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- PDB-6b44: Cryo-EM structure of Type I-F CRISPR crRNA-guided Csy surveillanc... -

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Basic information

Entry
Database: PDB / ID: 6b44
TitleCryo-EM structure of Type I-F CRISPR crRNA-guided Csy surveillance complex with bound target dsDNA
Components
  • (CRISPR-associated protein ...) x 3
  • CRISPR-associated endonuclease Cas6/Csy4
  • Non-target DNA strand (5'-D(P*CP*AP*GP*TP*CP*AP*TP*CP*AP*CP*CP*AP*A)-3')
  • Pseudomonas aeruginosa strain SMC4485 CRISPR repeat sequence
  • Target DNA strand (41-MER)
KeywordsIMMUNE SYSTEM/RNA/DNA / CRISPR-Cas / IMMUNE SYSTEM-RNA-DNA 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
CRISPR-associated endoribonuclease Cas6/Csy4 / CRISPR-associated protein Csy1 / CRISPR-associated protein (Cas_Csy1) / 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 Csy2 / CRISPR-associated protein (Cas_Csy2) / CRISPR-associated protein Csy3 / CRISPR-associated protein (Cas_Csy3) ...CRISPR-associated endoribonuclease Cas6/Csy4 / CRISPR-associated protein Csy1 / CRISPR-associated protein (Cas_Csy1) / 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 Csy2 / CRISPR-associated protein (Cas_Csy2) / CRISPR-associated protein Csy3 / CRISPR-associated protein (Cas_Csy3) / Alpha-Beta Plaits / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
: / DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated protein Csy1 / CRISPR-associated protein Csy2 / CRISPR-associated protein Csy3 / CRISPR-associated endonuclease Cas6/Csy4
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsGuo, T.W. / Bartesaghi, A. / Yang, H. / Falconieri, V. / Rao, P. / Merk, A. / Fox, T. / Earl, L. / Patel, D.J. / Subramaniam, S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 United States
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 1.1Dec 6, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Mar 13, 2024Group: Data collection / Database references / Source and taxonomy
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_related / pdbx_entity_src_syn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_related.content_type / _pdbx_entity_src_syn.ncbi_taxonomy_id / _pdbx_entity_src_syn.organism_scientific

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
A: CRISPR-associated protein Csy1
B: CRISPR-associated protein Csy2
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
L: CRISPR-associated endonuclease Cas6/Csy4
M: Pseudomonas aeruginosa strain SMC4485 CRISPR repeat sequence
N: Target DNA strand (41-MER)
O: Non-target DNA strand (5'-D(P*CP*AP*GP*TP*CP*AP*TP*CP*AP*CP*CP*AP*A)-3')


Theoretical massNumber of molelcules
Total (without water)376,77712
Polymers376,77712
Non-polymers00
Water00
1


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

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Components

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CRISPR-associated protein ... , 3 types, 8 molecules ABCDEFGH

#1: Protein CRISPR-associated protein Csy1


Mass: 49338.297 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: csy1, PA14_33330 / Plasmid: pRSF-Duet-SUMO / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q02ML9
#2: Protein CRISPR-associated protein Csy2


Mass: 36446.352 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: csy2, PA14_33320 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q02MM0
#3: 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

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DNA chain , 2 types, 2 molecules NO

#6: DNA chain Target DNA strand (41-MER)


Mass: 15191.747 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#7: DNA chain Non-target DNA strand (5'-D(P*CP*AP*GP*TP*CP*AP*TP*CP*AP*CP*CP*AP*A)-3')


Mass: 8216.318 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain , 2 types, 2 molecules LM

#4: 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
#5: 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 target dsDNA
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: .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, 2mM 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: 3064
Image scansMovie frames/image: 38

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
4CTFFIND4CTF correction
9FREALIGN9.11initial Euler assignment
10FREALIGN9.11final Euler assignment
11FREALIGN9.11classification
12FREALIGN9.113D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 39811 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00623327
ELECTRON MICROSCOPYf_angle_d0.96331964
ELECTRON MICROSCOPYf_dihedral_angle_d10.86312926
ELECTRON MICROSCOPYf_chiral_restr0.0583329
ELECTRON MICROSCOPYf_plane_restr0.0073961

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