Progress in research into radiation damage in cryo-cooled macromolecular crystals.
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ABSTRACT: The author's 2011 British Crystallographic Association Lonsdale Lecture included a tribute to Kathleen Lonsdale followed by detailed perspectives relevant to the title, with reference to the Synchrotron Radiation Source (SRS) and European Synchrotron Radiation Facility (ESRF). Detector initiatives have also been very important as have sample freezing cryomethods. The use of on-resonance anomalous scattering, smaller crystals, ultra-high resolution as well as the ability to handle large unit cells and the start of time-resolved structural studies have allowed a major expansion of capabilities. The reintroduction of the Laue method became a significant node point for separate development, and has also found wide application with neutron sources in biological and chemical crystallography. The UK's SRS has now been superseded by Diamond, a new synchrotron radiation source with outstanding capabilities. In Hamburg we now have access to the new ultra-low emittance PETRA III, the ultimate storage ring in effect. The ESRF Upgrade is also recently funded and takes us to sub-micrometre and even nanometre-sized X-ray beams. The very new fourth generation of the X-ray laser gives unprecedented brilliance for working with nanocrystals, and perhaps even smaller samples, such as the single molecule, with coherent X-rays, and at femtosecond time resolution.Crystallography Reviews 01/2011; · 2.32 Impact Factor
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ABSTRACT: Recent studies have defined a data-collection protocol and a metric that provide a robust measure of global radiation damage to protein crystals. Using this protocol and metric, 19 small-molecule compounds (introduced either by cocrystallization or soaking) were evaluated for their ability to protect lysozyme crystals from radiation damage. The compounds were selected based upon their ability to interact with radiolytic products (e.g. hydrated electrons, hydrogen, hydroxyl and perhydroxyl radicals) and/or their efficacy in protecting biological molecules from radiation damage in dilute aqueous solutions. At room temperature, 12 compounds had no effect and six had a sensitizing effect on global damage. Only one compound, sodium nitrate, appeared to extend crystal lifetimes, but not in all proteins and only by a factor of two or less. No compound provided protection at T=100 K. Scavengers are ineffective in protecting protein crystals from global damage because a large fraction of primary X-ray-induced excitations are generated in and/or directly attack the protein and because the ratio of scavenger molecules to protein molecules is too small to provide appreciable competitive protection. The same reactivity that makes some scavengers effective radioprotectors in protein solutions may explain their sensitizing effect in the protein-dense environment of a crystal. A more productive focus for future efforts may be to identify and eliminate sensitizing compounds from crystallization solutions.Acta Crystallographica Section D Biological Crystallography 10/2011; 67(Pt 10):881-93. · 12.67 Impact Factor
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ABSTRACT: As it is not possible to obtain an accurate point spread function (PSF) in remote sensing imaging, classic deconvolution methods such as Wiener filtering often introduce strong noise and ringing artifacts, which contaminate the restored images. In this paper, we modify the standard Richarson–Lucy (RL) algorithm with a piecewise local regularization term and combine it with residual deconvolution method. Experimental results show that it is effective in suppressing negative effects, and images with rich details and sharp edges are obtained.Optics & Laser Technology 01/2011; · 1.37 Impact Factor
J. Synchrotron Rad. (2007). 14, 1–3doi:10.1107/S0909049506053015
Progress in research into radiation damage in
cryo-cooled macromolecular crystals
Elspeth F. Garmana* and
Sean M. McSweeneyb
aDepartment of Biochemistry,
South Parks Road, Oxford OX1
3QU, UK, andbESRF, 6 rue Jules
Horowitz, 38043 Grenoble, France
The problem of radiation damage to cryo-cooled macromolecular crystals in intense
synchrotron beams has entered the mainstream of macromolecular crystallography (MX)
during the last few years. A growing body of researchers worldwide are now carrying out
systematic investigations into different aspects of this problem. The Fourth International
Workshop on X-ray Radiation Damage to Biological Crystalline Samples was held on
7–8 March 2006 at SPring-8 in Japan and was attended by 80 researchers, the meeting
being generously funded by SPring-8, Hyogo, Japan, and the Photon Factory in Tsukuba,
Japan. The series of workshops on X-ray Damage to Biological Crystalline Samples aims
to discuss and disseminate the latest research, often prior to publication, on the under-
standing, control, correction and possible use of radiation damage occurring during
macromolecular crystallography experiments. Papers from the second (2001) and third
(2003) workshops can be found in special issues of the Journal of Synchrotron Radiation
(RD2, 2002; RD3, 2005). In this issue of the Journal of Synchrotron Radiation, there are
12 papers covering various aspects of current research into this area, including nine
arising from the presentations made during the workshop.
