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.

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 Radiation damage in macromolecular crystallography: what is it and why should we care?

2010 ◽  
Vol 66 (4) ◽  
pp. 339-351 ◽  
Author(s):  
Elspeth F. Garman
Keyword(s):  
Data Collection ◽  
Data Quality ◽  
Radiation Damage ◽  
Current Literature ◽  
Diffraction Data ◽  
Absorbed Dose ◽  
Macromolecular Crystallography ◽  
Physical Processes ◽  
Quality Degradation ◽  
The Subject

Radiation damage inflicted during diffraction data collection in macromolecular crystallography has re-emerged in the last decade as a major experimental and computational challenge, as even for crystals held at 100 K it can result in severe data-quality degradation and the appearance in solved structures of artefacts which affect biological interpretations. Here, the observable symptoms and basic physical processes involved in radiation damage are described and the concept of absorbed dose as the basic metric against which to monitor the experimentally observed changes is outlined. Investigations into radiation damage in macromolecular crystallography are ongoing and the number of studies is rapidly increasing. The current literature on the subject is compiled as a resource for the interested researcher.

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.

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.

scholarly journals A tutorial for learning and teaching macromolecular crystallography

2008 ◽  
Vol 41 (6) ◽  
pp. 1161-1172 ◽  
Author(s):  
Annette Faust ◽  
Santosh Panjikar ◽  
Uwe Mueller ◽  
Venkataraman Parthasarathy ◽  
Andrea Schmidt ◽  
...  
Keyword(s):  
Data Collection ◽  
Data Processing ◽  
Diffraction Data ◽  
Structure Determination ◽  
Macromolecular Crystallography ◽  
Data Set ◽  
Learning And Teaching

Five experiments have been designed to be used for teaching macromolecular crystallography. The three proteins used in this tutorial are all commercially available; they can be easily and reproducibly crystallized and mounted for diffraction data collection. For each of the five experiments the raw images and the processed data of a sample diffraction data set as well as the refined coordinates and phases are provided for teaching the steps of data processing and structure determination.

scholarly journals XAS at the Canadian Macromolecular Crystallography Facility

2014 ◽  
Vol 70 (a1) ◽  
pp. C1525-C1525
Author(s):  
Julien Cotelesage ◽  
Pawel Grochulski ◽  
Michel Fodje ◽  
James Gorin ◽  
Kathryn Janzen ◽  
...  
Keyword(s):  
Data Collection ◽  
Diffraction Data ◽  
Three Dimensional ◽  
Minimal Risk ◽  
Macromolecular Crystallography ◽  
X Ray ◽  
Biologically Relevant ◽  
Wide Range ◽  
X Ray Absorption ◽  
Do So

Recent additions to the Canadian Macromolecular Crystallography Facility have expanded the capabilities of its bending magnet beamline. It is now possible to perform x-ray absorption spectroscopy (XAS) on crystals. A wide range of biologically relevant metals can be further studied, supplementing diffraction data. XAS can be used to determine if metalloproteins are photoreducing during diffraction data collection. The geometries of metal complexes can also be inferred with near-edge and EXAFS data, often more accurately than crystallography. CMCF-BM can be employed to do the abovementioned techniques on powder and solution samples that contain a metal of interest. One XAS-based technique that shows promise is single crystal plane polarized EXAFS. This technique combines crystallographic data with the findings from XAS to yield a high resolution three dimensional atomic model. More recently a number of the procedural steps required for the acquisition of XAS-based data have been automated in the MxDC software suite. These changes to data collection make it easier for users new to these disciplines to run the XAS-based experiments. By having the necessary equipment to do XAS at a protein crystallography facility, researchers who may not have had the opportunity delve into the field of XAS now can do so with minimal risk in terms of materials, funds and time.

scholarly journals RoboDiff: combining a sample changer and goniometer for highly automated macromolecular crystallography experiments

2016 ◽  
Vol 72 (8) ◽  
pp. 966-975 ◽  
Author(s):  
Didier Nurizzo ◽  
Matthew W. Bowler ◽  
Hugo Caserotto ◽  
Fabien Dobias ◽  
Thierry Giraud ◽  
...  
Keyword(s):  
Data Collection ◽  
Diffraction Data ◽  
High Capacity ◽  
High Accuracy ◽  
Macromolecular Crystallography ◽  
Sample Changer ◽  
High Level

