Conformational States of ABC Transporter MsbA in a Lipid Environment Investigated by Small-Angle Scattering Using Stealth Carrier Nanodiscs.

Josts I, Nitsche J, Maric S, Mertens HD, Moulin M, Haertlein M, Prevost S, Svergun DI, Busch S, Forsyth VT, Tidow H, Structure 26(8):1072-1079.e4 (2018) PubMed

SASDDB5 – MsbA in stealth nanodisc (SANS, 100% D2O)

Lipid A export ATP-binding/permease protein MsbA
Membrane scaffold protein 1D1 (deuterated, 75%)
1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl)
MWI(0) 145 kDa
MWexpected 183 kDa
VPorod 189 nm3
log I(s) 8.90×10-1 8.90×10-2 8.90×10-3 8.90×10-4
Lipid A export ATP-binding/permease protein MsbA Membrane scaffold protein 1D1 (deuterated, 75%) 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) small angle scattering data  s, nm-1
ln I(s)
Lipid A export ATP-binding/permease protein MsbA Membrane scaffold protein 1D1 (deuterated, 75%) 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) Guinier plot ln 8.90×10-1 Rg: 4 nm 0 (4 nm)-2 s2
(sRg)2I(s)/I(0)
Lipid A export ATP-binding/permease protein MsbA Membrane scaffold protein 1D1 (deuterated, 75%) 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) Kratky plot 1.104 0 3 sRg
p(r)
Lipid A export ATP-binding/permease protein MsbA Membrane scaffold protein 1D1 (deuterated, 75%) 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) pair distance distribution function Rg: 4.0 nm 0 Dmax: 13 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Lipid A export ATP-binding/permease protein MsbA Membrane scaffold protein 1D1 (deuterated, 75%) 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) SASREF CV model

SANS data from solutions of MsbA in stealth nanodisc (SANS, 100% D2O) in 30 mM Tris, 150 mM NaCl, pH 7.5 were collected on the D11 camera at the ILL storage ring (Grenoble, France) using a 3-He gas detector CERCA detector at a wavelength of λ = 0.46 nm (l(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). Solute concentrations ranging between 1 and 6 mg/ml were measured at 10°C. Two successive 4200 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.

MsbA ABC transporter protein encapsulated in a partially deuterated "stealth-nanodisc": (sND). Disc components (MSP1D1 scaffold protein - 75% deuterated, and POPC lipid - heads/tails 78% and 92% deuterated) are effectively matched out at 100% D2O. Thus the expected molecular weight should correspond to that of the MsbA protein (~132 kDa). Data measured in two configurations: 1.5 m / 1.5 m and 8 m / 8 m (collimation length / sample-detector distances) with a fixed neutron wavelength (λ) of 5 Å. Data is merged from these two configurations.

Lipid A export ATP-binding/permease protein MsbA (MsbA)
Mol. type   Protein
Organism   Escherichia coli (strain K12)
Olig. state   Dimer
Mon. MW   66.3 kDa
 
UniProt   P60752
Sequence   FASTA
 
Membrane scaffold protein 1D1 (deuterated, 75%) (d75-msp1d1)
Mol. type   Protein
Olig. state   Dimer
Mon. MW   24.7 kDa
Sequence   FASTA
 
1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) (d(78,92)-POPC)
Mol. type   Other
Organism   Escherichia coli. (AL95 strain)
Olig. state   Unknown
Mon. MW   0.8 kDa
Sequence   FASTA