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project…
(Traversing the mountain range of structure determination)
crystallization structure
protein protein determination
purification diffraction
expression
cloning
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
• 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
Metastable
zone
Overview
Basic principles of crystallisation
– Supersaturation
– Solubility
– Nucleation
– Crystal growth
– Factors that affect crystallisation
– Methods in crystallisation
– Precipitating agents
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
Reservoir
Protein (precipitant)
solution. solution.
Crystallization by vapor diffusion
A. Batch
B. Vapour diffusion
C. Dialysis
D. 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