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