Starting a crystallography project…

(Traversing the mountain range of structure determination)

cloning

proteinexpression

proteinpurification

crystallization

diffraction

structuredetermination

The method

X-rays}

1. Growing protein crystalsPrinciples, methods and optimization2. SymmetrySymmetry elements, point groups and space groups3. DiffractionIntroduction to diffraction of wavesThe reciprocal latticeDiffraction by crystals; Bragg equation4. Obtaining the diffraction patternInstrumentsData collection strategy/quality5. Deriving a trial structureMethods for solving phase problem6. Refining the structure7. Analysis of structural parameters - quality

Syllabus

Growing crystals

Protein Crystallisation

Principles and practice in crystallising biological

macromolecules

Learning objectives

• Understand the principles that govern crystal formation and growth

• Have knowledge of the different types of precipitant and how they work

• Be familiar with a number of different methods of crystallisation

• Be able to choose a suitable method for the crystallisation of your macromolecule and to design a crystallisation strategy

• Make decisions about screening results and selecting the best leads to follow

• Develop and/or modify existing methods to assist the crystallisation of your macromolecule

Overview

Basic principles of crystallisation– Supersatutation– Solubility– Nucleation– Crystal growth– Factors that affect crystallisation– Methods in crystallisation– Precipitating agents

Practical methods– Microbatch and other methods using oils

Introduction to the principles of crystallisation

• 3 steps in crystallisation: nucleation, growth and cessation of growth

• Macromolecule Crystallisation is a multi parameter process• The differences between protein crystallisation and the

crystallisation of small molecules are:– The physico-chemical properties– Conformational flexibility and chemical versatility– Origin of biological macromolecule

• To grow crystals molecules have to be brought into a supersaturated, thermodynamically unstable state, this may result in a crystalline or amorphous phase when it returns to equilibrium

Supersaturation

An unstable condition where more solutes (protein) are dissolved in a solvent that can

normally be held in solution under given conditions of temperature and pressure.

It can also be defined as when the chemical potential (change in free energy) of the solute

is greater in solution than in the crystal (solid).

Supersaturation

Supersaturation can be achieved by evaporation of the solvent or by varying any parameter that affects the chemical potential of the solute e.g.

– Protein concentration– Salt concentration– Temperature– Pressure

Supersaturation is the driving force for crystallisation – as such it is a key parameter in optimisation

Solubility

• Solubility is defined as: the amount of compound dissolved in a solution in equilibrium with an excess of undissolved compound

• There are different ways to define solubility– concentration values may be measured before complete equilibrium is reached– solubility may be measured in the presence of precipitate or crystals

Macromolecular structure

A biological macromolecule is a polymer of amino acids or nucleotides, which is folded into tertiary or quaternary structure held together

– mainly by dipole-dipole interaction, H-bonds and van der Waals interactions

– by some covalent bonds (S-S bridges)– occasionally by salt bridges

Water soluble proteins have mostly hydrophilic side-chains on their surface

Solubility

• Solubility is additionally defined by the characteristics of the solvent

• The additional chemicals contained in the solvent can affect the solubility of macromolecules by either:– interacting with the different functional groups of

the protein, perhaps modifying the conformation– modifying the properties of the solvent e.g.,

altering the pH or disrupting salt bridges

Solubility and the solid phase

aqueous solution crystal

Crystallisation is:– The transfer of molecules from the liquid phase (aqueous solution) to

an ordered solid phase– Thermodynamic factors govern the solubility– This is where the supersaturation becomes important, as only in the

supersaturated state will the equilibrium be shifted in favour of the formation of intermolecular bonds.

Solubility and temperature

• Entropic effects - An increase in temperature increases the disorder of solvent molecules

• During a temperature rise vapour will distil away from a drop increasing the degree of supersaturation - shower of xtals

• Decreases in temperature result in vapour condensing on the drop diluting it and increasing the volume (use microbatch or sitting drop)

Solubility and pH

• pH changes affect both solute and solvent but have a greater effect on the solute, potentially protonating or deprotonating the macromolecule

• Charged groups on the surface of the molecules may be affected by protons and different ions in the solution

Solubility and ionic strength

Salts are responsible for the ionic strength of a solution and affect macromolecular electrostatic interactions by charge shielding

This is achieved by acting in the following ways:–Forming direct electrostatic interactions with charged residues–Forming interactions with dipolar groups (e.g. peptide bonds, amino, hydroxyl or carboxyl groups)–Non-polar interactions of hydrophobic residues with organic salts –Association with binding sites

The effect of salts on solubility

• The change in protein solubility with increasing salt concentrations is described in terms of:

• Salting-in – increasing solubility at low salt concentration

• Salting-out – protein solubility is decreased at high ionic strength

How can we take advantage of these factors?

