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- EMDB-20129: Bacillus halodurans Cas4-Cas1-Cas2 symmetrical complex in presenc... -

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Basic information

Entry
Database: EMDB / ID: EMD-20129
TitleBacillus halodurans Cas4-Cas1-Cas2 symmetrical complex in presence of target CRISPR DNA
Map data
SampleBacillus halodurans Cas4-Cas1-Cas2 symmetrical complex with target CRISPR hairpin DNA:
Cas1 / Cas2Moose Lake (Lodge) Airport / Cas4 / nucleic-acidNucleic acid
SourceBacillus halodurans C-125 (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 19.7 Å
AuthorsSashital DG / Lee H / Dhingra Y
CitationJournal: Elife / Year: 2019
Title: The Cas4-Cas1-Cas2 complex mediates precise prespacer processing during CRISPR adaptation.
Authors: Hayun Lee / Yukti Dhingra / Dipali G Sashital /
Abstract: CRISPR adaptation immunizes bacteria and archaea against viruses. During adaptation, the Cas1-Cas2 complex integrates fragments of invader DNA as spacers in the CRISPR array. Recently, an additional ...CRISPR adaptation immunizes bacteria and archaea against viruses. During adaptation, the Cas1-Cas2 complex integrates fragments of invader DNA as spacers in the CRISPR array. Recently, an additional protein Cas4 has been implicated in selection and processing of prespacer substrates for Cas1-Cas2, although this mechanism remains unclear. We show that Cas4 interacts directly with Cas1-Cas2 forming a Cas4-Cas1-Cas2 complex that captures and processes prespacers prior to integration. Structural analysis of the Cas4-Cas1-Cas2 complex reveals two copies of Cas4 that closely interact with the two integrase active sites of Cas1, suggesting a mechanism for substrate handoff following processing. We also find that the Cas4-Cas1-Cas2 complex processes single-stranded DNA provided in cis or in trans with a double-stranded DNA duplex. Cas4 cleaves precisely upstream of PAM sequences, ensuring the acquisition of functional spacers. Our results explain how Cas4 cleavage coordinates with Cas1-Cas2 integration and defines the exact cleavage sites and specificity of Cas4.
DateDeposition: Apr 19, 2019 / Header (metadata) release: May 8, 2019 / Map release: May 8, 2019 / Update: May 8, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.21
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.21
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_20129.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.8 Å/pix.
x 80 pix.
= 304. Å
3.8 Å/pix.
x 80 pix.
= 304. Å
3.8 Å/pix.
x 80 pix.
= 304. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.8 Å
Density
Contour LevelBy AUTHOR: 0.21 / Movie #1: 0.21
Minimum - Maximum-0.077548474 - 0.39205563
Average (Standard dev.)0.005429223 (±0.03929943)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 304.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.83.83.8
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z304.000304.000304.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-0.0780.3920.005

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Supplemental data

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Sample components

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Entire Bacillus halodurans Cas4-Cas1-Cas2 symmetrical complex with targe...

EntireName: Bacillus halodurans Cas4-Cas1-Cas2 symmetrical complex with target CRISPR hairpin DNA
Number of components: 5

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Component #1: cellular-component, Bacillus halodurans Cas4-Cas1-Cas2 symmetrica...

Cellular-componentName: Bacillus halodurans Cas4-Cas1-Cas2 symmetrical complex with target CRISPR hairpin DNA
Recombinant expression: No
MassExperimental: 280 kDa
SourceSpecies: Bacillus halodurans C-125 (bacteria)

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Component #2: protein, Cas1

ProteinName: Cas1 / Recombinant expression: No
SourceSpecies: Bacillus halodurans C-125 (bacteria)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #3: protein, Cas2

ProteinName: Cas2Moose Lake (Lodge) Airport / Recombinant expression: No
SourceSpecies: Bacillus halodurans C-125 (bacteria)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #4: protein, Cas4

ProteinName: Cas4 / Recombinant expression: No
SourceSpecies: Bacillus halodurans C-125 (bacteria)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #5: nucleic-acid, target CRISPR hairpin DNA

nucleic acidName: target CRISPR hairpin DNA / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
GATTTTCGCT GTCGCACTCT TCATGGGTGC GTGGATTGAA ATATTGACGA TAGTCAATAT TTCAATCCAC GCACCCATGA AGAGTGCGAC AGCGAAAATC
SourceSpecies: Bacillus halodurans C-125 (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: negative staining
Sample solutionSpecimen conc.: 0.028 mg/mL / pH: 7.5
Support filmunspecified
Staining3 uL of sample was applied to a glow-discharged copper 400-mesh continuous carbon grid for one minute at room temperature. The excess sample was blotted with Whatman filter paper, followed by immediate application of 3 uL 2% (w/v) uranyl formate. The excess stain was blotted, followed by immediate application of 3 uL 2% uranyl formate. This step was repeated once more. The grids were allowed to dry for at least 5 minutes prior to imaging.
VitrificationCryogen name: NONE

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Electron microscopy imaging

ImagingMicroscope: JEOL 2100
Electron gunElectron source: LAB6 / Accelerating voltage: 200 kV / Electron dose: 30 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 200

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 18290
3D reconstructionAlgorithm: BACK PROJECTION / Software: RELION / Resolution: 19.7 Å / Resolution method: FSC 0.5 CUT-OFF

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