Rapid screening of in cellulo grown protein crystals via a small-angle X-ray scattering/X-ray powder diffraction synergistic approach

Lahey-Rudolph J, Schönherr R, Jeffries C, Blanchet C, Boger J, Ferreira Ramos A, Riekehr W, Triandafillidis D, Valmas A, Margiolaki I, Svergun D, Redecke L, Journal of Applied Crystallography 53(5) (2020) DOI

SASDH76 – In cellulo cathepsin B (CatB) protein crystals recombinantly expressed within High Five insect cells (cell-culture serial dilution series)

Cathepsin B-like cysteine protease
MWexperimental 37 kDa
MWexpected 37 kDa
log I(s) 3.50×105 3.50×104 3.50×103 3.50×102
Cathepsin B-like cysteine protease small angle scattering data  s, nm-1
Cathepsin B-like cysteine protease Kratky plot 1.104 0 3 sRg

Data validation

There are no models related to this curve.

Synchrotron SAXS data from High Five cell cultures containing in cellulo CatB protein crystals were collected on the EMBL P12 beam line at PETRA III (DESY; Hamburg, Germany) using a Pilatus 6M detector at a sample-detector distance of 3 m and at a wavelength of λ = 0.124 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). Data were collected at 7°C using a fixed sample position (no sample flow) in a 1.8 mm capillary. 40 successive 0.045 second frames were collected; the individual 2D images were summed (1.8 s total exposure) and then normalized to the intensity of the transmitted beam and radially averaged. The scattering of the solvent-blank was subtracted.

The SAXS profile displayed for this entry shows data measured from undiluted insect cell culture. Data spanning a serial dilution of the crystal-containing cell culture (100% - 1.6% v/v) are made available in the full entry zip archive. The serial dilution series was made by combining the crystal-containing cultures with a mockvirus-infected High Five insect cell control (e.g., SASDH46) so as to maintain the optical density of the final mixtures. All High Five cell cultures were suspended in TBS (20 mM Tris, 150 mM NaCl), pH 7, that was used for solvent-background scattering subtraction. Data validation metrics do not apply for this entry (Rg, I(0), MW, etc). The quoted 'experimental MW' is that of the monomeric protein calculated from the amino acid sequence.

Cathepsin B-like cysteine protease (CatB)
Mol. type   Protein
Organism   Trypanosoma brucei
Olig. state   Unknown
Mon. MW   37.2 kDa
UniProt   Q6R7Z5 (1-340)
Sequence   FASTA