It is now generally recognized that information deduced from three-dimensional
biological structures can be compromised because of radiation-induced effects. In
addition to the general degradation of diffraction properties associated with increasing
dose, specific structural damage is observed to occur at 100 K in a well defined sequence,
with the disulfide bond becoming disordered by breakage and delocalization, followed by
the decarboxylation of aspartates and glutamates (Weik et al., 2000; Burmeister, 2000;
Ravelli & McSweeney, 2000). Metal binding proteins are partly reduced during X-ray
exposure (Berglund et al., 2002; Yano et al., 2005), and active sites seem particularly
susceptible to damage (Weik et al., 2000). These observations imply that an awareness of
radiation damage effects is essential before any mechanistic conclusions are drawn from,
for instance, the oxidation state of a metal atom in an active site. Increasingly, the
processes of radiation damage are being used to enhance phase information and thereby
provide more accurate starting maps. As the trend towards multidisciplinary biological
research continues, it becomes important to consider how experience from other scien-
tific fields, for instance spectroscopic tools and electron microscopy, may aid research
Systematic experiments on the characteristics of radiation damage over the last five
years have resulted in the identification of a number of the important beam and crystal
parameters governing its rate of progression and character (reviewed by Ravelli &
Garman, 2006). Research reports in this Journal of Synchrotron Radiation issue can be
loosely grouped into five different areas: investigations into the general dependencies of
radiation damage on various parameters, the effects of damage on phasing power and
ways to correct for it, the use of optical and Raman spectroscopy to study aspects of
damage, the study of heating effects induced by the beam in cryo-cooled crystals, and,
lastly, experience in electron microscopy and how this might be applied to MX.
In this issue, Shimizu et al. (2007) report the analysis of various radiation damage
indicators in lysozyme crystals as a function of incident photon energy, concluding that
between 6.5 keV and 33 keV there is no observable energy dependence of radiation
damage on dose. The possible correlation between the rate of radiation damage and the
solvent accessibility of glutamates and aspartate residues in the halophilic enzyme malate
dehydrogenase is explored by Fioravanti et al. (2007), who find there to be no such
correlation. Borek et al. (2007) describe a range of diffraction experiments carried out on
crystals of several different proteins to investigate the effects of cryogen temperature on
various processing statistics and on the observed specific structural damage.
Aspects of the impact of radiation damage on the phasing of
macromolecular structures is addressed by three of the papers
presented here: Gonza ´lez (2007), who compares the relative
phasing power of a SAD experiment with a two-wavelength
MAD Se-Met experiment; Schiltz & Bricogne (2007), who
discuss different models for the description of site-specific
damage; and Holton (2007), who reports the use of XANES to
quantify the damage to selenium sites in proteins.
The use of various off-line and on-line spectroscopies
(UV-visible and Raman) to explore several phenomena are
described by three groups. Southworth-Davies & Garman
(2007) have used the 400 nm signature of the disulfide anion
and an on-line microspectrophotometer to screen for putative
MX scavengers, while Beitlich et al. (2007) investigate the
of radiation damage on the reduction rates of three heme-
containing proteins, again using on-line spectroscopy. Raman
spectroscopy has been successfully used to track damage to a
brominated DNA crystal by McGeehan et al. (2007), opening
up new possibilities for on-line monitoring of radiation
Experimental results on the heating of a glass bead imaged
with an infrared camera are described by Snell et al. (2007)
and promise the possibility of further measurements so that
detailed comparisons could be made with the theoretical
calculations already available (Mhaisekar et al., 2005).
Electron microscopists have long struggled with the
problem of the radiation damage caused by electron
bombardment, and Massover (2007) outlines strategies used
to minimize and circumvent its effects, pointing to some areas
which may be worth further investigation for MX. One of
these is in specimen preparation, and Ravelli et al. (2007)
describe first attempts to embed a protein crystal in plastic
using techniques developed for electron microscopy samples.
The various results of the work presented and the ensuing
discussions during the workshop could be broadly divided into
three categories: resolved issues, unresolved issues, and new
issues. The papers collected here address some of these. The
resolved issues were concluded to be:
(i) Dose/dose-rate effects, other than from crystal heating,
seem to be small (<10% on intensity) up to a flux density of
1015photons s?1mm?2(Sliz et al., 2003; Leiros et al., 2006;
Owen et al., 2006).
