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- EMDB-29226: Focused refinement for corner area1 of MapSPARTA -

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Basic information

Entry
Database: EMDB / ID: EMD-29226
TitleFocused refinement for corner area1 of MapSPARTA
Map data
Sample
  • Complex: Structure of tetramerized short MapSPARTA (short prokarytic Argonuate) system upon guide RNA-mediated target ssDNA binding
KeywordspAgo / SPARTA / Argonuate / Oligomerization / TIR domain / NAD / Immune system
Biological speciesMaribacter polysiphoniae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.74 Å
AuthorsShen ZF / Fu TM
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nature / Year: 2023
Title: Oligomerization-mediated activation of a short prokaryotic Argonaute.
Authors: Zhangfei Shen / Xiao-Yuan Yang / Shiyu Xia / Wei Huang / Derek J Taylor / Kotaro Nakanishi / Tian-Min Fu /
Abstract: Although eukaryotic and long prokaryotic Argonaute proteins (pAgos) cleave nucleic acids, some short pAgos lack nuclease activity and hydrolyse NAD(P) to induce bacterial cell death. Here we present ...Although eukaryotic and long prokaryotic Argonaute proteins (pAgos) cleave nucleic acids, some short pAgos lack nuclease activity and hydrolyse NAD(P) to induce bacterial cell death. Here we present a hierarchical activation pathway for SPARTA, a short pAgo consisting of an Argonaute (Ago) protein and TIR-APAZ, an associated protein. SPARTA progresses through distinct oligomeric forms, including a monomeric apo state, a monomeric RNA-DNA-bound state, two dimeric RNA-DNA-bound states and a tetrameric RNA-DNA-bound active state. These snapshots together identify oligomerization as a mechanistic principle of SPARTA activation. The RNA-DNA-binding channel of apo inactive SPARTA is occupied by an auto-inhibitory motif in TIR-APAZ. After the binding of RNA-DNA, SPARTA transitions from a monomer to a symmetric dimer and then an asymmetric dimer, in which two TIR domains interact through charge and shape complementarity. Next, two dimers assemble into a tetramer with a central TIR cluster responsible for hydrolysing NAD(P). In addition, we observe unique features of interactions between SPARTA and RNA-DNA, including competition between the DNA 3' end and the auto-inhibitory motif, interactions between the RNA G2 nucleotide and Ago, and splaying of the RNA-DNA duplex by two loops exclusive to short pAgos. Together, our findings provide a mechanistic basis for the activation of short pAgos, a large section of the Ago superfamily.
History
DepositionDec 19, 2022-
Header (metadata) releaseAug 23, 2023-
Map releaseAug 23, 2023-
UpdateSep 20, 2023-
Current statusSep 20, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29226.png

Downloads & links

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Map

FileDownload / File: emd_29226.map.gz / Format: CCP4 / Size: 307.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.95 Å
Density
Contour LevelBy AUTHOR: 0.888
Minimum - Maximum-5.554311 - 8.103028
Average (Standard dev.)0.0005955931 (±0.05727036)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions432432432
Spacing432432432
CellA=B=C: 410.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_29226_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_29226_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 : Structure of tetramerized short MapSPARTA (short prokarytic Argon...

EntireName: Structure of tetramerized short MapSPARTA (short prokarytic Argonuate) system upon guide RNA-mediated target ssDNA binding
Components
  • Complex: Structure of tetramerized short MapSPARTA (short prokarytic Argonuate) system upon guide RNA-mediated target ssDNA binding

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Supramolecule #1: Structure of tetramerized short MapSPARTA (short prokarytic Argon...

SupramoleculeName: Structure of tetramerized short MapSPARTA (short prokarytic Argonuate) system upon guide RNA-mediated target ssDNA binding
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Maribacter polysiphoniae (bacteria)

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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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: DIRECT ELECTRON APOLLO (4k x 4k) / Average electron dose: 50.0 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: RANDOM ASSIGNMENT
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.74 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 490999
FSC plot (resolution estimation)

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