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

Hilge, M. and Perrakis, A. and Abrahams, J. P. and Winterhalter, K. and Piontek, K. and Gloor, S. M.. (2001) Structure elucidation of beta-mannanase: from the electron-density map to the DNA sequence. Acta Crystallographica. Section D, Biological Crystallography, 57. pp. 37-43.

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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.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Nano-diffraction of Biological Specimen (Abrahams)
UniBasel Contributors:Abrahams, Jan Pieter
Item Type:Article, refereed
Article Subtype:Research Article
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:06 Nov 2020 07:38
Deposited On:06 Nov 2020 07:38

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