Triosephosphate isomerase (TIM) is a dimeric enzyme consisting of 2 identical subunits. Trypanosoma1 TIM can be crystallized in 4 different spacegroups: P212121, C2(big cell), C2(small cell), and P1. The P1 crystal form only grows in the presence of 1.4 M DMSO; there are 2 DMSO binding sites per subunit. The structures have been refined at a resolution of 1.83 A, 2.10 A, 2.13 A, and 1.80 A, respectively. In the 4 different spacegroups the TIM subunit can be observed in the context of 7 different crystallographic environments. In the C2 cells, the dimer 2-fold axis coincides with a crystallographic 2-fold axis. The similarities and differences of the 7 subunits are discussed. In 6 subunits the flexible loop (loop 6) is open, whereas in the P21212, cell, the flexible loop of subunit 2 is in an almost closed conformation. The crystal contacts in the 4 different crystal forms are predominantly generated by polar residues in loops. A statistical analysis of the residues involved in crystal contacts shows that, in particular, serines are frequently involved in these interactions; 19% of the exposed serines are involved in crystal contacts.Keywords: crystal contacts; DMSO; mutagenesis; flexible loop; TIM Crystal structures of trypanosomal triosephosphate isomerase (TIM) have already been described for 2 different crystal forms (Wierenga et al., 1991b;Noble et al., 1993b). The structures of TIM of chicken (Banner et al., 1975), yeast (Lolis et al., 1990, and Escherichia coli (Noble et al., 1993a) are also known. TIM is a dimeric glycolytic enzyme, consisting of 2 identical subunits. Each subunit has the regular (@a)8-fold, with 8 parallel @-strands in the center, covered by 8 a-helices on the outside. The loops between the @-strands and the subsequent helices, referred to as loop 1-loop 8, are important for dimer formation and catalysis. A characteristic property of TIM is the conformational flexibility of loop 6, also referred to as the flexible loop. This loop has been observed in open (unliganded) and closed (liganded) conformations. There is a 7-A movement at the tip of this loop between the open and closed conformations (Joseph et al., 1990 (Wierenga et al., 1991b). The comparison (Wierenga et al., 1992) of the open and closed TIM structures has shown that the open structures of chicken, yeast, and trypanosomal TIM are essentially identical to each other; also the closed structures of yeast and trypanosoma1 TIM are essentially identical. In the open structures of TIM, the tip of loop 6 is sometimes seen to be highly flexible. Despite the structural heterogeneity associated with this flexibility, unliganded trypanosomal TIM can be crystallized readily into well-diffracting crystals, under different conditions and in different crystal forms. Previously the refined crystal structures of a P212121 form, crystallized in spacegroup P212121, and a C2 form, crystallized in spacegroup C2, have been discussed. The asymmetric unit of the P212121 form is 1 dimer. Due to the coincidence of dimer and crystallographic 2-f...