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- EMDB-7347: CRISPR RNA-guided surveillance complex, pre-nicking -

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Basic information

Entry
Database: EMDB / ID: 7347
TitleCRISPR RNA-guided surveillance complex, pre-nicking
Map dataCRISPR RNA-guided surveillance complex with Cas3 bound in its pre-nicking state
SampleCRISPR RNA-guided surveillance complex with Cas3 bound in its pre-nicking state:
CRISPR-associated helicase, Cas3 family / (CRISPR-associated protein, ...) x 4 / Uncharacterized protein / (nucleic-acidNucleic acid) x 3 / ligand
Function / homologyCRISPR-associated protein, Cas5 / CRISPR-associated protein Cse3 / CRISPR associated protein / Helicase conserved C-terminal domain / Cse2 superfamily / CRISPR-associated Cas3-type HD domain superfamily / CRISPR-associated endonuclease/helicase Cas3 / P-loop containing nucleoside triphosphate hydrolase / CRISPR-associated protein Cas5, N-terminal / CRISPR-associated protein Cse2 ...CRISPR-associated protein, Cas5 / CRISPR-associated protein Cse3 / CRISPR associated protein / Helicase conserved C-terminal domain / Cse2 superfamily / CRISPR-associated Cas3-type HD domain superfamily / CRISPR-associated endonuclease/helicase Cas3 / P-loop containing nucleoside triphosphate hydrolase / CRISPR-associated protein Cas5, N-terminal / CRISPR-associated protein Cse2 / CRISPR-associated protein Cse1 / CRISPR-associated protein, CT1975 / CRISPR-associated protein Cse1 (CRISPR_cse1) / CRISPR-associated protein, CasD / CRISPR-associated Cas3-type HD domain / Helicase Cas3, CRISPR-associated, core / Helicase, C-terminal / CRISPR-associated protein Cse2 (CRISPR_cse2) / CRISPR-associated protein (Cas_Cas5) / HD Cas3-type domain profile. / CT1975-like protein / ATP-dependent DNA/RNA helicase activity / deoxyribonuclease activity / maintenance of CRISPR repeat elements / defense response to virus / RNA binding / ATP binding / identical protein binding / metal ion binding / CRISPR-associated helicase, Cas3 family / 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 (bacteria)
Methodsingle particle reconstruction / cryo EM / 3.66 Å resolution
AuthorsXiao Y / Luo M / Liao M / Ke A
CitationJournal: Science / Year: 2018
Title: Structure basis for RNA-guided DNA degradation by Cascade and Cas3.
Authors: Yibei Xiao / Min Luo / Adam E Dolan / Maofu Liao / Ailong Ke
Abstract: Type I CRISPR-Cas system features a sequential target-searching and degradation process on double-stranded DNA by the RNA-guided Cascade (CRISPR associated complex for antiviral defense) complex and ...Type I CRISPR-Cas system features a sequential target-searching and degradation process on double-stranded DNA by the RNA-guided Cascade (CRISPR associated complex for antiviral defense) complex and the nuclease-helicase fusion enzyme Cas3, respectively. Here, we present a 3.7-angstrom-resolution cryo-electron microscopy (cryo-EM) structure of the Type I-E Cascade/R-loop/Cas3 complex, poised to initiate DNA degradation. Cas3 distinguishes Cascade conformations and only captures the R-loop-forming Cascade, to avoid cleaving partially complementary targets. Its nuclease domain recruits the nontarget strand (NTS) DNA at a bulged region for the nicking of single-stranded DNA. An additional 4.7-angstrom-resolution cryo-EM structure captures the postnicking state, in which the severed NTS retracts to the helicase entrance, to be threaded for adenosine 5'-triphosphate-dependent processive degradation. These snapshots form the basis for understanding RNA-guided DNA degradation in Type I-E CRISPR-Cas systems.
Validation ReportPDB-ID: 6c66

SummaryFull reportAbout validation report
DateDeposition: Jan 17, 2018 / Header (metadata) release: Feb 7, 2018 / Map release: Jun 27, 2018 / Last update: Jun 27, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6c66
  • Surface level: 0.04
  • 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_7347.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
1.24 Å/pix.
= 317.44 Å
256 pix
1.24 Å/pix.
= 317.44 Å
256 pix
1.24 Å/pix.
= 317.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.24 Å
Density
Contour Level:0.03 (by author), 0.04 (movie #1):
Minimum - Maximum-0.10162261 - 0.21208328
Average (Standard dev.)-0.00003452048 (0.009961365)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions256256256
Origin0.0.0.
Limit255.255.255.
Spacing256256256
CellA=B=C: 317.44 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.241.241.24
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z317.440317.440317.440
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1020.212-0.000

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Supplemental data

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Sample components

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Entire CRISPR RNA-guided surveillance complex with Cas3 bound in its pre...

EntireName: CRISPR RNA-guided surveillance complex with Cas3 bound in its pre-nicking state
Number of components: 11

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Component #1: protein, CRISPR RNA-guided surveillance complex with Cas3 bound i...

ProteinName: CRISPR RNA-guided surveillance complex with Cas3 bound in its pre-nicking state
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 helicase, Cas3 family

ProteinName: CRISPR-associated helicase, Cas3 family / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 104.039219 kDa
SourceSpecies: Thermobifida fusca (strain YX) (bacteria) / Strain: YX
Source (engineered)Expression System: Escherichia coli (E. coli)

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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 (E. coli)

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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 (E. coli)

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Component #5: protein, Uncharacterized protein

ProteinName: Uncharacterized protein / 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 (E. coli)

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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 (bacteria)

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Component #7: nucleic-acid, Target strand

Nucleic-acidName: Target strand / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DG)(DC)(DG)(DT)(DC)(DC)(DA)(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)(DC)(DG)
MassTheoretical: 16.801725 kDa
SourceSpecies: Thermobifida fusca (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 (E. coli)

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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)(DC)(DG)(DG)(DA)(DC)(DG) (DA)(DA)(DG)(DC)(DC)(DA)(DG)(DT)(DG)(DA) (DT)(DA)(DA)(DG)(DT)(DG)(DG)(DA)(DA)(DT) (DG)(DC)(DC)(DA)(DT)(DG)(DT)(DG)(DG)(DG) (DC)(DT)(DG)(DT)(DC)(DG)(DC)(DC)(DT)(DG) (DG)(DA)(DC)(DG)(DC)
MassTheoretical: 17.09992 kDa
SourceSpecies: Thermobifida fusca (bacteria)

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Component #10: 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 (E. coli)

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Component #11: ligand, FE (III) ION

LigandName: FE (III) ION / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 5.584505 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 1 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: 1428

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 51889
3D reconstructionResolution: 3.66 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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