Modulating the Cascade architecture of a minimal Type I-F CRISPR-Cas system.

Gleditzsch D, Müller-Esparza H, Pausch P, Sharma K, Dwarakanath S, Urlaub H, Bange G, Randau L, Nucleic Acids Res 44(12):5872-82 (2016) Europe PMC

SASDBL4 – The 1:1:6:1 crRNA:Cas6f:Cas7fv:Cas5fv CRISPR/Cas Type I-F wild-type Cascade complex

Cas6f: CRISPR/Cas Type I-F Cascade component (CRISPR-associated protein, Csy4 family)
Cas5fv: CRISPR/Cas Type I-F Cascade component (Uncharacterized protein, Sputcn32_1822)
Hexameric Cas7fv: CRISPR/Cas Type I-F Cascade component (Uncharacterized protein, Sputcn32_1821)
wildtype-crRNA: CRISPR/Cas Type I-F Cascade component
MWexperimental 225 kDa
MWexpected 301 kDa
log I(s) 1.60×102 1.60×101 1.60×100 1.60×10-1
Cas6f: CRISPR/Cas Type I-F Cascade component (CRISPR-associated protein, Csy4 family) Cas5fv: CRISPR/Cas Type I-F Cascade component (Uncharacterized protein, Sputcn32_1822) Hexameric Cas7fv: CRISPR/Cas Type I-F Cascade component (Uncharacterized protein, Sputcn32_1821) wildtype-crRNA: CRISPR/Cas Type I-F Cascade component small angle scattering data  s, nm-1
ln I(s)
Cas6f: CRISPR/Cas Type I-F Cascade component (CRISPR-associated protein, Csy4 family) Cas5fv: CRISPR/Cas Type I-F Cascade component (Uncharacterized protein, Sputcn32_1822) Hexameric Cas7fv: CRISPR/Cas Type I-F Cascade component (Uncharacterized protein, Sputcn32_1821) wildtype-crRNA: CRISPR/Cas Type I-F Cascade component Guinier plot ln 1.61×102 Rg: 5.4 nm 0 (5.4 nm)-2 s2
(sRg)2I(s)/I(0)
Cas6f: CRISPR/Cas Type I-F Cascade component (CRISPR-associated protein, Csy4 family) Cas5fv: CRISPR/Cas Type I-F Cascade component (Uncharacterized protein, Sputcn32_1822) Hexameric Cas7fv: CRISPR/Cas Type I-F Cascade component (Uncharacterized protein, Sputcn32_1821) wildtype-crRNA: CRISPR/Cas Type I-F Cascade component Kratky plot 1.104 0 √3 sRg
p(r)
Cas6f: CRISPR/Cas Type I-F Cascade component (CRISPR-associated protein, Csy4 family) Cas5fv: CRISPR/Cas Type I-F Cascade component (Uncharacterized protein, Sputcn32_1822) Hexameric Cas7fv: CRISPR/Cas Type I-F Cascade component (Uncharacterized protein, Sputcn32_1821) wildtype-crRNA: CRISPR/Cas Type I-F Cascade component pair distance distribution function Rg: 5.4 nm 0 Dmax: 18.4 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Cas6f: CRISPR/Cas Type I-F Cascade component (CRISPR-associated protein, Csy4 family) Cas5fv: CRISPR/Cas Type I-F Cascade component (Uncharacterized protein, Sputcn32_1822) Hexameric Cas7fv: CRISPR/Cas Type I-F Cascade component (Uncharacterized protein, Sputcn32_1821) wildtype-crRNA: CRISPR/Cas Type I-F Cascade component DAMMIF model
log I(s)
 s, nm-1
Cas6f: CRISPR/Cas Type I-F Cascade component (CRISPR-associated protein, Csy4 family) Cas5fv: CRISPR/Cas Type I-F Cascade component (Uncharacterized protein, Sputcn32_1822) Hexameric Cas7fv: CRISPR/Cas Type I-F Cascade component (Uncharacterized protein, Sputcn32_1821) wildtype-crRNA: CRISPR/Cas Type I-F Cascade component DAMFILT model
Synchrotron SAXS data from solutions of the 1:1:6:1 crRNA:Cas6f:Cas7fv:Cas5fv CRISPR/Cas Type I-F Cascade complex in 50 mM HEPES 150 mM NaCl, 1mM DTT, 1mM EDTA, pH 7 were collected on the BM29 beam line at the ESRF (Grenoble, France) using a Pilatus 2M detector (I(s) vs s; s = 4π sin θ/λ, where 2θ is the scattering angle and λ=0.124 nm). The data displayed in this entry were measured from one sample at a solute concentration of 20 mg/ml using an exposure time of 10 s (collected as 10 x 1 s frames). The data were normalized to the intensity of the transmitted beam and radially averaged and the scattering from the matched solvent-blank was subtracted. The data were normalized to protein concentration.

Cas6f: CRISPR/Cas Type I-F Cascade component (CRISPR-associated protein, Csy4 family) (Cas6f)
Mol. type   Protein
Organism   Shewanella putrefaciens
Olig. state   Monomer
Mon. MW   20.7 kDa
 
UniProt   A4Y6G3 (1-183)
Sequence   FASTA
 
Cas5fv: CRISPR/Cas Type I-F Cascade component (Uncharacterized protein, Sputcn32_1822) (Cas5fv)
Mol. type   Protein
Organism   Shewanella putrefaciens
Olig. state   Monomer
Mon. MW   37.9 kDa
 
UniProt   A4Y6G2 (1-336)
Sequence   FASTA
 
Hexameric Cas7fv: CRISPR/Cas Type I-F Cascade component (Uncharacterized protein, Sputcn32_1821) (Cas7fv-hexamer)
Mol. type   Protein
Organism   Shewanella putrefaciens
Olig. state   Hexamer
Mon. MW   37.2 kDa
 
UniProt   A4Y6G1 (1-315)
Sequence   FASTA
 
wildtype-crRNA: CRISPR/Cas Type I-F Cascade component (wildtype-crRNA)
Mol. type   RNA
Olig. state   Monomer
Mon. MW   19.3 kDa
Sequence   FASTA