Introduction
The human body contains thousands of different proteins, which play essential roles in
maintaining life. Proteins consist of long macromolecule chains made up from 20
different amino acids. The chains can be several hundred residues long and fold into a 3dimensional structure.
The knowledge of accurate molecular structures is a prerequisite for rational drug design
and for structure based functional studies to aid the development of effective therapeutic
agents and drugs. Crystallography can reliably provide the answer to many structure
related questions, from global folds to atomic details of bonding. The price for the high
accuracy of crystallographic structures is that a good crystal must be found, and that only
limited information about the molecule's dynamic behavior is available from one single
diffraction experiment (1). A protein's structure determines the specific role that protein
plays in the human body; however, researchers lack detailed knowledge about the
3Dstructures of many proteins. With an improved understanding of the molecular
structures and interactions of proteins, drug designers may be able to develop new drug
treatments that target specific human, animal, and plant diseases.
Protein crystal growth is mainly a trail-and-error process but the primary rule is that the
protein should be as pure as possible. The higher purity, the better the chance of growing
crystals. In addition to some factors that require consideration are pH, concentration of
protein, temperature, and precipitants. The goal is to obtain a well-ordered crystal large
enough to provide a diffraction pattern when hit with x-ray beam.
There are several crystallization methods like vapor diffusion microbatch .The bestknown method is the vapor diffusion method in the hanging drop configuration. In this l a
droplet containing purified protein, buffer, and precipitant allowed to equilibrate with a
larger reservoir containing similar buffer and precipitant in higher concentrations.
Initially, the droplet of protein solution contains an insufficient concentration of
precipitant for crystallization, but as water vaporizes from the drop and transfers to the
reservoir, the precipitant concentration increases to a level optimal for crystallization.
Since the system is in equilibrium, these optimum conditions are maintained until the
crystallization is complete. (2)
Figure 1: Diagram of the hanging drop method
The microbatch methods under oil consist to pipette a small drop of protein combined
with the crystallography reagent, under a layer of oil. The benefits of microbatch under
oil include the use of very small amounts of protein and reagent volumes.
Figure 2: The microbatch method after Naomi Chayen.
X-ray crystallography is an experimental technique that exploits the fact that X-rays are
diffracted by crystals. X-rays have the proper wavelength (in the Angstrom range, ~10 -8
cm) to be scattered by the electron cloud of an atom of comparable size. Based on the
diffraction pattern obtained from X-ray scattering off the periodic assembly of molecules
or atoms in the crystal, the electron density can be reconstructed. A model is then
progressively built into the experimental electron density, refined against the data
resulting in the 3D molecular structure.
Synchrotrons are very big machines that allow a multitude of different experiments.
The synchrotron light is guided tangentially away from the storage ring through beam
lines were relativistic co are kept in a closed orbit. Beam lines are a collection of devices
that transport and condition the photon beam from the SR source to the experiment.
Beam line consist of front end, which contains slits, windows and safety components like
beam shutter and beam stopper. Windows, Apertures, Mirrors which can be focusing,
collimating, monochromators, which select a single wavelength It can either be a crystal
(for hard X-rays) or a grating (for soft X-rays), beam monitors (position and/or intensity),
end-station (sample manipulate environment control, detectors etc.). (1)
Goal
The goal of my experiment is to grow crystals in capillaries using microbatch method
under oil and to check if we can obtain high quality crystals that diffracted to high
resolution.
The experiment:
Sample:
Here we used a well known protein is Hen Egg white Lysozyme. Lysozyme’s 3D
molecular structure was first determined in 1965.
3
4
Figure 3: Buffer solutions in preparation for the experiment.
Figure 4: Microbatch plate used in this experiment.
We prepared 100 mg/ml protein solution and buffer of 50% Na Acetate, pH 4.7 and 50%
Glycerol to which we added enough NaCl to start the growth porcess.
We filed a microbatch plate with 2ml of mineral oil making sure that the entire surface
was covered by the oil. Next, we mixed the protein with the buffer and dispensed it in the
holes for crystallization. The plate was then kept at room temperature.
We used a variation of the microbatch method to grow crystals in a containerless
geometry in capillaries for diffraction. In this method developed by Dr. Naomi Chayen,
the sample has no contact with the surface of the crystal plate, Figure 5, or capillary.
Drops are dispensed at the interface between two layers of oil of different density. Highdensity oil is used in the bottom and low-density oil on the top. Figure 6, shows the
formation of one of these crystals to obtain the diffraction pattern.
5
Figure 5: The containerless method under two oils after N. Chayen.
Figure 6: Crystal grow in the capillary using containerless method.
6
We used two capillaries in this study:
1. Contained 6 crystals: only one was separated enough from the other crystals for data
collection.
2. Contained 6-7crystals: two crystals were separated from the others enough for data
collection.
Diffraction:
The capillaries were mounted on the X6A beam line, which is a typical protein
crystallography beam line at the NSLS. The sample to detector distance was optimized to
obtain the highest diffraction resolution. Figure7 shows a close-up of the end station.
.
Figure 7: Experimental setup on the X6A beam line of the NSLS.
360 degree was collected on one of the crystals, Figures 8. A typical example of the
diffraction patterns collected is shown in Figure 9. The patterns were indexed, the cell
parameters and space group were determined and the mosaicity was recorded.
Figure 8: Crystal in capillary at the beginning of the data collection
Figure 9: The diffraction pattern of the crystal shown in Figure 8, Resolution 1.4A,
Time/degree 3 sec, Distance 180mm
Conclusion:
One problem we encountered was that several crystals formed in the capillaries. It was
necessary to choose one crystal that was sufficiently separated from the others and did
not move during data collection. Finally it was possible to
 grow crystals by the batch method and containerless method in capillaries.
 show that high quality diffraction data can be collected on crystals grown at room
temperature in a viscous media.
AKNOWLEDGMENT
I would like to thank the US Department of Energy Initiative for the Middle East for the
financial support during this training. The support and incentive of Professor Brenda
Laster and Dr. Herman Winick. Jean Jakoncic for his guidance during the experimental
aspects of this work. Dr. Vivian Stojanoff, Dr. Zhong Zhong and Dr. Lisa Miller for
receiving us in their groups. I'm grateful to the NSLS staff without which this training
would not have been possible. The National Synchrotron Light Source is supported by
the U.S. Department of Energy, Office of Science, and Office of Basic Energy Sciences,
under Contract No. DE-AC02-98CH10886 ”. The X6A beam line is supported by a
NIH/NIGMS grant GM Y1-0080.
References:
1. http://www.lightsources.org/cms/?pid=1000147
2. http://www-structure.llnl.gov/Xray/101index.html
3.http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Kogoy/protein.
html
4. http://www.moleculardimensions.com/datasheets/md1-12.htm
5. http://www.hamptonresearch.com