Three-dimensional structures of two heavily N-glycosylated Aspergillus sp. family GH3 B-D-glucosidases

The industrial conversion of cellulosic plant biomass into useful products such as biofuels is a major societal goal. These technologies harness diverse plant degrading enzymes, classical exo- and endo-acting cellulases and, increasingly, cellulose-active lytic polysaccharide monooxygenases, to deconstruct the recalcitrant [beta]-D-linked polysaccharide. A major drawback with this process is that the exo-acting cellobiohydrolases suffer from severe inhibition from their cellobiose product. [beta]-D-Glucosidases are therefore important for liberating glucose from cellobiose and thereby relieving limiting product inhibition. Here, the three-dimensional structures of two industrially important family GH3 [beta]-D-glucosidases from Aspergillus fumigatus and A. oryzae, solved by molecular replacement and refined at 1.95 Å resolution, are reported. Both enzymes, which share 78% sequence identity, display a three-domain structure with the catalytic domain at the interface, as originally shown for barley [beta]-D-glucan exohydrolase, the first three-dimensional structure solved from glycoside hydrolase family GH3. Both enzymes show extensive N-glycosylation, with only a few external sites being truncated to a single GlcNAc molecule. Those glycans N-linked to the core of the structure are identified purely as high-mannose trees, and establish multiple hydrogen bonds between their sugar components and adjacent protein side chains. The extensive glycans pose special problems for crystallographic refinement, and new techniques and protocols were developed especially for this work. These protocols ensured that all of the D-pyranosides in the glycosylation trees were modelled in the preferred minimum-energy 4C1 chair conformation and should be of general application to refinements of other crystal structures containing O- or N-glycosylation. The Aspergillus GH3 structures, in light of other recent three-dimensional structures, provide insight into fungal [beta]-D-glucosidases and provide a platform on which to inform and inspire new generations of variant enzymes for industrial application

The Buccaneer software for automated model building

A new technique for the automated tracing of protein chains in experimental electron-density maps is described. The technique relies on the repeated application of an oriented electron-density likelihood target function to identify likely C positions. This function is applied both in the location of a few promising `seed' positions in the map and to grow those initial C positions into extended chain fragments. Techniques for assembling the chain fragments into an initial chain trace are discussed

Fast Fourier feature recognition

Various approaches have been demonstrated for the automatic interpretation of crystallographic data in terms of atomic models. The use of a masked Fourier-based search function has some benefits for this task. The application and optimization of this procedure is discussed in detail. The search function also acquires a statistical significance when used with an appropriate electron-density target and weighting, giving rise to improved results at low resolutions. Methods are discussed for building a library of protein fragments suitable for use with this procedure. These methods are demonstrated with the construction of a statistical target for the identification of short helical fragments in the electron density

On the combination of small and macro molecule techniques for the phase refinement of macromolecular structures

SIGLEAvailable from British Library Document Supply Centre- DSC:DX97536 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

The new CCP4 Coordinate Library as a toolkit for the design of coordinate-related applications in protein crystallography

The new CCP4 Coordinate Library is a development aiming to provide a common layer of coordinate-related functionality to the existing applications in the CCP4 suite, as well as a variety of tools that can simplify the design of new applications where they relate to atomic coordinates. The Library comprises a wide spectrum of useful functions, ranging from parsing coordinate formats and elementary editing operations on the coordinate hierarchy of biomolecules, to high-level functionality such as calculation of secondary structure, interatomic bonds, atomic contacts, symmetry transformations, structure superposition and many others. Most of the functions are available in a C++ object interface; however, a Fortran interface is provided for compatibility with older CCP4 applications. The paper describes the general principles of the Library design and the most important functionality. The Library, together with documentation, is available under the LGPL license from the CCP4 suite version 5.0 and higher.Peer reviewe