title: Structure elucidation of beta-mannanase: from the electron-density map to the DNA sequence.
abstract: The crystal structure of affinity-purified Thermomonospora fusca beta-mannanase 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 design a degenerate downstream PCR primer. Together with a specific primer previously derived from the N-terminus, 95.7% of the mannanase gene sequence was obtained from genomic T. fusca DNA by PCR. The structure-derived sequence was then compared with the DNA-derived sequence and corrected when necessary. Applying the presented protocol, there was no need to manually build a model at an early stage of structure determination, an erroneous and tedious process, especially in the absence of the amino-acid sequence. Using the DNA sequence information and the current version of ARP/wARP, 281 residues, or 93% of the polypeptide chain (including side chains), were built and refined to an R factor of 16.5% without any manual intervention.
authors.affiliation: Institute of Biochemistry, Federal Institute of Technology, ETH Center, CH-8092 Zürich, Switzerland. hilge@chem.leidenuniv.nl
authors.forename: M
authors.initials: M
authors.lastname: Hilge
authors.forename: A
authors.initials: A
authors.lastname: Perrakis
authors.forename: J P
authors.initials: JP
authors.lastname: Abrahams
authors.forename: K
authors.initials: K
authors.lastname: Winterhalter
authors.forename: K
authors.initials: K
authors.lastname: Piontek
authors.forename: S M
authors.initials: SM
authors.lastname: Gloor
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA; D004583:Electrons; D008361:Mannosidases / Q000737:chemistry*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D044902:beta-Mannosidase
chemical_list: D004247:DNA; D008361:Mannosidases; D044902:beta-Mannosidase