Abstract: The automated building of a protein model into an electron density map remains a challenging problem. In the ARP/wARP approach, model building is facilitated by initially interpreting a density map with free atoms of unknown chemical identity; all structural information for such chemically unassigned atoms is discarded. Here, this is remedied by applying restraints between free atoms, and between free atoms and a partial protein model. These are based on geometric considerations of protein structure and tentative (conditional) assignments for the free atoms. Restraints are applied in the REFMAC5 refinement program and are generated on an ad hoc basis, allowing them to fluctuate from step to step. A large set of experimentally phased and molecular replacement structures showcases individual structures where automated building is improved drastically by the conditional restraints. The concept and implementation we present can also find application in restraining geometries, such as hydrogen bonds, in low-resolution refinement.
Abstract: ProteinCCD (CCD for Crystallographic Construct Design) aims to facilitate a common practice in structural biology, namely the design of several truncation constructs of the protein under investigation, based on experimental data or on sequence analysis tools. ProteinCCD functions as a meta-server, available online at http://xtal.nki.nl/ccd, that collects information from prediction servers concerning secondary structure, disorder, coiled coils, transmembrane segments, domains and domain linkers. It then displays a condensed view of all results against the protein sequence. The user can study the output and choose interactively possible starts and ends for suitable protein constructs. Since the required input to ProteinCCD is the DNA and not the protein sequence, once the starts and ends of constructs are chosen, the software can automatically design the oligonucleotides needed for PCR amplification of all constructs. ProteinCCD outputs a comprehensive view of all constructs and all oligos needed for bookkeeping or for direct copy-paste ordering of the designed oligonucleotides.
Abstract: A procedure for analyzing and classifying publicly available crystal structures has been developed. It has been used to identify high-resolution protein-ligand complexes that can be assessed by reconstructing the electron density for the ligand using the deposited structure factors. The complexes have been clustered according to the protein sequences, and clusters have been discarded if they do not represent proteins thought to be of direct interest to the pharmaceutical or agrochemical industry. Rules have been used to exclude complexes containing non-drug-like ligands. One complex from each cluster has been selected where a structure of sufficient quality was available. The final Astex diverse set contains 85 diverse, relevant protein-ligand complexes, which have been prepared in a format suitable for docking and are to be made freely available to the entire research community (http://www.ccdc.cam.ac.uk). The performance of the docking program GOLD against the new set is assessed using a variety of protocols. Relatively unbiased protocols give success rates of approximately 80% for redocking into native structures, but it is possible to get success rates of over 90% with some protocols.
Abstract: An approach to automate protein-ligand crystallography is presented, with the aim of increasing the number of structures available to structure-based drug design. The methods we propose deal with the automatic interpretation of diffraction data for targets with known protein structures, and provide easy access to the results. Central to the system is a novel procedure that fully automates the placement of ligands into electron density maps. Automation provides an objective way to structure solution, whereas manual placement can be rather subjective, especially for data of low to medium resolution. Ligands are placed by docking into electron density, whilst taking care of protein-ligand interactions. The ligand fitting procedure has been validated on both public domain and in-house examples. Some of the latter deal with cocktails of low-molecular weight compounds, as used in fragment-based drug discovery by crystallography. For such library-screening experiments we show that the method can automatically identify which of the compounds from a cocktail is bound.