Automation of the mounting of cryocooled samples is now a feature of the majority of beamlines dedicated to macromolecular crystallography (MX). Robotic sample changers have been developed over many years, with the latest designs increasing capacity, reliability and speed. Here, the development of a new sample changer deployed at the ESRF beamline MASSIF-1 (ID30A-1), based on an industrial six-axis robot, is described. The device, named RoboDiff, includes a high-capacity dewar, acts as both a sample changer and a high-accuracy goniometer, and has been designed for completely unattended sample mounting and diffraction data collection. This aim has been achieved using a high level of diagnostics at all steps of the process from mounting and characterization to data collection. The RoboDiff has been in service on the fully automated endstation MASSIF-1 at the ESRF since September 2014 and, at the time of writing, has processed more than 20 000 samples completely automatically.

scholarly journals Radiation damage to nucleoprotein complexes in macromolecular crystallography

2015 ◽  
Vol 22 (2) ◽  
pp. 213-224 ◽  
Author(s):  
Charles Bury ◽  
Elspeth F. Garman ◽  
Helen Mary Ginn ◽  
Raimond B. G. Ravelli ◽  
Ian Carmichael ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Radiation Damage ◽  
Diffraction Data ◽  
Dose Range ◽  
Computational Method ◽  
Large Field ◽  
Macromolecular Crystallography ◽  
Damage Mechanisms ◽  
X Ray ◽  
Nucleoprotein Complexes

Significant progress has been made in macromolecular crystallography over recent years in both the understanding and mitigation of X-ray induced radiation damage when collecting diffraction data from crystalline proteins. In contrast, despite the large field that is productively engaged in the study of radiation chemistry of nucleic acids, particularly of DNA, there are currently very few X-ray crystallographic studies on radiation damage mechanisms in nucleic acids. Quantitative comparison of damage to protein and DNA crystals separately is challenging, but many of the issues are circumvented by studying pre-formed biological nucleoprotein complexes where direct comparison of each component can be made under the same controlled conditions. Here a model protein–DNA complex C.Esp1396I is employed to investigate specific damage mechanisms for protein and DNA in a biologically relevant complex over a large dose range (2.07–44.63 MGy). In order to allow a quantitative analysis of radiation damage sites from a complex series of macromolecular diffraction data, a computational method has been developed that is generally applicable to the field. Typical specific damage was observed for both the protein on particular amino acids and for the DNA on, for example, the cleavage of base-sugar N1—C and sugar-phosphate C—O bonds. Strikingly the DNA component was determined to be far more resistant to specific damage than the protein for the investigated dose range. At low doses the protein was observed to be susceptible to radiation damage while the DNA was far more resistant, damage only being observed at significantly higher doses.

scholarly journals Visualization of protein crystals by high-energy phase-contrast X-ray imaging

2019 ◽  
Vol 75 (11) ◽  
pp. 947-958 ◽  
Author(s):  
Maxim Polikarpov ◽  
Gleb Bourenkov ◽  
Irina Snigireva ◽  
Anatoly Snigirev ◽  
Sophie Zimmermann ◽  
...  
Keyword(s):  
Synchrotron Radiation ◽  
Data Collection ◽  
Diffraction Data ◽  
Phase Contrast ◽  
High Energy ◽  
Protein Crystals ◽  
Cubic Phase ◽  
Macromolecular Crystallography ◽  
X Ray ◽  
X Ray Imaging

For the extraction of the best possible X-ray diffraction data from macromolecular crystals, accurate positioning of the crystals with respect to the X-ray beam is crucial. In addition, information about the shape and internal defects of crystals allows the optimization of data-collection strategies. Here, it is demonstrated that the X-ray beam available on the macromolecular crystallography beamline P14 at the high-brilliance synchrotron-radiation source PETRA III at DESY, Hamburg, Germany can be used for high-energy phase-contrast microtomography of protein crystals mounted in an optically opaque lipidic cubic phase matrix. Three-dimensional tomograms have been obtained at X-ray doses that are substantially smaller and on time scales that are substantially shorter than those used for diffraction-scanning approaches that display protein crystals at micrometre resolution. Adding a compound refractive lens as an objective to the imaging setup, two-dimensional imaging at sub-micrometre resolution has been achieved. All experiments were performed on a standard macromolecular crystallography beamline and are compatible with standard diffraction data-collection workflows and apparatus. Phase-contrast X-ray imaging of macromolecular crystals could find wide application at existing and upcoming low-emittance synchrotron-radiation sources.

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