Starting a crystallography project

1
Starting a crystallography project
(Traversing the mountain range of structure
determination)
crystallization
structure determination
protein purification
protein expression
diffraction
cloning
2
The method
X-rays
3
Syllabus
1. Growing protein crystals Principles, methods
and optimization 2. Symmetry Symmetry elements,
point groups and space groups 3.
Diffraction Introduction to diffraction of
waves The reciprocal lattice Diffraction by
crystals Bragg equation 4. Obtaining the
diffraction pattern Instruments Data collection
strategy/quality 5. Deriving a trial
structure Methods for solving phase problem 6.
Refining the structure 7. Analysis of structural
parameters - quality

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Growing crystals
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Protein Crystallisation

• Principles and practice in crystallising
  biological macromolecules

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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

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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

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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

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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).
10
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
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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

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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

13
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

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Solubility and the solid phase

• 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.

15
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)

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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

17
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

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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

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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

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The phase diagram
21
Overview

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

22
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.
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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

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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)

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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.

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The results of nucleation

• Crystallike 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

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The phase diagram
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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)

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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

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Factors that affect nucleation
Factor Effect on nucleation
Seeding 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
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Overview

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

32
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

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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

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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

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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

36
Overview

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

37
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?

38
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

39
Overview

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

40
Methods in protein crystallisation

• Batch
• Microbatch (by hand and by robot)
• Vapour diffusion
• hanging drop
• sitting drop
• Equilibrium dialysis
• Free interface diffusion

41
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

42
Microbatch crystallisation

• A batch method where crystallization samples are
  dispensed as small drops (can be less than 1ml
  final volume) under oil.
• Enables systematic studies on very small
  quantities ml scale, of both protein and
  crystallizing agents.
• Applications include
• Screening
• Optimisation
• Control of nucleation and equilibration

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Dispensing Drops Under Oil
Chayen (1997) Structure 5, 1269-1274
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Phycocyanin crystal by microbatch
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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

46
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

Schematic diagram of a hanging drop
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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-up

Schematic diagram of a sitting drop
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Crystallization by vapor diffusion
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Crystallization by vapor diffusion
Sitting drop
Hanging drop
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Phase diagram for vapor diffusion(no crystals)
Airlie J McCoy, Protein Crystallography
course http//www-structmed.cimr.cam.ac.uk/Course/
Crystals/intro.html
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Phase diagram for vapor diffusion(crystals!!!)
Airlie J McCoy, Protein Crystallography
course http//www-structmed.cimr.cam.ac.uk/Course/
Crystals/intro.html
52
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

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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
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Revisiting the Phase Diagram

1. Batch
2. Vapour diffusion
3. Dialysis
4. Free interface diffusion

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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

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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

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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)

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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