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- PDB-5uz9: Cryo EM structure of anti-CRISPRs, AcrF1 and AcrF2, bound to type... -

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Basic information

Entry
Database: PDB / ID: 5uz9
TitleCryo EM structure of anti-CRISPRs, AcrF1 and AcrF2, bound to type I-F crRNA-guided CRISPR surveillance complex
Components
  • (Anti-CRISPR protein ...) x 2
  • (CRISPR-associated protein ...) x 3
  • CRISPR RNA (60-MER)
  • CRISPR-associated endonuclease Cas6/Csy4
KeywordsIMMUNE SYSTEM/RNA / CRISPR RNA-recognition-motif (RRM) / pseudo-helical / Type 1-F CRISPR / IMMUNE SYSTEM-RNA complex
Function / homology
Function and homology information


symbiont-mediated suppression of host CRISPR-cas system / maintenance of CRISPR repeat elements / defense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / RNA binding
Similarity search - Function
Viral Envelope Glycoprotein; domain 2 - #30 / CRISPR-associated endoribonuclease Cas6/Csy4 / : / Anti-CRISPR protein Acr30-35/AcrF1 / 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) / Viral Envelope Glycoprotein; domain 2 ...Viral Envelope Glycoprotein; domain 2 - #30 / CRISPR-associated endoribonuclease Cas6/Csy4 / : / Anti-CRISPR protein Acr30-35/AcrF1 / 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) / Viral Envelope Glycoprotein; domain 2 / 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
: / RNA / RNA (> 10) / Uncharacterized protein / CRISPR-associated protein Csy1 / CRISPR-associated protein Csy2 / CRISPR-associated protein Csy3 / CRISPR-associated endonuclease Cas6/Csy4 / Anti-CRISPR protein 30
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
Pseudomonas phage JBD30 (virus)
Pseudomonas phage D3112 (virus)
Pseudomonas aeruginosa UCBPP-PA14 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsChowdhury, S. / Carter, J. / Rollins, M.F. / Jackson, R.N. / Hoffmann, C. / Nosaka, L. / Bondy-Denomy, J. / Maxwell, K.L. / Davidson, A.R. / Fischer, E.R. ...Chowdhury, S. / Carter, J. / Rollins, M.F. / Jackson, R.N. / Hoffmann, C. / Nosaka, L. / Bondy-Denomy, J. / Maxwell, K.L. / Davidson, A.R. / Fischer, E.R. / Lander, G.C. / Wiedenheft, B.
Funding support United States, Canada, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P20GM103500, P30GM110732-03, R01GM110270, R01GM108888, P20GM103474, F32GM108436, DP2EB020402, DP5OD021344 United States
National Science Foundation (NSF, United States)EPS-110134 United States
Canadian Institutes of Health Research (CIHR)MOP-130482, MOP-136845 Canada
CitationJournal: Cell / Year: 2017
Title: Structure Reveals Mechanisms of Viral Suppressors that Intercept a CRISPR RNA-Guided Surveillance Complex.
Authors: Saikat Chowdhury / Joshua Carter / MaryClare F Rollins / Sarah M Golden / Ryan N Jackson / Connor Hoffmann / Lyn'Al Nosaka / Joseph Bondy-Denomy / Karen L Maxwell / Alan R Davidson / ...Authors: Saikat Chowdhury / Joshua Carter / MaryClare F Rollins / Sarah M Golden / Ryan N Jackson / Connor Hoffmann / Lyn'Al Nosaka / Joseph Bondy-Denomy / Karen L Maxwell / Alan R Davidson / Elizabeth R Fischer / Gabriel C Lander / Blake Wiedenheft /
Abstract: Genetic conflict between viruses and their hosts drives evolution and genetic innovation. Prokaryotes evolved CRISPR-mediated adaptive immune systems for protection from viral infection, and viruses ...Genetic conflict between viruses and their hosts drives evolution and genetic innovation. Prokaryotes evolved CRISPR-mediated adaptive immune systems for protection from viral infection, and viruses have evolved diverse anti-CRISPR (Acr) proteins that subvert these immune systems. The adaptive immune system in Pseudomonas aeruginosa (type I-F) relies on a 350 kDa CRISPR RNA (crRNA)-guided surveillance complex (Csy complex) to bind foreign DNA and recruit a trans-acting nuclease for target degradation. Here, we report the cryo-electron microscopy (cryo-EM) structure of the Csy complex bound to two different Acr proteins, AcrF1 and AcrF2, at an average resolution of 3.4 Å. The structure explains the molecular mechanism for immune system suppression, and structure-guided mutations show that the Acr proteins bind to residues essential for crRNA-mediated detection of DNA. Collectively, these data provide a snapshot of an ongoing molecular arms race between viral suppressors and the immune system they target.
History
DepositionFeb 25, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 26, 2017Provider: repository / Type: Initial release
Revision 1.1Sep 27, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.2Jul 18, 2018Group: Data collection / Experimental preparation / Category: em_sample_support / em_software
Item: _em_sample_support.grid_type / _em_software.image_processing_id / _em_software.name
Revision 2.0Nov 6, 2019Group: Atomic model / Data collection / Other / Category: atom_site / atom_sites / cell
Item: _atom_site.occupancy / _atom_sites.fract_transf_matrix[1][1] ..._atom_site.occupancy / _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB / _cell.length_a / _cell.length_b / _cell.length_c
Revision 2.1Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 2.2Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-8624
  • Imaged by UCSF Chimera
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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
I: Anti-CRISPR protein Acr30-35
J: Anti-CRISPR protein Acr30-35
K: Anti-CRISPR protein 30
L: CRISPR-associated endonuclease Cas6/Csy4
M: CRISPR RNA (60-MER)


