Crystallisation terminology used in C3
There are a number of concepts used in protein crystallisation – it is useful to be able to define what the various parts of an experiment are, and to have standard nomenclature for the different aspects of protein crystallisation. People have been trying to crystallise proteins for over 100 years, and during this long history lots of redundancy and ambiguity has crept into the field. Below we define terms that we use in C3 – there are very likely other terms in use for the same concepts, but the ones defined below should be well understood by all.
Block: A block is a 96 well deepwell container, which is used to store a screen, the physical interpretation of a design.
Buffer: This is often used interchangably with ‘formulation’, however, in C3 we use ‘buffer’ to mean a chemical and its conjugate which are used to make a solution that resists change in pH. Tris base with hydrochloric acid (tris chloride) is a buffer. A buffer has one or more pKas, and the buffering range of a buffer is accepted to be pKa +/- 1 pH unit.
Co-factor: An agent known to be important for biological activity of a protein – these can be as simple as a divalent cation or more complex small molecules. Addition of a co-factor to a sample may enhance a proteins crystallisability.
Crystal: Something that resembles a protein crystal. Until proven otherwise, it may be an ionic compound (salt), a phase interface concentration of protein, an insoluble additive, etc.
Crystallant: This is a mixture of chemicals, in C3 typically identical to a reservoir solution, but possibly containing chemicals (detergents, active molecules, etc) that are not present in the reservoir solution.
Crystallisation plate: A 96 well plate which contains a screen, and an array of droplets, consisting of sample mixed with reservoir solution, which is used to test for protein crystallisation. The ratio of the sample to the reservoir solution in the droplet can affect the outcome of the experiment, as can the total volume of the droplet.
Design: The description (in terms of chemicals and concentration) of a screen. For example, the design for a screen which contains just one reservoir solution might be:
- 20 %(w/v) polyethylene glycol 6000
- 0.20 M ammonium chloride
- 0.1 M sodium HEPES pH 7
(Design) Matrix_design: The term for a design used by the vendor Rigaku, so we use the term ‘matix_design’ on our online booking wizard
Differential Scanning Fluorimetry (DSF): See Thermofluor and Meltdown
Formulation: The combination of chemicals that is found in the sample, excluding the protein, is called the formulation. In C3 we will commonly talk about optimising a formulation to enhance the stability and longevity of a protein. A common formulation would be tris- or phosphate buffered saline.
Imaging schedule: The automated process that is used in C3 to take pictures (both visible and UV) of the drops on a crystallisation plate over a defined time course at a fixed incubation temperature.
Inhibitor: A chemical agent known to inhibit function of a protein – adding an inhibitor to a sample may increase conformational stability of the protein.
LCP: Lipidic cupic phase is used to create a physical scaffold that mimics a cell membrane, a method commonly used to aid the crystallisation process for membrane proteins.
Meltdown: Software developed by our UROP sponsored students that automatically processes the results from thermal shift assay (a.k.a. thermofluor, DSF).
Optimisation: To set up crystallisation plates with refined screens, modified to incorporate information obtained through the screening process. Optimisation screens may consist of additive screens, where a single reservoir solution (base condition) is doped with different chemicals, or might consist of a grid screen or random screen containing a more limited combination of stocks than is found in an initial screen.
Protein: A chain of amino acids. It can be described in terms of a sequence – the chain of amino acid residues that make up the protein.
Protein crystal: A crystal form that has been shown with some certainty to be made from protein.
Proteolysis: Application of a limited proteolytic agent (in C3 we use trypsin and chymotrypsin) to remove mobile (floppy) regions of a protein which may be decreasing the chance of crystallisation.
Recipe: A description of how to make a screen, in terms of stock solutions and volumes of those stock solutions. For a screen containing one reservoir solution, which has a design 20 %(w/v) polyethylene glycol 6000, 0.20 M ammonium chloride, 0.1 M sodium HEPES pH 7, a recipe (to make 1 ml) might be
- 400.0 µL of polyethylene glycol 6000 (50% w/v)
- 83.1 µL of sodium HEPES pH 6.5 (1M)
- 200.0 µL of ammonium chloride (1M)
- 16.9 µL of sodium HEPES pH 8.5 (1M)
- 300.0 µL of H2O
Reservoir Solution: This is a mixture of chemicals, most often consisting of a salt, a polymer such as polyethylene glycol and a buffer which is used in a crystallisation experiment.In C3, the reservoir is usually the primary crystallant.
Sample: A bio-macromolecule (typically a protein) ready for experiments. The sample will contain one or more protein(s) at a certain concentration, in protein crystallisation, the concentration is given as milligrams per millilitre (mg/mL). The sample may contain things other than protein – there may be general chemicals added to the sample (salt, or a buffer) or there might be specific small molecules (inhibitors, co-factors) added
Screen: A set of reservoir solutions, in a defined order. Essentially all of the screens used in C3 contain 96 reservoir solutions, arrayed in an 8 x 12 pattern, numbered from A01→ H12. This is interchangeable with the word Kit.
Screening: To set up one or more crystallisation plates, with general screens, to determine if a protein will crystallise, and the conditions under which it will crystallise.
Seeding: Dispensing small volumes of crushed protein crystals into the drops on a crystallisation plate; seeding may increase the probability of nucleation.
Stock solution: Chemicals in solution which are mixed together to form reservoir solutions. A stock solution most often contains a single chemical species, although buffer stocks will contain at least two chemicals (an acid and its conjugate base, for example) and some stocks solutions are defined mixtures of chemicals, (phosphate buffered saline, for example).
Thermofluor: A fluorescent dye based thermal melt analysis (differential scanning fluorimetry) used to optimise the formulation that a protein sample is stored in, with potential to define ligands that bind to the protein active site (co-factors, inhibitors).
Vapour diffusion: The process that C3 applies most commonly to drive proteins out of solution and into a protein crystal form.