The dDATcryst construct (Supplementary Fig. 1 ) was expressed in virus-infected mammalian cells and purified by affinity and size-exclusion chromatography. Fab 9D5 was added prior to crystallization along with nortriptyline (1mM) at a DAT:Fab molar ratio of 1:1.1 and concentrated down to 3 mg/ml. Crystals of the complex were obtained in the presence of 100 mM glycine pH 9 and 38% PEG 350 MME. The structure was solved by molecular replacement using a poly-alanine model of LeuT (PDB id. 3F3A) and an ensemble of Fab variable and constant domains. Data processing, model building, and refinement were performed using standard crystallographic software (Supplementary Table 1 ).
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Structural Determination of dDAT-Fab Complex
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A poly
Alanine
Cells
Crystallization
Crystallographic
Glycine
Mammalian
Molar
Nortriptyline
Size exclusion chromatography
Virus
Corresponding Organization :
Other organizations : Oregon Health & Science University, Vollum Institute
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Variable analysis
independent variables
- Expression of dDATcryst construct in virus-infected mammalian cells
- Addition of Fab 9D5 and nortriptyline (1mM) at a DAT:Fab molar ratio of 1:1.1
- Purification of the complex by affinity and size-exclusion chromatography
- Crystallization of the complex in the presence of 100 mM glycine pH 9 and 38% PEG 350 MME
- Solving the structure by molecular replacement
- Concentration of the complex down to 3 mg/ml
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