Capturing transient states of heterodimeric ABC transporter TM287/288 by Time-Resolved Small-Angle X-ray Scattering

Clement Blanchet.

SASDZ84 – ABC transporter TM287/288 heterodimer in presence of Sybody Sb#35

ABC transporter TM288 subunit
ABC transporter, ATP-binding protein
Synthetic nanobody Sb_TM#35 bound to TM287/288 ABC transporter
MWexperimental 186 kDa
MWexpected 146 kDa
log I(s) 3.03×104 3.03×103 3.03×102 3.03×101
ABC transporter TM288 subunit ABC transporter, ATP-binding protein Synthetic nanobody Sb_TM#35 bound to TM287/288 ABC transporter small angle scattering data  s, nm-1
ln I(s)
ABC transporter TM288 subunit ABC transporter, ATP-binding protein Synthetic nanobody Sb_TM#35 bound to TM287/288 ABC transporter Guinier plot ln 3.04×104 Rg: 5.4 nm 0 (5.4 nm)-2 s2
(sRg)2I(s)/I(0)
ABC transporter TM288 subunit ABC transporter, ATP-binding protein Synthetic nanobody Sb_TM#35 bound to TM287/288 ABC transporter Kratky plot 1.104 0 √3 sRg

Data validation

There are no models related to this curve.

Synchrotron SAXS data from solutions of ABC transporter TM287/288 heterodimer in presence of Sybody Sb#35 in 20 mM HEPES, 200 mM NaCl, 5 mM MgCl2, 20 μM DMM, 1 mM Mg2+ ATP, pH 7.6 were collected on the EMBL P12 beam line at the PETRA III storage ring (DESY; Hamburg, Germany) using a Eiger 4M detector at a sample-detector distance of 1.5 m and at a wavelength of λ = 0.124 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). at 20°C. 100 successive 10 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.

Time-resolved experiments: final scattering curves collected 240 s after mixing. The remaining kinetic curves are provided as supplementary files. The discrepancy between the theoretical and experimental molecular weight arises from the presence of DDM detergent molecules bound to the TM287/288 heterodimer for solubility.

ABC transporter TM288 subunit
Mol. type   Protein
Organism   Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8)
Olig. state   Monomers
Mon. MW   67.7 kDa
 
UniProt   Q9WYC4
Sequence   FASTA
 
ABC transporter, ATP-binding protein
Mol. type   Protein
Organism   ABC transporter TM287 subunit
Olig. state   Monomers
Mon. MW   64.4 kDa
 
UniProt   Q9WYC3
Sequence   FASTA
 
Synthetic nanobody Sb_TM#35 bound to TM287/288 ABC transporter (Sb_TM#35)
Mol. type   Other
Organism   synthetic (in vitro selected)
Olig. state   Monomer
Mon. MW   14 kDa