Data collection is one of the most important
processes in the determination of protein structure. The first step of data collection
is the mounting of the crystals. Once a suitable crystal has been grown it
needs to be mounted in order to be visualised. However, this step can be tricky as crystals
are small, fragile structures. Mounting can be done in two different ways; either at room
temperature or via freezing. Room temperature mounting is the more conventional
method. The crystal are mounted in a sealed thin-walled glass capillary tube. The surrounding
solution is then removed by filter paper, or by pipetting. The crystal adheres to the
walls of the capillary tube via surface tension and is sealed with oil, maintaining a stable
environment for water vapour to be added. It is necessary that the crystals are kept
in an environment saturated in water vapour, as being 50% water by volume themselves, they
are sensitive to dehydration. Maintaining this humidity prevents this water from evaporating,
thus avoiding crystals drying out and disordering. The crystals are mounted in a environment
which has a temperature of around 4 degrees celsius. The main advantage of room temperature mounting
is that there is less damage to the crystal in comparison to freeze mounting. The main disadvantage is that crystals experience
rapid radiation damage. This is comprised of two parts: a dose-dependent component due
to ionisation of protein and solution by X-ray photography, and the more dominant time-dependent
component, due to generation and propagation of free-energy radicals. Some crystals last
only 10 hours on a home source, which equates to approximately a minute of X-ray exposure
at X25. Freeze mounting reduces radiation damage from
the X-rays, in addition to noise in the Braggs peaks caused by thermal motion. The crystal
must be scooped up with a nylon loop to avoid unwanted dehydration prior to freezing. It
must then be coated with a cryoprotectant, such as glycerol, in order to remove as much
water as possible from the surrounding solution. The crystal is then flash-frozen. There are various different methods which can be used for freezing crystals, the most common being freezing with liquid nitrogen.
Other methods of freezing however, include using liquid propane, liquid freon or a cold
stream of nitrogen gas (at around 100K). Ice crystals are undesirable, as they would also diffract the X-rays and form unwanted rings on the pattern given by the detector. Flash-freezing ensures that the water inside the crystal will glassify, wherein the solvent molecules do not have crystalline order, but order is maintained for the crystal in lieu of ice crystal formation. There are many advantages of freeze mounting:
It is easier to store and transport frozen crystals.
The radiation damage to crystals is also reduced during data collection. Even though the X-rays
may ionise some of the atoms and form free radicals, these will have much less energy
to move around the frozen crystal and transfer the damage.
The cold temperatures used, to keep the crystal frozen, also reduce radiation scattering due
to the background. This reduction improves signal-to-noise ratio and thus improves the
resolution limit. Lastly, using frozen crystals allows for the
freeze trapping of reaction intermediates. However, there are also disadvantages:
As crystals are extremely fragile structures, they can be easily damaged through the process
of freezing. This is why crystals are pre-soaked in a cryoprotectant prior to freezing. However,
the cryoprotectant can also cause the crystal to crack, which ruins the crystal for crystallography.
Successful cryo-conditions are generally identified through trial-and-error. Today, crystals can also be mounted through
automated machines. Automated mounting, along with graphical computing have made calculating
and modelling crystals much easier. Here, we have a short video, which demonstrates
the automated-mounting process The next step is the generation of the data.
The crystal must be mounted in front of the x-ray source/generator, on a goniometer. This
allows the crystal to be rotated, with the detector remaining behind it. X-rays are then
generated. There are two common ways to generate X-rays; producing X-rays using the bombardment
of a copper anode, or by using a synchrotron. In producing the x-rays with a copper anode,
the anode is bombarded with electrons, to produce X-rays which are 1.54A, the
characteristic wavelength for copper. Using a synchrotron is the preferred alternate
method. Electrons are guided around a large ring by magnets. When they bend around the
circle, the electrons emit a spectrum of very intense radiation. This gives a brighter X-ray
beam, and also a choice of wavelengths between 0.5A and 1.6A. This can make it easier to
spot high resolution reflections. Detectors are then used, which allow us to
obtain the X-ray reflections from the crystal, which are used to deduce the protein structure. Detectors can traditionally be X-ray films,
image plates, or CCDs (Charge-Coupled Devices) which are found at the synchrotron and are
now increasingly more common at home. Image plates are a sensitive but cheap method
which are often used in labs. However, there is a lot of noise associated with this method
which is a major disadvantage comparing to CCDs. A charged-coupled device measures the movement
of electrical charge and is very sensitive. It produces data with the best resolution. These data collection techniques have been
used in multiple research projects. They have been used extensively from the 1970s, including
in the studies of viruses, such as the Satellite Tobacco Necrosis Virus. Now, onto some multiple choice questions,
to assess your knowledge. For room-temperature mounting, why do crystals
need to be kept in an environment where they are saturated in water vapour?
a) Because they are 50% water themselves. b)In order to adhere the crystal to the capillary
tube wall. c) To replace the surrounding solution of
the crystal. d) To induce the evaporation of the water
within the crystal structure. The answer is: a In terms of resolution achieved, what is the
best X-ray detection method to use? a) X-ray films.
b) CCDs. c) Image Plates.
d) None of the above. The answer is: b Which is of the following is NOT an advantage of freeze-mounting crystals? a) It is easier to store and transport frozen
crystals. b) Using frozen crystals allows freeze trapping
of reaction intermediates. c) This reduces the damage to the crystal,
in comparison to room temperature mounting. d) The radiation damage to crystals is reduced
during data collection. The answer is: c. Though radiation damage
is reduced, saying damage alone, would be too vague Why is the crystal mounted in front of the
x-ray source/generator, on a goniometer? a) This allows the crystal to be rotated,
with the detector remaining behind it. b) This allows the crystal to be held in one
position, with the detector remaining behind it.
c) This allows the X-rays to be directed at the crystal.
d) None of the above. The answer is: a How are X-rays generated?
a) By a synchrotron or bombardment of a copper anode with electrons.
b) By a synchrotron only. c) By bombardment of a copper anode with electrons
only. d) With a goniometer. The answer is: a These references can direct you to sources
which will help to extend your knowledge further. This presentation, along with the video, will
also be available. Thank you for listening.