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- EMDB-7049: Cryo-EM structure of Type I-F CRISPR crRNA-guided Csy surveillanc... -

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Basic information

Entry
Database: EMDB / ID: EMD-7049
TitleCryo-EM structure of Type I-F CRISPR crRNA-guided Csy surveillance complex
Map dataType I-F CRISPR crRNA-guided Csy surveillance complex
Sample
  • Complex: Type I-F CRISPR crRNA-guided Csy surveillance complex
    • Protein or peptide: CRISPR-associated protein Csy1
    • Protein or peptide: CRISPR-associated protein Csy2
    • Protein or peptide: CRISPR-associated protein Csy3
    • Protein or peptide: CRISPR-associated endonuclease Cas6/Csy4
    • RNA: Pseudomonas aeruginosa strain SMC4485 CRISPR repeat sequence
KeywordsCRISPR-Cas / IMMUNE SYSTEM - 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
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)
Similarity search - Domain/homology
CRISPR-associated protein Csy1 / CRISPR-associated protein Csy2 / CRISPR-associated protein Csy3 / CRISPR-associated endonuclease Cas6/Csy4
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria) / Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 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: 2.9
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 2.9
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6b45
  • Surface level: 2.9
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7049.png

Downloads & links

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Map

FileDownload / File: emd_7049.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
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 238 pix.
= 199.92 Å		0.84 Å/pix.
x 238 pix.
= 199.92 Å		0.84 Å/pix.
x 238 pix.
= 199.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.9 / Movie #1: 2.9
Minimum - Maximum-7.7016706 - 10.086831999999999
Average (Standard dev.)0.013592093 (±0.7144151)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions238238238
Spacing238238238
CellA=B=C: 199.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.840.840.84
M x/y/z238238238
origin x/y/z0.0000.0000.000
length x/y/z199.920199.920199.920
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS238238238
D min/max/mean-7.70210.0870.014

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Supplemental data

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

Fileemd_7049_additional.map
AnnotationUnsharpened map of Type I-F CRISPR crRNA-guided Csy surveillance complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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

EntireName: Type I-F CRISPR crRNA-guided Csy surveillance complex
Components
  • Complex: Type I-F CRISPR crRNA-guided Csy surveillance complex
    • Protein or peptide: CRISPR-associated protein Csy1
    • Protein or peptide: CRISPR-associated protein Csy2
    • Protein or peptide: CRISPR-associated protein Csy3
    • 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

SupramoleculeName: Type I-F CRISPR crRNA-guided Csy surveillance complex / 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 Csy1

MacromoleculeName: CRISPR-associated protein Csy1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Strain: UCBPP-PA14
Molecular weightTheoretical: 49.338297 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSMTSPLPTP TWQELRQFIE SFIQERLQGK LDKLQPDEDD KRQTLLATHR REAWLADAAR RVGQLQLVTH TLKPIHPDAR GSNLHSLPQ APGQPGLAGS HELGDRLVSD VVGNAAALDV FKFLSLQYQG KNLLNWLTED SAEALQALSD NAEQAREWRQ A FIGITTVK ...String:
GSMTSPLPTP TWQELRQFIE SFIQERLQGK LDKLQPDEDD KRQTLLATHR REAWLADAAR RVGQLQLVTH TLKPIHPDAR GSNLHSLPQ APGQPGLAGS HELGDRLVSD VVGNAAALDV FKFLSLQYQG KNLLNWLTED SAEALQALSD NAEQAREWRQ A FIGITTVK GAPASHSLAK QLYFPLPGSG YHLLAPLFPT SLVHHVHALL REARFGDAAK AAREARSRQE SWPHGFSEYP NL AIQKFGG TKPQNISQLN NERRGENWLL PSLPPNWQRQ NVNAPMRHSS VFEHDFGRTP EVSRLTRTLQ RFLAKTVHNN LAI RQRRAQ LVAQICDEAL QYAARLRELE PGWSATPGCQ LHDAEQLWLD PLRAQTDETF LQRRLRGDWP AEVGNRFANW LNRA VSSDS QILGSPEAAQ WSQELSKELT MFKEILEDER D

UniProtKB: CRISPR-associated protein Csy1

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Macromolecule #2: CRISPR-associated protein Csy2

MacromoleculeName: CRISPR-associated protein Csy2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Strain: UCBPP-PA14
Molecular weightTheoretical: 36.446352 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAMSVTDPEA LLLLPRLSIQ NANAISSPLT WGFPSPGAFT GFVHALQRRV GISLDIELDG VGIVCHRFEA QISQPAGKRT KVFNLTRNP LNRDGSTAAI VEEGRAHLEV SLLLGVHGDG LDDHPAQEIA RQVQEQAGAM RLAGGSILPW CNERFPAPNA E LLMLGGSD ...String:
MAMSVTDPEA LLLLPRLSIQ NANAISSPLT WGFPSPGAFT GFVHALQRRV GISLDIELDG VGIVCHRFEA QISQPAGKRT KVFNLTRNP LNRDGSTAAI VEEGRAHLEV SLLLGVHGDG LDDHPAQEIA RQVQEQAGAM RLAGGSILPW CNERFPAPNA E LLMLGGSD EQRRKNQRRL TRRLLPGFAL VSREALLQQH LETLRTTLPE ATTLDALLDL CRINFEPPAT SSEEEASPPD AA WQVRDKP GWLVPIPAGY NALSPLYLPG EVRNARDRET PLRFVENLFG LGEWLSPHRV AALSDLLWYH HAEPDKGLYR WST PRFVEH AIA

UniProtKB: CRISPR-associated protein Csy2

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Macromolecule #3: CRISPR-associated protein Csy3

MacromoleculeName: CRISPR-associated protein Csy3 / type: protein_or_peptide / ID: 3 / 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 #4: CRISPR-associated endonuclease Cas6/Csy4

MacromoleculeName: CRISPR-associated endonuclease Cas6/Csy4 / type: protein_or_peptide / ID: 4 / 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 #5: Pseudomonas aeruginosa strain SMC4485 CRISPR repeat sequence

MacromoleculeName: Pseudomonas aeruginosa strain SMC4485 CRISPR repeat sequence
type: rna / ID: 5 / 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
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 1784 / Average exposure time: 15.2 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.11) / Number images used: 31488
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: FREALIGN (ver. 9.11)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: FREALIGN (ver. 9.11)

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