Theoretical massNumber of molelcules
Total (without water)379,21613
Polymers379,21613
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, This RNA protein complex forms a stable assembly and has been observed as a single peak in size exclusion chromatography and also verified by electron microscopy.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Number of models5

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Components

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

#1: Protein CRISPR-associated protein Csy1 / Cas8F


Mass: 49194.168 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 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q02ML9
#2: Protein CRISPR-associated protein Csy2 / Cas5F


Mass: 36244.074 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 BL21(DE3) (bacteria) / References: UniProt: Q02MM0
#3: Protein
CRISPR-associated protein Csy3 / Cas7F


Mass: 37448.078 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 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q02MM1

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Anti-CRISPR protein ... , 2 types, 3 molecules IJK

#4: Protein Anti-CRISPR protein Acr30-35 / / AcrF1


Mass: 8693.734 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas phage JBD30 (virus) / Gene: JBD30_035 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: L7P7M1
#5: Protein Anti-CRISPR protein 30 / / Gene product 30 / gp30 / AcrF2


Mass: 10760.481 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas phage D3112 (virus) / Gene: orf30 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q6TM72

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Protein / RNA chain , 2 types, 2 molecules LM

#6: Protein CRISPR-associated endonuclease Cas6/Csy4 / Cas6F


Mass: 21691.848 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 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q02MM2, Hydrolases; Acting on ester bonds
#7: RNA chain CRISPR RNA (60-MER)


Mass: 19249.404 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Pseudomonas aeruginosa UCBPP-PA14 (bacteria)
References: GenBank: 115583796

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Details

Sequence detailsThe sequence was taken from the PDB entry 4AL5 used for rigid body fitting.