In order to initiate crystallisation we need to achieve supersaturation, effectively reducing the solubility of the protein.This can be done practically in a number of ways:

– Increase the concentration of protein – Alter the ionic strength of the solvent– Alter the pH of the solvent– Change the temperature

The phase diagram

Metastable zone

Overview

Basic principles of crystallisation– Supersaturation– Solubility– Nucleation– Crystal growth– Factors that affect crystallisation– Methods in crystallisation– Precipitating agents

Nucleation

the creation of a new (solid) phase – the formation of ordered aggregates.

It is essentially the coming together of solute molecules within a solution and requires that the energy barrier – the activation energy – is

overcome before the formation of intermolecular bonds can occur.

Nucleation

• Nucleation is the first step in crystallisation• To achieve nucleation supersaturation

must be induced• At supersaturation spontaneous nucleation

is a dynamic process• There is a lower energy requirement in

adding to an existing crystal surface than in creating a new nucleus

Types of nucleation

There are different types of nucleation:• Homogeneous – occurring within the

solution• Heterogeneous – occurring on solid

particles or surfaces• Primary – within a system containing no

crystalline matter• Secondary – when new nuclei originate

from an existing nucleus (to produce twinning or bunching)

Epitaxial nucleation• Epitaxial nucleation is where the regularity of the

surface facilitates nucleation. • Glass although siliconised can act as an

adhesion surface• The strength of interaction with the glass can be

stronger that the forces that bond the crystalline lattice

• Crystals or micro-crystals can also be nucleated on cellulose fibres which are accidentally present in the protein/precipitate drop

• The nucleation of crystals from aggregates and oils can be considered a case of epitaxial nucleation.

The results of nucleation

• Crystal–like precipitate– the nuclei form regular 3-dimensional structures.

(Shower of tiny crystals – too much nucleation but ordered)

• Non-crystalline precipitate– the solute molecules associate in a random

fashion by non-specific van der Waals forces.– Can be either gel-like precipitate or an amorphous

precipitate - there is nucleation in both cases, but it is random

The phase diagram

Metastable zone

Methods to induce nucleation

• Alter the protein and/or precipitant concentration

• Use an additive or add a nucleant• Use evaporation techniques• Use methods to separate

nucleation and growth (e.g. transfer methods)

Methods to reduce nucleation

• Slowing of equilibration:

– with dialysis set-ups

– by altering the major parameters of the vapour diffusion technique

• Use of silica based gels

• Use methods that involve dilution of protein and/or precipitant solutions

• Seed into the metastable zone

Factors that affect nucleation

Factor Effect on nucleationSeeding Limit/reduce nucleation

Epitaxy Induce nucleation

Charged surfaces ??

Magnetic/electric fields ??

Mechanical e.g. vibration, pressure

Induce nucleation in the metastable zone

Precipitant/protein/additive concentration

Effect control on nucleation

Container walls Induce heterogeneous nucleation

Kinetics (rates of equilibration) Induce or reduce

Overview

Basic principles of crystallisation– Supersatutation– Solubility– Nucleation– Crystal growth– Factors that affect crystallisation– Methods in crystallisation– Precipitating agents

Principles of crystal growth

To bring the system gradually into a state of supersaturation by:

– modifying the properties of the solvent

– altering a physical property such as temperature

Crystal growth

• Diffusion and convection play a major role - use gels

• Kinetic factors govern crystal growth• Events of crystal growth are quite different to

nucleation - uncouple growth from nucleation seeding is one such method

• To promote growth use an additive, add a nucleant or a seed crystal

• Crystal quality is affected by: rates of growth, internal order of the initial nucleus, purity of the sample

Optimising crystal growth

• Knowledge of the growth sequence is important– The time span for the first crystal to become

visible– Rates of equilibration and nucleation– An idea of an approximate growth rate– Exert some control of the kinetics of

supersaturation and nucleation

• Choose supersaturation conditions just above the border between metastable and nucleation

Cessation of crystal growth

• There is a limit to crystal growth• Cessation can be caused by:

– growth will naturally cease as the protein concentration drops to the solubility limit

– the random accumulation of defects as the crystal grows

– adsorption of impurities or denatured protein onto the surface – “poisoning” of the surface

Overview

Basic principles of crystallisation– Supersatutation– Solubility– Nucleation– Crystal growth– Factors that affect crystallisation– Methods in crystallisation– Precipitating agents

Factors that affect crystallisation

• What are the parameters that affect the thermodynamics of interactions between molecules?

• What factors affect the stability of proteins?

• Which biological parameters are involved?

