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- PDB-6b46: Cryo-EM structure of Type I-F CRISPR crRNA-guided Csy surveillanc... -

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Basic information

Entry
Database: PDB / ID: 6b46
TitleCryo-EM structure of Type I-F CRISPR crRNA-guided Csy surveillance complex with bound anti-CRISPR protein AcrF1
Components
  • Anti-CRISPR protein AcrF1
  • CRISPR-associated endonuclease Cas6/Csy4
  • CRISPR-associated protein Csy3
  • Pseudomonas aeruginosa strain SMC4485 CRISPR repeat sequence
KeywordsIMMUNE SYSTEM/HYDROLASE/RNA / CRISPR-Cas / IMMUNE SYSTEM-HYDROLASE-RNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / RNA binding
Similarity search - Function
Viral Envelope Glycoprotein; domain 2 - #30 / : / Anti-CRISPR protein Acr30-35/AcrF1 / CRISPR-associated endoribonuclease Cas6/Csy4 / Viral Envelope Glycoprotein; domain 2 / CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST / CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST superfamily / CRISPR-associated protein (Cas_Csy4) / CRISPR-associated protein Csy3 / CRISPR-associated protein (Cas_Csy3) ...Viral Envelope Glycoprotein; domain 2 - #30 / : / Anti-CRISPR protein Acr30-35/AcrF1 / CRISPR-associated endoribonuclease Cas6/Csy4 / Viral Envelope Glycoprotein; domain 2 / CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST / CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST superfamily / CRISPR-associated protein (Cas_Csy4) / CRISPR-associated protein Csy3 / CRISPR-associated protein (Cas_Csy3) / Alpha-Beta Plaits / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
: / RNA / RNA (> 10) / Uncharacterized protein / CRISPR-associated protein Csy3 / CRISPR-associated endonuclease Cas6/Csy4
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
Pseudomonas phage JBD30 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsGuo, T.W. / Bartesaghi, A. / Yang, H. / Falconieri, V. / Rao, P. / Merk, A. / Fox, T. / Earl, L. / Patel, D.J. / Subramaniam, S.
CitationJournal: Cell / Year: 2017
Title: Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex.
Authors: Tai Wei Guo / Alberto Bartesaghi / Hui Yang / Veronica Falconieri / Prashant Rao / Alan Merk / Edward T Eng / Ashleigh M Raczkowski / Tara Fox / Lesley A Earl / Dinshaw J Patel / Sriram Subramaniam /
Abstract: Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided ...Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition.
History
DepositionSep 25, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 18, 2017Provider: repository / Type: Initial release
Revision 1.1Mar 13, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_related
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_related.content_type

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Structure visualization

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  • EMDB-7050
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Assembly

Deposited unit
C: CRISPR-associated protein Csy3
D: CRISPR-associated protein Csy3
E: CRISPR-associated protein Csy3
F: CRISPR-associated protein Csy3
G: CRISPR-associated protein Csy3
H: CRISPR-associated protein Csy3
I: Anti-CRISPR protein AcrF1
J: Anti-CRISPR protein AcrF1
L: CRISPR-associated endonuclease Cas6/Csy4
M: Pseudomonas aeruginosa strain SMC4485 CRISPR repeat sequence


Theoretical massNumber of molelcules
Total (without water)285,52310
Polymers285,52310
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
CRISPR-associated protein Csy3


Mass: 37781.547 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Strain: UCBPP-PA14 / Gene: csy3, csy1-3, PA14_33310 / Plasmid: pCDF-Duet / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q02MM1
#2: Protein Anti-CRISPR protein AcrF1


Mass: 8969.060 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas phage JBD30 (virus) / Gene: JBD30_035 / Plasmid: pRSF-Duet-SUMO / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: L7P7M1
#3: Protein CRISPR-associated endonuclease Cas6/Csy4


Mass: 21629.777 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Strain: UCBPP-PA14 / Gene: cas6f, csy4, PA14_33300 / Plasmid: pACY-Duet / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)
References: UniProt: Q02MM2, Hydrolases; Acting on ester bonds
#4: RNA chain Pseudomonas aeruginosa strain SMC4485 CRISPR repeat sequence


Mass: 19265.404 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Plasmid: pACY-Duet / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: GenBank: 313291946

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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-F CRISPR crRNA-guided Csy surveillance complex with bound anti-CRISPR protein AcrF1
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.350 MDa / Experimental value: NO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.2
Details: 10 mM HEPES, pH 7.2, 150 mM NaCl, 2nM MgCl2, 1 mM DTT
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 98 %

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 15.2 sec. / Electron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of real images: 1975
Image scansMovie frames/image: 38

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
4CTFFIND4CTF correction
10FREALIGN9.11initial Euler assignment
11FREALIGN9.11final Euler assignment
12FREALIGN9.11classification
13FREALIGN9.113D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 55966 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00618788
ELECTRON MICROSCOPYf_angle_d0.84325695
ELECTRON MICROSCOPYf_dihedral_angle_d8.09310876
ELECTRON MICROSCOPYf_chiral_restr0.0542817
ELECTRON MICROSCOPYf_plane_restr0.0063220

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