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- PDB-8b2x: Structure of the type I-G CRISPR effector -

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Basic information

Entry
Database: PDB / ID: 8b2x
TitleStructure of the type I-G CRISPR effector
ComponentsType I-G CRISPR Cascade large subunit CSX17
KeywordsGENE REGULATION / CRISPR / Effector / Immune system
Function / homologyCRISPR-associated protein Csx17, subtype Dpsyc / Type I-U CRISPR-associated protein Csx17
Function and homology information
Biological speciesThioalkalivibrio sulfidiphilus HL-EbGr7 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8 Å
AuthorsShangguan, Q. / Graham, S. / Sundaramoorthy, R. / White, M.F.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/S000313/1 United Kingdom
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Structure and mechanism of the type I-G CRISPR effector.
Authors: Qilin Shangguan / Shirley Graham / Ramasubramanian Sundaramoorthy / Malcolm F White /
Abstract: Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 ...Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 enzyme to facilitate target DNA destruction, thus providing immunity against mobile genetic elements. Subtypes have been classified into families A-G, with type I-G being the least well understood. Here, we report the composition, structure and function of the type I-G Cascade CRISPR effector from Thioalkalivibrio sulfidiphilus, revealing key new molecular details. The unique Csb2 subunit processes pre-crRNA, remaining bound to the 3' end of the mature crRNA, and seven Cas7 subunits form the backbone of the effector. Cas3 associates stably with the effector complex via the Cas8g subunit and is important for target DNA recognition. Structural analysis by cryo-Electron Microscopy reveals a strikingly curved backbone conformation with Cas8g spanning the belly of the structure. These biochemical and structural insights shed new light on the diversity of type I systems and open the way to applications in genome engineering.
History
DepositionSep 15, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 9, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 16, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Jul 24, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
M: Type I-G CRISPR Cascade large subunit CSX17


Theoretical massNumber of molelcules
Total (without water)79,3721
Polymers79,3721
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area32300 Å2

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Components

#1: Protein Type I-G CRISPR Cascade large subunit CSX17


Mass: 79372.023 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thioalkalivibrio sulfidiphilus HL-EbGr7 (bacteria)
Strain: HL-EbGR7 / Gene: Tgr7_2441 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43 / References: UniProt: B8GLG4

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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: Type I-G cascade complex large subunit CSX17 protein / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.007926 MDa / Experimental value: NO
Source (natural)Organism: Thioalkalivibrio sulfidiphilus HL-EbGr7 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5 / Details: 20mM Tris-HCl, 250mM NaCl, pH 7.5
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Size exclusion purified monodisperse sample in solution
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 4 K / Details: blot time 3.5, blot force 4

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Calibrated magnification: 105000 X / Nominal defocus max: 2800 nm / Nominal defocus min: 1600 nm / Calibrated defocus min: 1600 nm / Calibrated defocus max: 2800 nm / Cs: 2 mm / C2 aperture diameter: 70 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 183 K / Temperature (min): 77 K
Image recordingAverage exposure time: 3 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 15710
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansSampling size: 0.84 µm / Width: 5000 / Height: 4000

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Processing

SoftwareName: PHENIX / Version: dev_4674: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4CTF correction
7Flex-EMmodel fitting
8ISOLDEmodel fitting
9UCSF ChimeraXmodel fitting
11PHENIX1.2model refinement
12RELION4initial Euler assignment
13RELION4final Euler assignment
14RELION4classification
15RELION43D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 1000
3D reconstructionResolution: 8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 415000 / Algorithm: FOURIER SPACE / Details: Multi-body refinement. / Symmetry type: POINT
Atomic model buildingB value: 188 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation Coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0025168
ELECTRON MICROSCOPYf_angle_d0.6777013
ELECTRON MICROSCOPYf_dihedral_angle_d12.7941955
ELECTRON MICROSCOPYf_chiral_restr0.038768
ELECTRON MICROSCOPYf_plane_restr0.006930

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