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- PDB-9fcv: Cas nuclease-CRISPR (cr)RNA ribonucleoprotein (RNP) complex -

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Basic information

Entry
Database: PDB / ID: 9fcv
TitleCas nuclease-CRISPR (cr)RNA ribonucleoprotein (RNP) complex
Components
  • Phage head-tail adaptor
  • crRNA
KeywordsRNA BINDING PROTEIN / AcrVIB1 / anti-CRISPR protein / Cas13b
Function / homologyRNA / RNA (> 10) / Phage head-tail adaptor
Function and homology information
Biological speciesSegatella buccae (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.09 Å
AuthorsSchmelz, S. / Lukat, P. / Blankenfeldt, W.
Funding support1items
OrganizationGrant numberCountry
Not funded
Citation
Journal: 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.
#1: Journal: Acta Crystallogr D Struct Biol / Year: 2019
Title: Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix.
Authors: Dorothee Liebschner / Pavel V Afonine / Matthew L Baker / Gábor Bunkóczi / Vincent B Chen / Tristan I Croll / Bradley Hintze / Li Wei Hung / Swati Jain / Airlie J McCoy / Nigel W Moriarty ...Authors: Dorothee Liebschner / Pavel V Afonine / Matthew L Baker / Gábor Bunkóczi / Vincent B Chen / Tristan I Croll / Bradley Hintze / Li Wei Hung / Swati Jain / Airlie J McCoy / Nigel W Moriarty / Robert D Oeffner / Billy K Poon / Michael G Prisant / Randy J Read / Jane S Richardson / David C Richardson / Massimo D Sammito / Oleg V Sobolev / Duncan H Stockwell / Thomas C Terwilliger / Alexandre G Urzhumtsev / Lizbeth L Videau / Christopher J Williams / Paul D Adams /
Abstract: Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological ...Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological processes and to develop new therapeutics against diseases. The overall structure-solution workflow is similar for these techniques, but nuances exist because the properties of the reduced experimental data are different. Software tools for structure determination should therefore be tailored for each method. Phenix is a comprehensive software package for macromolecular structure determination that handles data from any of these techniques. Tasks performed with Phenix include data-quality assessment, map improvement, model building, the validation/rebuilding/refinement cycle and deposition. Each tool caters to the type of experimental data. The design of Phenix emphasizes the automation of procedures, where possible, to minimize repetitive and time-consuming manual tasks, while default parameters are chosen to encourage best practice. A graphical user interface provides access to many command-line features of Phenix and streamlines the transition between programs, project tracking and re-running of previous tasks.
History
DepositionMay 16, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 12, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Phage head-tail adaptor
B: crRNA


Theoretical massNumber of molelcules
Total (without water)160,1812
Polymers160,1812
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area7980 Å2
ΔGint-71 kcal/mol
Surface area59140 Å2

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Components

#1: Protein Phage head-tail adaptor


Mass: 134285.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Segatella buccae (bacteria) / Gene: HMPREF6485_0083 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: E6K398
#2: RNA chain crRNA


Mass: 25895.355 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: unprocessed crRNA / Source: (synth.) synthetic construct (others)
Has protein modificationN

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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: Cas13b (cr)RNA complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Segatella buccae (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7 / Details: 20mM HEPES pH7.0 120 mM NaCl 0.5 mM DTT
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: for complex formation PubCas13b (1mg/ml) was mixed with crRNA in a ratio of 1:0.9 and kept on ice for 10 min prior vitrification.
Specimen supportDetails: 15 mA / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 40 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of real images: 3778
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv4.4.0particle selection
2EPU3.3.1.5184image acquisition
4cryoSPARCv4.4.0CTF correction
7UCSF ChimeraX1.71model fitting
8Coot0.9.6model fitting
10PHENIX1.21_5207model refinement
11cryoSPARCv4.4.0initial Euler assignment
12cryoSPARCv4.4.0final Euler assignment
14cryoSPARCv4.4.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.09 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 356629 / Symmetry type: POINT
Atomic model buildingPDB-ID: 6DTD

6dtd


Accession code: 6DTD / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 62.4 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.001810136
ELECTRON MICROSCOPYf_angle_d0.404413852
ELECTRON MICROSCOPYf_chiral_restr0.03281509
ELECTRON MICROSCOPYf_plane_restr0.00291619
ELECTRON MICROSCOPYf_dihedral_angle_d13.99171772

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