Computational protocol: Specific GFP-binding artificial proteins (αRep): a new tool for in vitro to live cell applications

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Protocol publication

[…] All crystallization experiments were carried out at 293 K using the vapour diffusion method. Initial crystallization screening was done at three different protein concentrations (15, 10 and 5 mg·ml−1) using commercially available kits (Qiagen Classic, MB Class I, PEG II, JCSG+). The two complexes behaved rather differently during the crystallization process. The bGFP-A–EGFP complex crystallized under many different crystallization conditions, whereas for the bGFP-C–EGFP complex only one hit was obtained. Optimization of the initial hits led to the following crystallization conditions: complex bGFP-A–EGFP (0.05M MgAc, 0.1M NaAc, 5%–15% PEG 8K) and complex bGFP-C–EGFP (50 mM Tricine, pH 6.9, 25% PEG4K).Crystals were flash-frozen in liquid nitrogen by two soaking steps using mother liquor supplemented with 15% and 30% glycerol as cryoprotectant. Diffraction data were collected at 100 K on beamline PROXIMA 1 at the SOLEIL synchrotron using a PILATUS detector. The images were integrated with the XDS program [] and processed using the CCP4 program suite []. For the resolution of the structure, molecular replacement phases were obtained with Phaser, implemented in the CCP4 program suite [] using the following search models: the structure of EGFP from Aequorea victoria, PDB ID 1JBZ [] and a six-helix motif from the αRep4 structure, PDB ID 3LTJ []. The experimental map was improved by solvent modification using the program DM []. The initial models were completed and adjusted with the program COOT []. Refinement was performed using REFMAC []. The crystal structure of the bGFP-A–EGFP (complex 1) at 2 Å (1 Å=0.1 nm) resolution was refined to R and Rfree crystallographic factors of 20.5% and 26.8% respectively (). The crystal structure of the bGFP-C–EGFP (complex 2) at 3.4 Å resolution was refined to R and Rfree crystallographic factors of 20.9% and 29.2% respectively (). Atomic co-ordinates and structure factor were deposited in the Protein Data Bank under accession codes 4XL5 for the complex 1 (bGFP-A–EGFP) and 4XVP for the complex 2 (bGFP-C–EGFP) respectively. […]

Pipeline specifications

Software tools XDS, CCP4, Coot
Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma