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- EMDB-40059: Homo-octamer of PbuCsx28 protein -

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Basic information

Entry
Database: EMDB / ID: EMD-40059
TitleHomo-octamer of PbuCsx28 protein
Map datasharpened map
Sample
  • Complex: octameric assembly of csx28
    • Protein or peptide: Accessory protein Csx28
KeywordsCRISPR-associated protein / ANTIVIRAL PROTEIN
Function / homologymembrane / Uncharacterized protein
Function and homology information
Biological speciesPrevotella buccae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.65 Å
AuthorsPark JU / Kellogg EH
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00 United States
CitationJournal: Science / Year: 2023
Title: Csx28 is a membrane pore that enhances CRISPR-Cas13b-dependent antiphage defense.
Authors: Arica R VanderWal / Jung-Un Park / Bogdan Polevoda / Julia K Nicosia / Adrian M Molina Vargas / Elizabeth H Kellogg / Mitchell R O'Connell /
Abstract: Type VI CRISPR-Cas systems use RNA-guided ribonuclease (RNase) Cas13 to defend bacteria against viruses, and some of these systems encode putative membrane proteins that have unclear roles in Cas13- ...Type VI CRISPR-Cas systems use RNA-guided ribonuclease (RNase) Cas13 to defend bacteria against viruses, and some of these systems encode putative membrane proteins that have unclear roles in Cas13-mediated defense. We show that Csx28, of type VI-B2 systems, is a transmembrane protein that assists to slow cellular metabolism upon viral infection, increasing antiviral defense. High-resolution cryo-electron microscopy reveals that Csx28 forms an octameric pore-like structure. These Csx28 pores localize to the inner membrane in vivo. Csx28's antiviral activity in vivo requires sequence-specific cleavage of viral messenger RNAs by Cas13b, which subsequently results in membrane depolarization, slowed metabolism, and inhibition of sustained viral infection. Our work suggests a mechanism by which Csx28 acts as a downstream, Cas13b-dependent effector protein that uses membrane perturbation as an antiviral defense strategy.
History
DepositionMar 13, 2023-
Header (metadata) releaseApr 26, 2023-
Map releaseApr 26, 2023-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40059.png

Downloads & links

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Map

FileDownload / File: emd_40059.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.33 Å/pix.
x 220 pix.
= 292.6 Å		1.33 Å/pix.
x 220 pix.
= 292.6 Å		1.33 Å/pix.
x 220 pix.
= 292.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.33 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.21349421 - 0.31146443
Average (Standard dev.)0.00012077732 (±0.0054890136)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 292.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: halfmap 1

Fileemd_40059_half_map_1.map
Annotationhalfmap 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap 2

Fileemd_40059_half_map_2.map
Annotationhalfmap 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : octameric assembly of csx28

EntireName: octameric assembly of csx28
Components
  • Complex: octameric assembly of csx28
    • Protein or peptide: Accessory protein Csx28

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Supramolecule #1: octameric assembly of csx28

SupramoleculeName: octameric assembly of csx28 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Prevotella buccae (bacteria)
Molecular weightTheoretical: 300 KDa

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Macromolecule #1: Accessory protein Csx28

MacromoleculeName: Accessory protein Csx28 / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Prevotella buccae (bacteria)
Molecular weightTheoretical: 21.910193 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MDYMELAKEA FSIICTFIAA YVAYYYAIKQ LHQKSVENIE YAKYQAVLQA HKSLYKLLRF TTNTENEDSI LIWEKTKDGK QEATYYFRK ENIRKFIKEL SKEIYNEGCG IFMSKEALSL ISEYRNIVYG FMLSAQNNPQ ETIRITNRES VERMKKIHQN L SIEIRQAI NLKKRDLRFE NLYFQ

UniProtKB: Uncharacterized 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

Concentration0.35 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4S4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
200.0 mMKClPotassium chloride
5.0 %C3H8O3Glycerol
1.0 mMC9H15O6PTCEP
0.1 %C24H46O11n-Dodecyl-beta-D-Maltoside
GridModel: C-flat-1.2/1.3 / Material: GRAPHENE OXIDE / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 47.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 247026
Startup modelType of model: INSILICO MODEL / In silico model: ab initio model generation / Details: cryoSPARC
Final reconstructionApplied symmetry - Point group: C8 (8 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.65 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 58694
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-8gi1:
Homo-octamer of PbuCsx28 protein

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