All mycobacteria with sequenced genomes, except M. leprae, have a second Single Stranded DNA Binding protein (SSBb) in addition to the canonical one (SSBa). This paralogue from M. smegmatis (MsSSBb) has been cloned, expressed and purified. The protein, which is probably involved in stress response, has been crystallized and X-ray analyzed in the first structure elucidation of a mycobacterial SSBb. In spite of the low sequence identity between SSBas and SSBbs in mycobacteria, the tertiary and quaternary structure of the DNA binding domain of MsSSBb is similar to that observed in mycobacterial SSBas. In particular, the quaternary structure is 'clamped' using a C-terminal stretch of the N-domain, which endows the tetrameric molecule with additional stability and its characteristic shape. Comparison involving available, rather limited, structural data on SSBbs from other sources, appears to suggest that SSBbs could exhibit higher structural variability than SSBas do. (C) 2016 Elsevier Inc. All rights reserved.