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- EMDB-29033: Inactivate state of Maribacter polysiphoniae Argonuate (short pAg... -

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Basic information

Entry
Database: EMDB / ID: EMD-29033
TitleInactivate state of Maribacter polysiphoniae Argonuate (short pAgo system)
Map data
Sample
  • Complex: MapSPARTA heterodimeric complex(short pAgo)
    • Protein or peptide: TIR-APAZ
    • Protein or peptide: short pAgo
KeywordsArgonuate / TIR domain / Oligomerization / NAD+ / IMMUNE SYSTEM
Function / homology
Function and homology information


nucleic acid binding / signal transduction
Similarity search - Function
TIR domain / TIR domain profile. / Toll/interleukin-1 receptor homology (TIR) domain / Toll/interleukin-1 receptor homology (TIR) domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
Piwi domain-containing protein / TIR domain-containing protein
Similarity search - Component
Biological speciesMaribacter polysiphoniae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.07 Å
AuthorsShen ZF / Yang XY / Fu TM
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
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 6, 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_29033.png

Downloads & links

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Map

FileDownload / File: emd_29033.map.gz / Format: CCP4 / Size: 307.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.53 Å/pix.
x 432 pix.
= 230.99 Å		0.53 Å/pix.
x 432 pix.
= 230.99 Å		0.53 Å/pix.
x 432 pix.
= 230.99 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.5347 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.6061678 - 0.7677637
Average (Standard dev.)0.0000011169984 (±0.01578458)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions432432432
Spacing432432432
CellA=B=C: 230.99039 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: sharped map

Fileemd_29033_additional_1.map
Annotationsharped map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_29033_half_map_1.map
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Half map: #1

Fileemd_29033_half_map_2.map
Projections & Slices
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Sample components

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Entire : MapSPARTA heterodimeric complex(short pAgo)

EntireName: MapSPARTA heterodimeric complex(short pAgo)
Components
  • Complex: MapSPARTA heterodimeric complex(short pAgo)
    • Protein or peptide: TIR-APAZ
    • Protein or peptide: short pAgo

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Supramolecule #1: MapSPARTA heterodimeric complex(short pAgo)

SupramoleculeName: MapSPARTA heterodimeric complex(short pAgo) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Maribacter polysiphoniae (bacteria)

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Macromolecule #1: TIR-APAZ

MacromoleculeName: TIR-APAZ / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Maribacter polysiphoniae (bacteria)
Molecular weightTheoretical: 53.270594 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MRNKIFISHA TPDDNDFTRW LALKLIGLGY EVWCDILFLD KGVDFWSNIE KVIREDTCKF LLVSSSYSNQ REGVLKELAV AAKVKKQLK DDKFIIPLAI DEQLSYDDIN IDIVRLNAID FKMSWARGLK DILEAFEKQK VPKEVADASK SNLLYQQIFL H DKSVIEKE ...String:
MRNKIFISHA TPDDNDFTRW LALKLIGLGY EVWCDILFLD KGVDFWSNIE KVIREDTCKF LLVSSSYSNQ REGVLKELAV AAKVKKQLK DDKFIIPLAI DEQLSYDDIN IDIVRLNAID FKMSWARGLK DILEAFEKQK VPKEVADASK SNLLYQQIFL H DKSVIEKE EIYDSNWLSI LSFPEELRFH EYNWMLPKRF DVRELTFPAV RYKNYLCTFA WAYDFTYHLP KTETYHKSKT IR IPTEEIL SGSYDSNFIR NAECKRLIVQ LLNKAFELRM KDKEVQEYEM SNKTAYWLEK GKLEKDKFEK TMLVGKQKDK NWH FAISGA SKLYPFPVLM ISSHIFFTAD GKKLIDSSSV QHSSRRRQGK NWWNNTWRTK LLAFIKYLSD DDTSFYLEMG SEEK VFVSN EPVKFKGNVS YNIPEKNTLE EEAELSGFNQ GEDIEELEEL IENLEAE

UniProtKB: TIR domain-containing protein

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Macromolecule #2: short pAgo

MacromoleculeName: short pAgo / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Maribacter polysiphoniae (bacteria)
Molecular weightTheoretical: 58.09141 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MKELIYIEEP KILFAHGQKC TDARDGLALF GPLNNLYGIK SGVIGTKQGL KIFRDYLDHI QKPIYNSNSI TRPMFPGFEA VFDCKWEST GITFKEVTNE DIGKFLYNSS THKRTYDLVS LFIDKIISAN KNEDENVDVW FVIVPDEIYK YCRPNSVLPK E MVQTKALM ...String:
MKELIYIEEP KILFAHGQKC TDARDGLALF GPLNNLYGIK SGVIGTKQGL KIFRDYLDHI QKPIYNSNSI TRPMFPGFEA VFDCKWEST GITFKEVTNE DIGKFLYNSS THKRTYDLVS LFIDKIISAN KNEDENVDVW FVIVPDEIYK YCRPNSVLPK E MVQTKALM SKSKAKSFRY EPSLFPDINI ELKEQEKEAE TYNYDAQFHD QFKARLLKHT IPTQIFREST LAWRDFKNAF GL PIRDFSK IEGHLAWTIS TAAFYKAGGK PWKLSDVRNG VCYLGLVYKK VEKSKNPRNA CCAAQMFLDN GDGTVFKGEV GPW YNPKNG QYHLEPKEAK ALLSQSLQSY KEQIGEYPKE VFIHAKTRFN HQEWDAFLEV TPKETNLVGV TISKTKPLKL YKTE GDYTI LRGNAYVVNE RSAFLWTVGY VPKIQTALSM EVPNPLFIEI NKGEADIKQV LKDILSLTKL NYNACIFADG EPVTL RFAD KIGEILTAST DIKTPPLAFK YYI

UniProtKB: Piwi domain-containing protein

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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 opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.07 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 217571
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: RANDOM ASSIGNMENT
FSC plot (resolution estimation)

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