![]() Candidate BAR domain proteins were identified using repeated iterations of Psi-BLAST ( against the arfaptin and amphiphysin BAR domains. ![]() We first obtained a general BAR sequence alignment by overlaying the Drosophila amphiphysin and arfaptin2 BAR structures (see Figs. Data were analyzed as described in detail in the supplementary information to (S10). The rotor was then accelerated to pull the macromolecule away from the meniscus, and further scans taken to provide initial estimates of the baseline for each cell. Scans (averaging 10 readings) were taken at 280nm at 24hr intervals, until no movement of the distribution was visible, when final scans (averaging 100 readings) were taken and assumed to be operationally at equilibrium. Long sample columns were used, with cells loaded at a variety of initial concentrations. Sedimentation was at 11,000 rev/min, 20.0☌, with initial overspeeding at 18,000 rev/min for 6hr, to reduce the time to reach equilibrium (S9). Sedimentation equilibrium experiments were performed in a Beckman Optima XL-A analytical ultracentrifuge with an An60-Ti rotor, in 10mM Tris Cl, pH 7.4, 200mM NaCl, 1mM TCEP. The model was built with O (S7) and refined with Refmac (S8). Phases were improved by solvent flattening with Solomon (S6). An additional two anomalous scattering sites at positions of the cysteine sulphur atoms were added during phasing by Sharp (S5). Two SeMet sites were located using the anomalous differences with the program Shake-and-Bake (S4). Images were integrated with Mosflm (S2) and scaled with Scala (S3). A single-wavelength X-ray diffraction dataset was collected to 2.6Å resolution at the Se edge at ESRF beamline ID29 ( Table S1). The crystal asymmetric unit contains one molecule, and the crystals belong to spacegroup P3121, cell dimensions a = b = 49.6Å, c = 190.3Å, g = 120°. Crystals were equilibrated in 20% glycerol for cooling to 100K. Crystals were grown by vapour diffusion from a 2.5mg/ml protein solution in 18% PEG4000, 200mM NaCl, 1mM DTT, 20mM HEPES pH 7.4 mixed with the well solution 18% PEG 4000, 0.2M ammonium acetate, 100mM sodium citrate pH 6.0. Clathrin was purified from rat brain as described on CD spectra were measured at 20☌ in 5mM HEPES, pH 7.4, 150mM NaCl using a Jobin Yvon CD6.ĭrosophila amphiphysin (residues 1-245) was expressed as an NH2-terminal GST fusion protein in minimal medium supplemented with selenomethionine. coli BL21 cells, purified by glutathione-affinity, cleaved with thrombin, and further purified by ion exchange chromatography before use. Derivatives and mutants were subcloned into pGEX4T1 or 4T2, expressed in E. Oligophrenin1 was cloned from a rat brain library, and amphiphysin1 (residues 1-377) and 2 (residues 1-422) were cloned from a rat brain library as previously described (S1). cDNAs for human BRAP1/Bin2 (5722815), mouse nadrin2 (3500822) were gifts from the I.M.A.G.E. cDNA for centaurinbeta2 (KIAA0041) was a gift from the Kazusa DNA research institute, Chiba, Japan. Full-length arfaptin2 in the expression vector pGEX6P2 was a gift from Steve Gamblin (National Institute for Medical Research, London, UK), and the cDNA for Drosophila amphiphysin was a gift from Cahir OKane (Univ.
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