213- After a few seconds collect the crystal from the cryo-solution
and immediately fl ash freeze by plunging into liquid nitrogen. - Cryo-cooled crystals can be stored at 100 K (−173 °C) indefi -
nitely, until required for diffraction studies and data collection.
The structure of TbMCA2 C213A was the fi rst crystal structure of a
metacaspase available in the Protein Data Bank ( http://www.pdb.
org [ 19 ]) and consequently the incorporation of a heavy atom
(HA) into the crystals was required to determine the structure
(various attempts at Molecular Replacement using caspase struc-
tures failed). However, searching for a HA to achieve this proved
to be time-consuming and laborious with all attempts at co-
crystallizing the enzyme with a heavy atom (or calcium) failing.
Crystal soaking with many of the more traditional HA compounds,
for example compounds containing mercury (e.g., pcmb, HgCl 2 ),
platinum (e.g., KPtCl 6 , KPtCl 4 ), lead (PbNO 3 ), and gold (AuCN),
proved unsuccessful typically resulting in crystal damage (and
unusable diffraction) as soon as the HA was incorporated. However,
lanthanides are often used to defi ne calcium-binding sites in pro-
teins [ 20 ] and consequently a lanthanide, in the form of samarium
acetate (SmAc), was tested as a HA derivative for the structure solu-
tion. This resulted in a fully occupied Sm 3+ binding site on the sur-
face of the molecule (Fig. 2 ), which allowed the structure to be
determined and also defi ned the Ca 2+ binding site of TbMCA2 [ 17 ].3.4.4 Sm 3+ Heavy
Atom Soak
Fig. 2 A samarium ion (Sm 3+ ) binds tightly on the surface of TbMCA2 C213A. Sm 3+
is coordinated by four aspartic acid residues (D173, D189, D190, and D220) and
defi nes the calcium-binding site in the enzymeTrypanosoma Metacaspases