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- EMDB-7050: Cryo-EM structure of Type I-F CRISPR crRNA-guided Csy surveillanc... -

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Basic information

Entry
Database: EMDB / ID: EMD-7050
TitleCryo-EM structure of Type I-F CRISPR crRNA-guided Csy surveillance complex with bound anti-CRISPR protein AcrF1
Map dataType I-F CRISPR crRNA-guided Csy surveillance complex with bound anti-CRISPR protein AcrF1
Sample
  • Complex: Type I-F CRISPR crRNA-guided Csy surveillance complex with bound anti-CRISPR protein AcrF1
    • Protein or peptide: CRISPR-associated protein Csy3
    • Protein or peptide: Anti-CRISPR protein AcrF1
    • Protein or peptide: CRISPR-associated endonuclease Cas6/Csy4
    • RNA: Pseudomonas aeruginosa strain SMC4485 CRISPR repeat sequence
KeywordsCRISPR-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
: / Anti-CRISPR protein Acr30-35/AcrF1 / 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)
Similarity search - Domain/homology
Uncharacterized protein / CRISPR-associated protein Csy3 / CRISPR-associated endonuclease Cas6/Csy4
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria) / Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria) / Pseudomonas phage JBD30 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsGuo TW / Bartesaghi A
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, 2017-
Header (metadata) releaseOct 18, 2017-
Map releaseOct 18, 2017-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.71
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 3.71
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6b46
  • Surface level: 3.71
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_7050.map.gz / Format: CCP4 / Size: 51.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationType I-F CRISPR crRNA-guided Csy surveillance complex with bound anti-CRISPR protein AcrF1
Voxel sizeX=Y=Z: 0.8254 Å
Density
Contour LevelBy AUTHOR: 3.71 / Movie #1: 3.71
Minimum - Maximum-7.799524 - 19.336175999999998
Average (Standard dev.)0.05566498 (±1.0823965)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions238238238
Spacing238238238
CellA=B=C: 196.4452 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.825399159663870.825399159663870.82539915966387
M x/y/z238238238
origin x/y/z0.0000.0000.000
length x/y/z196.445196.445196.445
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS238238238
D min/max/mean-7.80019.3360.056

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Supplemental data

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Additional map: Unsharpened map of Type I-F CRISPR crRNA-guided Csy...

Fileemd_7050_additional.map
AnnotationUnsharpened map of Type I-F CRISPR crRNA-guided Csy surveillance complex with bound anti-CRISPR protein AcrF1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Type I-F CRISPR crRNA-guided Csy surveillance complex with bound ...

EntireName: Type I-F CRISPR crRNA-guided Csy surveillance complex with bound anti-CRISPR protein AcrF1
Components
  • Complex: Type I-F CRISPR crRNA-guided Csy surveillance complex with bound anti-CRISPR protein AcrF1
    • Protein or peptide: CRISPR-associated protein Csy3
    • Protein or peptide: Anti-CRISPR protein AcrF1
    • Protein or peptide: CRISPR-associated endonuclease Cas6/Csy4
    • RNA: Pseudomonas aeruginosa strain SMC4485 CRISPR repeat sequence

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Supramolecule #1: Type I-F CRISPR crRNA-guided Csy surveillance complex with bound ...

SupramoleculeName: Type I-F CRISPR crRNA-guided Csy surveillance complex with bound anti-CRISPR protein AcrF1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 350 KDa

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Macromolecule #1: CRISPR-associated protein Csy3

MacromoleculeName: CRISPR-associated protein Csy3 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Strain: UCBPP-PA14
Molecular weightTheoretical: 37.781547 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAMSKPILST ASVLAFERKL DPSDALMSAG AWAQRDASQE WPAVTVREKS VRGTISNRLK TKDRDPAKLD ASIQSPNLQT VDVANLPSD ADTLKVRFTL RVLGGAGTPS ACNDAAYRDK LLQTVATYVN DQGFAELARR YAHNLANARF LWRNRVGAEA V EVRINHIR ...String:
MAMSKPILST ASVLAFERKL DPSDALMSAG AWAQRDASQE WPAVTVREKS VRGTISNRLK TKDRDPAKLD ASIQSPNLQT VDVANLPSD ADTLKVRFTL RVLGGAGTPS ACNDAAYRDK LLQTVATYVN DQGFAELARR YAHNLANARF LWRNRVGAEA V EVRINHIR QGEVARAWRF DALAIGLRDF KADAELDALA ELIASGLSGS GHVLLEVVAF ARIGDGQEVF PSQELILDKG DK KGQKSKT LYSVRDAAAI HSQKIGNALR TIDTWYPDED GLGPIAVEPY GSVTSQGKAY RQPKQKLDFY TLLDNWVLRD EAP AVEQQH YVIANLIRGG VFGEAEEK

UniProtKB: CRISPR-associated protein Csy3

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Macromolecule #2: Anti-CRISPR protein AcrF1

MacromoleculeName: Anti-CRISPR protein AcrF1 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas phage JBD30 (virus)
Molecular weightTheoretical: 8.96906 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSMKFIKYLS TAHLNYMNIA VYENGSKIKA RVENVVNGKS VGARDFDSTE QLESWFYGLP GSGLGRIENA MNEISRRENP

UniProtKB: Uncharacterized protein

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Macromolecule #3: CRISPR-associated endonuclease Cas6/Csy4

MacromoleculeName: CRISPR-associated endonuclease Cas6/Csy4 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Strain: UCBPP-PA14
Molecular weightTheoretical: 21.629777 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MAMDHYLDIR LRPDPEFPPA QLMSVLFGKL HQALVAQGGD RIGVSFPDLD ESRSRLGERL RIHASADDLR ALLARPWLEG LRDHLQFGE PAVVPHPTPY RQVSRVQAKS NPERLRRRLM RRHDLSEEEA RKRIPDTVAR ALDLPFVTLR SQSTGQHFRL F IRHGPLQV TAEEGGFTCY GLSKGGFVPW F

UniProtKB: CRISPR-associated endonuclease Cas6/Csy4

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Macromolecule #4: Pseudomonas aeruginosa strain SMC4485 CRISPR repeat sequence

MacromoleculeName: Pseudomonas aeruginosa strain SMC4485 CRISPR repeat sequence
type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 19.265404 KDa
SequenceString:
CUAAGAAAUU CACGGCGGGC UUGAUGUCCG CGUCUACCUG GUUCACUGCC GUGUAGGCAG

GENBANK: GENBANK: HQ326201.1

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.2
Details: 10 mM HEPES, pH 7.2, 150 mM NaCl, 2nM MgCl2, 1 mM DTT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 98 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 1975 / Average exposure time: 15.2 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: FREALIGN (ver. 9.11)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: FREALIGN (ver. 9.11)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.11) / Number images used: 55966

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