(ii) Experiments using an infrared camera to measure the
X-ray beam heating of small glass beads (Snell et al., 2007)
have indicated that the previous extensive theoretical
modelling of heating effects is likely to be reliable. These
models, when used to simulate heating of spherical crystals in
an X-ray beam, show that the temperature rise for the flux
densities currently used on most beamlines is not likely to be
larger than 15 K (Mhaisekar et al., 2005).
(iii) Helium cooling (15 K) versus nitrogen (100 K) does not
seem likely to give the factor of around five improvement in
radiation lifetime that would be necessary to justify both the
technical complications and the cost, although it may be useful
for special cases.
(iv) The maximum dose limit of 2 ? 107Gy, proposed by
analogy with the observed dose to reduce diffraction to half
the original intensity (Io) (hIi = 0.5Io) in electron microscopy
(Henderson, 1990), has been verified experimentally and
seems to have general applicability. A maximum dose limit for
macromolecular crystallography of 3 ? 107Gy has been
proposed (hIi = 0.7Io) after which the biological information
may be compromised (Owen et al., 2006).
Unresolved issues were identified as:
(i) The nature of any energy dependence of the rate of
radiation damage; recent experimental results indicate no
dependence (Weiss et al., 2005; Shimizu et al., 2007) in broad
agreement with theoretical predictions (Arndt, 1984; Murray
et al., 2004).
(ii) The identity of the gas(es) which escape the exposed
crystal after warming. Radiation chemistry models seem to
suggest that it might be hydrogen, but it may also be CO2.
Experimental verification of this for MX at 100 K is still
(iii) The mechanisms of scavenger action. There is a need
to understand the radiation chemistry involved in radio-
protection and scavenging; for instance, its pH dependence, so
that eventually rational design of scavengers will be possible
and will feed into experimental design.
(iv) There is a need to provide (more) user-friendly soft-
ware that takes radiation damage into account both for data
collection and structure determination.
(v) What is the pathway of energy dissipation after photo-
absorption by a (heavy) atom above the absorption edge
(compared with below the edge)?
(vi) There is a pivotal need to understand the biological
implications of radiation damage on structures, particularly to
metal centres which are damaged very fast (redox), and active
sites which appear particularly sensitive. It would be useful to
see the introduction of the possibility to flag radiation arte-
facts in the Protein Data Bank.
Several new issues had emerged since the last Radiation
(i) Have we properly correlated the experience available
from X-ray imaging, X-ray crystallography, and electron
microscopy and diffraction in terms of the limiting resolution,
dose deposited and specimen type?
(ii) There is a need to understand the nature of UV damage
compared with X-ray damage, especially since it has been
demonstrated that UV light can be used for crystal centring
(Nanao & Ravelli, 2006; Vernede et al., 2006).
(iii) Better methods for dealing with non-isomorphism
(both caused by unit-cell changes and specific structural
changes, including occupancy decrease of heavier atoms) are
A repeated theme from various contributors during the
Fourth Workshop was that there is an absolute requirement to
make a careful estimate of dose [e.g. using RADDOSE
(Murray et al., 2004)] whenever possible. We feel that this
information is an important consideration for experimental
design, and should, wherever practical, be provided by the
Garman and McSweeney
? Progress in research into radiation damage
J. Synchrotron Rad. (2007). 14, 1–3
synchrotron beamline control software. In practice this means
it is necessary to (i) measure and record the integrated in-line
beam counts for each image to allow proper quantitative
relative comparison between data taken on the same day to be
made and, if the beam characteristics (size, profile, flux) and
crystal size are known, the dose can be calculated and
measurements compared with those on other beamlines and
facilities; (ii) retain this information right through to scaling of
It was concluded that these international workshops
provide a valuable stimulus for new research and exchange of
information, so it is proposed to hold the fifth workshop in
Europe in 2008.
In establishing the stimulating programme we benefited
from the help and advice of the other organisers (Colin Nave,
Raimond Ravelli, Gerd Rosenbaum) and the local organisers
Masaki Yamamoto and Soichi Wakatsuki, who also took the
initiative in obtaining funding for the workshop. In addition,
Raimond Ravelli provided helpful comments on this short
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? Progress in research into radiation damage