Journal:

Article Title: Structural analysis of adenylate cyclases from Trypanosoma brucei in their monomeric state

doi: 10.1093/emboj/20.3.433

Figure Lengend Snippet: Fig. 1. (A) Overall structure of GRESAG4.1-(A884–T1131). The Δ-subdomain between α3 and β4 that is absent in other ACs is highlighted in orange. (B) Topologies of tACs, DNA polymerase and NDP kinase. The first half of the GRESAG4.1 AC domain (β1–β4, α1, α2) superimposes at 73 Cα positions with Taq DNA polymerase (r.m.s.d. 1.9 Å) and at 42 positions with NDP kinase from D.melanogaster (r.m.s.d. 2.3 Å). The active center is only in ACs and DNA polymerases placed along β1–β4, while NDP kinases harbor the active site in a loop region. (C) σA-weighted 2Fobs – Fcalc electron density map of GRESAG4.1 at 1.46 Å resolution (contouring level 1.7 σ). (D) Dendrogram showing the phylogenetic relationship of ACs and GCs. For its calculation, 40 protein sequences of nucleotidyl cyclases were aligned using the structural alignment between the mammalian C1A and C2A domains and GRESAG4.1 and the multiple and pairwise sequence alignments as computed by PSI-Blast (Altschul et al., 1997) and Clustal_X (Thompson et al., 1997). A non-redundant subset of 22 nucleotidyl cyclase sequences was selected from the resulting multiple sequence alignment to calculate the phylogenetic relationships using PAUPSCRIPT. The sequences of the AC domains of GRESAG4.1 and GRESAG4.3 correspond to the SWISSPROT entries cy41_trybb and cy43_trybb. Figure 1A and Figures ​Figures33–5 were made with MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merrit and Murphy, 1994).

Article Snippet: The DNA fragment of GRESAG4.3 (1.09 kbp) was digested with NheI and XhoI , and the 1.11 kbp fragment of ESAG4 with Nde I and Xho I and ligated into the expression vector pET-28a (Invitrogen) using standard cloning techniques.

Techniques: Sequencing