Structural insights into the substrate-binding proteins Mce1A and Mce4A from Mycobacterium tuberculosis

Asthana P, Singh D, Pedersen J, Hynönen M, Sulu R, Murthy A, Laitaoja M, Jänis J, Riley L, Venkatesan R, IUCrJ 8(5) (2021) DOI

SASDJY9 – Mammalian cell entry protein 4A (Mce4A39-320)

n-Dodecyl-β-D-Maltopyranoside
Mce-family protein Mce4A
MWexperimental 103 kDa
MWexpected 136 kDa
VPorod 189 nm3
log I(s) 1.68×10-1 1.68×10-2 1.68×10-3 1.68×10-4
n-Dodecyl-β-D-Maltopyranoside Mce-family protein Mce4A small angle scattering data  s, nm-1
ln I(s)
n-Dodecyl-β-D-Maltopyranoside Mce-family protein Mce4A Guinier plot ln 1.68×10-1 Rg: 4.8 nm 0 (4.8 nm)-2 s2
(sRg)2I(s)/I(0)
n-Dodecyl-β-D-Maltopyranoside Mce-family protein Mce4A Kratky plot 1.104 0 3 sRg
Dmax: 18.2 nm

Data validation


Fits and models


log I(s)
 s, nm-1
n-Dodecyl-β-D-Maltopyranoside Mce-family protein Mce4A CUSTOM IN-HOUSE model

log I(s)
 s, nm-1
n-Dodecyl-β-D-Maltopyranoside Mce-family protein Mce4A CUSTOM IN-HOUSE model

Synchrotron SAXS data from solutions of Mammalian cell entry protein 4A (Mce4A39-320) in 50mM Tris, 500mM NaCl, 10% Glycerol, 5mM DDM, 1mM Beta-ME, pH 8.5 were collected on the B21 beam line at the Diamond Light Source storage ring (Didcot, UK) using a Pilatus 2M detector at a wavelength of λ = 0.1 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 55.00 μl sample at 5 mg/ml was injected at a 0.06 ml/min flow rate onto a GE Superdex 200 Increase 3.2/300 column . The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

Synchrotron SAXS data of the Mce+Helical domain of Mammalian cell entry protein (Mce) 4A collected at DLS beamline B21 using SEC-SAXS. The molecular mass of the protein-detergent complex was determined using Size exclusion chromatography-multi angle light scattering (SEC-MALS) coupled to refractive index measurements. The model files contain the protein-detergent molecules. The protein models were generated using iTasser and fitted against the SAXS data using the in-house program, also taking in account the micelle contribution. The aggregation number for extended model (200) is provided with the sample data and the aggregation number for the coiled-coil model is 190.

n-Dodecyl-β-D-Maltopyranoside (DDM)
Mol. type   Other
Olig. state   0
Mon. MW   0.5 kDa
Chemical formula
 
Mce-family protein Mce4A (Mce4A)
Mol. type   Protein
Organism   Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Olig. state   Monomer
Mon. MW   34.4 kDa
 
UniProt   I6YC99 (39-320)
Sequence   FASTA