Structure determination and refinement of the Humicola insolens endoglucanase V at 1.5 angstrom resolution

G J Davies, G Dodson, M H Moore, S P Tolley, Z Dauter, K S Wilson, G Rasmussen, M Schulein

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Abstract

The structure of the catalytic core of the endoglucanase V (EGV) from Humicola insolens has been determined by the method of multiple isomorphous replacement at 1.5 Angstrom resolution. The final model, refined with X-PLOR and PROLSQ, has a crystallographic R factor of 0.163 (R(free) = 0.240) with deviations from stereochemical target values of 0.012 Angstrom and 0.037 degrees for bonds and angles, respectively. The model was further refined with SHELXL, including anisotropic modelling of the protein-atom temperature factors, to give a final model with an R factor of 0.105 and an R(free) of 0.154. The initial isomorphous replacement electron-density map was poor and uninterpretable but was improved by the use of synchrotron data collected at a wavelength chosen so as to optimize the f '' contribution of the anomalous scattering from the heavy atoms. The structure of H. insolens EGV consists of a six-stranded beta-barrel domain, similar to that found in a family of plant defence proteins, linked by a number of disulfide-bonded loop regions. A long open groove runs across the surface of the enzyme either side of which lie the catalytic aspartate residues. The 9 Angstrom separation of the catalytic carboxylate groups is consistent with the observation that EGV catalyzes the hydrolysis of the cellulose beta(1-->4) links with inversion of configuration at the anomeric C1 atom. This structure is the first representative from the glycosyl hydrolase family 45.

Original languageEnglish
Pages (from-to)717
Number of pages11
JournalActa Crystallographica. Section D, Biological Crystallography
Volume52
Publication statusPublished - 1 Jan 1996

Keywords

  • ACID-SEQUENCE SIMILARITIES
  • NUCLEAR-MAGNETIC-RESONANCE
  • CELLULOSE-BINDING DOMAIN
  • ELECTRON-DENSITY MAPS
  • C-TERMINAL DOMAIN
  • 3-DIMENSIONAL STRUCTURE
  • BARLEY SEED
  • TRICHODERMA-REESEI
  • PROTEIN MODELS
  • FAMILIES

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