HT1: Crystallization
L5 (Lecture): Radiation damage in macromolecular crystallography
Elspeth Garman
Biochemistry Department, Oxford University
South Parks Road, Oxford
OX1 3QU, U.K.
E-mail:elspeth.garman@bioch.ox.ac.uk
The three-dimensional structures of biological macromolecules are largely determined by crystallography: intense synchrotron produced X-ray beams of around 13 keV energy are used to measure the diffracted intensities of reflections from crystals of the molecule of interest, and the structure is solved by obtaining phase information by a variety of methods. The crystals typically contain between 30% and 70% solvent, confined in channels between the macromolecules.
For protein crystals at room temperature, radiation damage during the diffraction experiment is rapid even on a laboratory X-ray source. In the past, the required data had to be collected from several different crystals and merged together. The intense X-ray beams produced by third and fourth generation synchrotrons such as the EBS, ESRF, France and the NSLSII, U.S.A. destroy crystalline order in a matter of seconds. Over the last 30 years, the use of cryo-cooling techniques which allow X-ray data to be collected with the sample held in an open stream of cooled nitrogen gas at 100K, has become the norm; at 100K crystals can withstand around 70 times [1] the dose (energy lost/mass = J/kg = Gy) compared with room temperature, and the necessary data can usually be obtained from a single crystal.
However, observations of degradation of crystal diffraction with increasing radiation dose at 100K are commonplace at modern synchrotrons. Researchers have characterised the effects on the data in reciprocal space and on the resulting models in real space in order to understand the physical and chemical processes involved in this damage (reviewed in [2]). It manifests itself in a number of different ways, including: changes in crystal colour, decreasing diffraction power with dose, a small but measurable linear increase in unit cell volume, and specific structural damage to covalent bonds in the amino acids of the protein molecules. The bonds are broken in a reproducible order; firstly delocalisation of the sulphur atoms in disulphides, secondly decarboxylation of glutamate and aspartate residues, followed by the breaking of the C-S bond in methionines [3-5]. Enzyme active sites seem particularly sensitive to damage, so this phenomenon can lead to incorrect conclusions on biological mechanisms being drawn. Thus the issue of radiation damage during diffraction experiments has recently come to the fore as a concern for all structural biologists.
An outline of our current understanding of radiation effects and some current lines of investigations, including a recent survey of damage in deposited PDB structures [6] will be presented.
References:
[1] Nave, C. Garman, E.F. (2005) Towards an understanding of radiation damage in Cryo-cooled crystals. J. Synch. Radiat. 12, 257-260
[2] Garman, E.F., Weik M. (2018) Radiation Damage in Macromolecular Crystallography. Chapter 4 in ‘Protein Crystallography: Challenges and Practical Solutions’ Eds: Konstantinos Beis and Gwyndaf Evans, Published by the Royal Society of Chemistry, Pp 88-116
[3] Weik, M., Ravelli, R.G.B., Kryger, G., McSweeney, S, Raves, M,L,, Harel, M., Gros, P., Silman, I., Kroon, J., and Sussman, J.L. (2000) Specific chemical and structural damage to proteins produced by synchrotron radiation.PNAS 97, 623-628.
[4] Burmeister, W.P. (2000) Structural changes in a cryo-cooled protein crystal owing to radiation damage.Acta Cryst. D56, 328-341.
[5] Ravelli, R.G.B., McSweeney, S. (2000) The ‘fingerprint’ that X-rays can leave on structures.Structure 8, 315-328.
[6] Shelley, K.L, Garman, E.F. (2022) Quantifying radiation damage in the Protein Data Bank. Nature Communications 13:1314- 1325.