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- EMDB-8478: CRISPR RNA-guided surveillance complex -

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Basic information

Entry
Database: EMDB / ID: 8478
TitleCRISPR RNA-guided surveillance complex
Map data3.3 angstrom EM map of Type 1-E Cascade in the full R-loop state from Thermobifida fusca
SampleCRISPR RNA-guided surveillance complex:
(CRISPR-associated protein, ...) x 4 / Cse2Carbon diselenide / (nucleic-acidNucleic acid) x 3
Function / homologyCse2 superfamily / CRISPR-associated protein Cse2 (CRISPR_cse2) / CRISPR-associated protein, CasD / CRISPR-associated protein, CT1975 / CRISPR-associated protein Cse3 / CRISPR-associated protein Cse1 / CRISPR-associated protein Cse2 / CRISPR-associated protein Cas5, N-terminal / CRISPR-associated protein, Cas5 / CRISPR-associated protein (Cas_Cas5) ...Cse2 superfamily / CRISPR-associated protein Cse2 (CRISPR_cse2) / CRISPR-associated protein, CasD / CRISPR-associated protein, CT1975 / CRISPR-associated protein Cse3 / CRISPR-associated protein Cse1 / CRISPR-associated protein Cse2 / CRISPR-associated protein Cas5, N-terminal / CRISPR-associated protein, Cas5 / CRISPR-associated protein (Cas_Cas5) / CRISPR associated protein / CT1975-like protein / CRISPR-associated protein Cse1 (CRISPR_cse1) / maintenance of CRISPR repeat elements / defense response to virus / RNA binding / identical protein binding / CRISPR-associated protein, Cse1 family / Uncharacterized protein / CRISPR-associated protein, Cse4 family / CRISPR-associated protein, Cas5e family / CRISPR-associated protein, Cse3 family
Function and homology information
SourceThermobifida fusca (strain YX) (bacteria) / Thermobifida fusca YX (bacteria)
Methodsingle particle reconstruction / cryo EM / 3.3 Å resolution
AuthorsXiao Y / Luo M / Hayes RP / Kim J / Ng S / Ding F / Liao M / Ke A
CitationJournal: Cell / Year: 2017
Title: Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System.
Authors: Yibei Xiao / Min Luo / Robert P Hayes / Jonathan Kim / Sherwin Ng / Fang Ding / Maofu Liao / Ailong Ke
Abstract: Type I CRISPR systems feature a sequential dsDNA target searching and degradation process, by crRNA-displaying Cascade and nuclease-helicase fusion enzyme Cas3, respectively. Here we present two ...Type I CRISPR systems feature a sequential dsDNA target searching and degradation process, by crRNA-displaying Cascade and nuclease-helicase fusion enzyme Cas3, respectively. Here we present two cryo-EM snapshots of the Thermobifida fusca type I-E Cascade: (1) unwinding 11 bp of dsDNA at the seed-sequence region to scout for sequence complementarity, and (2) further unwinding of the entire protospacer to form a full R-loop. These structures provide the much-needed temporal and spatial resolution to resolve key mechanistic steps leading to Cas3 recruitment. In the early steps, PAM recognition causes severe DNA bending, leading to spontaneous DNA unwinding to form a seed-bubble. The full R-loop formation triggers conformational changes in Cascade, licensing Cas3 to bind. The same process also generates a bulge in the non-target DNA strand, enabling its handover to Cas3 for cleavage. The combination of both negative and positive checkpoints ensures stringent yet efficient target degradation in type I CRISPR-Cas systems.
Validation ReportPDB-ID: 5u0a

SummaryFull reportAbout validation report
DateDeposition: Nov 23, 2016 / Header (metadata) release: Feb 22, 2017 / Map release: Aug 9, 2017 / Last update: Jul 18, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.07
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-5u0a
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_8478.map.gz (map file in CCP4 format, 67109 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
256 pix
1.23 Å/pix.
= 314.88 Å
256 pix
1.23 Å/pix.
= 314.88 Å
256 pix
1.23 Å/pix.
= 314.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.23 Å
Density
Contour Level:0.07 (by author), 0.07 (movie #1):
Minimum - Maximum-0.29755154 - 0.47882083
Average (Standard dev.)0.000029935774 (0.017093083)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions256256256
Origin0.0.0.
Limit255.255.255.
Spacing256256256
CellA=B=C: 314.88 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.231.231.23
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z314.880314.880314.880
α/β/γ90.00090.00090.000
start NX/NY/NZ-34-26-36
NX/NY/NZ528549
MAP C/R/S123
start NC/NR/NS-25600
NC/NR/NS256256256
D min/max/mean-0.2980.4790.000

