Computational protocol: Solution Structure of the LIM-Homeodomain Transcription Factor Complex Lhx3/Ldb1 and the Effects of a Pituitary Mutation on Key Lhx3 Interactions

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Protocol publication

[…] Small-angle X-ray scattering data were collected from solutions of Ldb1-Lhx3 (4.5–9 mg mL−1) with 20 mM Na2HPO4, 40 mM NaCl, 1 mM DTT, pH 6.8 and a matched solvent blank at 283 K for 10 s intervals over 30 min using a line-collimated SAXSess scattering instrument (Anton Paar, Graz, Austria) equipped with a CCD detector . Scattering data were reduced to I(q) vs q (where q  =  (4πsinθ)/λ, 2θ is the scattering angle, and λ the X-ray wavelength, CuKα, 1.54 Å) using the SAXSQuant 2.0 software package (Anton Paar, Austria) that corrects for sample absorbance and detector sensitivity, and normalises and subtracts solvent from protein+solvent to yield I(q) versus q for the protein alone. The reduced scattering profiles were all placed on an absolute scale using the scattering from water . The programs GIFT and GNOM were used to calculate the probable distribution of atom-pair distances within Ldb1-Lhx3 (P(r) versus r profiles), accounting for the 10-mm slit-geometry of the instrument, from which the radius of gyration (Rg), maximum dimension (Dmax) and forward scattering intensity at zero angle (I(0)) were extracted; the two programs gave essentially the same results. A molecular weight estimate of Ldb1-Lhx3 was derived from I(0) as described in using values for the contrast (Δρ) and partial specific volume (υ) calculated in CONTRAST from the MULCh program suite . Although MULCh was designed for use in small-angle nuclear scattering (SANS), it can also be used to process SAXS data. An I(0) analysis of the 9 mg mL−1 sample was indicative of some aggregation and was not further analysed. Guinier analysis of the SAXS data was performed using PRIMUS . Ab initio shape restorations of Lhx3-Ldb1 were performed 10 times using the program DAMMIF and a consensus model developed via the spatial alignment and averaging of each solution combined with standard phase-occupancy and volume corrections . Rigid-body modelling against the SAXS data was performed using the high resolution structures of each LIM-half of the complex and the program BUNCH . CRYSOL was used to evaluate the fits against the scattering data of the final BUNCH model and each individual Ldb1-Lhx3 NMR structure derived from the NMR ensemble. CRYSOL was also used to calculate the theoretical scattering profiles of the resultant high-resolution models which were used to derive model P(r) vs r profiles for comparison with the experiment . […]

Pipeline specifications

Software tools ATSAS, DAMMIF, CRYSOL
Application Small-angle scattering
Organisms Dipturus trachyderma
Chemicals Zinc