scholarly journals Radiation damage and derivatization in macromolecular crystallography: a structure factor's perspective

2016 ◽  
Vol 72 (3) ◽  
pp. 388-394 ◽  
Author(s):  
Robin L. Owen ◽  
Darren A. Sherrell
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Relative Intensity ◽  
Macromolecular Crystallography ◽  
Site Specific ◽  
X Ray ◽  
Effects Of Radiation ◽  
Short Time ◽  
Approximate Rate ◽  
Time Dose

During, or even after, data collection the presence and effects of radiation damage in macromolecular crystallography may not always be immediately obvious. Despite this, radiation damage is almost always present, with site-specific damage occurring on very short time (dose) scales well before global damage becomes apparent. A result of both site-specific radiation damage and derivatization is a change in the relative intensity of reflections. The size and approximate rate of onset of X-ray-induced transformations is compared with the changes expected from derivatization, and strategies for minimizing radiation damage are discussed.

RADDOSE-3D: time- and space-resolved modelling of dose in macromolecular crystallography

2013 ◽  
Vol 46 (4) ◽  
pp. 1225-1230 ◽  
Author(s):  
Oliver B. Zeldin ◽  
Markus Gerstel ◽  
Elspeth F. Garman
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Diffraction Experiment ◽  
Macromolecular Crystallography ◽  
X Ray Diffraction ◽  
Time Resolved ◽  
X Ray ◽  
Online Methods ◽  
Crystal Volume ◽  
Number Of Iterations

RADDOSE-3D allows the macroscopic modelling of an X-ray diffraction experiment for the purpose of better predicting radiation-damage progression. The distribution of dose within the crystal volume is calculated for a number of iterations in small angular steps across one or more data collection wedges, providing a time-resolved picture of the dose state of the crystal. The code is highly modular so that future contributions from the community can be easily integrated into it, in particular to incorporate online methods for determining the shape of macromolecular crystals and better protocols for imaging real experimental X-ray beam profiles.

scholarly journals Computer-controlled liquid-nitrogen drizzling device for removing frost from cryopreserved crystals

2020 ◽  
Vol 76 (12) ◽  
pp. 616-622
Author(s):  
Yuki Nakamura ◽  
Seiki Baba ◽  
Nobuhiro Mizuno ◽  
Takaki Irie ◽  
Go Ueno ◽  
...  
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Liquid Nitrogen ◽  
Room Temperature ◽  
Temperature Data ◽  
Macromolecular Crystallography ◽  
X Ray ◽  
Low Visibility ◽  
Computer Controlled

Cryocrystallography is a technique that is used more often than room-temperature data collection in macromolecular crystallography. One of its advantages is the significant reduction in radiation damage, which is especially useful in synchrotron experiments. Another advantage is that cryopreservation provides simple storage of crystals and easy transportation to a synchrotron. However, this technique sometimes results in the undesirable adhesion of frost to mounted crystals. The frost produces noisy diffraction images and reduces the optical visibility of crystals, which is crucial for aligning the crystal position with the incident X-ray position. To resolve these issues, a computer-controlled device has been developed that drizzles liquid nitrogen over a crystal to remove frost. It was confirmed that the device works properly, reduces noise from ice rings in diffraction images and enables the centering of crystals with low visibility owing to frost adhesion.

scholarly journals Cool data: quantity AND quality

1999 ◽  
Vol 55 (10) ◽  
pp. 1641-1653 ◽  
Author(s):  
Elspeth Garman
Keyword(s):  
Experimental Data ◽  
Data Collection ◽  
Radiation Damage ◽  
Macromolecular Crystallography ◽  
X Ray ◽  
Vital Part ◽  
Data Collection Methods ◽  
Collection Methods

The use of cryo-techniques in macromolecular crystallography has increased enormously over the last eight years and has become a vital part of modern X-ray data-collection methods. This paper presents some reasons for the rise in popularity of cryo-techniques and a brief outline of the basic methods, followed by a detailed discussion of factors to be considered when trying to optimize both the quantity and quality of the data collected. As more experimenters at synchrotrons observe significant radiation damage to crystals held near 100 K, the available options for further prolonging crystal lifetime and extending the techniques become worth investigating. Some possibilities and parameters to be considered are presented, although these must remain speculative until more experimental data are available.

