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7/23/2019 Lysozyme Crystals Lab Report Chem 351
http://slidepdf.com/reader/full/lysozyme-crystals-lab-report-chem-351 1/7
Lysozyme Crystals 1
Lysozyme Crystals
Growth of Lysozyme Crystals
Abstract
For x-ray crystallography, ideal protein crystals are needed in order to get viable results
for the protein structure of protein molecules. The best crystals can be made by optimizing theconditions for making the best lysozyme crystals. This is done by using different precipitant
agents along with different concentrations of the precipitant concentrations and protein
concentrations. The best salt solutions found was NaCl and NaBr. This was discovered by using
different multiple wells with different conditions. The results were compiled from multiple
sources and compared.
Introduction
In order to determine the structure of the protein molecules by x-ray crystallography, it is
essential to grow crystals of the protein. By utilizing systematic trials in order to use optimal
growth conditions, the changes of growing good crystals goes up. One widely used technique is
the hanging-drop vapor-diffusion method. In this lab, lysozyme crystals were grown using this
technique. This is due to the exchange of water vapor between the drop and the reservoir. This
will result in the drop shrinking and leaves crystals in its place. Not only did this help to identify
the optimal conditions in which crystals will grow, but it helped to find no growth, nucleation,
nucleation transitioning to growth and precipitation.
Methods
In order to optimize the growth of the crystals, two different precipitating agents were
utilized in each of the trials. This was done so that the best precipitating agent was found in order
to get the best crystals. Two trials were performed so that the desired concentrations of the
protein and the salt would be found along with the best precipitating agent that could be pairedwith those ideal concentrations. Trial 1 utilized NaCl and NaI as the precipitating agents. Trial 2
used NaCl and NaBr as the precipitating agents. In order to get the desired crystals, a crystalizing
tray was prepared.
Preparation of Crystal Tray
A 24-well crystal tray was obtained, but only 12 of the wells were utilized. This was so
that the wells could be staggered and
ensured that the coverslips did not
overlap each other. This was so that thegrease applied around the wells created a
tight seal with the coverslips. This
method of using every other well can be
seen in Figure 1.
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Lysozyme Crystals 2
In each well, a reservoir was made at the bottom with a combined volume of 0.5mL. The
contents of the reservoirs contained the precipitant solution (NaCl (2M), NaI (2M), NaBr (2M)),
the sodium acetate buffer (1M, pH 4.5), and nanopure water. The concentration each of these
solutions were determined using the ranges in Table 1. After the concentrations were chosen for
each of the components, the concentrations were calculated using Equation 1 (C1V1=C2V2) from
the stock solutions given. The concentrations chosen for trial 1 and trial 2 can be seen in Table 2and Table 3. Then grease was used to rim the top of each of the wells.
After the reservoirs were completed, the drops that hung from the cover slips were made.
Each of the drops contained ½ protein solution (3uL) and ½ reservoir solution (3uL). The
concentration of each of these protein solutions were chosen using the ranges in Table 1 and
calculated using Equation 1. The drops were added to each of the coverslips. Tweezers were
used to grab the corner of the coverslips, quickly flipped over, and placed on top of each well.
This ensured that the drops did not slide off of the coverslips. The tweezers were then pushed
down onto the coverslip to create as seal with the grease on top of each well. The conditions for
trial 1 can be seen in Table 3 (NaCl) and Table 4 (NaI). The conditions for trial 2 can be seen in
Table 5 (NaCl) and Table 6 (NaBr).
Optimization of Crystals
The wells were then left and crystals were allowed to grow over time. The crystals were
checked as soon as possible so that the crystals did not re-dissolve and different crystals grew
instead. After crystals formed, they were put under the microscope. The crystals were stained
with a protein stain in order for the crystals to enhance the images of the crystals under the
microscope. The resulting crystals can be seen in Figure 3 and Figure 4. The results of the
crystal growth can be seen in Table 7. In order to better optimize the crystals, the data for the
crystal growth was collected from the whole class. The results can be seen for NaCl (Graph 1),
NaI (Graph 2), NaNO3 (Graph 3), and NaBr (Graph 4).
