Background: The cluster of orthologous group COG2042 has members in all sequenced Eukaryota as well as in many Archaea. The cellular function of these proteins of ancient origin remains unknown. PSI-BLAST analysis does not indicate a possible link with even remotely-related proteins that have been functionally or structurally characterized. As a prototype among COG2042 orthologs, SSO0551 protein from the hyperthermophilic archaeon Sulfolobus solfataricus was purified to homogeneity for biophysical characterization. Results: The untagged protein is thermostable and behaves as a monomeric protein in gel filtration experiment. Several mass spectrometry-based strategies were combined to obtain a set of low resolution structural information. Kinetic data from limited proteolysis with various endoproteases are concordant in pointing out that region Glu73-Arg78 is hyper-sensitive, and thus accessible and flexible. Lysine labeling with NHS-biotin and cross-linking with DTSSP revealed that the 35 amino acid RLI motif at the N terminus is solvent exposed. Cross-links between Lys10-Lys14 and Lys23- Lys25 indicate that these residues are spatially close and in adequate conformation to be crosslinked. These experimental data have been used to rank multiple three-dimensional models generated by a de novo procedure. Conclusion: Our data indicate that COG2042 proteins may share a novel fold. Combining biophysical, mass-spectrometry data and molecular model is a useful strategy to obtain structural information and to help in prioritizing targets in structural genomics programs