TrimethylamineN-oxide as a versatile cryoprotective agent in macromolecular crystallography

2011 ◽  
Vol 44 (2) ◽  
pp. 433-436 ◽  
Author(s):  
Christoph Mueller-Dieckmann ◽  
Brice Kauffmann ◽  
Manfred S. Weiss
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Diffraction Data ◽  
Test Cases ◽  
Third Generation ◽  
Diffraction Experiment ◽  
Macromolecular Crystallography ◽  
Cryoprotective Agents ◽  
The Past ◽  
Effects Of Radiation

The surge of macromolecular crystallography is intimately linked to the advent of methods for cryoprotecting macromolecular crystals. Only if crystals are kept cold during data collection can they withstand the effects of radiation damage during a diffraction experiment, especially at third-generation synchrotron sources. While a number of different cryoprotective agents and procedures have been described in the literature over the past three decades, it is still a time- and crystal-consuming process to establish and optimize a good cryo-condition for a specific crystal. In this study, trimethylamineN-oxide (TMAO) has been identified as a very versatile cryoprotectant for macromolecular crystals. In a few test cases it was shown that diffraction data collected from crystals treated with TMAO are of very good quality.

Intensity measurements

2002 ◽  
Author(s):  
David Blow
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Resolution Limit ◽  
Strategic Decisions ◽  
Experimental Conditions ◽  
X Ray ◽  
Intensity Measurements ◽  
Data Collection Technique ◽  
Molecular Structure Investigation ◽  
Disordered Crystal

Once a suitable crystal has been obtained, a molecular structure investigation requires measurement of the intensities of as many Bragg reflections as possible. In this chapter, some of the options that must be decided by the experimenter will be considered, and some of the criteria used to assess the accuracy and completeness of the data will be presented. The experimenter has to make a number of strategic decisions in collecting the crystal intensity data. These include: • What X-ray source should be used? • What X-ray detector should be used? • Under what conditions should the crystal be maintained? • How long should each crystal be exposed? • What data collection technique will be used? • What resolution limit should be applied? The choice of source and detector will depend largely on what is available, but the major decision is whether to use facilities in the home laboratory or whether to use a synchrotron at a central facility. The energy released by absorption of X-rays in a crystal inevitably damages it. The process of radiation damage increases crystal disorder and reduces the intensity of scattering. The experimenter will ultimately have to abandon data collection from the damaged and disordered crystal. Under ideal experimental conditions, all the useful diffraction data can be obtained from a crystal long before radiation damage takes its toll, and radiation damage does not create a practical problem. At the other end of the scale, it may be necessary to combine the measurements from many crystals in order to obtain a complete set of diffracted intensities. There is no definite criterion to decide when a crystal is so badly damaged that it must be discarded. But if the measurements are going to be of highest quality, any observable change is bad news. The most serious effects occur in the part of the diffraction pattern at the highest observed resolution, where the observed intensities of the Bragg reflections will be altered most rapidly. The first observable effect of radiation damage is usually a reduction of high angle intensities due to increased disorder.

Limiting radiation damage for high-brilliance biological solution scattering: practical experience at the EMBL P12 beamline PETRAIII

2015 ◽  
Vol 22 (2) ◽  
pp. 273-279 ◽  
Author(s):  
Cy M. Jeffries ◽  
Melissa A. Graewert ◽  
Dmitri I. Svergun ◽  
Clément E. Blanchet
Keyword(s):  
Radiation Damage ◽  
Practical Experience ◽  
Quality Data ◽  
Biological Macromolecules ◽  
Sample Handling ◽  
X Ray ◽  
X Ray Scattering ◽  
Sample Position ◽  
Effects Of Radiation

Radiation damage is the general curse of structural biologists who use synchrotron small-angle X-ray scattering (SAXS) to investigate biological macromolecules in solution. The EMBL-P12 biological SAXS beamline located at the PETRAIII storage ring (DESY, Hamburg, Germany) caters to an extensive user community who integrate SAXS into their diverse structural biology programs. The high brilliance of the beamline [5.1 × 1012 photons s−1, 10 keV, 500 (H) µm × 250 (V) µm beam size at the sample position], combined with automated sample handling and data acquisition protocols, enable the high-throughput structural characterization of macromolecules in solution. However, considering the often-significant resources users invest to prepare samples, it is crucial that simple and effective protocols are in place to limit the effects of radiation damage once it has been detected. Here various practical approaches are evaluated that users can implement to limit radiation damage at the P12 beamline to maximize the chances of collecting quality data from radiation sensitive samples.

scholarly journals Automated harvesting and processing of protein crystals through laser photoablation

