Protein Crystallisation Strategies, optimisation and non-standard methods

Overview First steps in crystallising proteins Available screens Screening strategies Considering the leads Optimisation experiment designs Non-standard methods Practical methods Simple non-standard techniques Microbatch gels

First steps in crystallising proteins Finding crystallising conditions for your protein It is necessary to screen a broad range of conditions to determine the solubility of your protein At this stage insoluble protein is observed as amorphous precipitate In the second optimisation stage potential conditions are explored Having outlined the theoretical areas I would like to introduce some practical aspects, how do we go about getting crystals of our material? In this section I will be considering what screens are available, their merits and drawbacks, suggesting how to identify leads and how best to follow them once you have identified them, and finally how to adapt the screens to manage problem proteins or shortage of material. Finding crystallising conditions for your protein It is necessary to screen a broad range of conditions this can be done by vapour diffusion or microbatch although vapour diffusion is more common to determine the solubility of your protein. The initial screens inform on the conditions which are soluble and those that are insoluble. At this stage insoluble protein is observed as amorphous precipitate. This usually means that the conditions are too severe to allow crystal growth. In the second optimisation stage, potential conditions are explored, e.g. conditions which gave precipitate in the initial screen are modified so that they allow a more gradual approach to insolubility.

Examination of plates Record the appearance of each well on a regular basis Observe immediately after setting up and again on the following day again after 2 or 3days and then once a week Use a stereomicroscope to make the observations Be careful not to shake or jolt plates when moving them We now move on the the second part of the teaching for today. Having outlined the theoretical areas I would like to introduce some practical aspects, how do we go about getting crystals of our material? In this section I will be considering what screens are available, their merits and drawbacks, suggesting how to identify leads and how best to follow them once you have identified them, and finally how to adapt the screens to manage problem proteins or shortage of material. Finding crystallising conditions for your protein It is necessary to screen a broad range of conditions this can be done by vapour diffusion or microbatch although vapour diffusion is more common to determine the solubility of your protein. The initial screens inform on the conditions which are soluble and those that are insoluble. At this stage insoluble protein is observed as amorphous precipitate. This usually means that the conditions are too severe to allow crystal growth. In the second optimisation stage, potential conditions are explored, e.g. conditions which gave precipitate in the initial screen are modified so that they allow a more gradual approach to insolubility.

What you should look for Crystals are transparent and have definite form recognisable by the planar faces Precipitate is irregular in shape without defined edges, opaque and often forms clumps Phase separation in the form of bubbles which may be disperse or quite dense. We now move on the the second part of the teaching for today. Having outlined the theoretical areas I would like to introduce some practical aspects, how do we go about getting crystals of our material? In this section I will be considering what screens are available, their merits and drawbacks, suggesting how to identify leads and how best to follow them once you have identified them, and finally how to adapt the screens to manage problem proteins or shortage of material. Finding crystallising conditions for your protein It is necessary to screen a broad range of conditions this can be done by vapour diffusion or microbatch although vapour diffusion is more common to determine the solubility of your protein. The initial screens inform on the conditions which are soluble and those that are insoluble. At this stage insoluble protein is observed as amorphous precipitate. This usually means that the conditions are too severe to allow crystal growth. In the second optimisation stage, potential conditions are explored, e.g. conditions which gave precipitate in the initial screen are modified so that they allow a more gradual approach to insolubility.

Identifying salt crystals Protein dye Crushing Dehydration Ultimately the x-ray beam Protein dyes are available and only stain protein. Crush test – using a fine needle such as an acupuncture needle, crush a sample crystal. Salt crystals usually crush with difficulty into relatively few pieces while protein crystals crush easily into a shower of very fine pieces. Dehydration test – allow a single crystal to dehydrate in air. Protein crystals will usually disintegrate while salt crystals usually dry intact. X-ray beam – the ultimate test since crystalline periodicities characteristic of proteins are readily distinguishable from those of salt crystals.

