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- EMDB-19404: Cryo-EM structure of the transmembrane anti-sigma factor DdvA -

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Basic information

Entry
Database: EMDB / ID: EMD-19404
TitleCryo-EM structure of the transmembrane anti-sigma factor DdvA
Map data
Sample
  • Complex: DdvA in complex with DdvS
    • Protein or peptide: CHAT domain-containing protein
KeywordsTransmembrane protein / anti-sigma factor / antiviral system / TPR-CHAT / SIGNALING PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.11 Å
AuthorsLopez-Alonso JP / Ochoa-Lizarralde B / Tascon I / Ubarretxena-Belandia I
Funding support Spain, 1 items
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, 2024-
Header (metadata) releaseNov 6, 2024-
Map releaseNov 6, 2024-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19404.png

Downloads & links

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Map

FileDownload / File: emd_19404.map.gz / Format: CCP4 / Size: 1000 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 640 pix.
= 413.568 Å		0.65 Å/pix.
x 640 pix.
= 413.568 Å		0.65 Å/pix.
x 640 pix.
= 413.568 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.6462 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.134
Minimum - Maximum-0.5190253 - 0.91623324
Average (Standard dev.)0.00019940363 (±0.011595878)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions640640640
Spacing640640640
CellA=B=C: 413.568 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19404_msk_1.map
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Half map: #2

Fileemd_19404_half_map_1.map
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Half map: #1

Fileemd_19404_half_map_2.map
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Sample components

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Entire : DdvA in complex with DdvS

EntireName: DdvA in complex with DdvS
Components
  • Complex: DdvA in complex with DdvS
    • Protein or peptide: CHAT domain-containing protein

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Supramolecule #1: DdvA in complex with DdvS

SupramoleculeName: DdvA in complex with DdvS / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Myxococcus xanthus (bacteria) / Strain: DK1050

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Macromolecule #1: CHAT domain-containing protein

MacromoleculeName: CHAT domain-containing protein / type: protein_or_peptide / ID: 1 / Details: DdvA / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Myxococcus xanthus (bacteria)
Molecular weightTheoretical: 108.640586 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSSSPCDQLQ SFADGDLPPM EAQAFGQHLA DCEKCQVELT RLLQLDQLGR GYIERHGPVD IPWHALPRNR WMAGGFALAS MVAVAFILL GTLRPQAPRA LPELWAQPQR TLEARVTYLA ADRYRRPPQV MMGGAAARSG PNRSLALLSE LEKRGDLHQL A VAYLATGV ...String:
MSSSPCDQLQ SFADGDLPPM EAQAFGQHLA DCEKCQVELT RLLQLDQLGR GYIERHGPVD IPWHALPRNR WMAGGFALAS MVAVAFILL GTLRPQAPRA LPELWAQPQR TLEARVTYLA ADRYRRPPQV MMGGAAARSG PNRSLALLSE LEKRGDLHQL A VAYLATGV PEPSSAKAIL EGMRSDLRWQ SADVLCDLGV AHYVASKPLD AARATEELRE ALRLFDTVLA MQPGHVQALW NR SLVYRDL GLPLSAMKDL TEFEHRETDE GWRSEARDRR ARLSSTLRRK ERWLAADQTG ADLINRGAQE LARALTFVDV PLL RRDFYH AVRARTSSTD VLALLPLAER LDASVGSGTV LADYVHQVAA RDFSRRAPLA EQYARLISGR IPESEQDALL QRFL TSDET DLALGALAHV MQRLPAYASE LVRRTQHDED PWFRVLGLQA QAMLERQQEH YKEALAPLEQ ALDICRRERL VYRCI FIEN DLSHVKSWLF RVNAAAQHAR DGLALARPNQ WDLEGVMLQA LGNVARQAAD VTLGRAYYGE ALLMAEGDKW STRNIH QNL AHLAIWALEL DEARASLDRA MDTGLPLTQH GVAALVDVAR TRRSPRDALM VEQALAREPG NTPGQRAYAK FLHGRIL VE VDPARGRMLL DEAIRQAEAL PLDDVSAAHA RAYSYTSLIF ADADTGDFIA ALARFGAELG FETPARCVLG LTADTERS L LVARGAQGQL LSAYVPLRSS RFEAASMEGA VPPEMLAALQ ACTLVDVLAR PPLQGRSGLL PPGIAWRYRT RAAAPPPPA GPGTHLVVNE VRYSEERNEV PLQWLPRTAP GAEARFLRDL AATPTQVLEA ISTATEIDLA THGKVDPDSN FAYLLLAPGA DGRDTLFED SIRASQLTGA PLVVLAACEG GRPNAALHRA GSLPSAFLAA GARAVLAATH PIPDLDSSAF FGAVRDRVLA G ASLAVAVR DERLQWLSAG GDSEWVNAVL VFE

UniProtKB: CHAT 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

Concentration7.5 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
50.0 mMTrisHCl
100.0 mMSodium ChlorideNaCl
0.2 %DDM
2.0 mMDTT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Pretreatment - Pressure: 0.035 kPa / Details: 9 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 37374 / Average exposure time: 1.37 sec. / Average electron dose: 60.033 e/Å2 / Details: 17.890 e/pix/s Illumination Area: 760 nm 50 frames
Electron beamAcceleration voltage: 300 kV / Electron source: LAB6
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 134788 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 20.0 µm / Nominal defocus min: 8.0 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2674718
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model in cryosparc
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.1) / Number images used: 339970
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1)
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. 4.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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