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- EMDB-4668: Cas1-Cas2-Csn2-DNA complex from the Type II-A CRISPR-Cas system -

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Basic information

Entry
Database: EMDB / ID: EMD-4668
TitleCas1-Cas2-Csn2-DNA complex from the Type II-A CRISPR-Cas system
Map data
SampleCas1-Cas2-Csn2-DNA monomer complex:
Cas1-Cas2-Csn2 / DNA / (CRISPR-associated ...) x 3 / (nucleic-acidNucleic acid) x 2 / ligand
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endodeoxyribonuclease activity / endoribonuclease activity / defense response to virus / Hydrolases, Acting on ester bonds / DNA binding / metal ion binding
Virulence-associated protein D / CRISPR associated protein Cas2 / CRISPR associated protein Cas1 / CRISPR associated protein Cas2 / CRISPR-associated protein Cas1 / CRISPR-associated protein, Csn2-type / CRISPR-associated protein (Cas_Csn2) / CRISPR-associated protein Cas1, NMENI subtype / CRISPR-associated endonuclease Cas2 / CRISPR-associated protein Csn2 superfamily
CRISPR-associated endonuclease Cas1 / CRISPR-associated endoribonuclease Cas2 / CRISPR-associated protein Csn2
SourceStreptococcus thermophilus (bacteria) / Escherichia coli BL21(DE3) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsWilkinson M / Drabavicius G / Silanskas A / Gasiunas G / Siksnys V / Wigley DB
CitationJournal: Mol. Cell / Year: 2019
Title: Structure of the DNA-Bound Spacer Capture Complex of a Type II CRISPR-Cas System.
Authors: Martin Wilkinson / Gediminas Drabavicius / Arunas Silanskas / Giedrius Gasiunas / Virginijus Siksnys / Dale B Wigley /
Abstract: CRISPR and associated Cas proteins function as an adaptive immune system in prokaryotes to combat bacteriophage infection. During the immunization step, new spacers are acquired by the CRISPR ...CRISPR and associated Cas proteins function as an adaptive immune system in prokaryotes to combat bacteriophage infection. During the immunization step, new spacers are acquired by the CRISPR machinery, but the molecular mechanism of spacer capture remains enigmatic. We show that the Cas9, Cas1, Cas2, and Csn2 proteins of a Streptococcus thermophilus type II-A CRISPR-Cas system form a complex and provide cryoelectron microscopy (cryo-EM) structures of three different assemblies. The predominant form, with the stoichiometry Cas1-Cas2-Csn2, referred to as monomer, contains ∼30 bp duplex DNA bound along a central channel. A minor species, termed a dimer, comprises two monomers that sandwich a further eight Cas1 and four Cas2 subunits and contains two DNA ∼30-bp duplexes within the channel. A filamentous form also comprises Cas1-Cas2-Csn2 units (typically 2-6) but with a different Cas1-Cas2 interface between them and a continuous DNA duplex running along a central channel.
Validation ReportPDB-ID: 6qxf

SummaryFull reportAbout validation report
DateDeposition: Mar 7, 2019 / Header (metadata) release: May 8, 2019 / Map release: May 8, 2019 / Update: May 22, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6qxf
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_4668.map.gz / Format: CCP4 / Size: 98.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 296 pix.
= 321.16 Å
1.09 Å/pix.
x 296 pix.
= 321.16 Å
1.09 Å/pix.
x 296 pix.
= 321.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.085 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.044958826 - 0.14251357
Average (Standard dev.)0.000005108034 (±0.0063090026)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions296296296
Spacing296296296
CellA=B=C: 321.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0851.0851.085
M x/y/z296296296
origin x/y/z0.0000.0000.000
length x/y/z321.160321.160321.160
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS296296296
D min/max/mean-0.0450.1430.000

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Supplemental data

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Sample components

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Entire Cas1-Cas2-Csn2-DNA monomer complex

EntireName: Cas1-Cas2-Csn2-DNA monomer complex / Number of components: 9

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Component #1: protein, Cas1-Cas2-Csn2-DNA monomer complex

ProteinName: Cas1-Cas2-Csn2-DNA monomer complex / Recombinant expression: No
MassTheoretical: 530 kDa

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Component #2: protein, Cas1-Cas2-Csn2

ProteinName: Cas1-Cas2-Csn2 / Recombinant expression: No
SourceSpecies: Streptococcus thermophilus (bacteria)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #3: protein, DNA

ProteinName: DNA / Recombinant expression: No
SourceSpecies: Escherichia coli BL21(DE3) (bacteria)

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Component #4: protein, CRISPR-associated protein Csn2

ProteinName: CRISPR-associated protein Csn2 / Number of Copies: 8 / Recombinant expression: No
MassTheoretical: 25.533473 kDa
SourceSpecies: Streptococcus thermophilus (bacteria)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #5: protein, CRISPR-associated endonuclease Cas1

ProteinName: CRISPR-associated endonuclease Cas1 / Number of Copies: 8 / Recombinant expression: No
MassTheoretical: 35.363461 kDa
SourceSpecies: Streptococcus thermophilus (bacteria)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #6: protein, CRISPR-associated endoribonuclease Cas2

ProteinName: CRISPR-associated endoribonuclease Cas2 / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 13.43156 kDa
SourceSpecies: Streptococcus thermophilus (bacteria)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #7: nucleic-acid, DNA (25-MER)

nucleic acidName: DNA (25-MER) / Class: DNA
Details: Ordered portion representing the mixed DNA fragments bound to the complex after sonicating cells and treating with DNaseI.
Structure: OTHER / Synthetic: No
Sequence:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)
MassTheoretical: 7.559863 kDa
SourceSpecies: Escherichia coli BL21(DE3) (bacteria)

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Component #8: nucleic-acid, DNA (25-MER)

nucleic acidName: DNA (25-MER) / Class: DNA
Details: Ordered portion representing the mixed DNA fragments bound to the complex after sonicating cells and treating with DNaseI.
Structure: OTHER / Synthetic: No
Sequence:
(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)
MassTheoretical: 7.785214 kDa
SourceSpecies: Escherichia coli BL21(DE3) (bacteria)

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Component #9: ligand, CALCIUM ION

LigandName: CALCIUM IONCalcium / Number of Copies: 8 / Recombinant expression: No
MassTheoretical: 4.007805 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.05 mg/mL / Buffer solution: Buffer solution was filtered / pH: 7.5
Support filmGraphene oxide sheets were applied to the grid prior to sample application
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 90 % / Details: 15 s wait time, 1 s blot time..

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 92.8 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 75000.0 X (nominal), 129032.0 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1300.0 - 2800.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: FEI FALCON III (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 4908 / Sampling size: 14 µm / Details: Images were collected in movie mode with 39 frames

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 306470
3D reconstructionSoftware: RELION / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Target criteria: Correlation coefficient and visual inspection
Refinement space: REAL
Details: Rounds of manual fitting in Coot, jelly-body refinement in REFMAC and real-space refinement in PHENIX
Input PDB model: 3V7F, 5XVN, 5XVN, 5XVN
Chain ID: A, B, E
Output model

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