All SNARE monomers were separately expressed from pET28a as His6-tagged proteins in E. coli Bl21 (DE3) cells and purified by Ni2+-NTA and ion exchange chromatography. SNARE complexes were then assembled from monomers and purified by ion exchange and size exclusion chromatography. Diffraction data were collected on beamline X10SA at the Swiss Light Source of the Paul Scherrer Institut (Switzerland) and processed with HKL2000. Initial phases were obtained from single-wavelength anomalous dispersion data from crystals containing selenomethionine-labelled syntaxin 1A that diffracted to 4.3 Å resolution. For the final model building native diffraction data to a resolution of 3.4 Å were used. Model building and refinement were performed using the programs COOT and Phenix, respectively.
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Structural Determination of SNARE Complex
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Cells
E coli proteins
Ion exchange
Ion exchange chromatography
Light
Selenomethionine
Size exclusion chromatography
Snare
Syntaxin 1a
Corresponding Organization : Institute of Neurobiology
Other organizations : Freie Universität Berlin, Max Planck Institute for Biophysical Chemistry
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Variable analysis
independent variables
- Expression of SNARE monomers from pET28a as His6-tagged proteins in E. coli Bl21 (DE3) cells
- Purification of SNARE monomers by Ni2+-NTA and ion exchange chromatography
- Assembly of SNARE complexes from monomers
- Purification of SNARE complexes by ion exchange and size exclusion chromatography
- Diffraction data collected on beamline X10SA at the Swiss Light Source
- Initial phases obtained from single-wavelength anomalous dispersion data from crystals containing selenomethionine-labelled syntaxin 1A
- Final model building and refinement using native diffraction data to a resolution of 3.4 Å
- Maintenance of E. coli Bl21 (DE3) cells
- Chromatography techniques (Ni2+-NTA, ion exchange, size exclusion)
- Positive control: Crystals containing selenomethionine-labelled syntaxin 1A used for initial phase determination
- Negative control: Not explicitly mentioned
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