Abstract: We present a novel atom-atom potential derived from a database of protein-ligand complexes. First, we clarify the similarities and differences between two statistical potentials described in the literature, PMF and Drugscore. We highlight shortcomings caused by an important factor unaccounted for in their reference states, and describe a new potential, which we name the Astex Statistical Potential (ASP). ASP's reference state considers the difference in exposure of protein atom types towards ligand binding sites. We show that this new potential predicts binding affinities with an accuracy similar to that of Goldscore and Chemscore. We investigate the influence of the choice of reference state by constructing two additional statistical potentials that differ from ASP only in this respect. The reference states in these two potentials are defined along the lines of Drugscore and PMF. In docking experiments, the potential using the new reference state proposed for ASP gives better success rates than when these literature reference states were used; a success rate similar to the established scoring functions Goldscore and Chemscore is achieved with ASP. This is the case both for a large, general validation set of protein-ligand structures and for small test sets of actives against four pharmaceutically relevant targets. Virtual screening experiments for these targets show less discrimination between the different reference states in terms of enrichment. In addition, we describe how statistical potentials can be used in the construction of targeted scoring functions. Examples are given for cdk2, using four different targeted scoring functions, biased towards increasingly large target-specific databases. Using these targeted scoring functions, docking success rates as well as enrichments are significantly better than for the general ASP scoring function. Results improve with the number of structures used in the construction of the target scoring functions, thus illustrating that these targeted ASP potentials can be continuously improved as new structural data become available.
Abstract: This study addresses a number of topical issues around the use of protein-ligand docking in virtual screening. We show that, for the validation of such methods, it is key to use focused libraries (containing compounds with one-dimensional properties, similar to the actives), rather than "random" or "drug-like" libraries to test the actives against. We also show that, to obtain good enrichments, the docking program needs to produce reliable binding modes. We demonstrate how pharmacophores can be used to guide the dockings and improve enrichments, and we compare the performance of three consensus-ranking protocols against ranking based on individual scoring functions. Finally, we show that protein-ligand docking can be an effective aid in the screening for weak, fragment-like binders, which has rapidly become a popular strategy for hit identification. All results presented are based on carefully constructed virtual screening experiments against four targets, using the protein-ligand docking program GOLD.
Abstract: The first collaborative workshop on crystal structure prediction (CSP1999) has been followed by a second workshop (CSP2001) held at the Cambridge Crystallographic Data Centre. The 17 participants were given only the chemical diagram for three organic molecules and were invited to test their prediction programs within a range of named common space groups. Several different computer programs were used, using the methodology wherein a molecular model is used to construct theoretical crystal structures in given space groups, and prediction is usually based on the minimum calculated lattice energy. A maximum of three predictions were allowed per molecule. The results showed two correct predictions for the first molecule, four for the second molecule and none for the third molecule (which had torsional flexibility). The correct structure was often present in the sorted low-energy lists from the participants but at a ranking position greater than three. The use of non-indexed powder diffraction data was investigated in a secondary test, after completion of the ab initio submissions. Although no one method can be said to be completely reliable, this workshop gives an objective measure of the success and failure of current methodologies.
Abstract: A collaborative workshop was held in May 1999 at the Cambridge Crystallographic Data Centre to test how well currently available methods of crystal structure prediction perform when given only the atomic connectivity for an organic compound. A blind test was conducted on a selection of four compounds and a wide range of methodologies representing, the principal computer programs currently available were used. There were 11 participants who were allowed to propose at most three structures for each compound. No program gave consistently reliable results. However, seven proposed structures were close to an experimental one and were classified as "correct". One compound occurred in two polymorphs, but only one form was predicted correctly among the calculated structures. The basic problem with lattice energy based methods of crystal structure prediction is that many structures are found within a few kJ mol(-1) of the global minimum. The fine detail of the force-field methodology and parametrization influences the energy ranking within each method. Nevertheless, present methods may be useful in providing a set of structures as possible polymorphs for a given molecular structure.
Abstract: ossible crystal structures of benzene were generated without any prior crystallographic information, using a systematic grid search method. Only structures with one molecule in the asymmetric unit were considered. 31 space groups were investigated, and the resulting structures were clustered and checked for stability upon removal of the space-group symmetry. In the enthalpy range 10 kJ mol-1 30 structures were found at zero pressure and 20 structures at a pressure of 30 kbar. Their pressure-dependent rankings and interconversions are discussed. The results are compared to previous, less complete, investigations. A possible structure for the high-pressure phase benzene (II), on which only limited powder diffraction data have been published, is suggested.