Factors affecting crystal growth

• Protein purity and homogeneity• Precipitating solution• The pH of the crystallisation solution• Crystallisation temperature• Chemical or biochemical modifications to the

protein• Stability of the protein or macromolecule• Surface charge of the macromolecule

Overview

Basic principles of crystallisation– Supersatutation– Solubility– Nucleation– Crystal growth– Factors that affect crystallisation– Methods in crystallisation– Precipitating agents

Methods in protein crystallisation

a) Batch b) Microbatch (by hand and by robot)c) Vapour diffusion

i. hanging dropii. sitting drop

d) Equilibrium dialysise) Free interface diffusion

Batch crystallisation

• The method involves mixing the biological macromolecule and the crystallising solution to achieve supersaturation instantaneously.

• Since experiment starts at supersaturation – nucleation tends to be too large

• Large crystals can be obtained when working close to metastable

Microbatch crystallisation

• A batch method where crystallization samples are dispensed as small drops (can be less than 1l final volume) under oil.

• Enables systematic studies on very small quantities – l scale, of both protein and crystallizing agents.

• Applications include:• Screening• Optimisation • Control of nucleation and equilibration

Chayen (1997) Structure 5, 1269-1274

Dispensing Drops Under Oil

Phycocyanin crystal by microbatch

Vapour diffusion

• A good method for screening large numbers of crystallisation conditions

• Evaporation of water from the sample droplet accompanied by net condensation into the reservoir solution so as to equalise the concentrations of the two solutions

• This migration of water from the droplet results in concentration of both the protein and the precipitating agent lowering the solubility of the protein and if the condition are right inducing the formation of crystals

Vapour diffusion – by hanging drop

• The macromolecule and crystallising agent equilibrate against the reservoir which is at a higher - generally twice - concentration than that of the drop

• Equilibration proceeds by evaporation of the volatile species (water or organic solvent) until the vapour pressure in the droplet equals that of the reservoir

Hanging drop

droplet

Reservoir

Schematic diagram of a hanging drop

Vapour diffusion – by sitting drop

• The same principle applies in the hanging drop as in the sitting drop the difference is in the experimental set-upSchematic

diagram of a sitting drop

Sitting drop

droplet

Reservoir

Crystallization by vapor diffusion

Protein solution.

Reservoir (precipitant) solution.

Sitting drop Hanging drop

Crystallization by vapor diffusion

Phase diagram for vapor diffusion

(no crystals)

Airlie J McCoy, Protein Crystallography coursehttp://www-structmed.cimr.cam.ac.uk/Course/Crystals/intro.html

Phase diagram for vapor diffusion

(crystals!!!)

Airlie J McCoy, Protein Crystallography coursehttp://www-structmed.cimr.cam.ac.uk/Course/Crystals/intro.html

Dialysis

• In dialysis the biological macromolecule is separated from a large volume of solvent by a semi-permeable membrane which allows small molecules (such as ions, additives, buffer etc.) to pass through but prevents the passage of the macromolecule

• The kinetics of the equilibrium will depend on the membrane cut-off, the ratio of the concentration of crystallising agent on either side of the membrane and the temperature and design of dialysis set up

Free interface diffusion• Also known as the liquid/liquid

diffusion method• Equilibration occurs by

diffusion of the crystallising agent into the biological macromolecule volume

• To avoid rapid mixing:– Less dense solution is poured on

more dense (salt usually)– Crystallising agent is frozen and

protein layered on top

• Use tubes of small inner diameter to reduce convection

Diagram of a liquid/liquid set-

up

Wax

Protein solution

Crystallising solution

Revisiting the Phase Diagram

A. BatchB. Vapour diffusionC. DialysisD. Free interface diffusion

Precipitating agents

Chemical precipitants are used to achieve supersaturation in order to induce crystallisation, they can be divided into the following categories:

–Salts–Straight chain polymers (e.g. PEG)–Organic solvents

The highest numbers of macromolecular crystals have been obtained using:

Ammonium sulphate, PEGs, Na/K phosphate, sodium chloride, MPD and magnesium chloride

Salts as precipitants

Salts work by disrupting the hydration shell of proteins minimising the attractive protein-solvent interactions and maximising the attractive protein-protein interactions

Organic precipitants

• Organic precipitants function primarily by lowering the dielectric constant of the solution to reduce the electrostatic shielding of charged and polar functional groups on proteins

• Most commonly used organic solvents are:– 2-methyl-2,4-pentanediol (MPD)– Polyethylene glycols (PEGs)

Poly(ethylene glycol)s (PEGs)

• PEGs are very large polymers produced from a mixture of ethylene

• Like other organic solvents PEGs lower the dielectric constant of the solution but they also affect the structure of water

• PEGs may be contaminated with things such as aldhydes and peroxides – use crystallisation grade PEGs