Crystallization and preliminary X-ray analysis of a monomeric mutant of Azami-Green (mAG), an Aequorea victoria green fluorescent protein-like green-emitting fluorescent protein from the stony coral Galaxea fascicularis

Abstract

Monomeric Azami-Green (mAG) from the stony coral Galaxea fascicularis is the first monomeric green-emitting fluorescent protein that is not a derivative of Aequorea victoria green fluorescent protein (avGFP). mAG and avGFP are 27% identical in amino-acid sequence. Diffraction-quality crystals of recombinant mAG were obtained by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The mAG crystal diffracted X-rays to 2.20 A resolution on beamline AR-NW12A at the Photon Factory (Tsukuba, Japan). The crystal belonged to space group P1, with unit-cell parameters a = 41.78, b = 51.72, c = 52.89 A, alpha = 90.96, beta = 103.41, gamma = 101.79 degrees. The Matthews coefficient (V(M) = 2.10 A(3) Da(-1)) indicated that the crystal contained two mAG molecules per asymmetric unit.

Figure 1

Amino-acid sequence alignment of the mAG mutant that was used in this study, mAG, AG and avGFP. The three point mutations, Y123T, Y188A and F190K, introduced to produce mAG from AG are indicated by white arrowheads. The four amino-acid substitutions, R149E, A160R, D182E and V191I, between mAG and the mAG mutant are indicated by black arrowheads. This figure was generated by ESPript (Gouet et al., 1999 ▶).

Figure 2

A diffraction-quality crystal of mAG (indicated by an arrow) together with thin crystals.

Figure 3

(a) A diffraction image of the mAG crystal after annealing. The ring corresponds to a resolution of 2.20 Å. (b) A diffraction image of the mAG crystal before annealing.