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- PDB-8rot: Cryo-EM structure of the transmembrane anti-sigma factor DdvA -

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Basic information

Entry
Database: PDB / ID: 8rot
TitleCryo-EM structure of the transmembrane anti-sigma factor DdvA
ComponentsCHAT domain-containing protein
KeywordsSIGNALING PROTEIN / Transmembrane protein / anti-sigma factor / antiviral system / TPR-CHAT
Function / homologyAnti-sigma factor, zinc-finger domain superfamily / Putative zinc-finger / Putative zinc-finger / CHAT domain / CHAT domain / Tetratricopeptide-like helical domain superfamily / membrane / CHAT domain-containing protein
Function and homology information
Biological speciesMyxococcus xanthus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.11 Å
AuthorsLopez-Alonso, J.P. / Ochoa-Lizarralde, B. / Tascon, I. / Ubarretxena-Belandia, I.
Funding support Spain, 1items
OrganizationGrant numberCountry
Ministerio de Ciencia e Innovacion (MCIN)PID2022-143177NB-I00 Spain
CitationJournal: Sci Adv / Year: 2024
Title: Structural basis for regulation of a CBASS-CRISPR-Cas defense island by a transmembrane anti-σ factor and its ECF σ partner.
Authors: Diego Bernal-Bernal / David Pantoja-Uceda / Jorge Pedro López-Alonso / Alfonso López-Rojo / José Antonio López-Ruiz / Marisa Galbis-Martínez / Borja Ochoa-Lizarralde / Igor Tascón / ...Authors: Diego Bernal-Bernal / David Pantoja-Uceda / Jorge Pedro López-Alonso / Alfonso López-Rojo / José Antonio López-Ruiz / Marisa Galbis-Martínez / Borja Ochoa-Lizarralde / Igor Tascón / Montserrat Elías-Arnanz / Iban Ubarretxena-Belandia / S Padmanabhan /
Abstract: How CRISPR-Cas and cyclic oligonucleotide-based antiphage signaling systems (CBASS) are coordinately deployed against invaders remains unclear. We show that a locus containing two CBASS and one type ...How CRISPR-Cas and cyclic oligonucleotide-based antiphage signaling systems (CBASS) are coordinately deployed against invaders remains unclear. We show that a locus containing two CBASS and one type III-B CRISPR-Cas system, regulated by the transmembrane anti-σ DdvA and its cognate extracytoplasmic function (ECF) σ DdvS, can defend against a phage. Cryo-electron microscopy reveals DdvA-DdvS pairs assemble as arrow-shaped transmembrane dimers. Each DdvA periplasmic domain adopts a separase/craspase-type tetratricopeptide repeat (TPR)-caspase HetF-associated with TPR (TPR-CHAT) architecture with an incomplete His-Cys active site, lacking three α-helices conserved among CHAT domains. Each active site faces the dimer interface, raising the possibility that signal-induced caspase-like DdvA autoproteolysis in trans precedes RseP-mediated intramembrane proteolysis and DdvS release. Nuclear magnetic resonance reveals a DdvA cytoplasmic CHCC-type zinc-bound three-helix bundle that binds to DdvS σ and σ domains, undergoing σ-induced helix extension to trap DdvS. Altogether, we provide structural-mechanistic insights into membrane anti-σ-ECF σ regulation of an antiviral CBASS-CRISPR-Cas defense island.
History
DepositionJan 12, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 6, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 27, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: CHAT domain-containing protein
A: CHAT domain-containing protein


Theoretical massNumber of molelcules
Total (without water)217,2812
Polymers217,2812
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein CHAT domain-containing protein


Mass: 108640.586 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: DdvA / Source: (gene. exp.) Myxococcus xanthus (bacteria) / Gene: MXAN_7288 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q1CW22
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: DdvA in complex with DdvS / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Myxococcus xanthus (bacteria) / Strain: DK1050
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTrisHCl1
2100 mMSodium ChlorideNaCl1
30.2 %DDM1
42 mMDTT1
SpecimenConc.: 7.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 9 mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: LAB6 / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Calibrated magnification: 134788 X / Nominal defocus max: 20000 nm / Nominal defocus min: 8000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.37 sec. / Electron dose: 60.033 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 37374 / Details: 17.890 e/pix/s Illumination Area: 760 nm 50 frames
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.1particle selectionTemplate picker
2EPU3.4.0.5704RELimage acquisition
4cryoSPARC4.1CTF correction
9cryoSPARC4.1initial Euler assignment
10cryoSPARC4.1final Euler assignment
11cryoSPARC4.1classification
12cryoSPARC4.13D reconstruction
13PHENIX1.21model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2674718
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 339970 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00414114
ELECTRON MICROSCOPYf_angle_d0.57719204
ELECTRON MICROSCOPYf_dihedral_angle_d4.4842016
ELECTRON MICROSCOPYf_chiral_restr0.0382160
ELECTRON MICROSCOPYf_plane_restr0.0052558

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