PROTEIN CRYSTALLOGRAPHY
Xenon and Krypton Derivatives for Phasing
We have facilities for using xenon derivatives (at room temp. and 100K) for MIR and Anomalous Dispersion experiments on the protein crystallography stations at the SRS, Daresbury.
Why use xenon? | Properties | History
Developments
of the method @ Daresbury | Useful links | Reference
list
Why Use XENON?
- Derivative protein crystals can be produced by pressurising native crystals with xenon gas. Modification of the mother liquor to determine soaking conditions is avoided.
- The number of binding sites, and their occupancies can be changed by altering the gas pressure, thus, several derivatives could be produced from the same crystal. These sites often differ from metal binding sites.
- Xenon atoms interact weakly with protein; isomorphism of the derivative with the native is high.
- Xenon binding is often reversible; the same crystal could be used for a further heavy atom soak.
Problems
- Systematic errors in measured intensities due to absorption by the pressurising gas.
- Accelerated radiation damage to unfrozen crystals.
- Safety implications associated with high pressure equipment.
- Possible formation of hydrates at low temperatures and high pressures.
XENON Properties
| XENON (Xe) | |
|---|---|
| Atomic no | 54 |
| Melting point | -112 |
| Boiling point | -108 |
| Relative density (Gas) | 4.5 |
| Relative density (liquid) | 1.5 |
| Molecular weight | 131 |
| Solubility in water | 644 mg/l |
| Absorption edges (Å and (kev)) | ||||
|---|---|---|---|---|
| K | LI | LII | LIII | MI |
| 0.3587 | 2.2738 | 2.4293 | 2.5926 | 10.8321 |
| (34.5614) | (5.4528) | (5.1037) | (4.7822) | (1.1446) |
Text file of anomalous scattering coefficients f' and f'' as a Function of energy
HISTORY
- 1965
- Schoenborn et al determine a xenon binding site in sperm whale metmyoglobin by collecting x-ray diffraction data to a resolution of 2.8Å from crystals pressurised to 2.5 atmospheres.
- 1984
- Tilton et al varied the number of bound xenon atoms by altering the sample pressure.
- 1991
- Vitali et al demonstrate the use of xenon as a heavy atom, for the first time, in determining phases for the structure of sperm whale metmyoglobin, with the technique of isomorphous replacement with anomalous scattering (SIRAS).
- 1994
- Schiltz et al use xenon as an alternative to heavy metal derivatives; for the first time calculate phases of a protein of unknown structure using a xenon derivative.
- 1997
- Soltis et al for the first time, demonstrate a method for preparing cry-cooled xenon derivatised protein crystals.
Devlopments @ Daresbury
We now have the equipment to produce xenon derivatives from crystals and collect the anomalous data at room temperature or 100K. The equipment for freezing crystals or producing xenon derivatives can be borrowed for use on any of the PX stations.
For Capilliary Mounted Crystals
We are developing a xenon cell at Daresbury Laboratory for collectiong room temp. data from xenon derivative crystals in the 0 to 25 bar pressure range. It is a very simple but effective device based on a commercially available compression fitting.
left-The Room Temperature Cell Mounted on a Goniometer
Head.
right - The Various Components of the Room Temperature Xenon Cell
Schematic Diagram of the Xenon Cell
The Xenon Cell Mounted on the Goniostat of Station 7.2 at the SRS.
For Frozen Crystals
We have acquired an Oxford Cryosystems Xcell for pressurising crystals prior to freezing.
Users of the xenon pressure cell at Daresbury
are required to complete a class 3 risk assessment, including a description
of the hazard and safe system of work, prior to their beamtime.
http://www.dl.ac.uk/srs/PX/xenon_cell/new_risk.html
Instructions for Using the Oxford Cryosystems Xcell
- Unscrew the Xcell filter paper cap and replace the old filter with one soaked in mother liquor. Refit the filter paper cap and tighten finger tight. Do not remove the filter paper cap once the Xcell is pressurised.
- Unlock the Xcell slider mechanism and fit a magnetic base to the sample mount.
- Connect the gas regulator to the xenon gas bottle.
- Connect the gas hose to the regulator and the Xcell inlet valve. Do not disconnect the gas hose from the regulator once the hose is pressurised. Do not use the Xcell on uneven surfaces.
- Ensure: the Xcell vent valve is closed; the slider mechanism is locked; the filter paper cap is fitted and the inlet valve is closed.
