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Radiation Damage To Biological Macromolecules

Radiation damage to biological macromolecules: some answers and more questions Elspeth F. Garmana * and Martin Weikb,c,d,e* a Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK, bComissariat a` l’Energie Atomique, Insti...

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radiation damage
Journal of
Synchrotron
Radiation damage to biological macromolecules:
Radiation some answers and more questions
ISSN 0909-0495

Elspeth F. Garmana* and Martin Weikb,c,d,e*
Received 11 December 2012
Accepted 11 December 2012 a
Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks
Road, Oxford OX1 3QU, UK, bComissariat à l’Energie Atomique, Institut de Biologie Structurale,
F-38054 Grenoble, France, cCNRS, UMR5075, F-38027 Grenoble, France, dUniversité Joseph
Fourier, F-38000 Grenoble, France, and eESRF, 6 rue Jules Horowitz, BP 220, 38043 Grenoble
Cedex, France. E-mail: ,

Research into radiation damage in macromolecular crystallography has matured
over the last few years, resulting in a better understanding of both the processes
and timescales involved. In turn this is now allowing practical recommendations
for the optimization of crystal dose lifetime to be suggested. Some long-standing
questions have been answered by recent investigations, and from these answers
new challenges arise and areas of investigation can be proposed. Six papers
published in this volume give an indication of some of the current directions of
this field and also that of single-particle cryo-microscopy, and the brief summary
below places them into the overall framework of ongoing research into
macromolecular crystallography radiation damage.

# 2013 International Union of Crystallography Keywords: X-ray and electron radiation damage; macromolecular crystallography;
Printed in Singapore – all rights reserved radical scavenger; temperature dependence; XFEL.

It is now widely recognized that radiation damage (RD) the processes involved in RD progression [see, for example,
during macromolecular crystallography (MX) experiments is X-ray-excited optical luminescence of protein crystals
a mainstream concern for structural biologists, since it can be (XEOL) (Owen et al., 2012a) and a recent special issue enti-
a major limiting factor in structure determination and in tled ‘Protein Structure and Function in the Crystalline State:
obtaining high-resolution information, as well as sometimes From X-ray to Spectroscopy’ of Biochimica et Biophysica Acta
compromising the biological interpretation of observed elec- (BBA, 2011)].
tron density. Concerted research into the character and For some aspects of RD, the accumulated knowledge from
progression rates of radiation damage in MX has now been such research is now allowing practical recommendations for
ongoing for nearly fifteen years (see, for instance, papers from optimizing crystal dose lifetime to be suggested and tested
the second, third, fourth, fifth and sixth radiation damage experimentally. Five papers published in this volume give an
workshops in special issues of the Journal of Synchrotron indication of some of the current directions of the field, and
Radiation in 2002, 2005, 2007, 2009 and 2011, respectively). the brief summary below places them into the overall frame-
Although much more is now understood and there are work of ongoing research into MX radiation damage. The
answers to some of the questions posed in these earlier arti- remaining paper describes progress in combining the indivi-
cles, there are still many areas where further investigation dual frames of a cryo-electron microscopy exposure series, in
and more thorough characterization could greatly benefit order to reduce the beam-induced blurring, a phenomenon
the practising crystallographer. To obtain useful statistically caused by beam-induced particle movements.
significant results, RD experiments must involve more than The vast majority of systematic radiation damage studies
one sample investigated under nominally the same conditions: have so far been carried out at 100 K, the temperature at
this means that the studies have been both labour intensive which approximately 95% of all synchrotron MX datasets are
and time consuming. However, the recent widespread and currently collected because the radiation sensitivities of
ongoing automation at synchrotron beamlines involving macromolecular crystals is reduced by up to two orders of
robotic crystal mounting from liquid-nitrogen dewars, and magnitude (Garman, 2010) compared with at room tempera-
implementation of data collection and processing pipelines, as ture (RT). However, there is currently renewed interest in RD
well as the staggering increase in detector speed, has opened effects at RT since RT data collection at synchrotrons is at the
up new and exciting possibilities for multi-sample systematic beginnings of a renaissance, due to a number of factors such
studies which are now bearing fruit. Additionally, many as the facility to mount crystallization plates directly on some
complementary methods are increasingly being employed in goniometers (Jacquamet et al., 2004), the availability of
concert with crystallography to gain a deeper understanding of convenient devices to control the humidity (Sanchez-Weath-

