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.

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 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 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 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.

scholarly journals Anaerobic fixed-target serial crystallography

IUCrJ ◽  
2020 ◽  
Vol 7 (5) ◽  
pp. 901-912
Author(s):  
Patrick Rabe ◽  
John H. Beale ◽  
Agata Butryn ◽  
Pierre Aller ◽  
Anna Dirr ◽  
...  
Keyword(s):  
Data Collection ◽  
Room Temperature ◽  
Temperature Data ◽  
Protein Crystal ◽  
Light Sources ◽  
Strictly Anaerobic ◽  
X Ray ◽  
Synchrotron Light ◽  
Synchrotron Light Sources ◽  
Anaerobic Environment

Cryogenic X-ray diffraction is a powerful tool for crystallographic studies on enzymes including oxygenases and oxidases. Amongst the benefits that cryo-conditions (usually employing a nitrogen cryo-stream at 100 K) enable, is data collection of dioxygen-sensitive samples. Although not strictly anaerobic, at low temperatures the vitreous ice conditions severely restrict O2 diffusion into and/or through the protein crystal. Cryo-conditions limit chemical reactivity, including reactions that require significant conformational changes. By contrast, data collection at room temperature imposes fewer restrictions on diffusion and reactivity; room-temperature serial methods are thus becoming common at synchrotrons and XFELs. However, maintaining an anaerobic environment for dioxygen-dependent enzymes has not been explored for serial room-temperature data collection at synchrotron light sources. This work describes a methodology that employs an adaptation of the `sheet-on-sheet' sample mount, which is suitable for the low-dose room-temperature data collection of anaerobic samples at synchrotron light sources. The method is characterized by easy sample preparation in an anaerobic glovebox, gentle handling of crystals, low sample consumption and preservation of a localized anaerobic environment over the timescale of the experiment (<5 min). The utility of the method is highlighted by studies with three X-ray-radiation-sensitive Fe(II)-containing model enzymes: the 2-oxoglutarate-dependent L-arginine hydroxylase VioC and the DNA repair enzyme AlkB, as well as the oxidase isopenicillin N synthase (IPNS), which is involved in the biosynthesis of all penicillin and cephalosporin antibiotics.

scholarly journals Room-temperature serial crystallography at synchrotron X-ray sources using slowly flowing free-standing high-viscosity microstreams

2015 ◽  
Vol 71 (2) ◽  
pp. 387-397 ◽  
Author(s):  
Sabine Botha ◽  
Karol Nass ◽  
Thomas R. M. Barends ◽  
Wolfgang Kabsch ◽  
Beatrice Latz ◽  
...  
Keyword(s):  
High Resolution ◽  
Data Collection ◽  
High Throughput ◽  
Room Temperature ◽  
High Viscosity ◽  
Temperature Data ◽  
High Dose ◽  
X Ray ◽  
Serial Data ◽  
Serial Crystallography

Recent advances in synchrotron sources, beamline optics and detectors are driving a renaissance in room-temperature data collection. The underlying impetus is the recognition that conformational differences are observed in functionally important regions of structures determined using crystals kept at ambient as opposed to cryogenic temperature during data collection. In addition, room-temperature measurements enable time-resolved studies and eliminate the need to find suitable cryoprotectants. Since radiation damage limits the high-resolution data that can be obtained from a single crystal, especially at room temperature, data are typically collected in a serial fashion using a number of crystals to spread the total dose over the entire ensemble. Several approaches have been developed over the years to efficiently exchange crystals for room-temperature data collection. These includein situcollection in trays, chips and capillary mounts. Here, the use of a slowly flowing microscopic stream for crystal delivery is demonstrated, resulting in extremely high-throughput delivery of crystals into the X-ray beam. This free-stream technology, which was originally developed for serial femtosecond crystallography at X-ray free-electron lasers, is here adapted to serial crystallography at synchrotrons. By embedding the crystals in a high-viscosity carrier stream, high-resolution room-temperature studies can be conducted at atmospheric pressure using the unattenuated X-ray beam, thus permitting the analysis of small or weakly scattering crystals. The high-viscosity extrusion injector is described, as is its use to collect high-resolution serial data from native and heavy-atom-derivatized lysozyme crystals at the Swiss Light Source using less than half a milligram of protein crystals. The room-temperature serial data allowde novostructure determination. The crystal size used in this proof-of-principle experiment was dictated by the available flux density. However, upcoming developments in beamline optics, detectors and synchrotron sources will enable the use of true microcrystals. This high-throughput, high-dose-rate methodology provides a new route to investigating the structure and dynamics of macromolecules at ambient temperature.

