Room-temperature scavengers for macromolecular crystallography: increased lifetimes and modified dose dependence of the intensity decay

2009 ◽  
Vol 16 (2) ◽  
pp. 205-216 ◽  
Author(s):  
Adam I. Barker ◽  
Robert J. Southworth-Davies ◽  
Karthik S. Paithankar ◽  
Ian Carmichael ◽  
Elspeth F. Garman
Keyword(s):  
Room Temperature ◽  
Dose Dependence ◽  
Protein Crystals ◽  
Alternative Mechanism ◽  
X Ray ◽  
Dose Rates ◽  
Striking Change ◽  
Observed Damage ◽  
Dose Tolerance ◽  
Intensity Decay

The advent of highly intense wiggler and undulator beamlines has reintroduced the problem of X-ray radiation damage in protein crystals even at cryogenic temperatures (100 K). Although cryocrystallography can be utilized for the majority of protein crystals, certain macromolecular crystals (e.g. of viruses) suffer large increases in mosaicity upon flash cooling and data are still collected at room temperature (293 K). An alternative mechanism to cryocooling for prolonging crystal lifetime is the use of radioprotectants. These compounds are able to scavenge the free radical species formed upon X-ray irradiation which are thought to be responsible for part of the observed damage. Three putative radioprotectants, ascorbate, 1,4-benzoquinone and 2,2,6,6-tetramethyl-4-piperidone (TEMP), were tested for their ability to prolong lysozyme crystal lifetimes at 293 K. Plots of relative summed intensity against dose were used as a metric to assess radioprotectant ability: ascorbate and 1,4-benzoquinone appear to be effective, whereas studies on TEMP were inconclusive. Ascorbate, which scavenges OH^{\bullet} radicals (k OH = 8 × 109 M −1 s−1) and electrons with a lower rate constant (k e-(aq) = 3.0 × 108 M −1 s−1), doubled the crystal dose tolerance, whereas 1,4-benzoquinone, which also scavenges both OH^{\bullet} radicals (k OH = 1.2 × 109 M −1 s−1) and electrons (k e-(aq) = 1.2 × 1010 M −1 s−1), offered a ninefold increase in dose tolerance at the dose rates used. Pivotally, these preliminary results on a limited number of samples show that the two scavengers also induced a striking change in the dose dependence of the intensity decay from a first-order to a zeroth-order process.

scholarly journals To scavenge or not to scavenge, that is STILL the question

2012 ◽  
Vol 20 (1) ◽  
pp. 23-36 ◽  
Author(s):  
Elizabeth G. Allan ◽  
Melissa C. Kander ◽  
Ian Carmichael ◽  
Elspeth F. Garman
Keyword(s):  
Crystal Size ◽  
Chemical Species ◽  
Absorbed Dose ◽  
Dose Dependence ◽  
Radiation Chemistry ◽  
X Ray ◽  
On Line ◽  
Crystallization Conditions ◽  
Effects Of Radiation ◽  
Dose Tolerance

An extensive radiation chemistry literature would suggest that the addition of certain radical scavengers might mitigate the effects of radiation damage during protein crystallography diffraction data collection. However, attempts to demonstrate and quantify such an amelioration and its dose dependence have not yielded consistent results, either at room temperature (RT) or 100 K. Here the information thus far available is summarized and reasons for this lack of quantitative success are identified. Firstly, several different metrics have been used to monitor and quantify the rate of damage, and, as shown here, these can give results which are in conflict regarding scavenger efficacy. In addition, significant variation in results from data collected from crystals treated in nominally the same way has been observed. Secondly, typical crystallization conditions contain substantial concentrations of chemical species which already interact strongly with some of the X-ray-induced radicals that the added scavengers are intended to intercept. These interactions are probed here by the complementary technique of on-line microspectrophotometry carried out on solutions and crystals held both at 100 K and RT, the latter enabled by the use of a beamline-mounted humidifying device. With the help of computational chemistry, attempts are made to assign some of the characteristic spectral features observed experimentally. A further source of uncertainty undoubtedly lies in the challenge of reliably measuring the parameters necessary for the accurate calculation of the absorbed dose (e.g.crystal size and shape, beam profile) and its distribution within the volume of the crystal (an issue addressed in detail in another article in this issue). While microspectrophotometry reveals that the production of various species can be quenched by the addition of scavengers, it is less clear that this observation can be translated into a significant gain in crystal dose tolerance for macromolecular crystallographers.

scholarly journals The rise of neutron cryo-crystallography

2018 ◽  
Vol 74 (8) ◽  
pp. 792-799 ◽  
Author(s):  
Hanna Kwon ◽  
Patricia S. Langan ◽  
Leighton Coates ◽  
Emma L. Raven ◽  
Peter C. E. Moody
Keyword(s):  
Data Collection ◽  
Liquid Nitrogen ◽  
Room Temperature ◽  
Protein Crystallography ◽  
Protein Crystals ◽  
X Ray ◽  
Problems And Solutions ◽  
Neutron Protein Crystallography ◽  
Direct Extrapolation ◽  
Neutron Crystallography

