Structure elucidation of beta-mannanase: from the electron-density map to the DNA sequence

The crystal structure of affinity-purified Thermomonospora fusca beta -mann anase has been solved despite the lack of the major part of the amino-acid sequence. A high-quality electron-density map allowed the identification of a stretch of eight amino acids close to the C-terminus which was used to d esign a degenerate downstream PCR primer. Together with a specific primer p reviously derived from the N-terminus, 95.7% of the mannanase gene sequence was obtained from genomic T. fusca DNA by PCR. The structure-derived seque nce was then compared with the DNA-derived sequence and corrected when nece ssary. Applying the presented protocol, there was no need to manually build a model at an early stage of structure determination, an erroneous and ted ious process, especially in the absence of the amino-acid sequence. Using t he DNA sequence information and the current version of ARP/wARP, 281 residu es, or 93% of the polypeptide chain (including side chains), were built and refined to an R factor of 16.5% without any manual intervention.