protein crystallisation strategies, optimisation and non-standard methods

Protein Crystallisation

Strategies, optimisation and non-standard

methods

Overview

First steps in crystallising proteinsAvailable screensScreening strategiesConsidering the leadsOptimisation experiment designsNon-standard methodsPractical 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

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

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.

Identifying salt crystals

• Protein dye

• Crushing

• Dehydration

• Ultimately the x-ray beam

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

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

Overview




–Interpreting results



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?

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:

1. crystals with one or more conditions2. amorphous precipitates or precrystalline

aggregates with one or more conditions3. 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

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

Overview




–Interpreting results



Non-standard methods

• Microbatch crystallisation with gels

• Microbatch controlled evaporation

• Oil barrier methods

• Containerless crystallisation

• Separation of nucleation and growth

Adapting the screens

Adapting the screens– Using dilution– Using evaporation– Using oils– Screen at a different temperature– Screen at a different pH– Using gels

Microbatch crystallisation

Drops between 3l and 0.3l are dispensed under oil either by hand or by robot under oil.

Chayen and Saridakis (2002) Acta Cryst. D58, 921-927

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.

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:i. choosing the oil which covers the trialsii. varying the thickness of the oil layer covering

the trialsiii. 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 0.8

Na/K 0.8

Na/K 0.8

Na/K 0.8

Na/K 0.8

Na/K 1.0

Na/K 1.0

Na/K 1.0

Na/K 1.0

Na/K 1.0

Na/K 1.0

Na/K 1.2

Na/K 1.2

Na/K 1.2

Na/K 1.2

Na/K 1.2

Na/K 1.2

Na/K 1.4

Na/K 1.4

Na/K 1.4

Na/K 1.4

Na/K 1.4

Na/K 1.4

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

A constant temperature

crystallization room.

Inside a crystallization roomSetting up crystallization trials

Looking at crystallization trialsMany conditions are screened

Optimization of Crystallization Conditions

Here are some good crystals.

In difficult cases it is typical to try homologous proteins from several different species.

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.

protein crystallisation strategies, optimisation and non-standard methods

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