Structure elucidation of beta-mannanase: from the electron-density map to the DNA sequence.
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.
Hilge
M
M
Institute of Biochemistry, Federal Institute of Technology, ETH Center, CH-8092 Zürich, Switzerland. hilge@chem.leidenuniv.nl
Perrakis
A
A
Abrahams
J P
JP
Winterhalter
K
K
Piontek
K
K
Gloor
S M
SM
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
D004247:DNA; D008361:Mannosidases; D044902:beta-Mannosidase