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- EMDB-51513: PbuCas13b Helical-2 domain in complex with anti-CRISPR protein (A... -

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Basic information

Entry
Database: EMDB / ID: EMD-51513
TitlePbuCas13b Helical-2 domain in complex with anti-CRISPR protein (AcrVIB1)
Map data
Sample
  • Complex: PbuCas13b Helical-2 domain in complex with AcrVIB1
    • Protein or peptide: PbuCas13b Helical-2 domain
    • Protein or peptide: AcrVIB1
KeywordsAcrVIB1 / anti-CRISPR protein / Cas13b / RNA BINDING PROTEIN
Biological speciesSegatella buccae (bacteria) / Riemerella anatipestifer (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.11 Å
AuthorsSchmelz S / Lukat P / Blankenfeldt W
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Mol Cell / Year: 2025
Title: AcrVIB1 inhibits CRISPR-Cas13b immunity by promoting unproductive crRNA binding accessible to RNase attack.
Authors: Katharina G Wandera / Stefan Schmelz / Angela Migur / Anuja Kibe / Peer Lukat / Tatjana Achmedov / Neva Caliskan / Wulf Blankenfeldt / Chase L Beisel /
Abstract: Anti-CRISPR proteins (Acrs) inhibit CRISPR-Cas immune defenses, with almost all known Acrs acting on the Cas nuclease-CRISPR (cr)RNA ribonucleoprotein (RNP) complex. Here, we show that AcrVIB1 from ...Anti-CRISPR proteins (Acrs) inhibit CRISPR-Cas immune defenses, with almost all known Acrs acting on the Cas nuclease-CRISPR (cr)RNA ribonucleoprotein (RNP) complex. Here, we show that AcrVIB1 from Riemerella anatipestifer, the only known Acr against Cas13b, principally acts upstream of RNP complex formation by promoting unproductive crRNA binding followed by crRNA degradation. AcrVIB1 tightly binds to Cas13b but not to the Cas13b-crRNA complex, resulting in enhanced rather than blocked crRNA binding. However, the more tightly bound crRNA does not undergo processing and fails to activate collateral RNA cleavage even with target RNA. The bound crRNA is also accessible to RNases, leading to crRNA turnover in vivo even in the presence of Cas13b. Finally, cryoelectron microscopy (cryo-EM) structures reveal that AcrVIB1 binds a helical domain of Cas13b responsible for securing the crRNA, keeping the domain untethered. These findings reveal an Acr that converts an effector nuclease into a crRNA sink to suppress CRISPR-Cas defense.
History
DepositionSep 10, 2024-
Header (metadata) releaseFeb 12, 2025-
Map releaseFeb 12, 2025-
UpdateApr 2, 2025-
Current statusApr 2, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51513.png

Downloads & links

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Map

FileDownload / File: emd_51513.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.91 Å/pix.
x 150 pix.
= 136.5 Å		0.91 Å/pix.
x 150 pix.
= 136.5 Å		0.91 Å/pix.
x 150 pix.
= 136.5 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.91 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.11
Minimum - Maximum-0.3681377 - 0.5439884
Average (Standard dev.)0.00037651608 (±0.024136078)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 136.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_51513_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_51513_half_map_2.map
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Sample components

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Entire : PbuCas13b Helical-2 domain in complex with AcrVIB1

EntireName: PbuCas13b Helical-2 domain in complex with AcrVIB1
Components
  • Complex: PbuCas13b Helical-2 domain in complex with AcrVIB1
    • Protein or peptide: PbuCas13b Helical-2 domain
    • Protein or peptide: AcrVIB1

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Supramolecule #1: PbuCas13b Helical-2 domain in complex with AcrVIB1

SupramoleculeName: PbuCas13b Helical-2 domain in complex with AcrVIB1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Segatella buccae (bacteria)

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Macromolecule #1: PbuCas13b Helical-2 domain

MacromoleculeName: PbuCas13b Helical-2 domain / type: protein_or_peptide / ID: 1 / Details: PbuCas13b Helical-2 domain (residues 579 - 747) / Enantiomer: DEXTRO
Source (natural)Organism: Segatella buccae (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GGGRRCKDMG KEAE RKIGE MIDDTQRRLD LLCKQTNQKI RIGKRNAGLL KSGKI ADWL VNDMMRFQPV QKDQNNIPIN NSKANSTEYR MLQRAL ALF GSENFRLKAY FNQMNLVGND NPHPFLAETQ WEHQTNI LS FYRNYLEARK KYLKGLKPQN WKQYQHFLIL KVQKCNR

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Macromolecule #2: AcrVIB1

MacromoleculeName: AcrVIB1 / type: protein_or_peptide / ID: 2 / Enantiomer: DEXTRO
Source (natural)Organism: Riemerella anatipestifer (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GGGRMKDLDL SKLKGEEIAQ WLLNNKKA T AIQLSSERTD TDDGFMHILV HKDEYVEIIY SYLKIDEDD VMQNFTIYSK RWGNIDNSYF ELQTFEGEIF TGESDKILCG VLSLGDLTT LK

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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

Concentration0.1 mg/mL
BufferpH: 7 / Details: 20mM HEPES pH7.5 150 mM NaCl
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 100 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 4.0 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV
DetailsThe SEC co-purified complex of PbuCas13b_ Helical-2:AcrVIB1 was diluted to 0.1 mg/ml and used for grid preparation

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Electron microscopy

MicroscopeTFS GLACIOS
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
SoftwareName: EPU (ver. 3.3.1.5184)
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Number real images: 10707 / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.6 µm
Sample stageCooling holder cryogen: NITROGEN

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Image processing

Startup modelType of model: INSILICO MODEL / Details: insilco model generated with cryoSPARC in C1
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.11 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.5.3) / Number images used: 1078020
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.5.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.5.3)

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