain protein / Piwi


Mass: 53325.566 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacter sulfurreducens (bacteria) / Gene: GSU1361 / Production host: Escherichia coli (E. coli) / References: UniProt: Q74DF5
#3: DNA chain 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')


Mass: 6728.394 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: RNA chain 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')


Mass: 6998.139 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: Chemical ChemComp-NAD / NICOTINAMIDE-ADENINE-DINUCLEOTIDE / Nicotinamide adenine dinucleotide


Mass: 663.425 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H27N7O14P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: NAD*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1the Sir2/Ago/Guide RNA/recognition DNA complexCOMPLEX#1-#40MULTIPLE SOURCES
2Sir2Sirtuin 1COMPLEX#11RECOMBINANT
3AgoCOMPLEX#21RECOMBINANT
4DNA/guide RNA duplexCOMPLEX#3-#41RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
41NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Geobacter sulfurreducens PCA (bacteria)243231
32Geobacter sulfurreducens PCA (bacteria)243231
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
21Escherichia coli (E. coli)562BL21 (DE3)
32Escherichia coli (E. coli)562BL21 (DE3)
Buffer solutionpH: 8
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
2EPUFEIimage acquisition
12cryoSPARC3D reconstruction
13PHENIX1.20.1_4487:model refinement
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 100134 / Symmetry type: POINT
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0049553
ELECTRON MICROSCOPYf_angle_d0.58513155
ELECTRON MICROSCOPYf_dihedral_angle_d14.0571619
ELECTRON MICROSCOPYf_chiral_restr0.0411462
ELECTRON MICROSCOPYf_plane_restr0.0041524

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