Journal:Acta Cryst D:S2059798319009008

Combining rMMS and the Magic Triangle for the efficient structure solution of a bacteriophage P68 lysin

Jia Quyen Truong, Santosh Panjikar, Linda Shearwin, John Bruning and Keith Shearwin ^[1]

Molecular Tour
Solving a protein crystal structure without a homologous template involves experimental phasing. Ultimately, this requires the incorporation of heavy atoms into the lattice of the protein crystal. However, this process can be very laborious as suitable heavy atom compounds and conditions are found empirically through screening. A method to efficiently derivatize crystals was developed, by performing a Random Microseed Matrix screen (rMMS) in the presence of 5-amino-2,4,6-triiodoisophthalic acid (I3C).

In a random microseed matrix, a crystal or crystalline precipitate is crushed up, diluted and added to new crystallization conditions from a sparse matrix screen. This often generates new crystallization where crystals can grow. I3C is compatible with many crystallization reagents and provides a large phasing power.

In the two test proteins, the Orf11 NTD from Staphylococcus phage P68 (PDB entry 6o43) and Hen Egg White Lysozyme (PDB entry 6pbb), the rMMS technique in the presence of I3C generated numerous new conditions where derivatized crystals could grow. The I3C molecules in the lattice were sufficient to completely solve the structure using anomalous phasing methods.

I3C was present in the tested crystals in the rMMS screens containing I3C, where they often sit at the junctions between protein molecules in the lattice. These bridging contacts could improve the crystal lattice or generate new conditions where crystals can grow.

I3C can mediate contacts between protein molecules in a crystal:

• One I3C molecule from the lysozyme crystal.
• I3C molecule from Orf11 NTD crystal (1st orientation).
• I3C molecule from Orf11 NTD crystal (2nd orientation).

The same I3C molecule from the Orf11 NTD crystal is displayed in two different orientations for clarity. I3C-protein interactions include hydrogen bonding, π-π stacking interactions, salt bridges, water bridges and halogen bonding. In both cases, each I3C molecule forms interactions with three different proteins molecules (each shown in a different color), which could assist in lattice packing.

PDB references: Crystal structure of a lysin protein from Staphylococcus phage P68 6o43; Crystal structure of Hen Egg White Lysozyme in complex with I3C 6pbb.

References

1. ↑ Truong JQ, Panjikar S, Shearwin-Whyatt L, Bruning JB, Shearwin KE. Combining random microseed matrix screening and the magic triangle for the efficient structure solution of a potential lysin from bacteriophage P68. Acta Crystallogr D Struct Biol. 2019 Jul 1;75(Pt 7):670-681. doi:, 10.1107/S2059798319009008. Epub 2019 Jul 2. PMID:31282476 doi:http://dx.doi.org/10.1107/S2059798319009008