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- EMDB-50055: Cryo-EM structure of SAVED-Lon protease CCaCalpL filament bound to cA4 -

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Basic information

Entry
Database: EMDB / ID: EMD-50055
TitleCryo-EM structure of SAVED-Lon protease CCaCalpL filament bound to cA4
Map data
Sample
  • Complex: Filament fragment of cA4 bound CCaCalpL
    • Complex: CCaCalpL
      • Protein or peptide: SMODS-associated and fused to various effectors domain-containing protein
    • Complex: cA4
      • RNA: cA4
KeywordsLON PROTEASE / SAVED DOMAIN / CRISPR / HYDROLASE
Function / homologySMODS-associated and fused to various effectors / SMODS-associated and fused to various effectors sensor domain / SMODS-associated and fused to various effectors domain-containing protein
Function and homology information
Biological speciesCandidatus Cloacimonas acidaminovorans (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsTamulaitiene G / Sasnauskas G / Smalakyte D / Tamulaitis G
Funding supportLithuania, 1 items
OrganizationGrant numberCountry
Research Council of LithuaniaS-MIP-22-9Lithuania
CitationJournal: Mol Cell / Year: 2024
Title: Filament formation activates protease and ring nuclease activities of CRISPR Lon-SAVED.
Authors: Dalia Smalakyte / Audrone Ruksenaite / Giedrius Sasnauskas / Giedre Tamulaitiene / Gintautas Tamulaitis
Abstract: To combat phage infection, type III CRISPR-Cas systems utilize cyclic oligoadenylates (cA) signaling to activate various auxiliary effectors, including the CRISPR-associated Lon-SAVED protease CalpL, ...To combat phage infection, type III CRISPR-Cas systems utilize cyclic oligoadenylates (cA) signaling to activate various auxiliary effectors, including the CRISPR-associated Lon-SAVED protease CalpL, which forms a tripartite effector system together with an anti-σ factor, CalpT, and an ECF-like σ factor, CalpS. Here, we report the characterization of the Candidatus Cloacimonas acidaminovorans CalpL-CalpT-CalpS. We demonstrate that cA binding triggers CalpL filament formation and activates it to cleave CalpT within the CalpT-CalpS dimer. This cleavage exposes the CalpT C-degron, which targets it for further degradation by cellular proteases. Consequently, CalpS is released to bind to RNA polymerase, causing growth arrest in E. coli. Furthermore, the CalpL-CalpT-CalpS system is regulated by the SAVED domain of CalpL, which is a ring nuclease that cleaves cA in a sequential three-step mechanism. These findings provide key mechanistic details for the activation, proteolytic events, and regulation of the signaling cascade in the type III CRISPR-Cas immunity.
History
DepositionApr 9, 2024-
Header (metadata) releaseOct 2, 2024-
Map releaseOct 2, 2024-
UpdateDec 18, 2024-
Current statusDec 18, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50055.png

Downloads & links

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Map

FileDownload / File: emd_50055.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesX (Sec.)Y (Row.)Z (Col.)
1.1 Å/pix.
x 240 pix.
= 264. Å		1.1 Å/pix.
x 240 pix.
= 264. Å		1.1 Å/pix.
x 240 pix.
= 264. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 7.0
Minimum - Maximum-22.984999999999999 - 43.563408000000003
Average (Standard dev.)0.000000000003838 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 264.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_50055_msk_1.map
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Additional map: #1

Fileemd_50055_additional_1.map
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Half map: #2

Fileemd_50055_half_map_1.map
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Half map: #1

Fileemd_50055_half_map_2.map
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Sample components

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Entire : Filament fragment of cA4 bound CCaCalpL

EntireName: Filament fragment of cA4 bound CCaCalpL
Components
  • Complex: Filament fragment of cA4 bound CCaCalpL
    • Complex: CCaCalpL
      • Protein or peptide: SMODS-associated and fused to various effectors domain-containing protein
    • Complex: cA4
      • RNA: cA4

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Supramolecule #1: Filament fragment of cA4 bound CCaCalpL

SupramoleculeName: Filament fragment of cA4 bound CCaCalpL / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: CCaCalpL

SupramoleculeName: CCaCalpL / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Candidatus Cloacimonas acidaminovorans (bacteria)

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Supramolecule #3: cA4

SupramoleculeName: cA4 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: SMODS-associated and fused to various effectors domain-containing...

MacromoleculeName: SMODS-associated and fused to various effectors domain-containing protein
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Candidatus Cloacimonas acidaminovorans (bacteria) / Strain: Evry
Molecular weightTheoretical: 63.294172 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGGSHHHHHH GMASWSHPQF EKGGHHHHHH GGSGENLYFQ GGSGPKFNET ADKYLKSGSA EAELIILQYI QQDRVSEDDE EWVYNLLEK ANNPYIKLNA LLWLSAKRKY LTQLSKLWGI SENELKSLSQ QQPKIGLFPA VDSRKNAFLA KVFVYKLKSE E PIALAILG ...String:
MGGSHHHHHH GMASWSHPQF EKGGHHHHHH GGSGENLYFQ GGSGPKFNET ADKYLKSGSA EAELIILQYI QQDRVSEDDE EWVYNLLEK ANNPYIKLNA LLWLSAKRKY LTQLSKLWGI SENELKSLSQ QQPKIGLFPA VDSRKNAFLA KVFVYKLKSE E PIALAILG DKIENFSYLA QLGKQNCLIG FNKNIQGNSW QLAVLATLLV KDEKIISKIA YSGIVLPSGE IITAEEIEYK KR CCQNLVH RIKKIEQLDA WLNTETIPLP VIQYQGEENE LKRWQKAMEQ KVQEKFSWFS YELLEDFYGI TNSDLAIFGN GIL PFEANA WQKLLQEQVK DKFKLLEDKV MPKKVLWFYA GQISTLQLGI GALFGFKRAV SILQMEFSNT TYHEVFILYG KENA RQLKN VSVKKEDYQY IQSELLINEP HKNELGFIIY LGSHNPIGEA KAYCQKQLQI NNFLIIQARE NQGVMETSQN WLPYL QEIN SALNTARQEY HWERIHLFQT APTALCMALG IAVGHFLPVD VYHYQFNAEE PKYRCVFSLD KMLNL

UniProtKB: SMODS-associated and fused to various effectors domain-containing protein

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Macromolecule #2: cA4

MacromoleculeName: cA4 / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 1.271866 KDa
SequenceString:
AAAA

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 92000

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.4.1) / Number images used: 124470
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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