Computational protocol: Structural and dynamic changes associated with beneficial engineered single-amino-acid deletion mutations in enhanced green fluorescent protein

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Protocol publication

[…] Purified EGFPD190Δ and EGFPA227Δ (15 mg ml−1 in 50 mM Tris–HCl pH 8.0, 150 mM NaCl) were screened for crystal formation by the sitting-drop vapour-diffusion method with incubation at 18°C. Drops were set up with equal volumes of protein and precipitant solution (0.5 µl each). Crystals of EGFPD190Δ were obtained from 0.1 M sodium cacodylate pH 6.5, 0.2 M NaCl, 1 M sodium citrate. Mother liquor (0.5 µl) supplemented with 15–25%(v/v) ethylene glycol was added to the crystal-containing drops as a cryoprotectant and crystals were mounted and vitrified in liquid nitrogen. Crystals of EGFPA227Δ were obtained from 0.1 M MMT buffer (malic acid, MES and Tris) pH 4.0, 25%(w/v) PEG 1500. Crystals were mounted directly from mother liquor with no cryoprotectant and were vitrified. Data were collected on beamlines I03 (EGFPD190Δ) or I04 (EGFPA227Δ) at the Diamond Light Source.Data were reduced with the xia2 package (Winter, 2009), space-group assignment was performed by POINTLESS (Evans, 2006) and scaling and merging were completed with SCALA (Evans, 2006) and TRUNCATE from CCP4 (Winn et al., 2011). Initial molecular replacement for the EGFP deletion-variant structures was performed using a previously determined EGFP structure (PDB entry 4eul; Arpino, Rizkallah et al., 2012) as the search model using Phaser (McCoy et al., 2007). The structures of the EGFP deletion variants were adjusted manually using Coot (Emsley et al., 2010) and refinement of the completed molecule was carried out using REFMAC (Murshudov et al., 2011). Protein atoms were refined isotropically and anisotropically. All nonprotein atoms were refined isotropically. The above routines were used within the CCP4 package (Winn et al., 2011; http://www.ccp4.ac.uk). Graphical representations were generated with PyMOL (Schrödinger). […]

Pipeline specifications

Software tools xia2, CCP4, Coot, PyMOL
Application Protein structure analysis
Chemicals Amino Acids