Crystallization, data collection, and structure determination
This protocol is extracted from research article:
Dynamic tuning of FRET in a green fluorescent protein biosensor
Sci Adv, Aug 7, 2019; DOI: 10.1126/sciadv.aaw4988

Crystals of Twitch-2B and Twitch-6 were obtained by vapor diffusion mixing of 1 μl of protein solution with 1 μl of well solution [0.2 M Na-formiate (pH 7.0), 5 mM CaCl2, and 18 to 20% polyethylene glycol 3350]. Crystals were cryoprotected by transferring them to a well solution supplemented with 16 to 18% glycerol for 1 min and flash-cooled by plunging them into liquid nitrogen.

Data collection was performed at PXII, SLS, Switzerland, using a PILATUS 6M detector (Dectris). Native data were collected at 100 K at a wavelength of 1 Å. Selenomethionine derivative data were measured at 0.98 Å. All data were processed with x-ray detector software (XDS) (21) and scaled with SADABS (Bruker AXS). Space group determination and statistical analysis were carried out using XPREP (Bruker AXS). Phasing was performed with AutoSol (22).

The initial model was built with AutoBuild and refined twice with phenix.refine (23) with intermediate manual model building with Coot (24). The final model was obtained by combined manual tracing (Coot) and refinement using Refmac5 (25). In the Ramachandran plot, 96.69% of the residues were located in the favored region, 2.72% in the allowed region, and 0.58% of the residues were outliers. The crystal structure of Twitch-6 was solved with PHASER (26) using PDB (Protein Data Bank) entry 6GEL as the search model. Model building and refinement were performed as described for Twitch-2B. For this mutant, 96.68% of the residues fell into the favored region of the Ramachandran plot, 2.83% fell into the allowed region, and 0.49% were outliers.

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