The use of boiled-off liquid nitrogen to maintain protein crystals at 100 K during X-ray data collection has become almost universal. Applying this to neutron protein crystallography offers the opportunity to significantly broaden the scope of biochemical problems that can be addressed, although care must be taken in assuming that direct extrapolation to room temperature is always valid. Here, the history to date of neutron protein cryo-crystallography and the particular problems and solutions associated with the mounting and cryocooling of the larger crystals needed for neutron crystallography are reviewed. Finally, the outlook for further cryogenic neutron studies using existing and future neutron instrumentation is discussed.

scholarly journals Dose dependence of radiation damage for protein crystals studied at various X-ray energies

2006 ◽  
Vol 14 (1) ◽  
pp. 4-10 ◽  
Author(s):  
Nobutaka Shimizu ◽  
Kunio Hirata ◽  
Kazuya Hasegawa ◽  
Go Ueno ◽  
Masaki Yamamoto
Keyword(s):  
Radiation Damage ◽  
Dose Dependence ◽  
Protein Crystals ◽  
X Ray

scholarly journals Damaged goods? Evaluating the impact of X-ray damage on conformational heterogeneity in room temperature and cryo-cooled protein crystals

2021 ◽  
Author(s):  
Filip Yabukarski ◽  
Tzanko Doukov ◽  
Daniel A. Mokhtari ◽  
Siyuan Du ◽  
Daniel Herschlag
Keyword(s):  
Protein Function ◽  
Room Temperature ◽  
Proteinase K ◽  
Protein Crystals ◽  
Conformational Ensemble ◽  
Conformational Heterogeneity ◽  
Critical Question ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Impact

X-ray crystallography is a cornerstone of biochemistry. Traditional freezing of protein crystals to cryo-temperatures mitigates X-ray damage and facilitates crystal handling but provides an incomplete window into the ensemble of conformations at the heart of protein function and energetics. Room temperature (RT) X-ray crystallography provides more extensive ensemble information, and recent developments allow conformational heterogeneity, the experimental manifestation of ensembles, to be extracted from single crystal data. However, high sensitivity to X-ray damage at RT raises concerns about data reliability. To systematically address this critical question, we obtained increasingly X-ray-damaged high-resolution datasets (1.02–1.52 Å) from single thaumatin, proteinase K, and lysozyme crystals. Heterogeneity analyses indicated a modest increase in conformational disorder with X-ray damage. Nevertheless, these effects do not alter overall conclusions and can be minimized by limiting the extent of X-ray damage or eliminated by extrapolation to obtain heterogeneity information free from X-ray damage effects. To compare these effects to damage at cryo temperature and to learn more about damage and heterogeneity in cryo-cooled crystals, we carried out an analogous analysis of increasingly damaged proteinase K cryo datasets (0.9–1.16 Å). We found X-ray damage-associated heterogeneity changes that were not observed at RT. This observation and the scarcity of reported X-ray doses and damage extent render it difficult to distinguish real from artifactual conformations, including those occurring as a function of temperature. The ability to aquire reliable heterogeneity information from single crystals at RT provides strong motivation for further development and routine implementation of RT X-ray crystallography to obtain conformational ensemble information.

scholarly journals An automated platform for in situ serial crystallography at room temperature

IUCrJ ◽  
2020 ◽  
Vol 7 (6) ◽  
pp. 1009-1018
Author(s):  
Zhong Ren ◽  
Cong Wang ◽  
Heewhan Shin ◽  
Sepalika Bandara ◽  
Indika Kumarapperuma ◽  
...  
Keyword(s):  
Data Collection ◽  
Room Temperature ◽  
Protein Structures ◽  
Routine Data ◽  
Protein Crystals ◽  
X Ray Diffraction ◽  
X Ray ◽  
Serial Crystallography ◽  
Routine Data Collection

Direct observation of functional motions in protein structures is highly desirable for understanding how these nanomachineries of life operate at the molecular level. Because cryogenic temperatures are non-physiological and may prohibit or even alter protein structural dynamics, it is necessary to develop robust X-ray diffraction methods that enable routine data collection at room temperature. We recently reported a crystal-on-crystal device to facilitate in situ diffraction of protein crystals at room temperature devoid of any sample manipulation. Here an automated serial crystallography platform based on this crystal-on-crystal technology is presented. A hardware and software prototype has been implemented, and protocols have been established that allow users to image, recognize and rank hundreds to thousands of protein crystals grown on a chip in optical scanning mode prior to serial introduction of these crystals to an X-ray beam in a programmable and high-throughput manner. This platform has been tested extensively using fragile protein crystals. We demonstrate that with affordable sample consumption, this in situ serial crystallography technology could give rise to room-temperature protein structures of higher resolution and superior map quality for those protein crystals that encounter difficulties during freezing. This serial data collection platform is compatible with both monochromatic oscillation and Laue methods for X-ray diffraction and presents a widely applicable approach for static and dynamic crystallographic studies at room temperature.

scholarly journals High-throughput in situ X-ray screening of and data collection from protein crystals at room temperature and under cryogenic conditions

