Xenon derivatization

The use of soft X-rays offers the opportunity to employ a bigger set of atoms for derivatisation: those already naturally present in biological macromolecules (sulfur and phosphorus in particular) as well as more rare ones (Xe, I, U, Cs, Fe, Ca ...) .

Among these the use of xenon as derivative has recently increased a lot, thanks to the easy procedure to obtain derivatives and their high isomorphism. Xenon presents a strong anomalous signal in the range 1.5-3.0 A, with L-I, L-II and L-III edges at 2.27, 2.43 and 2.59 A respectively.

Xenon derivatized crystals can be obtained by subjecting a native protein crystal to a Xenon gas atmosphere. The appreciable solubility of the Xenon in water allows the rapid diffusion of atoms towards the interaction sites via the solvent channels present in protein crystal. To obtain Xenon derivatives, a pressurization cell has been developed and tested at xenon gas pressures in the range 5-70 bars (see picture below). The cell can also be used to obtain derivatives with other gases (e.g. Kr), or to study the interaction between enzymes and their substrates (e.g. CO2).

Due to the weak Xenon binding to the proteins, the pressurized crystals must be quickly transferred in liquid nitrogen (transfer time is usually lower than 3 seconds). XRD1 beamline at the present offers the possibility to produce xenon derivatives using a pressurization custom cell designed starting from Paul Tucker's one or a commercially available cell from Oxford Cryosystems, which is more limited in the pressure range (max 25 bars), but uses much less xenon gas. Both cells can use a pump to use all the gas in the cylinder, even when the available pressure is lower than the target one, and both mount the standard Hampton magnetic pins (the recommended height is 24-25 mm - from the pin bottom to the top of the loop). In the figures below, a picture of the custom pressurization cell (based on P. Tucker's cell): the cell can be safety used in the range 5-70 bars. Last image refers to the commercial Oxford Cryosystems pressurization cell.



There are few considerations about the methods to obtain your gas derivatives (xenon, krypton...). Two main parameters should be taken in to account: the time of pressurization and the value of the pressure itself. As a general rule, the time should be as short as possible to avoid the sample suffering, while the pressure should be as high as possible to ensure a strong and certain derivatization.

Against these points, some initial tests showed the mosaicity increases increasing the pressure, with the risk to loose the isomorphism between the native and the derivative crystals. On the other hand too short times could produce sites (too) weakly occupied, and so useless.

The experience in the pressurization of different crystals suggested the starting values of 10 minutes and 10 bars for time and pressure for the initial pressurization. If the crystal suffers the cryosolution environment, this time should be shorter. A test on a crystal mounted in the closed cell (without any pressurization) can easily indicate such kind of behavior.

The cell routinely used on the beamline is the commercial one (Oxford Cryosystems) for the easiest use and smaller consumptions. The in-house one is rather used for special purposes and can be modified in order to solve individual requirements.
 
Here's the list of procedures in order to obtain the xenon derivative of your protein.

1. Check your crystal survives in the cell for 10 minutes without any pressurization as indicated previously.
2. Close the 2 valves (Inlet and Vent) of the cell.
3. Connect the cell to the cylinder through the inlet valve. If the gas pressure in the cylinder is lower than the pressurization target pressure, fit the pump between the cylinder and the cell inlet valve.
4. Open the cell using the switch at the top of it (turn it right), then pull out the crystal holder using the slider handle.
5. Mount your crystal and close back the cell (do not forget to close the cell turning left the switch at the top).
6. Open the cylinder main valve, and then open the cell vent valve.
7. Open a while (few seconds) the Inlet valve, purging the cell then close it.
8. Close the vent valve and open the inlet one again, reaching inside the cell the pressure you want (control it on the meter at the top of the cell). At the end close the inlet valve. If the pressure in the cylinder is not enough, use the manual pump to reach the pressure value you want (suggested value: 10 bars). Do not use more then 25 bars.
9. Close the cylinder main valve. If you want to mount the crystal directly on the spindle, unplug the tube from the cell inlet valve.
10. Wait the pressurization time (10 minutes suggested), preparing a container with liquid nitrogen or move with the cell close to the beamline spindle (on the experimental table).
11. As fast as possible, open the vent valve. When the meter at the top indicates there is no extra-pressure inside, open the cell rotating the switch to the right and pulling out the crystal using the slider handle.
12. Use some tongs to pick up the pin and plug it into the liquid nitrogen (or use your fingers to put it directly onto the goniometer head).
13. Once in liquid nitrogen, use the Hampton magnetic pen avaiable to handle the crystal and put it in its cap. Working close to the liquid nitrogen, do not forget to use the proper gloves and glasses.
14. Use the mounting arc or the cryo tongs to mount the crystal on the goniometer head or store it in the dewar for a future data collection.
15. During the data collection, check the presence of the heavy atom on the Harker sections.