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- EMDB-4670: Cas1-Cas2-Csn2-DNA dimer complex from the Type II-A CRISPR-Cas system -

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Basic information

Entry
Database: EMDB / ID: EMD-4670
TitleCas1-Cas2-Csn2-DNA dimer complex from the Type II-A CRISPR-Cas system
Map dataCas1-Cas2-Csn2-DNA dimeric complex. Refined with C1 symmetry.
Sample
  • Complex: Cas1-Cas2-Csn2-DNA monomer complex
    • Complex: Cas1-Cas2-Csn2
      • Protein or peptide: CRISPR-associated protein Csn2
      • Protein or peptide: CRISPR-associated endonuclease Cas1
      • Protein or peptide: CRISPR-associated endoribonuclease Cas2
    • Complex: DNA
      • DNA: DNA (64-MER)
      • DNA: DNA (64-MER)
  • Ligand: CALCIUM ION
KeywordsCRISPR / Cas / DNA binding / Nuclease / Adaptation / Spacer acquisition / DNA BINDING PROTEIN / Protein complex / Protein-DNA complex / Calcium / Metal-binding / Antiviral
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / RNA endonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / metal ion binding
Similarity search - Function
CRISPR-associated protein, Csn2-type / CRISPR-associated protein Csn2 superfamily / CRISPR-associated protein (Cas_Csn2) / CRISPR-associated protein Cas1, NMENI subtype / CRISPR-associated endonuclease Cas2 / Virulence-associated protein D / CRISPR associated protein Cas2 / CRISPR associated protein Cas2 / : / CRISPR-associated protein Cas1 / CRISPR-associated endonuclease Cas1, C-terminal domain / CRISPR associated protein Cas1
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas1 / CRISPR-associated endoribonuclease Cas2 / CRISPR-associated protein Csn2
Similarity search - Component
Biological speciesStreptococcus thermophilus (bacteria) / Escherichia coli BL21(DE3) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.9 Å
AuthorsWilkinson M / Drabavicius G
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.
History
DepositionMar 8, 2019-
Header (metadata) releaseMay 8, 2019-
Map releaseMay 8, 2019-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6qxt
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4670.png

Downloads & links

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Map

FileDownload / File: emd_4670.map.gz / Format: CCP4 / Size: 134.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCas1-Cas2-Csn2-DNA dimeric complex. Refined with C1 symmetry.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 328 pix.
= 355.88 Å		1.09 Å/pix.
x 328 pix.
= 355.88 Å		1.09 Å/pix.
x 328 pix.
= 355.88 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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.034462504 - 0.080922656
Average (Standard dev.)0.000028035374 (±0.007907804)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions328328328
Spacing328328328
CellA=B=C: 355.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0851.0851.085
M x/y/z328328328
origin x/y/z0.0000.0000.000
length x/y/z355.880355.880355.880
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS328328328
D min/max/mean-0.0340.0810.000

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Supplemental data

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Additional map: Supplementary D2-symmetrised map used to aid model building.

Fileemd_4670_additional.map
AnnotationSupplementary D2-symmetrised map used to aid model building.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cas1-Cas2-Csn2-DNA monomer complex

EntireName: Cas1-Cas2-Csn2-DNA monomer complex
Components
  • Complex: Cas1-Cas2-Csn2-DNA monomer complex
    • Complex: Cas1-Cas2-Csn2
      • Protein or peptide: CRISPR-associated protein Csn2
      • Protein or peptide: CRISPR-associated endonuclease Cas1
      • Protein or peptide: CRISPR-associated endoribonuclease Cas2
    • Complex: DNA
      • DNA: DNA (64-MER)
      • DNA: DNA (64-MER)
  • Ligand: CALCIUM ION

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Supramolecule #1: Cas1-Cas2-Csn2-DNA monomer complex

SupramoleculeName: Cas1-Cas2-Csn2-DNA monomer complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Molecular weightTheoretical: 1.4 MDa

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Supramolecule #2: Cas1-Cas2-Csn2

SupramoleculeName: Cas1-Cas2-Csn2 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#3
Details: Represents a small proportion of the Cas1-Cas2-Csn2-DNA particles.
Source (natural)Organism: Streptococcus thermophilus (bacteria)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #4-#5
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #1: CRISPR-associated protein Csn2

MacromoleculeName: CRISPR-associated protein Csn2 / type: protein_or_peptide / ID: 1 / Number of copies: 16 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus thermophilus (bacteria)
Molecular weightTheoretical: 25.533473 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKINFSLLDE PMEVNLGTVL VIEDVSVFAQ LVKEFYQYDE QSNLTIFDSK IRSIRSSELL LITDILGYDI NTSQVLKLLH TDIVSQLND KPEVRSEIDS LVSLITDIIM AECIENELDI EYDEITLLEL IKALGVRIET KSCTVFEKIF EILQIFKYLV K KRILVFVN ...String:
MKINFSLLDE PMEVNLGTVL VIEDVSVFAQ LVKEFYQYDE QSNLTIFDSK IRSIRSSELL LITDILGYDI NTSQVLKLLH TDIVSQLND KPEVRSEIDS LVSLITDIIM AECIENELDI EYDEITLLEL IKALGVRIET KSCTVFEKIF EILQIFKYLV K KRILVFVN SLSYFSKDEI YQILEYTKLS QADVLFLEPR QIEGIQQFIL DKDYILMPYN N

