Extended Data Fig. 4: Crystal structure of StayGold.
From: StayGold variants for molecular fusion and membrane-targeting applications
StayGold crystallized at pH 8.5 in space group P21 and diffracted to 1.56 Å. The final model has an Rwork of 18.63% and an Rfree of 21.53%. StayGold also crystallized at pH 5.6 in space group P61 and diffracted to 2.2 Å with the final model having an Rwork of 15.47% and an Rfree of 18.97%. See Supplementary Table 1 for a summary of the refinement statistics and model quality. The coordinates and structure factors have been deposited in the PDB data base (pH 8.5, entry ID: 8ILK; pH 5.6, entry ID: 8ILL). a, Superposition of the α-carbon backbone and chromophore (A protomer) of the two crystal structures (pH 8.5, cyan; pH 5.8, magenta). The root-mean-square deviation value was calculated to be 0.664 Å. M1 and L217 indicate the N- and C-terminal ends, respectively. Because of its higher resolution, the pH 8.5 crystal structure is used hereafter for characterizing the atomic structure of StayGold. b, The chromophore and surrounding side chains represented in stick format. Hydrogen bonds are represented as broken lines with indicated distances in angstroms. Water molecules are indicated as red spheres. c, Four proline residues (Pro136, Pro143, Pro151, and Pro157) located on the seventh and eighth β-strands, which are close to the hydroxy group of the p-hydroxyphenyl ring of the chromophore in the cis conformation (stick representation). These prolines may rigidify this region of the β-barrel, thus stabilizing the chromophore in the cis state. It is noted that most fluorescent proteins have only one proline in the corresponding region. The side chain of Val152 may also contribute to the stabilization of the chromophore. During the crystal structure refinement, placement of a chloride ion was found to account for the peak observed in the Fo–Fc map near the chromophore. The chloride ion (indicated as a magenta sphere) is expected to sterically prevent the chromophore from adopting a trans conformation. d, The chromophore (represented in the space-filling format) is positioned in its cis (left) or trans (right) conformation. In the latter case, there is an apparent collision against the chloride ion. b–d, The atoms are color-coded as follows: carbon, white; oxygen, red; nitrogen, blue; sulfur, yellow.
