The Second International Workshop on X-ray Damage to Crystalline Biological Samples, funded by DOE-OBER and NIH-NCRR and co-sponsored by the Structural Biology Center, ANL and the APS User Program Division, was held on 1-2nd December 2001 at the APS, Argonne National Laboratory, Illinois, U.S.A. The Workshop was organised by Elspeth Garman, [University of Oxford, U.K.], Colin Nave, [CCRLC Daresbury, U.K.] and Gerd Rosenbaum, [University of Georgia, U.S.A.]. Local arrangements were made by APS Conference Services in consultation with Susan Strasser of the APS Users Office and Gerd Rosenbaum.

There were 49 registered participants 17 of whom gave presentations. As in the spirit of a workshop, interruptions requesting factual information from the audience were encouraged, but detailed scientific debate was saved for the end of each talk and the main discussion session on the second morning.

The specific topics discussed at the Second Workshop included:

1) Experience of data collection at very high intensities.

2) Radiation damage at specific sites in a protein.

3) Effect of radiation damage on structure determination by MAD and SAD.

4) Modelling studies of putative heating effects at high intensities.

5) Experience of use of helium for cryo-cooling.

6) Assessment of the onset of increasing degrees of radiation damage as a function of dose and dose rate.

After a welcome and introduction to the remit of the Workshop by the Chair for the day, Elspeth Garman, some aspects of data collection at very high intensities on an undulator beamline were reported by Andrzej Joachimiak [APS, U.S.A.] followed by a talk on the radiation damage process from the perspective of radiation chemistry given by Peter O'Neill [MRC Harwell, U.K.]. Preliminary experimental results on the addition of radical scavengers as a possible damage mitigation strategy were presented by James Murray [University of Oxford, U.K.] and James Penner-Hahn [University of Michigan, Ann Arbor, U.S.A.] described radiation damage of redox sensitive transition metal ions in catalase during EXAFS measurements, both at cryogenic and room temperature.

The question of sample heating and cryogen temperature was then addressed. The theoretical modelling and analysis of the heating of crystals in high flux density X-ray beams was outlined by Michael Kazmierczak [University of Cinncinati, U.S.A.] and Eddie Snell [NASA Huntsville, U.S.A.] reported the first experimental results of using an infrared camera to image the cryo-cooling of protein crystals. Experiments testing the radiation damage induced at temperatures alternating between 40K and 150K were described by Tsu-yi Teng [University of Chicago, U.S.A.] and at 16K by Leif Hanson [Oak Ridge, U.S.A.]. Edgar Weckert [Universität Karlsruhe, Germany] reported some experiments to investigate the use of accurate d-spacing measurements to track temperature changes and radiation damage in the crystal sample. Measurements on the variation of specific structural damage to the protein as a function of temperature were described by Martin Weik [IBS Grenoble, France].

One of the most serious results of radiation damage is that it causes the failure of the Multi-wavelength Anomalous Dispersion [MAD] method of phasing because the diffraction data quality becomes compromised before enough information has been collected. Luke Rice [University of California, San Fransisco, U.S.A.] had analysed the radiation damage in failed MAD experiments and investigated the potential of using a single wavelength to obtain the necessary phase information. Raimond Ravelli [EMBL Grenoble, France] presented a novel way of phasing proteins using a method he named RIP [Radiation Damage Induced Phasing] whereby data were collected before and after significant radiation damage. For the second data set the sulphurs had suffered structural damage and difference in the signal from with and without sulphur in an `inverse heavy atom experiment' could give phase information. He presented a successful test of this method for bovine trypsin. Masaki Yamamoto [RIKEN, Japan] described the design and installation of a trichromator at SPring-8 used to try and optimise MAD experiments, and some experimental results from it.

The Workshop then moved on to consider dose/dose rate effects. Observations of radiation damage at different doses/dose rates to lipid membranes and mesophases were summarised by Martin Caffrey [Ohio State University, U.S.A.], and Colin Nave [CCLRC Daresbury, U.K.] reviewed the current status of our knowledge of dose limits and the consequences for the use of protein microcrystals. An experimental study on 5 different proteins of the dose/dose rate question was presented by Piotr Sliz [Harvard University, U.S.A.] and Sean McSweeney [ESRF, France] described extensive investigations into the same question.

