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Elspeth F Garman - One of the best experts on this subject based on the ideXlab platform.
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to scavenge or not to scavenge that is still the question
Journal of Synchrotron Radiation, 2013Co-Authors: Elizabeth G Allan, Melissa C Kander, Ian Carmichael, Elspeth F GarmanAbstract: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.
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radiation damage in macromolecular crystallography what is it and why should we care
Acta Crystallographica Section D-biological Crystallography, 2010Co-Authors: Elspeth F GarmanAbstract: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.
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experimental determination of the radiation dose limit for cryocooled protein crystals
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Robin L Owen, Enrique Rudinopinera, Elspeth F GarmanAbstract:Radiation damage to cryocooled protein crystals during x-ray structure determination has become an inherent part of macromolecular Diffraction Data Collection at third-generation synchrotrons. Generally, radiation damage is an undesirable component of the experiment and can result in erroneous structural detail in the final model. The characterization of radiation damage thus has become an important area for structural biologists. The calculated dose limit of 2 × 107 Gy for the diffracting power of cryocooled protein crystals to drop by half has been experimentally evaluated at a third-generation synchrotron source. Successive Data sets were collected from four holoferritin and three apoferritin crystals. The absorbed dose for each crystal was calculated by using the program raddose after measurement of the incident photon flux and determination of the elemental crystal composition by micro-particle-induced x-ray emission. Degradation in Diffraction quality and specific structural changes induced by synchrotron radiation then could be compared directly with absorbed dose for different dose/dose rate regimes: a 10% lifetime decrease for a 10-fold dose rate increase was observed. Remarkable agreement both between different crystals of the same type and between apoferritin and holoferritin was observed for the dose required to reduce the diffracted intensity by half (D1/2). From these measurements, a dose limit of D1/2 = 4.3 (±0.3) ×107 Gy was obtained. However, by considering other Data quality indicators, an intensity reduction to Iln2 = ln2 × I0, corresponding to an absorbed dose of 3.0 × 107 Gy, is recommended as an appropriate dose limit for typical macromolecular crystallography experiments.
Christoph Mueller-dieckmann - One of the best experts on this subject based on the ideXlab platform.
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Attaining atomic resolution from in situ Data Collection at room temperature using counter-diffusion-based low-cost microchips
Acta crystallographica. Section D Structural biology, 2020Co-Authors: J. A. Gavira, Isaac Rodriguez Ruiz, Sergio Martínez-rodríguez, Shibom Basu, Sébastien Teychené, Andrew Mccarthy, Christoph Mueller-dieckmannAbstract:Sample handling and manipulation for cryoprotection currently remain critical factors in X-ray structural determination. While several microchips for macromolecular crystallization have been proposed during the last two decades to partially overcome crystal-manipulation issues, increased background noise originating from the scattering of chip-fabrication materials has so far limited the attainable resolution of Diffraction Data. Here, the conception and use of low-cost, X-ray-transparent microchips for in situ crystallization and direct Data Collection, and structure determination at atomic resolution close to 1.0 Å , is presented. The chips are fabricated by a combination of either OSTEMER and Kapton or OSTEMER and Mylar materials for the implementation of counter-diffusion crystallization experiments. Both materials produce a sufficiently low scattering background to permit atomic resolution Diffraction Data Collection at room temperature and the generation of 3D structural models of the tested model proteins lysozyme, thaumatin and glucose isomerase. Although the high symmetry of the three model protein crystals produced almost complete Data sets at high resolution, the potential of in-line Data merging and scaling of the multiple crystals grown along the microfluidic channels is also presented and discussed.
Brian K Kobilka - One of the best experts on this subject based on the ideXlab platform.
