Structure Determination and Analysis : X-ray Crystallography

Understanding biology through structures Course work 2006

 Energy = hc
λ

Understanding biology through structures Course work 2006

 X-rays
• Unlike using a light microscope,
there is no way of re-focusing
diffracted x-rays.
• Instead we must collect a
diffraction pattern (spots).

•It is possible to translate information in the diffraction pattern

into atomic structure using Bragg’s law, which predicts the angle
of reflection of any diffracted beam from specific atomic planes

Understanding biology through structures Course work 2006

 A typical crystallography experiment
Pure protein

Grow crystal

Characterize crystals

Collect diffraction data

Solve phase problem

Calculate electron density map

Build/rebuild model

Refine model

Analyze structure
Understanding biology through structures Course work 2006
Principles of X-ray diffraction
What is a crystal?

•The unit cell is the basic building block of the crystal

•The unit cell can contain multiple copies of the same molecule
whose positions are governed by symmetry rules

Understanding biology through structures Course work 2006

 Proteins and crystallisation
•What type of protein is it? Has
anything similar been crystallized
before?

•Proteins must be pure (> 99%) & fully

folded Check the activity of your protein
if you have an assay Check folding by
other spectroscopic methods
•Proteins must be homogenous & monodispersed.
•Need large amount (mg quantities)
•Is it stable ( salt, pH, temp)
•Will modifications have to be made?

Understanding biology through structures Course work 2006

 •Crystallisation of proteins ‘controlled’ precipitation of the
protein.
•Protein aggregates associate & form intermolecular contacts
that resemble those found in the final crystal. Aggregates reach
the critical nuclear size, growth proceeds by addition of
molecules to the crystalline lattice.
•The processes of nucleation and crystal growth both occur in
supersaturated solutions.
Cover-slip
Protein in
sealed with
“Hanging drop”
Process controlled by: vacuum grease
•Temp
•pH
•Salt conc Precipitant

•Precipitants (PEG, ethanol)

Understanding biology through structures Course work 2006
 Diffraction Apparatus

Understanding biology through structures Course work 2006

 Diffraction

•Each image represents the

rotation of the crystal 1
degree in the X-ray beam.
•Each images gives us the
position of each spot relative
to all the others & there
intensity.
•Intensity = square of
amplitude.

Understanding biology through structures Course work 2006

 Resolution
6Å: Outline of the model, feature
such as helices can be identified.
3Å: Can trace polypeptide chain
using sequence data, establish
folding topology. Assign side
chains.
2Å: Accurately establish
mainchain conformation, assign
sidechains without sequence data,
I.d water molecules.
1.5Å : Individual atoms are almost
resolved, detailed discription of
water structure.
1.2Å: Hydrogen atoms may
become visible.
Understanding biology through structures Course work 2006
 Final Structure

But the work is not over yet!

Understanding biology through structures Course work 2006

 Refinement
• The process of building and rebuilding a model can cause
many errors in the structure.
1. Bond length,
2. Bond angle
3. Atomic clashes etc
• It is necessary to subject the structure to refinement in order
to remove these errors and produce a better structure.
• Minimization
• Thermal parameters
• In order to further improve the model, it is refined using a
simulated annealing protocol
• Refinement progress is monitored by following the agreement
between the the observed data ( data collected) and the
calculated data (data calculated from current model) = R
factor
Understanding biology through structures Course work 2006
 Quality of the structure?

• R-factor The agreement between the the observed data (data

collected) and the calculated data (data calculated from
current model) the lower the number the better; typically
around 20%
• Resolution The higher the resolution the more detail that can
be seen 3.0Å is fairly low whilst 1.1Å is approaching atomic
resolution
• B-factor Measure of thermal motion. i.e. how much energy
each atom contains. Gives us information on mobility &
stability
• Rms deviation Deviation of bond lengths & angles from ideal

Understanding biology through structures Course work 2006

Understanding biology through structures Course work 2006
 Where can Circular Dichroism be used?

Understanding biology through structures Course work 2006