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- EMDB-39028: Cryo-EM structure of the tetrameric SPARSA gRNA-ssDNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-39028
TitleCryo-EM structure of the tetrameric SPARSA gRNA-ssDNA complex
Map data
Sample
  • Complex: Tetrameric SPARSA gRNA-ssDNA complex
    • Protein or peptide: Sir2 superfamily protein
    • Protein or peptide: Piwi domain protein
    • RNA: RNA (21-mer)
    • DNA: DNA (25-mer)
  • Ligand: MAGNESIUM ION
KeywordsRNA BINDING PROTEIN-RNA-DNA COMPLEX / STRUCTURAL PROTEIN / STRUCTURAL PROTEIN-RNA-DNA complex
Function / homologySIR2-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 (bacteria) / Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.38 Å
AuthorsZhang JT / Cui N / Wei XY / Jia N
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2024
Title: Tetramerization-dependent activation of the Sir2-associated short prokaryotic Argonaute immune system.
Authors: Ning Cui / Jun-Tao Zhang / Zhuolin Li / Xin-Yang Wei / Jie Wang / Ning Jia /
Abstract: Eukaryotic Argonaute proteins (eAgos) utilize short nucleic acid guides to target complementary sequences for RNA silencing, while prokaryotic Agos (pAgos) provide immunity against invading plasmids ...Eukaryotic Argonaute proteins (eAgos) utilize short nucleic acid guides to target complementary sequences for RNA silencing, while prokaryotic Agos (pAgos) provide immunity against invading plasmids or bacteriophages. The Sir2-domain associated short pAgo (SPARSA) immune system defends against invaders by depleting NAD and triggering cell death. However, the molecular mechanism underlying SPARSA activation remains unknown. Here, we present cryo-EM structures of inactive monomeric, active tetrameric and active NAD-bound tetrameric SPARSA complexes, elucidating mechanisms underlying SPARSA assembly, guide RNA preference, target ssDNA-triggered SPARSA tetramerization, and tetrameric-dependent NADase activation. Short pAgos form heterodimers with Sir2-APAZ, favoring short guide RNA with a 5'-AU from ColE-like plasmids. RNA-guided recognition of the target ssDNA triggers SPARSA tetramerization via pAgo- and Sir2-mediated interactions. The resulting tetrameric Sir2 rearrangement aligns catalytic residue H186 for NAD hydrolysis. These insights advance our understanding of Sir2-domain associated pAgos immune systems and should facilitate the development of a short pAgo-associated biotechnological toolbox.
History
DepositionFeb 5, 2024-
Header (metadata) releaseOct 16, 2024-
Map releaseOct 16, 2024-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_39028.png

Downloads & links

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Map

FileDownload / File: emd_39028.map.gz / Format: CCP4 / Size: 371.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 460 pix.
= 380.42 Å		0.83 Å/pix.
x 460 pix.
= 380.42 Å		0.83 Å/pix.
x 460 pix.
= 380.42 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.827 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.375
Minimum - Maximum-1.0841378 - 2.0024376
Average (Standard dev.)0.0016953376 (±0.052274268)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions460460460
Spacing460460460
CellA=B=C: 380.42 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_39028_msk_1.map
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Half map: #2

Fileemd_39028_half_map_1.map
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Half map: #1

Fileemd_39028_half_map_2.map
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Sample components

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Entire : Tetrameric SPARSA gRNA-ssDNA complex

EntireName: Tetrameric SPARSA gRNA-ssDNA complex
Components
  • Complex: Tetrameric SPARSA gRNA-ssDNA complex
    • Protein or peptide: Sir2 superfamily protein
    • Protein or peptide: Piwi domain protein
    • RNA: RNA (21-mer)
    • DNA: DNA (25-mer)
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Tetrameric SPARSA gRNA-ssDNA complex

SupramoleculeName: Tetrameric SPARSA gRNA-ssDNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Geobacter sulfurreducens (bacteria)

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Macromolecule #1: Sir2 superfamily protein

MacromoleculeName: Sir2 superfamily protein / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Geobacter sulfurreducens (bacteria)
Molecular weightTheoretical: 66.645922 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
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: 4 / Enantiomer: LEVO
Source (natural)Organism: Geobacter sulfurreducens (bacteria)
Molecular weightTheoretical: 53.325566 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
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 (21-mer)

MacromoleculeName: RNA (21-mer) / type: rna / ID: 3 / Number of copies: 4
Source (natural)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Molecular weightTheoretical: 6.651949 KDa
SequenceString:
AUUCCUGUUA GAGCUGUUAA C

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Macromolecule #4: DNA (25-mer)

MacromoleculeName: DNA (25-mer) / type: dna / ID: 4 / Number of copies: 4 / Classification: DNA
Source (natural)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Molecular weightTheoretical: 7.70804 KDa
SequenceString:
(DA)(DA)(DG)(DT)(DT)(DA)(DA)(DC)(DA)(DG) (DC)(DT)(DC)(DT)(DA)(DA)(DC)(DA)(DG)(DG) (DA)(DA)(DT)(DA)(DA)

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 4 / 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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 36656
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: RANDOM ASSIGNMENT
FSC plot (resolution estimation)

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