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Supplemental data

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Sample components

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Entire CRISPR RNA-guided surveillance complex

EntireName: CRISPR RNA-guided surveillance complex / Number of components: 9

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Component #1: protein, CRISPR RNA-guided surveillance complex

ProteinName: CRISPR RNA-guided surveillance complex / Recombinant expression: No
SourceSpecies: Thermobifida fusca (strain YX) (bacteria)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria) / Vector: pcdf

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Component #2: protein, CRISPR-associated protein, Cse3 family

ProteinName: CRISPR-associated protein, Cse3 family / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 26.327938 kDa
SourceSpecies: Thermobifida fusca (strain YX) (bacteria) / Strain: YX
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #3: protein, CRISPR-associated protein, Cse1 family

ProteinName: CRISPR-associated protein, Cse1 family / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 61.433297 kDa
SourceSpecies: Thermobifida fusca (strain YX) (bacteria) / Strain: YX
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #4: protein, CRISPR-associated protein, Cse4 family

ProteinName: CRISPR-associated protein, Cse4 family / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 41.043043 kDa
SourceSpecies: Thermobifida fusca (strain YX) (bacteria) / Strain: YX
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #5: protein, Cse2

ProteinName: Cse2Carbon diselenide / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 27.446613 kDa
SourceSpecies: Thermobifida fusca (strain YX) (bacteria) / Strain: YX
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #6: nucleic-acid, crRNA

Nucleic-acidName: crRNA / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
AUGGACCGCC AGUGAUAAGU GGAAUGCCAU GUGGGCUGUC GUGAGCCCCA CGCACGUGGG G
MassTheoretical: 19.790793 kDa
SourceSpecies: Thermobifida fusca YX (bacteria)

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Component #7: nucleic-acid, Target Strand

Nucleic-acidName: Target Strand / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DG)(DC)(DC)(DT)(DG)(DG)(DC)(DG)(DA)(DC) (DA)(DG)(DC)(DC)(DC)(DA)(DC)(DA)(DT)(DG) (DG)(DC)(DA)(DT)(DT)(DC)(DC)(DA)(DC)(DT) (DT)(DA)(DT)(DC)(DA)(DC)(DT)(DG)(DG)(DC) (DT)(DT)(DC)(DG)(DT)(DC)(DC)(DG)(DC)(DG)
MassTheoretical: 15.251741 kDa
SourceSpecies: Thermobifida fusca YX (bacteria)

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Component #8: protein, CRISPR-associated protein, Cas5e family

ProteinName: CRISPR-associated protein, Cas5e family / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 28.27926 kDa
SourceSpecies: Thermobifida fusca (strain YX) (bacteria) / Strain: YX
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #9: nucleic-acid, Nontarget Strand

Nucleic-acidName: Nontarget Strand / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DC)(DG)(DC)(DG)(DG)(DA)(DC)(DG)(DA)(DA) (DG)(DC)(DC)(DA)(DG)(DT)(DG)(DA)(DC)(DC) (DA)(DT)(DG)(DT)(DG)(DG)(DG)(DC)(DT)(DG) (DT)(DC)(DG)(DC)(DC)(DA)(DG)(DG)(DC)
MassTheoretical: 12.076723 kDa
SourceSpecies: Thermobifida fusca YX (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.8 mg/ml / Buffer solution: 10 mM HEPES pH 7.5, 150 mM NaCl, 5 mM DTT / pH: 7.5
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Humidity: 85 %

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 8 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 31000. X (nominal) / Cs: 2 mm / Imaging mode: BRIGHT FIELD
Specimen HolderModel: GATAN LIQUID NITROGEN / Temperature: K ( 80.0 - 105.0 K)
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 1072

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 131292
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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