Tamburrini KC,
Kodama S,
Grisel S,
Haon M,
Nishiuchi T,
Bissaro B,
Kubo Y,
Longhi S,
Berrin JG,
Proc Natl Acad Sci U S A
121(13):e2319998121
(2024)
Europe PMC
Synchrotron SAXS data from solutions of AA9 LPMO (C286A) in 50 mM MES, 150 mM NaCl, pH 6.5 were collected on the BM29 beam line at the ESRF (Grenoble, France) using a Pilatus3 2M detector at a sample-detector distance of 2.9 m and at a wavelength of λ = 0.0992 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: A 50.00 μl sample at 9.5 mg/ml was injected at a 0.35 ml/min flow rate onto a Agilent Bio SEC-3, 300 Å column at 20°C. 500 successive 1 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.
*The position of the quoted amino acid mutation - C286A - is relative to the canonical amino acid sequence of UniProt N4UN01 that includes a twenty amino acid signal-secretion peptide at the N-terminus that is otherwise cleaved in the mature protein. Therefore, for the mature protein, the mutation occurs at C266A as described in the accompanying manuscript. The CoAA9A was heterologously expressed in Pichia pastoris. The sample was N-deglycosylated before recording SAXS data, however it was not possible to remove O-glycosylation. The difference between the experimental molecular weight and the expected molecular weight is due to O-glycosylation.
s, nm-1