Overview First steps in crystallising proteins Available screens Screening strategies Considering the leads Optimisation experiment designs Non-standard methods

Available screens Principles behind screening – to determine solubility and for optimisation. Types of screen available Sparse matrix screens Clear strategy PEG ion Detergent screens Additive screens We will look at how to use them. Get feedback from group ask them to say how they use the screen which ones they prefer etc. Say we will look at more systematic approaches later in the course on Wednesday in the session dealing with optimisation. Principles behind screening – to determine solubility and for optimisation. Types of screen available Sparse matrix screens Clear strategy PEG ion Detergent screens Additive screens

Screening strategies Since it is impossible to predict the conditions for nucleation, screening is a good way of determining the crystallising conditions. Random screens Trial and error sparse matrix approach Systematic screens Selected variation of two parameters

Sparse matrix screens Sparse matrix screens are composed of a collection of conditions which have been used successfully for crystallisation of other proteins Within the screens the following parameters are varied: pH, precipitating agent, type of buffer and salt components Mentions systematic screens that you design them yourself.

Overview First steps in crystallising proteins Available screens Screening strategies Interpreting results Optimisation experiment designs Non-standard methods

Interpreting results Skills in crystallisation are: Describing your observations - hampton score sheet Interpreting the results of an experiment Deciding what to do next Identifying and following the leads Precipitation - types of precipitate, granular, microcrystalline Phase separation - phase separation bubbles, gel precipitate Micro crystals - is it protein or salt? Since the important skills in protein crystallisation are: Describing your observations Interpreting the results of an experiment Deciding what to do next

What the leads might mean Amorphous or granular precipitate May/may not be the ideal crystallisation condition, concentration of protein or precipitant too high Phase separation and phase gel If all observations are phase separation related select those that have a gelatinous appearance Microcrystalline precipitate and crystals Likely to be the correct conditions concentration of protein or precipitant too high

Using the leads to gain understanding Gathering information from the screens to gain an understanding of the solubility of your macromolecule Make a note of the pH of the screen condition, are there any trends regarding pH? Any trends regarding salts? Difference in results with high salt and low salt Hofmeister series – ranking of ions in order of their ability to precipitate proteins Any common appearances e.g. lots of precipitate or only phase separation

Hofmeister series Cations: Li+ > Na+>K+>NH+4 >Mg2+ Anions: sulphate 2- > phosphate2- > acetate- > citrate3- > tartrate2- > bicarbonate- > chromate2- > chloride-> nitrate- >> chlorate - > thiocyanate-

Optimisation experiment designs After completing an initial screen you may have one of the following results: crystals with one or more conditions amorphous precipitates or precrystalline aggregates with one or more conditions no crystals, precipitate or aggregates with any of the conditions in the screen If you obtained results 1 or 2 you may want to fine tune your screen. After completing an initial screen you may have one of the following results: crystals with one or more conditions amorphous precipitates or precrystalline aggregates with one or more conditions no crystals, precipitate or aggregates with any of the conditions in the screen If you obtained results 1 or 2 you may want to fine tune your screen.

Fine tuning the sparse matrix conditions The sparse matrix screen has yielded a number of conditions in which your protein is insoluble (crystals or precipitate) Design a narrow-range grid screen based on varying the pH, and the concentrations of each component systematically observing whether one or more of the variations gives good crystals The sparse matrix screen has yielded a number of conditions in which your protein is insoluble Design a narrow-range grid screen based on varying the pH, and the concentrations of each component systematically observing whether one or more of the variations gives good crystals In addition this would be a time to begin to include a test of the effects of ligands on the crystallisation of your protein by using an additive screen. It is best to start with the conditions that have given crystals. But also good to test as many conditions as possible.

Expanding the initial screen It is possible that none of the conditions from your first screen gave any leads to expand your screening it is worth trying other sparse matrix screens which are commercially available or a grid screening kit

Exercise to practice optimisation skills How would you go about optimising the crystallisation to achieve the following and why: larger crystals fewer crystals improve the diffraction quality Here I would like to (i) test the students understanding of the principles taught on the first day and (ii) give the students the opportunity to apply the knowledge they have acquired to real problems. Doing it this way there is a break from my teaching Series of set exercises, to try in small groups, based on real crystallisation projects. How would you go about optimising the crystallisation to achieve the following and why: larger crystals fewer crystals improve the diffraction quality Summary of the important issues relating to optimisation.