J. Synchrotron Rad. (2013). 20, 1–6 doi:10.1107/S0909049512050418 1

, radiation damage
erby et al., 2009) and the recent discovery that RD could be two-parameter model which at RT is found to be resolution
outrun at RT (Owen et al., 2012b; Warkentin et al., 2013) using independent and to give a linear increase in Debye–Waller
the latest generation of pixel detectors (e.g. Broennimann et factors. The authors introduce a new global damage metric
al., 2006). In fact a series of case studies showing the power of called the normalized half-dose, which is a modified version
this in situ RT approach was published recently (Axford et al., of the conventional D1/2 (the dose required to reduce the
2012). In addition, RT collection is used for protein crystals intensity of the diffraction to half of its original value). This
which do not tolerate cryo-cooling (e.g. virus crystals) and also new metric is found to be a better descriptor of the intensity
because biologically relevant conformational heterogeneity decay since D1/2 was observed to be dependent on the B-
can be preserved (Fraser et al., 2011). Studies of RD at RT factor, B0, of the first wedge of data, with higher B0 giving
started in 1962 with the seminal work of Blake and Phillips higher D1/2 due to the fact that the weak high-resolution
(Blake & Phillips, 1962), but were followed by comparatively reflections were not present even at the start for these cases.
few investigations [all work until 2007 reviewed by South- The usual D1/2 is thus normalized here to DN1=2 corresponding
worth-Davies et al. (2007)] until more recently when the to the decay of the sum of intensities when adjusted to a
renewed interest in RT data collection has resulted in a starting value representing a standard overall B-factor of
number of important observations and developments. 20 Å2. DN1=2 is reported to vary by a factor of more than ten
In this issue, Warkentin et al. (2013) provide a compre- over the range of crystal types studied. In the study, no dose-
hensive review of recent progress made in describing global rate effects were observed over the range investigated, in
radiation damage at RT and how it evolves as the temperature contrast to some previous studies within the same dose-rate
is decreased down to cryo-conditions. A transition in radiation range [e.g. Rajendran et al. (2011): 1.3 to 8.4 kGy s 1]. Inter-
sensitivity has been observed at 200 K (Warkentin & Thorne, estingly, a correlation was established between crystal sensi-
2010), at which both protein and the surrounding solvent tivity at RT and solvent content with the approximate
undergo a coupled glass transition (Weik & Colletier, 2010). relationship DN1=2 / (solvent content) 1 1. The normalized
Above 200 K, global radiation damage is thus dominated by DN1=2 will be calculated in BEST (Bourenkov & Popov, 2010).
diffusive motions in the protein and the solvent (Warkentin & RT crystallography is also attracting renewed attention
Thorne, 2010) and a ‘dark progression’ of damage is observed owing to the recent exciting and promising results emerging
when X-rays are turned off (Warkentin et al., 2011). As from using X-ray free-electron lasers (XFELs) on biological
opposed to data collection at 100 K where only small dose-rate samples, where outrunning the damage has now been
effects have been observed at the flux densities currently used successfully achieved on a grand scale. The latest experiments
[global (Owen et al., 2006; Sliz et al., 2003), specific (Leiros et impressively expand the field beyond the seminal paper of
al., 2006)], a dose-rate effect is present at temperatures above Chapman et al. (2011) which first proved that the diffraction-
200 K and, for example, half of the global damage can be before-destruction concept (Neutze et al., 2000) is applicable
outrun at 260 K by collecting data in 1 s at a dose rate of to protein structure determination [reviewed by Spence et al.
680 kGy s 1 (gray: energy absorbed per mass of crystal, Gy = (2012), Schlichting & Miao (2012)]. In these so-called serial
J kg 1) (Warkentin et al., 2012a). At even higher dose rates femtosecond crystallography experiments (SFX) performed
(i.e. 1 MGy s 1) a significant fraction of global damage can be at RT, several tens of thousands of diffraction images are
outrun even at RT (Owen et al., 2012b). Consequently, further collected, each one from a small (typically 1 mm) protein
increases in synchrotron-radiation brilliance and detector crystal. Short (5–100 fs) and very brilliant X-ray pulses from
readout speeds are predicted (Warkentin et al., 2013; Owen et an XFEL are used with the crystals streaming across the beam
al., 2012b) to raise the RT dose limit close to the 30 MGy path in a free jet. The dose absorbed (as calculated using
(dose to reach 0.7 of the initial diffraction intensity) value RADDOSE v2) by each crystal during the XFEL pulse equals
determined for data collection at 100 K (Owen et al., 2006). or even exceeds (Boutet et al., 2012; Kern et al., 2012) the
Synchrotron-based data collection at RT is also the corner- experimental dose limit of 30 MGy determined for one or
stone of time-resolved Laue crystallography, a technique several complete datasets in MX at 100 K (Owen et al., 2006).
which aims to provide structural snapshots of conformational It should be noted that since RADDOSE currently does not
changes after reaction initiation in crystalline proteins. A take into account the possibility of photoelectrons escaping
recent paper addressed RT RD in time-resolved Laue studies from the irradiated crystal, it has limited applicability in esti-
on photoactive yellow protein crystals (Schmidt et al., 2012). mating absorbed doses for XFEL experiments, during which
The authors found that refinement of a kinetic reaction this escape of energy is highly probable from the nano-crystals
mechanism was only possible from data collected up to 40% of used. SFX has now even successfully been applied to structure
the dose limit applicable to determining reliable static struc- determination from in vivo grown protein crystals (Redecke et
tures. al., 2012; Koopmann et al., 2012). Furthermore, time-resolved
A further RT study is presented in this issue (Leal et al., pump–probe SFX (Neutze & Moffat, 2012) is beginning to be
2013), in which a comprehensive analysis of the RD rates at successfully applied (Aquila et al., 2012) and might eventually
dose rates between 0.05 and 300 kGy s 1 for 15 different provide molecular movies of proteins at work with femto-
model proteins using previously established automated tech- second time resolution.
niques (Leal et al., 2011) was carried out. The decay of Returning to more conventional MX experiments, one of
diffracted intensity as a function of dose was explored using a the possible strategies for extending the crystal dose lifetime is

2 Garman and Weik Radiation damage to biological macromolecules J. Synchrotron Rad. (2013). 20, 1–6

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