Structure elucidation of beta-mannanase: from the electron-density map to the DNA sequence.
Acta Crystallogr D Biol Crystallogr
; 57(Pt 1): 37-43, 2001 Jan.
Article
em En
| MEDLINE
| ID: mdl-11134925
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Manosidases
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Acta Crystallogr D Biol Crystallogr
Ano de publicação:
2001
Tipo de documento:
Article
País de afiliação:
Suíça