Transcript Basic Principles of Protein Crystallography

Starting a crystallography
project…
(Traversing the mountain range of structure determination)
cloning
protein
expression
protein
purification
crystallization
diffraction
structure
determination
The method
X-rays
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
Growing crystals
Protein Crystallisation
Principles and practice in
crystallising biological
macromolecules
Learning objectives
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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
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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, Hbonds 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
crystal
aqueous solution
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
Seeding
Epitaxy
Charged surfaces
Magnetic/electric fields
Mechanical e.g. vibration,
pressure
Precipitant/protein/additive
concentration
Container walls
Effect on nucleation
Limit/reduce nucleation
Induce nucleation
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Induce nucleation in the
metastable zone
Effect control on
nucleation
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
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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 drop
ii. sitting drop
d) Equilibrium dialysis
e) 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 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
Dispensing Drops Under Oil
Chayen (1997) Structure 5, 1269-1274
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
droplet
Reservoir
Hanging drop
Schematic
diagram of a
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
Vapour diffusion – by sitting
drop
droplet
Reservoir
Sitting drop
Schematic
diagram of a
sitting drop
• The same principle
applies in the
hanging drop as in
the sitting drop the
difference is in the
experimental set-up
Crystallization by vapor diffusion
Protein
solution.
Reservoir
(precipitant)
solution.
Crystallization by vapor diffusion
Sitting drop
Hanging drop
Phase diagram for vapor diffusion
(no crystals)
Airlie J McCoy, Protein Crystallography course
http://www-structmed.cimr.cam.ac.uk/Course/Crystals/intro.html
Phase diagram for vapor diffusion
(crystals!!!)
Airlie J McCoy, Protein Crystallography course
http://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
Crystallising
solution
agent into the biological
macromolecule
volume
Protein
• To avoid rapid mixing:
solution
Wax
Diagram of a
liquid/liquid setup
– 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
Revisiting the Phase Diagram
A.
B.
C.
D.
Batch
Vapour diffusion
Dialysis
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