2018 ◽  
Vol 13 (2) ◽  
pp. 260-292 ◽  
Author(s):  
Jana Broecker ◽  
Takefumi Morizumi ◽  
Wei-Lin Ou ◽  
Viviane Klingel ◽  
Anling Kuo ◽  
...  
Keyword(s):  
Data Collection ◽  
High Throughput ◽  
Room Temperature ◽  
Protein Crystals ◽  
X Ray ◽  
Cryogenic Conditions

scholarly journals Diffraction before destruction

2014 ◽  
Vol 369 (1647) ◽  
pp. 20130313 ◽  
Author(s):  
Henry N. Chapman ◽  
Carl Caleman ◽  
Nicusor Timneanu
Keyword(s):  
Diffraction Pattern ◽  
Structure Determination ◽  
High Dose ◽  
X Ray ◽  
Dose Rates ◽  
Structure Factors ◽  
Serial Crystallography ◽  
The Many ◽  
Dose Tolerance

X-ray free-electron lasers have opened up the possibility of structure determination of protein crystals at room temperature, free of radiation damage. The femtosecond-duration pulses of these sources enable diffraction signals to be collected from samples at doses of 1000 MGy or higher. The sample is vaporized by the intense pulse, but not before the scattering that gives rise to the diffraction pattern takes place. Consequently, only a single flash diffraction pattern can be recorded from a crystal, giving rise to the method of serial crystallography where tens of thousands of patterns are collected from individual crystals that flow across the beam and the patterns are indexed and aggregated into a set of structure factors. The high-dose tolerance and the many-crystal averaging approach allow data to be collected from much smaller crystals than have been examined at synchrotron radiation facilities, even from radiation-sensitive samples. Here, we review the interaction of intense femtosecond X-ray pulses with materials and discuss the implications for structure determination. We identify various dose regimes and conclude that the strongest achievable signals for a given sample are attained at the highest possible dose rates, from highest possible pulse intensities.

A simple and rapid method for mounting protein crystals at room temperature

2003 ◽  
Vol 36 (1) ◽  
pp. 165-166 ◽  
Author(s):  
Aengus Mac Sweeney ◽  
Allan D'Arcy
Keyword(s):  
Data Collection ◽  
Room Temperature ◽  
Protein Crystals ◽  
Freezing Process ◽  
Routine Method ◽  
X Ray ◽  
Significant Damage ◽  
Simplified Method ◽  
Intrinsic Quality

Cryocooling of protein crystals for X-ray data collection has now become a routine method in the majority of biostructural laboratories. The improvement of facilities at synchrotron sources and their increased use has made it essential to have properly frozen crystals for optimal data collection. Although in general crystals can be cooled without significant damage, there are often cases in which crystals with slight disorder or twinning problems suffer considerably during the freezing process. In other cases, poor or mosaic diffraction may be blamed on the cryoprotectant or cooling protocol. Many crystals may be wasted in searching for the best freezing conditions when the intrinsic quality of the crystals is poor. In principle, the collection of room-temperature diffraction data would provide a reference that would allow the detection of crystal damage caused by addition of cryoprotectant or by cryocooling. In practice, however, many investigators are reluctant to do this, one reason being that capillary mounting of crystals is a tedious method, especially for those who are new to crystallography. Here a simplified method for mounting crystals at room temperature is reported, which requires little expertise and no expensive equipment.

scholarly journals Instrumentation and experimental procedures for robust collection of X-ray diffraction data from protein crystals across physiological temperatures

2020 ◽  
Vol 53 (6) ◽  
pp. 1493-1501
Author(s):  
Tzanko Doukov ◽  
Daniel Herschlag ◽  
Filip Yabukarski
Keyword(s):  
Radiation Damage ◽  
Diffraction Data ◽  
Protein Function ◽  
Room Temperature ◽  
Three Dimensional ◽  
Proteinase K ◽  
Protein Crystals ◽  
X Ray Diffraction ◽  
X Ray ◽  
Conformational Ensembles

Traditional X-ray diffraction data collected at cryo-temperatures have delivered invaluable insights into the three-dimensional structures of proteins, providing the backbone of structure–function studies. While cryo-cooling mitigates radiation damage, cryo-temperatures can alter protein conformational ensembles and solvent structure. Furthermore, conformational ensembles underlie protein function and energetics, and recent advances in room-temperature X-ray crystallography have delivered conformational heterogeneity information that can be directly related to biological function. Given this capability, the next challenge is to develop a robust and broadly applicable method to collect single-crystal X-ray diffraction data at and above room temperature. This challenge is addressed herein. The approach described provides complete diffraction data sets with total collection times as short as ∼5 s from single protein crystals, dramatically increasing the quantity of data that can be collected within allocated synchrotron beam time. Its applicability was demonstrated by collecting 1.09–1.54 Å resolution data over a temperature range of 293–363 K for proteinase K, thaumatin and lysozyme crystals at BL14-1 at the Stanford Synchrotron Radiation Lightsource. The analyses presented here indicate that the diffraction data are of high quality and do not suffer from excessive dehydration or radiation damage.

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