MIR with Xenon

Protein Crystallography: Xenon Derivative

We are developing facilities allowing the use of 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 crystal, mother liquor and pressurising gas.
Accelerated radiation damage to unfrozen crystals.
Safety implications associated with high pressures.
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.

DEVELOPMENTS @ DARESBURY

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. The Room Temperature Cell Mounted on a Goniometer Head.

Schematic Diagram of the Xenon Cell.

The Various Components of the Room Temperature Xenon Cell.
The Xenon Cell Mounted on the Goniostat of
Station 7.2 at the SRS.

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.

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.
Develop a cell for freeze-trapping xenon in protein crystals.

ACKNOWLEDGEMENTS

Ethan A Merritt �1996-1999/ merritt@u.washington.edu / Biomolecular Structure Center at UW

USEFUL LINKS

Tutorial by Mike Soltis of the SSRL

Xenon and Krypton at LURE

Xenon Pressure Cells at the SSRL

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.