During the extensive and lively discussion session, it was apparent that our understanding of some of the above areas had increased since the first Workshop at the ESRF in June 1999, but there is still substantial work to be done if reliable mitigation strategies are to be identified and implemented. There was a protracted debate on the best radiation damage indicators, and further questions requiring experimental answers were posed. Some participants offered to undertake the necessary investigations, as well as further theoretical studies of, in particular, crystal heating.

The question of experimental practice for radiation damage investigations was also addressed. Necessary information for systematic studies to be fruitful is: the careful estimation of incident and absorbed radiation dose for all measurements, all parameters of experiment (flow rate of cryogen, exact cryo-protocol of crystal treatment, crystal size) to be recorded to allow isolation of the variable in question, and comparison experiments where possible to be performed under identical physical conditions (beamline, slits, wavelength, attenuation).

The following conclusions were drawn (by the authors of this report) from the various presentations. They do not necessarily refer to published work subject to peer review. Any reference to these conclusions, where relevant, should be taken from the refereed papers published in the special section of Journal of Synchrotron Radiation [Sep or Oct 2002] and those published elsewhere.

1. A large number of damage sites per protein molecule are created during typical X-ray experiments. These can cause effects at specific sites, observed by analysis of protein crystals. The identification of "sensitive" sites is important as it means that caution should be used in interpreting structural features in these regions if the specimen has been subject to a high dose. This is especially true of active sites of enzymes which by their nature are usually solvent accessible and therefore more susceptible to damage. However, ionisation and free radical formation is likely to occur elsewhere without producing identifiable changes in the positions of the atoms.

2. Radiation damage can cause reduction at metal sites. This is an important consideration during X-ray spectroscopy (and diffraction) investigations of metallo-proteins.

3. The radiation damage can alter phase transitions (in lipid systems), even though it is not always possible to identify the onset of radiation damage from individual X-ray diffraction patterns.

4. It is possible to model heating effects in protein crystals subject to intense X-ray beams. Measurements of actual heating can be made by observing small changes in d-spacing or by thermal imaging. It would be useful to combine all these methods.

5. Published work and data presented at the workshop indicate that radiation damage depends only on the absorbed dose for flux densities up to 10¹⁵ photons/s/mm². No clear evidence for a dose rate effect was presented.

6. The effect of ascorbate as a scavenger to reduce radiation damage at cryo-temperatures appears promising and merits further investigation.

7. Cooling to a temperature of 40K appears to decrease the atomic displacement factors (B values) relative to 100K (Teng's presentation and Teng and Moffat (2002) J. Synchrotron Rad. (2002). 9, 198-201). However the increase in B factor with X-ray exposure occurs at the same rate for exposures up to 2 * 10⁷ Gy. It is not clear whether the potential gains merit adoption of helium cooling as a routine technique. Teng and Moffatt concluded that the benefit of cooling to 40K was only significant at doses greater than 1.5 * 10⁷ Gy which is much larger than commonly needed for a full data set.

8. Radiation damage can have a serious effect on MAD experiments and this has led to the suggestion that collecting complete, high quality data at the "peak" wavelength should be a priority. Other wavelengths could then be used as an "insurance" if the SAD methods fail. This advice will depend on the details (e.g. is there a strong "white line"). Some work on using the changes produced by radiation damage at specific sites as a phasing method was presented.

Clearly the issue of radiation damage to biological specimens still merits further investigation. It is possible in the future, as for electron microscopy, that low dose techniques with multiple specimens will become more popular for examining weakly diffracting specimens. However, the X-ray structural biology community are now more aware of the issue and, like the electron microscopists, are designing their experiments to take account of the effect of intense doses on the specimens.

The organisers of the workshop gratefully acknowledge all the contributors for a stimulating meeting in which these, and other topics were discussed in the formal and informal sessions, as well as Susan Strasser, the staff of the APS User Office for making all the local arrangements, and Roland Hirsch (DoE-OBER) and Amy Swain (NIH-NCRR) for funding. They would hope to organise a follow up Third Workshop in 2003, probably hosted by the ESRF.