Overview First steps in crystallising proteins Available screens Screening strategies Interpreting results Optimisation experiment designs Non-standard methods

Non-standard methods Microbatch crystallisation with gels Microbatch controlled evaporation Oil barrier methods Containerless crystallisation Separation of nucleation and growth The influence of gels on crystal nucleation Heterogeneous nucleation – Gels tend to have a cleansing effect on the growth solution, as the gel polymerises heterogeneities in the growth solution (such as fibres, gas bubbles and dust) tend to get trapped in the gel and are no longer available to generate crystal nuclei. Secondary nucleation – since solid and fluid movements are prevented in gels, secondary nucleation that arises from particles pulled out of a primary crystal by convection or by impact of crystals against each other or the vessel wall cannot take place. Homogeneous nucleation – solubility curves remain unchanged, critical supersaturation may be significantly changed, nucleation rate may change (increasing in agarose gels and inhibiting in silica gels). See chapter 6 Ducruix and Giege

Adapting the screens Adapting the screens Using dilution Using evaporation Using oils Screen at a different temperature Screen at a different pH Using gels The non-standard method use these themes to modify the screens.

Microbatch crystallisation Drops between 3ml and 0.3ml are dispensed under oil either by hand or by robot under oil.

Controlled Evaporation Evaporation methods can be applied to both microbatch and vapour diffusion methods. In the case of microbatch, the drops are dispensed under a thin layer of oil is to allow limited evaporation. After a predetermined time the tray is filled with oil to prevent any further evaporation. Chayen and Saridakis (2002) Acta Cryst. D58, 921-927

Advantages of crystallisation in microbatch Under Oil Some crystals will ONLY grow in oil Hanging drops tend to spread over the surface of siliconised cover slips Mechanically batch is the simplest crystallisation method which lends itself readily to HTP Very small drop volumes down to 1nl Crystals can be grown under controlled nucleation conditions in three ways by: choosing the oil which covers the trials varying the thickness of the oil layer covering the trials applying a ‘container-less’ crystallisation set-up Some crystals will ONLY grow in oil Hanging drops tend to spread over the surface of siliconised cover slips because of the decrease in surface tension caused by the detergent, however in batch drops are round an symmetrical. Mechanically batch is the simplest crystallization method which lends itself readily to HTP so that trials can be dispensed automatically by a robot Very small volumes down to 1nl Crystals can be grown under controlled nucleation conditions in three ways – start below nucleation and allow some evaporation. By choosing the oil which covers the trials By varying the thickness of the oil layer covering the trials By applying a ‘container-less’ crystallisation set-up

Problems Associated with Microbatch Crystallisation Shock nucleation Use of organic components Stabilising / harvesting crystals

Oil barrier methods Method to control crystallisation by altering the rate of vaporisation from the reservoir and therefore the rate at which the drop equilibrates.

Methods which utilise separation of nucleation and growth This is achieved by transferring the cover slip with the drop from a reservoir with crystallisation agent at higher concentration to one with a reservoir at lower concentration.

Approaches to aid crystallisation Trial and error Screening and fine tuning conditions for crystal growth Systematic studies Understanding the fundamental principles of crystal growth Designing experiments using these principles to produce better crystals

The systematic approach As an example of the screening process suppose you found that condition 35 of the Hampton crystal screen (0.1M HEPES, pH 7.5; 1.6M Na/K phosphate) gave amorphous precipitate, you might set up the following grid screen:

The systematic approach pH 7.0 pH 7.2 pH 7.4 pH 7.6 pH 7.8 pH 8.0 Na/K 0.8 Na/K 1.0 Na/K 1.2 Na/K 1.4 Notice that pH and precipitant concentration are being varied and the pH range includes that of the screen however the precipitant concentration is below that of the initial screen. It may take several cycles of fine-tuning to get the optimal conditions it is important to be patient. Crystal growth is slower that precipitation which may appear after only a few minutes. Crystal growth can take anything from hours to weeks.

Designing your own screens Have a go and use your Intuition! Screen with salt and PEG Grid screen with buffered ammonium sulphate pH screen with one precipitant For this exercise you could give the students some parameters to start with. Get examples from your own work

A constant temperature crystallization room. Setting up crystallization trials Inside a crystallization room A constant temperature crystallization room. Looking at crystallization trials Many conditions are screened

Optimization of Crystallization Conditions The need to grow crystals is often the limiting step in structure determination. This is an empirical process that involves much trial and error. Commercial kits are used to sample hundreds of trial conditions. When encouraging leads are found (e.g. small crystals), the initial conditions are refined. Good crystals will be about 0.1 - 0.5 mm in diameter, and have no flaws, such as cracks, or two crystals growing together. Here are some good crystals. In difficult cases it is typical to try homologous proteins from several different species.