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- EMDB-39031: Cryo-EM structure of the monomeric SPARSA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-39031
TitleCryo-EM structure of the monomeric SPARSA complex
Map data
Sample
  • Complex: Monomeric SPARSA complex
    • Protein or peptide: Sir2 domain-containing protein
    • Protein or peptide: Piwi domain-containing protein
KeywordsRNA BINDING PROTEIN-RNA-DNA COMPLEX / STRUCTURAL PROTEIN
Biological speciesGeobacter sulfurreducens (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.66 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_39031.png

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Map

FileDownload / File: emd_39031.map.gz / Format: CCP4 / Size: 64 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 256 pix.
= 211.712 Å		0.83 Å/pix.
x 256 pix.
= 211.712 Å		0.83 Å/pix.
x 256 pix.
= 211.712 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.827 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-1.1419652 - 1.6382157
Average (Standard dev.)-0.0003918442 (±0.04358654)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 211.712 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_39031_msk_1.map
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Half map: #1

Fileemd_39031_half_map_1.map
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Half map: #2

Fileemd_39031_half_map_2.map
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Sample components

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Entire : Monomeric SPARSA complex

EntireName: Monomeric SPARSA complex
Components
  • Complex: Monomeric SPARSA complex
    • Protein or peptide: Sir2 domain-containing protein
    • Protein or peptide: Piwi domain-containing protein

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Supramolecule #1: Monomeric SPARSA complex

SupramoleculeName: Monomeric SPARSA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Geobacter sulfurreducens (bacteria)

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Macromolecule #1: Sir2 domain-containing protein

MacromoleculeName: Sir2 domain-containing protein / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Geobacter sulfurreducens (bacteria)
SequenceString: MDVLTDNEFY QHYLQNSQHM MWFLGAGTSR SAGLPTASDI IWDLKHRYYC LHENQDYQKH DINNHAIKSK IQSYMDSKGF PLQWSPEEYS FYFELVFRDD YEAQRKYLLE ALASRKVSLN IGHRVLAALL EMNQTKVVFT TNFDDVIETA FSDISGKHLS VYHLEGSYAA ...String:
MDVLTDNEFY QHYLQNSQHM MWFLGAGTSR SAGLPTASDI IWDLKHRYYC LHENQDYQKH DINNHAIKSK IQSYMDSKGF PLQWSPEEYS FYFELVFRDD YEAQRKYLLE ALASRKVSLN IGHRVLAALL EMNQTKVVFT TNFDDVIETA FSDISGKHLS VYHLEGSYAA LSALNTEAFP IYAKIHGDFR YQKIKNLTPD LQTNDREIHK CFLAAAIRFG LVVSGYSGRD ENVMTMLRAA IDQNNAFPHG LYWTVPSISK SEPAVQDLIT YAQGKGVRAY LVETGTFDEM LSKIWRQVKD KPAAIDAKVR TARVCPVSIP LPGPGKSFPA LRTNALPVVT QSIRCGVVTL ASPITFSELK ERISQKSPKA LLTYTEKVLF LGGEPEIRKI FSNDEINSIG QYYIDEIAQS VAASTFLKSF VEEAILTALL REKPILHRVR HRTHYAVIPN ASAKDDRFLD LRKAVGFKGD LGYITGNVTN AKELSWAEAV SIRLEERGGK LWIMLKPEIW IKPLDRREEA TDFIRSRRRY RFNQCSYQIL DAWIKILFGS IGGGGTVNIS CFPDAEFKAE FEIGTRTAFS LGVGYGR

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Macromolecule #2: Piwi domain-containing protein

MacromoleculeName: Piwi domain-containing protein / type: protein_or_peptide / ID: 2 / Enantiomer: DEXTRO
Source (natural)Organism: Geobacter sulfurreducens (bacteria)
SequenceString: MADNLSQLAA HSTIPEPLLL FKDNRTDTHP LRGLSQYGPY SACFNLPGQV RLAYLAPTEH MRKLDAIVRE LQNPATPKEA TNYYVEYGGF EKVFKVPLVM PQEHLRCLAL DECHGVAANG NGLALADKIV QSMSGLFRQK HAFDVLLVYL PASWKKCFEY DGFDLHDRIK ...String:
MADNLSQLAA HSTIPEPLLL FKDNRTDTHP LRGLSQYGPY SACFNLPGQV RLAYLAPTEH MRKLDAIVRE LQNPATPKEA TNYYVEYGGF EKVFKVPLVM PQEHLRCLAL DECHGVAANG NGLALADKIV QSMSGLFRQK HAFDVLLVYL PASWKKCFEY DGFDLHDRIK AKVAPLNLPI QIINDTALTR QCRANVMWGV SVALYAKAGG IPWKLADWDK DEAYIGLSYA IKKNAEGQEY TTCCSQVFDP DGTGFEFVAY DTREFITDRK GNPYLSYQEM QSVLSKSLHL YQSSHNGRMP RKIFIHKTTH FTEDEIQGAF DSFSSSTEIE LVQIIQSTNW YGLKVDGKKG DKPVAPASYP VDRGLYQPLT ESECLLWTQG SVMGVNQQNP GQPVFKEAAL TPLPNPIMLR RFSGNGGWHA TCSSILALTK VDWNNNTLYK KLPVTLVYSQ VFADVVKQTP EIVNEIYDYR FFM

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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: 3.66 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 75576
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: RANDOM ASSIGNMENT
FSC plot (resolution estimation)

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