Protein Crystallisation

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Protein Crystallisation

• Strategies, optimisation and non-standard methods

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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

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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

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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

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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.

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Identifying salt crystals

• Protein dye
• Crushing
• Dehydration
• Ultimately the x-ray beam

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Overview

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

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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

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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

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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

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Overview

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

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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?

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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

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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

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Hofmeister series

• Cations Li gt NagtKgtNH4 gtMg2
• Anions sulphate 2- gt phosphate2- gt acetate- gt
  citrate3- gt tartrate2- gt bicarbonate- gt
  chromate2- gt chloride-gt nitrate- gtgt chlorate - gt
  thiocyanate-

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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.

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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

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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

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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

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Overview

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

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Non-standard methods

• Microbatch crystallisation with gels
• Microbatch controlled evaporation
• Oil barrier methods
• Containerless crystallisation
• Separation of nucleation and growth

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Adapting the screens

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

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Microbatch crystallisation

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

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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
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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

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Problems Associated with Microbatch
Crystallisation

• Shock nucleation
• Use of organic components
• Stabilising / harvesting crystals

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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.

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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.

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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

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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

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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
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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

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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.