“Growing lysozyme crystals to the size of mm3 for neutron diffraction”
Jens-Chr. N. Poulsen, Tobias Tandrup, Annette E. Langkilde, Sine Larsen
Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Kbh. Ø, Denmark
jnp@chem.ku.dk
Crystallization remains a bottleneck for protein structure determination, and it can be a challenge to get crystals that are of a size that enables structure determination by use of X-ray crystallography. Crystals of a size around 105 µm3 are sufficient for data collections at most synchrotrons, but with the use of microfocus beamlines crystals smaller than 103 µm3 are used for data collection and structure determination. The X-ray synchrotron beamlines have been automated and improved so data collections can be performed in minutes. Compared to this there are major challenges associated with obtaining crystals of a size that will enable structural studies at high resolution of proteins by neutron diffraction. Here mm3 size protein crystals are needed and it requires days of data collection.
To compare high resolution data (better than 1.2 Å resolution) collected on crystals of Hen Egg White Lysozyme (HEWL) from both an X-ray source and a neutron source, triclinic crystals of HEWL are required that should be larger than 5.0 mm3 in size.
Triclinic crystals of HEWL are difficult to obtain, but following the procedures by Heijna et al and Legrand et al a reproducible path for making triclinic HEWL was achieved. The procedure involves a precipitation step at 4°C, before crystal growth at room temperature.
Optimizing batch crystallization conditions starting with 0.2 M NaNO3, 0.05 M Na-acetate pH 4.5 and a protein concentration in the drop of 10 mg/ml, using repeated serial macro seeding and months of growing we have obtained more than 10 crystals bigger than 5 mm3 for planned neutron diffraction experiments at ILL, Grenoble.
