Structural basis for activation of plasma-membrane Ca2+-ATPase by calmodulin.

Nitsche J, Josts I, Heidemann J, Mertens HD, Maric S, Moulin M, Haertlein M, Busch S, Forsyth VT, Svergun DI, Uetrecht C, Tidow H, Commun Biol 1:206 (2018) Europe PMC

SASDET4 – ACA8 complex with Calmodulin (75% deuterated) in stealth nanodisc (SANS, 100% D2O)

Membrane scaffold protein 1D1 (deuterated, 75%)
1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl)
Calcium-transporting ATPase 8, plasma membrane-type
Calmodulin-7 (deuterated 75%)
MWexperimental 136 kDa
MWexpected 168 kDa
VPorod 217 nm3
log I(s) 9.20×10-2 9.20×10-3 9.20×10-4 9.20×10-5
Membrane scaffold protein 1D1 (deuterated, 75%) 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) Calcium-transporting ATPase 8, plasma membrane-type Calmodulin-7 (deuterated 75%) small angle scattering data  s, nm-1
ln I(s)
Membrane scaffold protein 1D1 (deuterated, 75%) 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) Calcium-transporting ATPase 8, plasma membrane-type Calmodulin-7 (deuterated 75%) Guinier plot ln 9.20×10-2 Rg: 4.3 nm 0 (4.3 nm)-2 s2
(sRg)2I(s)/I(0)
Membrane scaffold protein 1D1 (deuterated, 75%) 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) Calcium-transporting ATPase 8, plasma membrane-type Calmodulin-7 (deuterated 75%) Kratky plot 1.104 0 3 sRg
p(r)
Membrane scaffold protein 1D1 (deuterated, 75%) 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) Calcium-transporting ATPase 8, plasma membrane-type Calmodulin-7 (deuterated 75%) pair distance distribution function Rg: 4.6 nm 0 Dmax: 15 nm

Data validation


Fits and models


log I(s)
 s, nm-1
ACA8 complex with Calmodulin (75% deuterated) in stealth nanodisc (SANS, 100% D2O) Rg histogram Rg, nm
Membrane scaffold protein 1D1 (deuterated, 75%) 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) Calcium-transporting ATPase 8, plasma membrane-type Calmodulin-7 (deuterated 75%) EOM/GAJOE model

SANS data from solutions of ACA8 complex with Calmodulin (75% deuterated) in stealth nanodisc (SANS, 100% D2O) in 30 mM Tris, 150 mM NaCl, 1mM MgCl2, 1 mM CaCl2, pH 7.5 were collected using a MLZ detector at a sample-detector distance of 5.5 m and at a wavelength of λ = 5 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). Solute concentrations ranging between 3.2 and 3.2 mg/ml were measured at 10°C. One 7200 second frame was collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

ACA8 protein complex with Calmodulin (75% deuterated) encapsulated in a partially deuterated "stealth-nanodisc": (sND). The Calmodulin and 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 ACA8 protein alone (~118 kDa). Data measured using a single instrument configuration: 5.5 m (sample-detector distances) with a fixed neutron wavelength (λ) of 5 Å.

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
Chemical formula
 
Calcium-transporting ATPase 8, plasma membrane-type (ACA8)
Mol. type   Protein
Organism   Arabidopsis thaliana
Olig. state   Monomer
Mon. MW   118.2 kDa
 
UniProt   Q9LF79
Sequence   FASTA
 
Calmodulin-7 (deuterated 75%) (d75-Cam7)
Mol. type   Protein
Organism   Arabidopsis thaliana
Olig. state   Monomers
 
UniProt   P59220