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- PDB-6b44: Cryo-EM structure of Type I-F CRISPR crRNA-guided Csy surveillanc... -
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Open data
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Basic information
Entry | Database: PDB / ID: 6b44 | ||||||
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Title | Cryo-EM structure of Type I-F CRISPR crRNA-guided Csy surveillance complex with bound target dsDNA | ||||||
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![]() | IMMUNE SYSTEM/RNA/DNA / CRISPR-Cas / IMMUNE SYSTEM-RNA-DNA complex | ||||||
Function / homology | ![]() maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / RNA binding Similarity search - Function | ||||||
Biological species | ![]() ![]() synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å | ||||||
![]() | Guo, T.W. / Bartesaghi, A. / Yang, H. / Falconieri, V. / Rao, P. / Merk, A. / Fox, T. / Earl, L. / Patel, D.J. / Subramaniam, S. | ||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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PDBx/mmCIF format | ![]() | 526.5 KB | Display | ![]() |
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PDB format | ![]() | 413.2 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 7048MC ![]() 7049C ![]() 7050C ![]() 7051C ![]() 7052C ![]() 6anvC ![]() 6anwC ![]() 6b45C ![]() 6b46C ![]() 6b47C ![]() 6b48C M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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1 |
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Components
-CRISPR-associated protein ... , 3 types, 8 molecules ABCDEFGH
#1: Protein | Mass: 49338.297 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Strain: UCBPP-PA14 / Gene: csy1, PA14_33330 / Plasmid: pRSF-Duet-SUMO / Production host: ![]() ![]() |
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#2: Protein | Mass: 36446.352 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Strain: UCBPP-PA14 / Gene: csy2, PA14_33320 / Production host: ![]() ![]() |
#3: Protein | Mass: 37781.547 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Strain: UCBPP-PA14 / Gene: csy3, csy1-3, PA14_33310 / Plasmid: pCDF-Duet / Production host: ![]() ![]() |
-DNA chain , 2 types, 2 molecules NO
#6: DNA chain | Mass: 15191.747 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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#7: DNA chain | Mass: 8216.318 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-Protein / RNA chain , 2 types, 2 molecules LM
#4: Protein | Mass: 21629.777 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Strain: UCBPP-PA14 / Gene: cas6f, csy4, PA14_33300 / Plasmid: pACY-Duet / Production host: ![]() ![]() References: UniProt: Q02MM2, Hydrolases; Acting on ester bonds |
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#5: RNA chain | Mass: 19265.404 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Type I-F CRISPR crRNA-guided Csy surveillance complex with bound target dsDNA Type: COMPLEX / Entity ID: all / Source: RECOMBINANT |
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Molecular weight | Value: .350 MDa / Experimental value: NO |
Source (natural) | Organism: ![]() ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 7.2 Details: 10 mM HEPES, pH 7.2, 150 mM NaCl, 2mM MgCl2, 1 mM DTT |
Specimen | Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: 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 |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average 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 scans | Movie frames/image: 38 |
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Processing
Software | Name: PHENIX / Version: 1.12_2829: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 39811 / Symmetry type: POINT | ||||||||||||||||||||||||
Refine LS restraints |
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