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in meso in situ serial x ray crystallography of soluble and membrane proteins at cryogenic temperatures
Acta Crystallographica Section D-biological Crystallography, 2016Co-Authors: Chia Ying Huang, Vincent Olieric, Nicole Howe, L Vogeley, Xiangyu Liu, R Warshamanage, Tobias Weinert, Ezequiel Panepucci, Brian K KobilkaAbstract:Here, a method for presenting crystals of soluble and membrane proteins growing in the lipid cubic or sponge phase for in situ Diffraction Data Collection at cryogenic temperatures is introduced. The method dispenses with the need for the technically demanding and inefficient crystal-harvesting step that is an integral part of the lipid cubic phase or in meso method of growing crystals. Crystals are dispersed in a bolus of mesophase sandwiched between thin plastic windows. The bolus contains tens to hundreds of crystals, visible with an in-line microscope at macromolecular crystallography synchrotron beamlines and suitably disposed for conventional or serial crystallographic Data Collection. Wells containing the crystal-laden boluses are removed individually from hermetically sealed glass plates in which crystallization occurs, affixed to pins on goniometer bases and excess precipitant is removed from around the mesophase. The wells are snap-cooled in liquid nitrogen, stored and shipped in Dewars, and manually or robotically mounted on a goniometer in a cryostream for Diffraction Data Collection at 100 K, as is performed routinely with standard, loop-harvested crystals. The method is a variant on the recently introduced in meso in situ serial crystallography (IMISX) method that enables crystallographic measurements at cryogenic temperatures where crystal lifetimes are enormously enhanced whilst reducing protein consumption dramatically. The new approach has been used to generate high-resolution crystal structures of a G-protein-coupled receptor, α-helical and β-barrel transporters and an enzyme as model integral membrane proteins. Insulin and lysozyme were used as test soluble proteins. The quality of the Data that can be generated by this method was attested to by performing sulfur and bromine SAD phasing with two of the test proteins.
J. A. Gavira - One of the best experts on this subject based on the ideXlab platform.
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Attaining atomic resolution from in situ Data Collection at room temperature using counter-diffusion-based low-cost microchips
Acta crystallographica. Section D Structural biology, 2020Co-Authors: J. A. Gavira, Isaac Rodriguez Ruiz, Sergio Martínez-rodríguez, Shibom Basu, Sébastien Teychené, Andrew Mccarthy, Christoph Mueller-dieckmannAbstract:Sample handling and manipulation for cryoprotection currently remain critical factors in X-ray structural determination. While several microchips for macromolecular crystallization have been proposed during the last two decades to partially overcome crystal-manipulation issues, increased background noise originating from the scattering of chip-fabrication materials has so far limited the attainable resolution of Diffraction Data. Here, the conception and use of low-cost, X-ray-transparent microchips for in situ crystallization and direct Data Collection, and structure determination at atomic resolution close to 1.0 Å , is presented. The chips are fabricated by a combination of either OSTEMER and Kapton or OSTEMER and Mylar materials for the implementation of counter-diffusion crystallization experiments. Both materials produce a sufficiently low scattering background to permit atomic resolution Diffraction Data Collection at room temperature and the generation of 3D structural models of the tested model proteins lysozyme, thaumatin and glucose isomerase. Although the high symmetry of the three model protein crystals produced almost complete Data sets at high resolution, the potential of in-line Data merging and scaling of the multiple crystals grown along the microfluidic channels is also presented and discussed.
Jan Pieter Abrahams - One of the best experts on this subject based on the ideXlab platform.
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a medipix quantum area detector allows rotation electron Diffraction Data Collection from submicrometre three dimensional protein crystals
Acta Crystallographica Section D-biological Crystallography, 2013Co-Authors: Igor Nederlof, Eric Van Genderen, Jan Pieter AbrahamsAbstract:When protein crystals are submicrometre-sized, X-ray radiation damage precludes conventional Diffraction Data Collection. For crystals that are of the order of 100 nm in size, at best only single-shot Diffraction patterns can be collected and rotation Data Collection has not been possible, irrespective of the Diffraction technique used. Here, it is shown that at a very low electron dose (at most 0.1 e− A−2), a Medipix2 quantum area detector is sufficiently sensitive to allow the Collection of a 30-frame rotation series of 200 keV electron-Diffraction Data from a single ∼100 nm thick protein crystal. A highly parallel 200 keV electron beam (λ = 0.025 A) allowed observation of the curvature of the Ewald sphere at low resolution, indicating a combined mosaic spread/beam divergence of at most 0.4°. This result shows that volumes of crystal with low mosaicity can be pinpointed in electron Diffraction. It is also shown that strategies and Data-analysis software (MOSFLM and SCALA) from X-ray protein crystallography can be used in principle for analysing electron-Diffraction Data from three-dimensional nanocrystals of proteins.