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- EMDB-40679: Incomplete map of Maribacter polysiphoniae Argonaute (MapSPARTA) ... -

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Basic information

Entry
Database: EMDB / ID: EMD-40679
TitleIncomplete map of Maribacter polysiphoniae Argonaute (MapSPARTA) bound with guide RNA and target DNA duplex.
Map data
Sample
  • Complex: Complex of MapSPARTA binding with gRNA tDNA duplex
    • Other: Map Argonaute
KeywordsgRNA mediated DNA binding / Microbiology / Argonaute / microbic immune system / DNA-RNA HYBRID
Biological speciesMaribacter polysiphoniae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsShen ZF / Yang XY / Fu TM
Funding support United States, 1 items
OrganizationGrant numberCountry
Not funded 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
DepositionMay 3, 2023-
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_40679.png

Downloads & links

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Map

FileDownload / File: emd_40679.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.)
0.95 Å/pix.
x 256 pix.
= 243.2 Å		0.95 Å/pix.
x 256 pix.
= 243.2 Å		0.95 Å/pix.
x 256 pix.
= 243.2 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.95 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0249
Minimum - Maximum-0.061871365 - 2.1975942
Average (Standard dev.)0.0009176363 (±0.02007194)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 243.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_40679_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_40679_half_map_2.map
Projections & Slices
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Histogram

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Sample components

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Entire : Complex of MapSPARTA binding with gRNA tDNA duplex

EntireName: Complex of MapSPARTA binding with gRNA tDNA duplex
Components
  • Complex: Complex of MapSPARTA binding with gRNA tDNA duplex
    • Other: Map Argonaute

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Supramolecule #1: Complex of MapSPARTA binding with gRNA tDNA duplex

SupramoleculeName: Complex of MapSPARTA binding with gRNA tDNA duplex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Maribacter polysiphoniae (bacteria)

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Macromolecule #1: Map Argonaute

MacromoleculeName: Map Argonaute / type: other / ID: 1 / Details: UGACGGCUCUAAUCUAUUAGU CAACTAATAGATTAGAGCCGTCAAT / Classification: other
Source (natural)Organism: Maribacter polysiphoniae (bacteria)
SequenceString: RNKIFISHAT PDDNDFTRWL ALKLIGLGYE VWCDILFLDK GVDFWSNIEK VIREDTCKFL LVSSSYSNQR EGVLKELAV AAKVKKQLKD DKFIIPLAID EQLSYDDINI DIVRLNAIDF KMSWARGLKD ILEAFEKQKV PKEVADASKS NLLYQQIFLH DKSVIEKEE ...String:
RNKIFISHAT PDDNDFTRWL ALKLIGLGYE VWCDILFLDK GVDFWSNIEK VIREDTCKFL LVSSSYSNQR EGVLKELAV AAKVKKQLKD DKFIIPLAID EQLSYDDINI DIVRLNAIDF KMSWARGLKD ILEAFEKQKV PKEVADASKS NLLYQQIFLH DKSVIEKEE IYDSNWLSIL SFPEELRFHE YNWMLPKRFD VRELTFPAVR YKNYLCTFAW AYDFTYHLPK TETYHKSKTI R IPTEEILS GSYDSNFIRN AECKRLIVQL LNKAFELRMK DKEVQEYEMS NKTAYWLEKG KLEKDKFEKT MLVGKQKDKN WH FAISGAS KLYPFPVLMI SSHIFFTADG KKLIDSSSVQ HSSRRRQGKN WWNNTWRTKL LAFIKYLSDD DTSFYLEMGS EEK VFVSNE PVKFKGNVSY NIPEKNTLEE EAELSGFNQG EDIEELEELI ENLEAE MKE LIYIEEPKIL FAHGQKCTDA RDGLALFGPL NNLYGIKSGV IGTKQGLKIF RDYLDHIQKP IYNSNSITRP MFPGFEA VF DCKWESTGIT FKEVTNEDIG KFLYNSSTHK RTYDLVSLFI DKIISANKNE DENVDVWFVI VPDEIYKYCR PNSVLPKE M VQTKALMSKS KAKSFRYEPS LFPDINIELK EQEKEAETYN YDAQFHDQFK ARLLKHTIPT QIFRESTLAW RDFKNAFGL PIRDFSKIEG HLAWTISTAA FYKAGGKPWK LSDVRNGVCY LGLVYKKVEK SKNPRNACCA AQMFLDNGDG TVFKGEVGPW YNPKNGQYH LEPKEAKALL SQSLQSYKEQ IGEYPKEVFI HAKTRFNHQE WDAFLEVTPK ETNLVGVTIS KTKPLKLYKT E GDYTILRG NAYVVNERSA FLWTVGYVPK IQTALSMEVP NPLFIEINKG EADIKQVLKD ILSLTKLNYN ACIFADGEPV TL RFADKIG EILTASTDIK TPPLAFKYYI
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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: FEI FALCON IV (4k 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: 2.1 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: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 534729
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: RANDOM ASSIGNMENT
FSC plot (resolution estimation)

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