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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: Anti-CRISPRs AcrF1 and AcrF2 bound P. aeruginosa crRNA-guided CRISPR surveillance(Csy)complex.
Type: COMPLEX
Details: Cas5F, Cas6F, Cas7F, Cas8F and CRISPR RNA (crRNA) forms the Csy surveillance complex. Cas5F, Cas8F and 5'crRNA handle forms the "tail" of the complex, and 3' crRNA stem-loop along with Cas6F ...Details: Cas5F, Cas6F, Cas7F, Cas8F and CRISPR RNA (crRNA) forms the Csy surveillance complex. Cas5F, Cas8F and 5'crRNA handle forms the "tail" of the complex, and 3' crRNA stem-loop along with Cas6F forms the "head". Six copies Cas7F along with crRNA spacer forms the "core" of the complex.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.45 MDa / Experimental value: NO
Source (natural)Organism: Pseudomonas aeruginosa UCBPP-PA14 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5 / Details: Buffer was filtered before use.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
2100 mMPotassium ChlorideKCl1
31 mMTCEPC9H15O6P1
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was free from any aggregation and monodisperse.
Specimen supportDetails: Holey grid was coated with an amorphous carbon film.
Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/2
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 98 % / Chamber temperature: 277 K
Details: Sample was applied to poly-L-lysine hydrobromide pre-treated amorphous carbon film coated over a holey grid. Excess sample was blotted with Whatman-1 filter paper and plunge frozen. Manual ...Details: Sample was applied to poly-L-lysine hydrobromide pre-treated amorphous carbon film coated over a holey grid. Excess sample was blotted with Whatman-1 filter paper and plunge frozen. Manual plunge freezing was performed in a cold room.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Objective astigmatism was corrected at nominal magnification of 29000X, using Thon rings visualized with a K2 camera.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 29000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 79 K / Temperature (min): 77 K
Image recordingAverage exposure time: 6 sec. / Electron dose: 46 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 2261
Details: Images were collected in movie mode using Leginon automated data acquisition software. Movie frames were aligned using MotionCorr program.
Image scansSampling size: 15 µm / Width: 3838 / Height: 3710 / Movie frames/image: 30 / Used frames/image: 1-30

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Processing

SoftwareName: PHENIX / Version: dev_2621: / Classification: refinement
EM software
IDNameVersionCategoryDetailsFitting-ID
1FindEMparticle selectionImplemented within Appion processing pipeline
2Leginonimage acquisitionAutomated data acquisition
4CTFFIND33CTF correctionCTF parameters were calculated using the aligned micrographs
7Coot0.8.7model fittingAb initio model buiding1
9RELION1.4initial Euler assignment
10RELION1.4final Euler assignment
11RELION1.4classification
12RELION1.43D reconstruction
13PHENIX1.11.1model refinementReal space refinement1
21UCSF Chimera1.12model fittingInitially rigid body fitted to a low resolution map2
26PHENIX1.11.1model refinementRigid body refinement2
34UCSF Chimera1.12model fittingInitially rigid body fitted into the map3
39PHENIX1.11.1model refinementReal Space refinement3
Image processingDetails: Raw movie frames were aligned using MotionCorr.
CTF correctionDetails: Particles were ctf-corrected during 2D and 3D processing.
Type: NONE
Particle selectionNum. of particles selected: 199348
Details: Template based particle picking was done using FindEM program implemented in Appion processing package.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51212 / Algorithm: FOURIER SPACE
Details: Per-frame dose weighting was performed using the RELION particle polishing method. A B-factor of -71 square angstrom was applied to sharpen the final map. Signal subtracted focused ...Details: Per-frame dose weighting was performed using the RELION particle polishing method. A B-factor of -71 square angstrom was applied to sharpen the final map. Signal subtracted focused classification and refinement was performed for the "tail" region of the complex. This lead to a 4 angstrom (Gold standard FSC 0.143) map.
Num. of class averages: 1 / Symmetry type: POINT
Atomic model building
IDProtocolSpaceDetails
1AB INITIO MODELREALTop scoring five models out of two hundred models generated using Rosetta were refined using Phenix and deposited as an ensemble atomic model for the final EM map.
2RIGID BODY FITREALThe docked Cas6F structure in the "head" region of the complex was reduced to C-alpha backbone.
3RIGID BODY FITREALInitially two copies were fitted using Chimera and then backbones and side chains were fixed using Coot, followed by real space refinement in Phenix.
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00624948
ELECTRON MICROSCOPYf_angle_d0.94234087
ELECTRON MICROSCOPYf_dihedral_angle_d6.73114566
ELECTRON MICROSCOPYf_chiral_restr0.053750
ELECTRON MICROSCOPYf_plane_restr0.0064350

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