scholarly journals Radiation damage effects on protein conformation at room temperature and 100K

2014 ◽  
Vol 70 (a1) ◽  
pp. C344-C344
Author(s):  
Silvia Russi ◽  
Shawn Kann ◽  
Henry van den Bedem ◽  
Ana M. González
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Crystal Structures ◽  
Room Temperature ◽  
Relative Rate ◽  
Absorbed Dose ◽  
Conformational Ensemble ◽  
Data Sets ◽  
Cryogenic Temperatures ◽  
Full Data

Protein crystallography data collection at synchrotrons today is routinely carried out at cryogenic temperatures to mitigate radiation damage to the crystal. Although damage still takes place, at 100 K and below, the immobilization of free radicals increases the lifetime of the crystals by orders of magnitude. Increasingly, experiments are carried out at room temperature. The lack of adequate cryo-protectants, the induced lattice changes or internal disorders during the cooling process, and the convenience of collecting data directly from the crystallization plates, are some of the reasons. Moreover, recent studies have shown that flash-freezing affects the conformational ensemble of crystal structures [1], and can hide important functional mechanisms from observation [2]. While there has been a considerable amount of effort in studying radiation damage at cryo-temperatures, its effects at room temperature are still not well understood. We investigated the effects of data collection temperature on secondary local damage to the side chain and main chain from different proteins. Data were collected from crystals of thaumatin and lysozyme at 100 K and room temperature. To carefully control the total absorbed dose, full data sets at room temperature were assembled from a few diffraction images per crystal. Several data sets were collected at increasing levels of absorbed dose. Our analysis shows that while at cryogenic temperatures, radiation damage increases the conformational variability, _x0004_at room temperature it has the opposite effect_x0005_. We also observed that disulfide bonds appear to break up at a different relative rate at room temperature, perhaps because of a more active repair mechanism. Our analysis suggests that elevated conformational heterogeneity in crystal structures at room temperature is observed despite radiation damage, and not as a result thereof.

scholarly journals Room-temperature macromolecular crystallography using a micro-patterned silicon chip with minimal background scattering

2016 ◽  
Vol 49 (3) ◽  
pp. 968-975 ◽  
Author(s):  
Philip Roedig ◽  
Ramona Duman ◽  
Juan Sanchez-Weatherby ◽  
Ismo Vartiainen ◽  
Anja Burkhardt ◽  
...  
Keyword(s):  
Data Collection ◽  
Room Temperature ◽  
Structural Changes ◽  
Crystalline Silicon ◽  
Background Level ◽  
Sample Holder ◽  
Macromolecular Crystallography ◽  
Recent Success ◽  
Millisecond Range ◽  
Humidified Air

Recent success at X-ray free-electron lasers has led to serial crystallography experiments staging a comeback at synchrotron sources as well. With crystal lifetimes typically in the millisecond range and the latest-generation detector technologies with high framing rates up to 1 kHz, fast sample exchange has become the bottleneck for such experiments. A micro-patterned chip has been developed from single-crystalline silicon, which acts as a sample holder for up to several thousand microcrystals at a very low background level. The crystals can be easily loaded onto the chip and excess mother liquor can be efficiently removed. Dehydration of the crystals is prevented by keeping them in a stream of humidified air during data collection. Further sealing of the sample holder, for example with Kapton, is not required. Room-temperature data collection from insulin crystals loaded onto the chip proves the applicability of the chip for macromolecular crystallography. Subsequent structure refinements reveal no radiation-damage-induced structural changes for insulin crystals up to a dose of 565.6 kGy, even though the total diffraction power of the crystals has on average decreased to 19.1% of its initial value for the same dose. A decay of the diffracting power by half is observed for a dose ofD1/2= 147.5 ± 19.1 kGy, which is about 1/300 of the dose before crystals show a similar decay at cryogenic temperatures.

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.

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.

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