Abstract
BackgroundComparative genomic analysis has revealed that in each genome a large number of open reading frames have no homologues in other species. Such singleton genes have attracted the attention of biochemists and structural biologists as a potential untapped source of new folds. Cthe_2751 is a 15.8 kDa singleton from an anaerobic, hyperthermophile Clostridium thermocellum. To gain insights into the architecture of the protein and obtain clues about its function, we decided to solve the structure of Cthe_2751.ResultsThe protein crystallized in 4 different space groups that diffracted X-rays to 2.37 Å (P3121), 2.17 Å (P212121), 3.01 Å (P4122), and 2.03 Å (C2221) resolution, respectively. Crystal packing analysis revealed that the 3-D packing of Cthe_2751 dimers in P4122 and C2221 is similar with only a rotational difference of 2.69° around the C axes. A new method developed to quantify the differences in packing of dimers in crystals from different space groups corroborated the findings of crystal packing analysis. Cthe_2751 is an all α-helical protein with a central hydrophobic core providing thermal stability via π:cation and π: π interactions. A ProFunc analysis retrieved a very low match with a splicing endonuclease, suggesting a role for the protein in the processing of nucleic acids.ConclusionsNon-Pfam singleton Cthe_2751 folds into a known all α-helical fold. The structure has increased sequence coverage of non-Pfam proteins such that more protein sequences can be amenable to modelling. Our work on crystal packing analysis provides a new method to analyze dimers of the protein crystallized in different space groups. The utility of such an analysis can be expanded to oligomeric structures of other proteins, especially receptors and signaling molecules, many of which are known to function as oligomers.
Highlights
One of the perplexing outcomes of sequencing of a number of genomes is the discovery of a large set of open reading frames (ORFs) in each genome that have no homologues in other species
Singletons have attracted the attention of evolutionary biologists, biochemists and structural biologists regarding their origin, functional significance and the possibility that they may carry a relatively untapped source of new folds
A mutation in the singleton fln gene that encodes protein for a thick filament in flight muscle results in a viable but flightless fly [10]; a mutation in the circadian rhythm to gene produces a rhythm defective fly [11]. All these functions of singletons are expected to play a role in the fly’s response to specific ecological or environmental challenges. These examples underscore the fact that singletons are expressed as proteins and of 2-fold symmetry related molecules for space group P4122 and C2221
Summary
One of the perplexing outcomes of sequencing of a number of genomes is the discovery of a large set of open reading frames (ORFs) in each genome that have no homologues in other species. A mutation in the singleton fln gene that encodes protein for a thick filament in flight muscle results in a viable but flightless fly [10]; a mutation in the circadian rhythm to gene produces a rhythm defective fly [11] All these functions of singletons are expected to play a role in the fly’s response to specific ecological or environmental challenges. Comparative genomic analysis has revealed that in each genome a large number of open reading frames have no homologues in other species Such singleton genes have attracted the attention of biochemists and structural biologists as a potential untapped source of new folds. To gain insights into the architecture of the protein and obtain clues about its function, we decided to solve the structure of Cthe_2751
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