1. Presented To: Prof. Tayyab
Presented By: Group # 01
Tayyaba fayaz
Hira akram
Rida fatima
Sadia iqbal
2. Historical background
X-ray crystallography and its principle
Why we prefer x-ray
Why do we need crystals
Reflection
Protein purification
Crystallization
Tissue culture
Testing crystal
Data collection
High resolution data collection
Structural solution
Model building
Refinement
3. Discovery of X-rays:
CONRAD RONTGEN
Diffraction of X-ray discovery :
Maxvon Laue ( wave nature)
X-ray crystallography:
BRAGS (father and son)
Determine the atomic structure of matter
First protein structure determination :
MAX PERUTZ AND JOHN KENDREW
Chemistry noble prize in 1962
myoglobin
4. ‽ It is a form of very high resolution
microscopy.
‽ It enables us to visualize protein
structures at atomic level and
enhances our understanding of
protein function.
‽ It tells about the interaction of
proteins with other molecule and
their conformational changes
mechanisms etc.
5. ‽ In x-ray crystallography an x-ray
beam is diffracted by a crystal.
The diffraction pattern can be
recorded as spots where the
diffracted x-rays strike on a
photographic plate.
8. ‽ In all form of microscopy,
resolution is limited by the
wavelength of electro-magnetic
radiations used.
‽ With light microscopy, where the
shortest wavelength is about
300nm, we can study individual
cells and sub-cellular organelles.
9. ‽ With electron microscopy,
where wavelength is below
10nm, we can see detailed
cellular structure and shape of
large protein molecules.
‽ In X-ray it is not possible to
focus physically on diffraction
pattern, so it is done by
mathematically and computers
are used.
10. A three dimensional array of elements to
form a solid structure.
11. ‽ Diffraction from single molecule
would be too weak to be
measurable so we use an ordered
three-dimensional array of
molecules (crystals).
‽ X-rays are diffracted by the
electrons in structure results in
three-dimensional map showing
the distribution of electrons in the
structure.
12. ‽ A crystal behaves like a three-
dimensional diffraction grating,
with give rise to both constructive
and destructive interference
effects in diffraction pattern.
‽ It appears on the detector as a
series of discrete spots which are
known as “reflections”
13. ‽ Each reflection contains information
on all atoms in the structure.
‽ As X-rays have wave properties so,
they have both an amplitude and a
phase.
‽ For the recombination of diffraction
pattern both of above parameters are
require for each reflection.
14. ‽ Unfortunately only amplitudes can
be recorded experimentally and all
phase information in lost!
‽ This is known as “the phase
problem”
‽ When crystallographer says that they
solve a structure it means they solve
phase problem and obtain phase
information to describe electron
density map.
15. ‽ Firstly we need to obtain a pure
sample of our target protein.
‽ We can do this by;
1. Isolating
2. Cloning its gene into a
high
expression system.
17. ‽ Before beginning the trials
sample needs to be concentrated
and transferred to dilute buffer.
‽ It is done by centrifugal
concentrator.
‽ in order to screen a reasonable
number of conditions we at least
200ml of protein at 10mg/ml.
18. ‽ If the similar protein has already
been crystallized then it is worth
trying the conditions used to grow
crystals of this protein.
19. ‽ Normally tissue culture trays are
used to set up crystallization with
up to 24 different conditions per
tray.
‽ The method used is hanging drop
vapour diffusion.
20. ‽ The well is prepared usually
contains 1ml of buffer precipitant
solution such as polyethylene
glycol or ammonium sulfate.
‽ Sometimes additives are also
included such as detergents or
metal ions which may enhance the
crystallization.
21. ‽ 1ml of protein sample is pipetted
onto a siliconized coverslip,
followed by 1ml of well solution
‽ Then the coverslip is inverted
over the well and sealed using a
bead of vaccum grease.
‽ This is then left for at least 24
hours to equilibrate.
