Structure Determination

Crystal Structure of MraY 

As explained in the paper ‘Crystal Structure of MraY, an Essential Membrane Enzyme for Bacterial Cell Wall Synthesis' by (Chung et al., 2013) it explains the process they took to develop a stable and crystalline crystal of MraY which was then suitable to X-ray diffraction in order to build an electron density map and determine the structure.

Aquifex aeolicus (platinum shadowed).
K.O. Stetter & Reinhard Rachel, University of Regensburg

They first screened 19 different species of bacteria in order to identify a strain that produced the MraY which was most biochemically stable and thus suitable for structural and functional studies. Aquifex aeolicus was the chosen bacterium and thus the protein from this strain is termed MraY_AA.

Previous papers describe the intrinsic refractory nature to overexpression of this protein (Bouhss et al., 2004) . Chung et al achieved this, by modifying the gene at the molecular level so when the protein is expressed it is infact a fusion protein, consisting of a decahistidine-maltose binding protein domain (His-MBP). This poly-histidine tag aids in the purification of the protein through the highly specific reversible interactions that it forms with chelating Nickel ions (Ni²⁺), or in this case Cobalt ions (Co²⁺), immobilized on an affinity chromatography column. This separates the target protein from the rest of the bacterial lysate, which can then be eluted by the addition of excess elution buffer containing imidazole. This elution buffer acts as a metal ion ligand, out-competing the histidine side chains for the cobalt on the column. 

Figure 1: Affinity chromatography procedure used in the purification of MraYAA

The Maltose binding protein region was added to aid in the solubility of the protein, to prevent its aggregation once it is removed from its native cell membrane when the bacterial cell is lysed. Just after this region on the protein there was a PreScission protease cleavage site added, which enabled the removal of the His-MBP after purification, which is not necessary for the crystallization step and not a representation of the protein structure. The final purification technique undertaken was Gel Filtration, which essentially discriminates against size and shape to ensure maximum homogeneity of the MraY_AA protein (Chung et al., 2013) .

Figure 2: Gel Filtration chromatography procedure used in the final purification steps of MraYAA

The crystallization process was tried and tested using numerous conditions and they found that magnesium based crystallization solution gave optimum results, when the protein was crystallized over the course of 7 to 14 days at 17^oC (Chung et al., 2013) . This produced large crystals containing the MraY_AA which were then suitable for X-ray diffraction analysis. 

The results of the diffraction pattern with a resolution of 3.9 Å were interpreted using the RESOLVE program to formulate an electron density map, which contained 10 discernible transmembrane helices. Then through further manipulation, refinement and calculation of phases using single anomalous dispersion (MR-SAD) the overall 3 dimensional structure was created.

The crystal structure of MraY revealed that the asymmetric unit consists of MraYAA as a dimer (Chung et al., 2013) . As is the case with X-ray crystallization, protein conformation can be disrupted, due to the high protein concentration used in the process which often results in interactions between adjacent molecules which otherwise would not occur. Thus, to ensure that protein function was not compromised and prove that the shape has not drastically changed they soaked the crystal in its binding ligands (Mg²⁺ and Ni²⁺) and proved that it still had a non-disrupted active site (Chung et al., 2013) .