2016 ◽  
Vol 72 (4) ◽  
pp. 454-466 ◽  
Author(s):  
Ulrich Zander ◽  
Guillaume Hoffmann ◽  
Irina Cornaciu ◽  
Jean-Pierre Marquette ◽  
Gergely Papp ◽  
...  
Keyword(s):  
Data Collection ◽  
Macromolecular Crystallography ◽  
X Ray Diffraction ◽  
X Ray ◽  
New Methods ◽  
Repeated Sample ◽  
Sample Recovery ◽  
Through Diffusion ◽  
X Ray Background ◽  
Low X

Currently, macromolecular crystallography projects often require the use of highly automated facilities for crystallization and X-ray data collection. However, crystal harvesting and processing largely depend on manual operations. Here, a series of new methods are presented based on the use of a low X-ray-background film as a crystallization support and a photoablation laser that enable the automation of major operations required for the preparation of crystals for X-ray diffraction experiments. In this approach, the controlled removal of the mother liquor before crystal mounting simplifies the cryocooling process, in many cases eliminating the use of cryoprotectant agents, while crystal-soaking experiments are performed through diffusion, precluding the need for repeated sample-recovery and transfer operations. Moreover, the high-precision laser enables new mounting strategies that are not accessible through other methods. This approach bridges an important gap in automation and can contribute to expanding the capabilities of modern macromolecular crystallography facilities.

Optimizing the spatial distribution of dose in X-ray macromolecular crystallography

2012 ◽  
Vol 20 (1) ◽  
pp. 49-57 ◽  
Author(s):  
Oliver B. Zeldin ◽  
Markus Gerstel ◽  
Elspeth F. Garman
Keyword(s):  
Spatial Distribution ◽  
Data Collection ◽  
Rotation Axis ◽  
Macromolecular Crystallography ◽  
X Ray ◽  
Crystal Shapes ◽  
Peak Dose ◽  
Wide Range ◽  
Helical Scan ◽  
Crystal Volume

X-ray data collection for macromolecular crystallography can lead to highly inhomogeneous distributions of dose within the crystal volume for cases when the crystal is larger than the beam or when the beam is non-uniform (Gaussian-like), particularly when crystal rotation is fully taken into account. Here the spatial distribution of dose is quantitatively modelled in order to compare the effectiveness of two dose-spreading data-collection protocols: helical scanning and translational collection. Their effectiveness in reducing the peak dose per unit diffraction is investigatedviasimulations for four common crystal shapes (cube, plate, long and short needles) and beams with a wide range of full width half maximum values. By inspection of the chosen metric, it is concluded that the optimum strategy is always to use as flat (top-hat) a beam as possible and to either match the beam size in both dimensions to the crystal, or to perform a helical scan with a beam which is narrow along the rotation axis and matched to the crystal size along the perpendicular axis. For crystal shapes where this is not possible, the reduction in peak dose per unit diffraction achieved through dose spreading is quantified and tabulated as a reference for experimenters.

scholarly journals A multicrystal diffraction data-collection approach for studying structural dynamics with millisecond temporal resolution

IUCrJ ◽  
2016 ◽  
Vol 3 (6) ◽  
pp. 393-401 ◽  
Author(s):  
Robin Schubert ◽  
Svetlana Kapis ◽  
Yannig Gicquel ◽  
Gleb Bourenkov ◽  
Thomas R. Schneider ◽  
...  
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Temporal Resolution ◽  
Diffraction Data ◽  
Structural Changes ◽  
Structural Information ◽  
Data Sets ◽  
Enzymatic Reactions ◽  
Time Resolved ◽  
Site Specific

Many biochemical processes take place on timescales ranging from femtoseconds to seconds. Accordingly, any time-resolved experiment must be matched to the speed of the structural changes of interest. Therefore, the timescale of interest defines the requirements of the X-ray source, instrumentation and data-collection strategy. In this study, a minimalistic approach forin situcrystallization is presented that requires only a few microlitres of sample solution containing a few hundred crystals. It is demonstrated that complete diffraction data sets, merged from multiple crystals, can be recorded within only a few minutes of beamtime and allow high-resolution structural information of high quality to be obtained with a temporal resolution of 40 ms. Global and site-specific radiation damage can be avoided by limiting the maximal dose per crystal to 400 kGy. Moreover, analysis of the data collected at higher doses allows the time-resolved observation of site-specific radiation damage. Therefore, our approach is well suited to observe structural changes and possibly enzymatic reactions in the low-millisecond regime.

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