Anti60 rotor

Sedimentation equilibrium (SEQ) analytical ultracentrifugation experiments were performed at 20 °C using a Beckman XL-I instrument with an AnTi60 rotor. The sample chambers used Epon double sector centerpieces with sapphire windows. Samples of wild type and 5-F-Trp Ud NS1A ED (each 175 μL containing 15 μM total protein) were prepared in low salt pH 8 NMR buffer [50 mM Tris-HCl pH 8.0, 30 mM NaCl, 2.5% (v/v) glycerol, 10 mM DTT, 10% (v/v) ²H₂O]. Sedimentation equilibrium scans were recorded after 72 h at three rotor speeds: 15,000, 25,000, and 31,000 rpm. The protein partial specific volume, solvent density and solvent viscosity were calculated using Sednterp 1.09 (Laue et al., 1992 ). Data analysis was performed using Sedphat 10.40 (Schuck, 2003 (link); Vistica et al., 2004 (link)). For each protein, the sedimentation equilibrium profiles were globally fitted to a monomer-dimer equilibrium model using mass conservation and rotor stretch restraints. An F-statistics error mapping approach (Johnson and Straume, 1994 ) was used to determine the 95% confidence intervals for the homodimerization dissociation constant.

Sedimentation velocity (SV) experiments were carried out in a Beckman Optima XL-I analytical ultracentrifuge (Beckman Coulter, Indianapolis, IN) equipped with a 4-hole AnTi-60 rotor and standard Epon 2-channel centerpieces. The samples of ERF-DNA complexes and DNA alone in analytical ultracentrifugation (AUC) runs were in 20 mM Tris-HCl pH 8.0, 50 mM NaCl, the sample of ERF without DNA was in 40 mM Tris-HCl pH 8.0, 400 mM NaCl, to avoid protein aggregation. AUC-SV studies were performed with the ERF DBD at 2 μM and DNA at 1 or 2 μM, for a 2-site or a 1-site DNA, respectively. The sequences of the DNA ligands used in this study are given in Table S2. AUC-SV experiments were performed with a rotor speed of 40,000 rpm at 20°C. The data collection for the ERF DBD alone was performed in the intensity mode at 280 nm. For the ERF-DNA complexes and DNA alone, the data were collected in the intensity mode at 260 nm. About 200 scans were collected for each sample. Van Holde-Weischet analysis was performed using the Ultrascan III software.^{27 (link), 28 (link)} Theoretical calculations of sedimentation coefficients were performed in Ultrascan using calculated partial specific volumes of 0.74 and 0.55 mL/g for the ERF DBD and DNA, respectively.