Sample optimizations to enable the structure determination of biotin-dependent carboxylases.

Biotin-dependent carboxylases have central roles in the metabolisms of fatty acids, amino acids and other compounds. Their functional importance is underscored by their strong conservation from bacteria to humans. These enzymes are large, multi-domain or multi-subunit complexes, and can have molecular weights of 500 to 750 kDa. Despite their large sizes, the first structures of most of these enzymes were determined using X-ray crystallography. This chapter presents various technical challenges that were overcome during their structure determination, which involves extensive optimization of the protein samples and their crystals. The cryo electron microscopy resolution revolution has made it easier to study these large complexes at the atomic level.