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

MW^experimental	225	kDa
MW^expected	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)

ln 1.61×10² R_g: 5.4 nm 0 (5.4 nm)^-2 s²

(sR_g)²I(s)/I(0)

1.104 0 √3 sR_g

p(r)	R_g: 5.4 nm 0 D_max: 18.4 nm

Data validation

Experimental data range

p(r) fit to data

Metric

Value

s_min D_max / π

0.5

N_Shannon

Worse

Better

Metric

Value

p_cormap

χ²

0.000
2.093

Worse

Better

Fits and models

log I(s)	s, nm^-1
+3 Δ/σ -3	s, nm^-1

log I(s)	s, nm^-1
+3 Δ/σ -3	s, nm^-1

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