22. ‽ At the start of the experiment,
the precipitant concentration in
the drop is half that of the well.
‽ Then equilibrium takes place via
vapour phase, give relatively large
volume of the well, its
concentration equals to that of the
well
‽ However, drop loses water
vapours to well until the
precipitant concentration equals
to that of the well.
23. ‽ If the conditions have been
favorable, at some point during
this process the protein has
become supersaturated and
driven out of the solution in the
form of the crystals.
Success rate at this stage is less
than 0.1%...!!!
24. ‽ However if we are lucky enough
to get one or more hits then we
do some betterment, which are
variations in conditions so that we
obtain a large single crystal.
‽ These changings include using
additives, slight change in pH and
varying concentrations of all
components.
25.
26. ‽ To test, the crystal mounted in a
capillary at room temperature or
flash-cooled to 100K in a loop and
then attached to a device known as
Goniometer head.
‽ This enables the sample to be
accurately positioned in x-ray beam
by means of number of adjustment
screws.
27. ‽ For Cryogenic data collection ,
cold nitrogen gas stream keeps
the crystal at 100K.
‽ Focused x-ray s emerge from a
narrow tube called collimator and
strike a crystal to produce a
diffraction pattern
‽ This is record on x-ray detector.
28. ‽ All being well, we should see clean
sharp spots; one lattice of spot
indicating a single crystal.
‽ Ideally crystal should diffract to
better than 4Å.
‽ If these criteria are not satisfied,
then check another crystal, if all
crystals have same result then go
back to crystallization step.
29.
30. ‽ Firstly, we need to know
‽ The method we use is to rotate the
crystal through a small angle,
typically 1 degree and record the x-
ray diffraction pattern.
‽ If the diffraction pattern is very
crowded then resolution angle
should be reduced so that each spot
can be resolved on the image.
31. ‽ This is repeated until the crystal
moved through at least 30 degrees
and sometimes 180 degrees,
depending upon crystal symmetry.
‽ The lower the symmetry, then more
data are required.
‽ A typical medium resolution data set
may take up to 3 days using an in-
house x-ray source.
32. ‽ For high resolution data
collection we need greater x-ray
intensity so that data collection
time are shorter – sometimes as
fast as 10 minutes!
‽ For this point we become
computer dependent where every
spot on each image is measured.
33. Some of nitrogenase data sets
contain around 300 images with
over 5000 spots per image!!!
34. ‽ In order to visualize structure we need
to solve phase problem, for protein
structure determination we can use
following ways;
1. If we already have the coordinates of
similar protein then we can solve the
structure by using Molecular
Replacement which involves rotating
and translating it into new crystal
system until we get a good match of
our experimental data.
35. ‽ If we are successful then we
calculate amplitude and phases
from this solution then which can
be combined with our data to
produce electron density map.
36. 2. If we have no starting model,
then we use Isomorphous
Replacement Method where one
or more heavy atoms are
introduced into specific sites
within the unit cell without
disturbing the crystal lattice.
37. ‽ This is the process where electron
density map is describes in terms of
a set of atomic coordinates.
‽ This is more better in molecular
replacement case because we already
have coordinate set to work with.
‽ Normal procedure is to fit a protein
backbone first then if resolution
permits insert the sequence.
38. ‽ The amount of detail that is
visible is dependent on the
resolution and quality of the
phases.
‽ Often regions of high flexibility
are not visible at all due to static
disorder, where structure varies
from one molecule to next within
the crystal.
39.
40. ‽ After having a model we can
refine it against our data.
‽ This improves the phases which
results in clearer maps and
therefore better models.
Editor's Notes
1- if internal order of crystal is poor then x-ray will not be diffracted to high resolution and data will not yield detailed structure…….
If no crystals are form then we change conditions like temperature etc.
Typical and good crystals take days to weeks to grow.
Once we have crystals then its time to test them with x-rays. (start)
Solve phase problem means obtain some phase information