UniProtKB: CRISPR-associated protein Csn2

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Macromolecule #2: CRISPR-associated endonuclease Cas1

MacromoleculeName: CRISPR-associated endonuclease Cas1 / type: protein_or_peptide / ID: 2 / Number of copies: 24 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus thermophilus (bacteria)
Molecular weightTheoretical: 35.363461 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAGWRTVVVN IHSKLSYKNN HLIFRNSYKT EMIHLSEIDI LLLETTDIVL TTMLVKRLVD ENILVIFCDD KRLPTAFLTP YYARHDSSL QIARQIAWKE NVKCEVWTAI IAQKILNQSY YLGECSFFEK SQSIMELYHG LERFDPSNRE GHSARIYFNT L FGNDFTRE ...String:
MAGWRTVVVN IHSKLSYKNN HLIFRNSYKT EMIHLSEIDI LLLETTDIVL TTMLVKRLVD ENILVIFCDD KRLPTAFLTP YYARHDSSL QIARQIAWKE NVKCEVWTAI IAQKILNQSY YLGECSFFEK SQSIMELYHG LERFDPSNRE GHSARIYFNT L FGNDFTRE SDNDINAALD YGYTLLLSMF AREVVVCGCM TQIGLKHANQ FNQFNLASDI MEPFRPIIDR IVYQNRHNNF VK IKKELFS IFSETYLYNG KEMYLSNIVS DYTKKVIKAL NQLGEEIPEF RILESGWSHP QFEKA

UniProtKB: CRISPR-associated endonuclease Cas1

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Macromolecule #3: CRISPR-associated endoribonuclease Cas2

MacromoleculeName: CRISPR-associated endoribonuclease Cas2 / type: protein_or_peptide / ID: 3 / Number of copies: 12 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus thermophilus (bacteria)
Molecular weightTheoretical: 13.43156 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MSYRYMRMIL MFDMPTDTAE ERKAYRKFRK FLLSEGFIMH QFSVYSKLLL NHTANTAMVG RLKANNPKKG NITILTVTEK QFARMIYLY GDKNTSIANS EERLVFLGDN YCDED

UniProtKB: CRISPR-associated endoribonuclease Cas2

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Macromolecule #4: DNA (64-MER)

MacromoleculeName: DNA (64-MER) / type: dna / ID: 4
Details: Mixed sequences of cell DNA fragments bound represented as poly(A)
Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 19.423369 KDa
SequenceString: (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)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT) (DT)(DT)(DT)(DT)(DT)(DT) ...String:
(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)(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)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT) (DT)(DT)(DT)(DT)

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Macromolecule #5: DNA (64-MER)

MacromoleculeName: DNA (64-MER) / type: dna / ID: 5
Details: Mixed sequences of cell DNA fragments bound represented as poly(T)
Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 20.000281 KDa
SequenceString: (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)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA) (DA)(DA)(DA)(DA)(DA)(DA) ...String:
(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)(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)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA) (DA)(DA)(DA)(DA)

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Macromolecule #6: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 6 / Number of copies: 16 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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

Concentration0.05 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
25.0 mMTris
150.0 mMSodium chlorideNaCl
1.0 mMTCEP

Details: Buffer solution was filtered
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 15 s wait time, 1 s blot time..
DetailsSample was DNaseI treated followed by gel filtration. Peak fractions were concentrated prior to making grids.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 4908 / Average exposure time: 1.0 sec. / Average electron dose: 92.8 e/Å2 / Details: Images were collected in movie mode with 39 frames
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 129032 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 75000
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

Particle selectionNumber selected: 426924
Details: Reproductions of an initial ab initio generated 3D reconstruction were used as templates for auto picking. Most of the picked particles were monomers or filament particles that were later excluded
Startup modelType of model: INSILICO MODEL
In silico model: Generated ab initio from the data with cryoSPARC
Details: Reconstruction was low-pass filtered to at least 30 Angstroms prior to starting classification/refinement runs
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 3109
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 3 / Avg.num./class: 3000 / Software - Name: RELION (ver. 3.0)
Details: Focused 3D classification without alignment after running 3D refinement. A low-pass filtered soft mask around the central part of the complex was used with D2 symmetry enforced.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6qxf

chain_id: A, source_name: PDB, initial_model_type: experimental model

6qxf

chain_id: B, source_name: PDB, initial_model_type: experimental model

6xqf

chain_id: I, source_name: PDB, initial_model_type: experimental model

6xqf

chain_id: J, source_name: PDB, initial_model_type: experimental model

6xqf

chain_id: K, residue_range: 100-110, source_name: PDB, initial_model_type: experimental model

5zyf

chain_id: A, source_name: PDB, initial_model_type: experimental model
DetailsRounds of manual fitting in Coot, jelly-body refinement in REFMAC and rigid-body refinement in PHENIX
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Target criteria: Correlation coefficient and visual inspection
Output model

PDB-6qxt:
Cas1-Cas2-Csn2-DNA dimer complex from the Type II-A CRISPR-Cas system

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