Transcription-export complex 2 (TREX-2 complex) facilitates the localization of actively transcribing genes to the nuclear periphery and also functions to contribute to the generation of export-competent mRNPs through interactions with the general mRNA nuclear export factor Mex67:Mtr2. The TREX-2 complex is based on a Sac3 scaffold to which Thp1, Sem1, Cdc31, and Sus1 bind. TREX-2 can be subdivided into two modules: one, in which Thp1 and Semi bind to the Sac3(M) region (residues similar to 100-551), and the other in which Cdc31 and two Sus1 chains bind to the Sac3(CID) region (residues similar to 710-805). Complementary structural analyses using X-ray crystallography, electron microscopy, and small-angle X-ray scattering of the Saccharomyces cerevisiae TREX-2 complex, expressed using Baculovirus-infected Sf9 cells, have indicated that the TPR-like repeats of the Sac3(M) region extend considerably further towards the N-terminus than previously thought, and also indicate that this region and Sac3(CID):Sus1: Cdc31 region of the S. cerevisiae complex are structurally independent. Although the density visible accounted for only similar to 100 kDa, a 5.3 angstrom resolution cryo-EM reconstruction was obtained of the M-region of TREX-2 that showed an additional three putative a-helices extending towards the Sac3 N-terminus and these helices were also seen in a 4.9 angstrom resolution structure obtained by X-ray crystallography. Summary statement: We describe the expression, purification and structural characterization of the S. cerevisiae TREX-2 complex and demonstrate that the Sac3 TPR-like repeats are more extensive than previously thought and that the M- and CID-regions do not appear to have a defined spatial orientation. (C) 2016 MRC Laboratory of Molecular Biology. Published by Elsevier Inc.