Biomedical subjects
Publications and source records attributed to Janet Newman.
There are a number of published reports of techniques that may extend the diffraction limits or otherwise improve the quality of the X-ray diffraction data from a crystal. This review touches on some of these techniques to provide a way forward for the experimentalist burdened with less than ideal crystals.
A database application has been developed for the collection of crystallographic information. This database (the BDP) has been populated with the information found in the Protein Data Bank (PDB). The tool has been used to store crystallization data parsed out of the PDB and these data may be used to extend the crystallization information found in the Biological Macromolecule Crystallization Database (BMCD) and could be used to refine crystallization methodology. A standard is proposed for describing a crystallization experiment that will ease future crystallization data collations and analyses.
A crystallization screening process is presented that was developed for a small academic laboratory. Its underlying concept is to combine sparse-matrix screening with systematic screening in a minimum number of crystallization conditions. The sparse-matrix screen is the cherry-picked combination of conditions from the Joint Center for Structural Genomics (JCSG) extended using conditions from other screens. Its aim is to maximize the coverage of crystallization parameter space with no redundancy. The systematic screen, a pH-, anion- and cation-testing (PACT) screen, aims to decouple the components of each condition and to provide information about the protein, even in the absence of crystals, rather than cover a wide crystallization space. This screening strategy is combined with nanolitre-volume dispensing hardware and a small but practical experiment-tracking system. The screens have been tested both at the NKI and in other laboratories and it is concluded that they provide a useful minimal screening strategy.
Setting up vapor-diffusion crystallization experiments against four different reservoir solutions showed that the reservoir solution may have a profound effect on the outcome of a crystallization experiment. This suggests that a facile way to increase crystallization space through screening is not to add more crystallization conditions to the process, but to set up the same conditions over different reservoirs.
In protein crystallization, screening is initially performed to obtain an indication of the conditions under which a macromolecule might crystallize. These preliminary conditions are then optimized to produce (in a perfect world) well diffracting crystals; this process of optimization often involves fine grid screening around the initial conditions. An issue in optimization is to find factors which are independent, so as to simplify the analysis of the results of optimization trials. This is necessarily difficult with buffers, as a buffer and its pH range tend to be very highly correlated. Multi-buffer systems for pH modulation are presented which enable a broad pH range to be sampled without changing the chemical composition of the buffering component.
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In contrast to academic pursuits of structural genomics, Structural GenomiX (SGX) solves protein structures at high throughput for the main purpose of enhancing drug-discovery projects, either internally or in partnership with pharmaceutical/biotechnology companies. This involves a radical redesign of the pipeline of methods that turn a gene sequence into a three-dimensional protein structure. The various processes all report electronically to a Laboratory Information Management System (LIMS) to make sure all the parameters of the experiment are recorded in an accessible and 'mineable' form, helping guarantee reproducibility of results. Quality control at several key points keeps the process from branching out on a wrong hypothesis. Protein annotation, in a broad sense, takes care of the interpretation of a protein crystal structure or the crystal structure of one or several protein-ligand complexes. This interpretation both gathers all necessary biological information (protein function, mechanism, specific features within a protein family etc.) and hands over this information in a form accessible to medicinal chemistry teams designing specific small-molecule agonists or antagonists.