- Vent the Xcell and gas hose by: opening the xenon gas bottle valve; setting the gas regulator to the working pressure; slowly opening the Xcell inlet valve; slowly opening the Xcell vent valve to bleed air from the system. Do not exceed 25 bar when pressurising the Xcell
- Close the Xcell inlet and vent valve.
- Disconnect the Xcell from the gas hose and position it close to the sample. Do not use the Xcell on uneven surfaces.
- Slowly open the Xcell vent valve to de-pressurise the chamber.
- Unlock the Xcell slider mechanism and expose the magnetic base.Do not open the chamber while the Xcell is pressurised.
- Mount the loop with crystal on the magnetic base.
- Shut and lock the Xcell slider mechanism.
- Carefully take the Xcell back to the xenon gas bottle.
- Reconnect the Xcell to the gas hose. Do not use the Xcell on uneven surfaces.
- Ensure: the Xcell vent valve is closed; the slider mechanism is locked; the filter paper cap is finger tight and the inlet valve is closed.
- Slowly open the Xcell inlet valve pressurising the chamber to the working pressure. Do not exceed 25 bar.
- Slowly bleed air from the Xcell chamber using the vent valve.
- Ensure the Xcell has reached the working pressure then close the inlet vale. Do not exceed 25 bar.
- Close the xenon gas bottle valve.
- Leave the crystal under pressure for the desired length of time.
- Disconnect the Xcell from the gas hose.
- Carefully position the Xcell close to the freezing agent. Do not use the Xcell on uneven surfaces.
- Slowly open the Xcell vent valve to de-pressurise the chamber.
- Unlock the slider mechanism, carefully remove the loop and crystal for freezing. Do not open the chamber while the Xcell is pressurised.
Future Developments
Explore the possibilities of exploiting absorption edges for MAD and SAD experiments. Xe K-edge 0.3587 Å, Kr K-edge 0.86552 Å.Acknowledgements
Ethan A Merritt ©1996-1999/ merritt@u.washington.edu / Biomolecular Structure Center at UW
Useful Links
- Xenon as a Heavy Atom? by Mike Soltis of the SSRL
- Xenon and Krypton at LURE
- Xenon Pressure Cell at the SSRL
- Another Xenon Pressure Cell at the SSRL
- MAD Phasing of MB and SP18 with Kr by Mike Soltis
REFERENCES
- Binding of Xenon to Sperm Whale
Myoglobin. Schoenborn B.P.; Watson, H.C.; Kendrew, J.C. (1965). Nature, 207,
28-30.
- Cavities in proteins: structure
of a metmyoglobin-xenon complex solved to 1.9&Angring. Tilton,
R.F.; Kuntz, L.D.; Pesko, G.A. (1984) Biochemistry 23. 2849-2857.
- Using Xenon as a Heavy Atom
for Determining Phases in Sperm Whale Metmyoglobin. Vitali, J.; Robbins,
A.H.; Almo, S.C.; Tilton, R.F. (1991). Journal of Applied Crystallography, 24,
931-935.
- On the Preparation and X-ray
Data Collection of Isomorphous Xenon Derivatives. Schiltz, M.; Prange,
T.; Fourme, R. (1994). Journal of Applied Crystallography, 27,
950-960.
- Successful flash-cooling of
xenon-derivatised myoglobin crystals. Soltis, S.M.; Stowell, M.H.B.;
Wiener, M.C.; Philips, G.N.; Rees, D.C. (1997).Journal of Applied
Crystallography, 30, 190-194.
- Freeze-Trapping Isomorphous
Xenon Derivatives of Protein Crystals. Sauer, O.; Schmidt, A.; Kratky,
C. (1997). Journal of Applied Crystallography, 30,
476-486.
- Protein Crystallography at Ultra-Short
Wavelengths: Feasibility Study of Anomalous Dispersion Experiments
at the Xenon K-Edge. Schiltz, M., Kvick, A., Svensson, O., Shepard,
W., De LaFortelle, E., Prange, T., Kahn, R. & Fourme, R. (1997). Journal
of Synchrotron Radiation , 4, 287-297.
- A method to stabilize reduced
and/or gas treated protein crystals by flash cooling under a controlled
atmospher
Xavier Vermede et.al. J. App. Cryst, (1999) 32(3) 505-509 - Better structures from better data through better methods: a review of developments in de novo macromolecular phasing techniques and associated instrumentation at LURE. Fourme, R. et al (1999) J. Synch. Rad. 6(4) 834-844
OTHER REFERENCES
http://www.dl.ac.uk/srs/PX/xenon_cell/reference_list.txt
- Teng et al (1990) J. Appl. Cryst. 23. 387-391
