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- EMDB-41947: Locally refined (symmetry based) cryo-EM map of the second dimer ... -

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Basic information

Entry
Database: EMDB / ID: EMD-41947
TitleLocally refined (symmetry based) cryo-EM map of the second dimer of the SPARTA oligomer with guide RNA and target DNA
Map datalocally refined map
Sample
  • Complex: SPARTA complex with gRNA/tDNA
    • Protein or peptide: Ago
    • Protein or peptide: TIR-APAZ
KeywordsRNA / DNA / Argonaute / TIR / immune system / RNA BINDING PROTEIN
Biological speciesThermoflavifilum thermophilum (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.17 Å
AuthorsMalik R / Kottur J / Aggarwal AK
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI) United States
CitationJournal: Nat Commun / Year: 2024
Title: Nucleic acid mediated activation of a short prokaryotic Argonaute immune system.
Authors: Jithesh Kottur / Radhika Malik / Aneel K Aggarwal /
Abstract: A short prokaryotic Argonaute (pAgo) TIR-APAZ (SPARTA) defense system, activated by invading DNA to unleash its TIR domain for NAD(P) hydrolysis, was recently identified in bacteria. We report the ...A short prokaryotic Argonaute (pAgo) TIR-APAZ (SPARTA) defense system, activated by invading DNA to unleash its TIR domain for NAD(P) hydrolysis, was recently identified in bacteria. We report the crystal structure of SPARTA heterodimer in the absence of guide-RNA/target-ssDNA (2.66 Å) and a cryo-EM structure of the SPARTA oligomer (tetramer of heterodimers) bound to guide-RNA/target-ssDNA at nominal 3.15-3.35 Å resolution. The crystal structure provides a high-resolution view of SPARTA, revealing the APAZ domain as equivalent to the N, L1, and L2 regions of long pAgos and the MID domain containing a unique insertion (insert57). Cryo-EM structure reveals regions of the PIWI (loop10-9) and APAZ (helix αN) domains that reconfigure for nucleic-acid binding and decrypts regions/residues that reorganize to expose a positively charged pocket for higher-order assembly. The TIR domains amass in a parallel-strands arrangement for catalysis. We visualize SPARTA before and after RNA/ssDNA binding and uncover the basis of its active assembly leading to abortive infection.
History
DepositionSep 13, 2023-
Header (metadata) releaseJun 12, 2024-
Map releaseJun 12, 2024-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41947.png

Downloads & links

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Map

FileDownload / File: emd_41947.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationlocally refined map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 480 pix.
= 519.84 Å		1.08 Å/pix.
x 480 pix.
= 519.84 Å		1.08 Å/pix.
x 480 pix.
= 519.84 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.083 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.7
Minimum - Maximum-2.9894373 - 4.856839
Average (Standard dev.)-0.00041309706 (±0.07413297)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 519.83997 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_41947_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map

Fileemd_41947_half_map_2.map
Annotationhalf-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 : SPARTA complex with gRNA/tDNA

EntireName: SPARTA complex with gRNA/tDNA
Components
  • Complex: SPARTA complex with gRNA/tDNA
    • Protein or peptide: Ago
    • Protein or peptide: TIR-APAZ

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Supramolecule #1: SPARTA complex with gRNA/tDNA

SupramoleculeName: SPARTA complex with gRNA/tDNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Thermoflavifilum thermophilum (bacteria)

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Macromolecule #1: Ago

MacromoleculeName: Ago / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermoflavifilum thermophilum (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKELIYIEEP SILFAHGQKC TDPRDGLALF GPLNQIYGIK SGVVGTQKGL QIFKSYLDKI QKPIYNHNN ITRPMFPGFE AVFGCKWESQ NIVFKEITDE EIRRYLFNAS THKRTYDLVT L FNDKIITA NKNDEERVDV WFVIVPEEIY KYCRPNSVLP NELVQTKSLI ...String:
MKELIYIEEP SILFAHGQKC TDPRDGLALF GPLNQIYGIK SGVVGTQKGL QIFKSYLDKI QKPIYNHNN ITRPMFPGFE AVFGCKWESQ NIVFKEITDE EIRRYLFNAS THKRTYDLVT L FNDKIITA NKNDEERVDV WFVIVPEEIY KYCRPNSVLP NELVQTKSLI SKSKAKSFRY TP TLFEEFN KKLKEVEKEA KTYNYDAQFH DQLKARLLEH TIPTQILRES TLAWRDFKNT FGA PIRDFS KIEGHLAWTI STAAYYKAGG KPWKLGDIRP GVCYLGLVYK KIEKSKNPQN ACCA AQMFL DNGDGTVFKG EVGPWYNPEK GEYHLKPKEA KALLTQALES YKEQNKSYPK EVFIH ARTR FNDEEWNAFN EVTPKNTNLV GVTITKSKPL KLYKTEGAFP IMRGNAYIVD EKKAFL WTL GFVPKLQSTL SMEVPNPIFI EINKGEAEIQ QVLKDILALT KLNYNACIYA DGEPVTL RF ANKIGEILTA STEIKTPPLA FKYYI

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Macromolecule #2: TIR-APAZ

MacromoleculeName: TIR-APAZ / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Thermoflavifilum thermophilum (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRNKIFISHA TPEDDDFTRW LSLKLIGLGY EVWCDILFLD KGVDFWSTIE KEIRENTCKF LIVSSTAGN KREGVLKELA VATKVKKHLQ DDMFIIPLAI DENLSYDDIN IEIVRLNAID F KKSWAKGL QDLLDAFEKQ NVPKKPPDHS KSNLLYQQIF LHDKQAIEKE ...String:
MRNKIFISHA TPEDDDFTRW LSLKLIGLGY EVWCDILFLD KGVDFWSTIE KEIRENTCKF LIVSSTAGN KREGVLKELA VATKVKKHLQ DDMFIIPLAI DENLSYDDIN IEIVRLNAID F KKSWAKGL QDLLDAFEKQ NVPKKPPDHS KSNLLYQQIF LHDKQAIEKE ETYDSNWFPI IS FPNELRF HRYDWRLPKQ FDVRTLAFPA IRYKEYLCTF AWEYDFIHQL PKTETYNGQE SIR ISTSDI LSGRYDTDFI RNYECQRLIV QLINKAFELR MKDKNVREYQ MSKTFAYWIE KGKL EKDKF EKIKLVGKQK NKYWHFGISA AGKLYPSPVL MVSSHIIFTM DGINLIKSKS IQHSS RRKQ GKNWWNDKWR EKLLAFIRFL SDDQNAIYLN VGSEEKILIS NKPLKFFGKM SYVTPS EVT LEEESVLADI NNFEEDTEDL DELEDIE

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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: 7.5
Sugar embeddingMaterial: Vitreous ice
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 51.11 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 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: 3.17 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 238432
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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