How does the HC1b work?
The device produces an airstream around the sample at controlled relative humidity (RH). This airstream is generated by using DewPoint to remove water from a hot, saturated airstream, to give a specific RH when it reaches the crystal. This can be used to change the hydration state of macromolecular crystals.
In practice what you will see at the beamline is a device next to the diffractometer with a nozzle that looks like a standard cryo-stream. Your crystals can be directly mounted by hand, just as if you were cryo-cooling directly on the beamline, and from that point onwards there is nothing different to a standard data collection.
Why use dehydration?
Dehydration is a factor known to affect crystal lattice. The changes it produces in the packing can, in some cases, lead to improved diffraction properties. Unit cell contraction, decreased mosaicity, space group changes and even improved diffraction limit are among the documented effects.
Why use a device and not chemical methods?
The advantages of using a device to control dehydration are that we can couple dehydration with data collection, and we can precisely control the speed at which the experiment happens. In this way you have a direct measurement of the effect dehydration has on your diffraction with no added treatment, like cryo-protection, prior to data collection.
Why use this device and not others?
The new HC1b device is easy to mount on a standard beamline without disturbing any of the other equipment. It is easy to use and to monitor progress for anyone familiar with normal operation in any standard beamline. Currently there are two other options to carry out this sort of experiments (REFS). Despite being perfectly suitable, they are not very user-friendly and are not available for use at a synchrotron beamline.
Why perform this experiment at the beamline rather than at my home source?
In principle your home source is perfectly good for this sort of experiment: it will definitely pose less threat to your crystals in terms of radiation damage, and you will not waste a day of good beamtime. Despite this, many badly diffracting crystals only give any hint of diffraction once they are tested on a synchrotron, and thus will not be available for use at a home lab thereafter. Furthermore, the time required for collecting a single frame in house (5-30 minutes) gives very little opportunity to record changes that happen over a shorter time span. At a beamline the typical exposure time (0.1 - 2 seconds) permits accurate characterisation of even the most subtle changes. Lastly, despite being time-consuming, these experiments are often performed as part of a broader approach to obtain suitable data. In many important cases a number of synchrotron shifts is required to test hundreds of crystals that have been harvested over weeks or months of careful work in order to obtain that elusive dataset. If this beamtime is used responsibly it can only help in achieving that final goal.
What do I need to look for in my crystals?
This is a difficult question as no thorough studies have been undertaken. There are several indicators to suggest the likelihood of your crystals changing.
In principle the solvent content may be a good indicator that there is room for improvement. The more solvent in your crystals, the more solvent can be excluded, and the more space there is for rearrangements. Despite this, keep in mind that if the crystals harbour a membrane protein with detergent, or a membrane between the different protein molecules, there is not as much removable solvent and thus dehydration may not encourage the required rearrangement.
The second thing to look for is a protein capable of crystallising more than one alternate spacegroup and/or unit cell. Also look for those crystals capable of changing packing upon the addition of substrate, additives, cryos, etc, or which change harvesting, soaking or cryo-protection times vary. These kinds of changes are indicate the possibility of movement within the crystal, and therefore dehydration is potentially very useful.
Symmetry may also be important. In order to improve the internal order of the crystal, local rearrangements are needed to realign the molecules or to stabilise flexible areas of the individual protein molecules. If the arrangement of the molecules is such that they establish a great number of equivalent crystal contacts, there may be very little hope for such an increase in contacts. Instead they would need to be broken and reformed differently. On the contrary, if the symmetry of the crystal is such that, small compressions or rearrangements create new symmetry interactions, a greater number of previously unrelated molecules can now contribute to the overall scattering, thus increasing the diffraction power/order of the crystal. Furthermore, a similar effect may be triggered by dehydration if the asymmetric unit is constituted by a number of non-symmetric monomers.
What do I do after dehydrating?
Dehydration may actually be an alternative method of cryo-protection. Dehydration will exclude water while increasing the concentration of the mother liquor in the solvent channels, and can prevent ice formation. Furthermore, much of the mechanical stress, which crystals undergo upon flash cooling, can be minimised by having contracted the lattice to a minimum, prior to cryo-cooling.
What if I cannot cryo-cool my crystals after dehydration?
If all attempts to cryo-cool the crystals fail to yield a suitable dataset, an alternative option is to collect them at room temperature. Despite the device being stable enough to permit collecting a full dataset, it will have to be collected with great caution. First, radiation damage is a major worry and the data collection strategy has to be meticulously optimised. It may even be necessary to collect data from several crystals in order to get a full dataset. Secondly, as crystals are not cooled they may move, thus complicating data processing.
How do I cite the HC1?
If you've got results using the device that you would like to publish please site the following references.
Diamond Light Source is the UK's national synchrotron science facility, located at the Harwell Science and Innovation Campus in Oxfordshire.
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