Results
Stock solutions used
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Lysozyme Crystals 3
Conditions for Trial 1
Conditions for Trial 2
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Lysozyme Crystals 4
Resulting lysozyme crystal growth for Trial 1
Resulting lysozyme crystal growth for Trial 2
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Lysozyme Crystals 5
Crystal growth results
Class lysozyme growth results
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Lysozyme Crystals 6
Discussion
In this lab, we performed 2 trials to determine the best conditions for the ideal crystalsthat we want for x-ray crystallography. The conditions that stayed the same for all of the trials
was the amount of acetate buffer that was used. This was done to make the conditions consistent
for all of the trials.
For trial 1, the precipitant concentration that was used for the wells of NaCl was
configured using the range from Table 1. Each well of NaCl had a different concentration of
precipitant solution from the lowest end of the range to the highest end of the range from 0.4M-
1.2M. This was done so that the right concentration of the NaCl could be chosen. In order to do
this, the protein concentration used was the middle of the range of the protein of concentration
shown in Table 1 from 20mg/mL-60mg/mL. This was done at a 30 mg/mL protein concentration
so that the protein concentration would not be too high or too low. The resulting crystals inFigure 3 were small, so they were not the ideal crystals needed.
The precipitant concentration used for each well of NaI in trial 1 was different for each
well using the range in Table 1. The range used was 0.10M-0.40M with concentrations from the
lowest end to the highest end of the range. The protein concentration used for NaI was 40mg/mL
because this concentration was in the middle of the range of protein concentration in Table 1.
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Lysozyme Crystals 7
There were no resulting crystals in trial 1 for NaI, because mold had grown instead. This negated
NaI as an ideal precipitant solution that would be used for x-ray crystallography.
For trial 2, we used NaCl and NaBr. We used NaCl because it produced good crystals
from trial 1, but the crystals were too small. Trial 2 was used as a way to optimize the crystal
growth of NaCl. NaCl in the previous trial had small crystals. For this trial, the concentration ofthe precipitation solution stayed the same for NaCl for trial 2. The change in the NaCl wells were
changed by using the lowest amount of protein concentration in the range for NaCl which is
20mg/mL for half of the NaCl wells. The other half of the wells of NaCl used 60mg/mL. The
reasoning behind this was that the protein concentration could be optimized by seeing if a high
concentration or a low concentration of protein would be ideal. This would allow for larger
crystals. The resulting crystals seen in Figure 4 are much larger than the crystals in trial 1 in well
4 and well 5. This could mean that a larger protein concentration would produce a larger crystal.
The second precipitant in trial 2 was NaBr. This was decided because NaBr produced
mold instead of crystals. The precipitant concentration used for each well of NaBr in trial 2 was
different for each well using the range in Table 1. The range used was 0.30M-0.60M withconcentrations from the lowest end to the highest end of the range. The NaBr wells were
changed by using the lowest amount of protein concentration in the range for NaBr which is
50mg/mL for half of the NaBr wells. The other half of the wells of NaBr used 80mg/mL. This
allowed us to see if the crystals formed from the NaBr wells would be larger with a smaller or
larger protein concentration. The resulting crystals were large as well in NaBr as seen in Figure
4 for well 1, well 2, and well 5. Well 5 had only one crystal, but well 1 and well 2 had larger
crystals. This could mean that a smaller protein concentration for NaBr would produce larger
crystals.
When all of the data was compiled from the whole class, it displayed which of the
precipitants would produce the best crystals as well as which protein concentration wouldenhance that. NaI had no growth for the whole class. NaNO3 produced very little crystals. The
best crystals were seen in NaBr and NaCl. For NaBr, the best conditions seem to be a protein
concentration of ~80mg/mL and a precipitant concentration of ~0.4M. For NaCl, the best
conditions seem to be a protein concentration of ~60mg/mL and a precipitant concentration of
~1M.