Syllabus of Structural Biology Course
This course is for graduate students who want to understand the basic theory of protein
crystallography
and
to
know
usual
technique
for
the
determination
of
three-dimensional structure of proteins. Total of eight classes are scheduled, as follows.
However, a student can select the classes which he/she wants. He/she does not need to
have the subject that has already been acquired.
1. Overexpression of recombinant protein (more than half a day, but not all the time)
To crystallize a protein, it is important to prepare relatively high concentration of the
protein. In order to prepare a large amount of the target protein, overexpression system
using microorganisms such Escherichia coli (E. coli)and yeast are well used.
1-1. Construction of an overexpression system (no experiment)
・ Cloning the gene encoding the target protein in cloning vector
・ Overexpression of the protein as a recombinant protein using expression vector in
appropriate host cells
In vivo expression system
Escherichia coli, yeast (S. cerevisiae, P. pastries etc.), insect cells
In vitro expression system
cell-free system
1-2. Cultivation of the cells containing the cloned expression vector
In this course, we will cultivate a recombinant E. coli overproducing any protein as a
conventional method.
2. Protein purification (more than half a day)
It is also important to prepare high quality of pure protein for its crystallization. In this
step, chromatography is ordinarily used.
2-1. Sonication of cultivated cells and preparation of cell free extract
2-2. Chromatography
・ Affinity chromatography
Affinity chromatography (AC) is a technique enabling purification of a biomolecule
with respect to biological function or individual chemical structure.
・ Gel filtration
Gel filtration (GF) is a simple and reliable chromatographic method for separating
molecules according to size.
・ Hydrophobic interaction chromatography
The technique of hydrophobic interaction chromatography (HIC) is a technique for
the purification and separation of biomolecules based on differences in their surface
hydrophobicity.
・ Ion exchange chromatography (anion exchange, cation exchange)
Ion exchange chromatography (IEX) separates biomolecules based on differences in
their anionic or cationic charge characteristics. High resolution can be achieved during
gradient elution by optimizing the ionic strength or pH of the buffer.
2-3. Confirmation of the purity of the protein
・ SDS-PAGE
In this course, we will perform affinity chromatography for purification of a
recombinant protein as a simple purification procedure.
3. Crystallization (60-90 min.)
For crystallization of protein, following procedures are usually performed.
・ Check the purity of the protein
・ Concentration of the protein in a suitable solvent
・ Screening a crystallization condition and finding a suitable precipitant
・ Optimization of the concentration of precipitant, pH, temperature, etc.
3-1. Crystallization techniques
There are several techniques for setting up crystallization experiments.
・ Sitting drop vapor diffusion
The advantages of the sitting drop technique include speed and simplicity. The
disadvantages are that crystals can sometimes adhere to the sitting drop surface
making removal difficult. The sitting drop is an excellent method for screening and
optimization
・ Hanging drop vapor diffusion
The advantages of the hanging drop technique include the ability to view the drop
through glass without the optical interference from plastic, flexibility, reduced chance
of crystals sticking to the hardware, and easy access to the drop. The disadvantage
is that a little extra time is required for set ups.
・ Microbatch under oil
The benefits of Crystallization Under Oil include the use of very small sample and
reagent volumes with less concern for unwanted evaporation, the minimization of
surface interaction with the sample, the ability to precisely control sample and
reagent concentrations during the experiment, and the minimization of condensation
during temperature fluctuations.
・ Dialysis
The advantage of dialysis is that the precipitating solution can be easily changed.
In this course, crystallization of protein will be performed by hanging drop vapor
diffusion method. As a model protein, lysozyme will be used here.
4. Data collection (180 min.)
If you have a good crystals of proteins, the next step is to collect X-ray diffract data.
As the theory of data collection is somewhat difficult, only the basic of the theory is
lectured. The best way to know how to collect data is to see the practical data collection
experiment procedure. We will go to the Faculty of technology, Kagawa University, and
see the data collection using an X-ray diffractometer.
5. Phase determination and calculation of electron density (90 min.)
The phase determination is the big hurdle that must be overcome for X-ray crystal
analysis. There are many of methods for phase determination, depending on the
proteins. In this class, they are lectured briefly, and we will determine the initial phase
of lysozyme by a molecular replacement method, and calculate the electron density for
lysozyme.
6. Modeling and Structure Refinement (90 min.)
Now, many of excellent programs for modeling are available. In this class, you will
learn these programs, and do modeling procedure in the classical way on a computer.
The theory of the structure refinement is difficult, but the excellent programs are also
available. You also will learn the refinement calculations using the program CNS.
7. Use of Protein Data Bank and Drawing of model (90 min.)
The structural information must be deposited to Protein Data Bank. In this class,
you will learn the use of PDB and how to draw the beautiful protein structure which we
see in the journals. To draw an original picture of protein structure is home work.
8. Presentations and free discussion (60 min.)
Show your original work drawing the protein structure, using PowerPoint slides.
After presentations, we will have free discussion about this course with tea and cakes.