scholarly journals X-ray-excited optical luminescence of protein crystals: a new tool for studying radiation damage during diffraction data collection

2012 ◽  
Vol 68 (5) ◽  
pp. 505-510 ◽  
Author(s):  
Robin L. Owen ◽  
Briony A. Yorke ◽  
Arwen R. Pearson
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Diffraction Data ◽  
Protein Crystals ◽  
X Ray ◽  
Optical Luminescence

scholarly journals A Medipix quantum area detector allows rotation electron diffraction data collection from submicrometre three-dimensional protein crystals

2013 ◽  
Vol 69 (7) ◽  
pp. 1223-1230 ◽  
Author(s):  
Igor Nederlof ◽  
Eric van Genderen ◽  
Yao-Wang Li ◽  
Jan Pieter Abrahams
Keyword(s):  
Electron Diffraction ◽  
Data Collection ◽  
Diffraction Data ◽  
Three Dimensional ◽  
Protein Crystal ◽  
Protein Crystals ◽  
Electron Diffraction Data ◽  
Single Shot ◽  
X Ray ◽  
Area Detector

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− Å−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 Å) 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 (MOSFLMandSCALA) from X-ray protein crystallography can be used in principle for analysing electron-diffraction data from three-dimensional nanocrystals of proteins.

A hands-free capillary system for protein crystallography from crystallization to data collection

2008 ◽  
Vol 42 (1) ◽  
pp. 129-133
Author(s):  
Michihiro Sugahara ◽  
Yuichi Kageyama ◽  
Naoki Kunishima
Keyword(s):  
Data Collection ◽  
Diffraction Data ◽  
Thermus Thermophilus ◽  
Heavy Atom ◽  
Experimental System ◽  
Protein Crystals ◽  
Glass Capillary ◽  
Free System ◽  
X Ray ◽  
Capillary System

With the goal of producing a fully automated experimental system for protein X-ray crystallography, a hands-free system is presented in which all aspects of crystallographic experiments, from crystallization to diffraction data collection, are performed within a modified X-ray glass capillary. Because it eliminates the manual handling of crystals, this capillary system allows the user to evaluate the quality of protein crystals more accurately. The capabilities of this capillary system were examined using the TTHB049 protein fromThermus thermophilusHB8 and xylanase fromTrichoderma longibrachiatum. Each protein was successfully crystallized in the capillary and three complete diffraction data sets were collected at 100 K without direct manual intervention. The diffraction data from the capillary system were superior, in terms of both quality and reproducibility, when compared with data from conventional cryoloop systems. In addition, the capillary system allows thein situheavy-atom derivatization of protein crystals,i.e.the TTHB049 crystals were successfully derivatized with K2PtCl4within the capillary.

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.

scholarly journals Identification of the point of diminishing returns in high-multiplicity data collection for sulfur SAD phasing

2017 ◽  
Vol 24 (1) ◽  
pp. 19-28 ◽  
Author(s):  
Selina L. S. Storm ◽  
Fabio Dall'Antonia ◽  
Gleb Bourenkov ◽  
Thomas R. Schneider
Keyword(s):  
Data Collection ◽  
Data Quality ◽  
Radiation Damage ◽  
Diffraction Data ◽  
High Multiplicity ◽  
X Ray ◽  
Anomalous Data ◽  
Diminishing Returns ◽  
Sad Phasing

High-quality high-multiplicity X-ray diffraction data were collected on five different crystals of thaumatin using a homogeneous-profile X-ray beam at E = 8 keV to investigate the counteracting effects of increased multiplicity and increased radiation damage on the quality of anomalous diffraction data collected on macromolecular crystals. By comparing sulfur substructures obtained from subsets of the data selected as a function of absorbed X-ray dose with sulfur positions in the respective refined reference structures, the doses at which the highest quality of anomalous differences could be obtained were identified for the five crystals. A statistic σ{ΔF} D , calculated as the width σ of the normalized distribution of a set {ΔF} of anomalous differences collected at a dose D, is suggested as a measure of anomalous data quality as a function of dose. An empirical rule is proposed to identify the dose at which the gains in data quality due to increased multiplicity are outbalanced by the losses due to decreases in signal-to-noise as a consequence of radiation damage. Identifying this point of diminishing returns allows the optimization of the choice of data collection parameters and the selection of data to be used in subsequent crystal structure determination steps.

scholarly journals Practical macromolecular cryocrystallography

2015 ◽  
Vol 71 (6) ◽  
pp. 622-642 ◽  
Author(s):  
J. W. Pflugrath
Keyword(s):  
Glass Transition ◽  
Data Collection ◽  
Single Crystal ◽  
Radiation Damage ◽  
Diffraction Data ◽  
Cryogenic Temperatures ◽  
X Ray Diffraction ◽  
X Ray ◽  
Flash Cooling ◽  
Solvent Fraction

Cryocrystallography is an indispensable technique that is routinely used for single-crystal X-ray diffraction data collection at temperatures near 100 K, where radiation damage is mitigated. Modern procedures and tools to cryoprotect and rapidly cool macromolecular crystals with a significant solvent fraction to below the glass-transition phase of water are reviewed. Reagents and methods to help prevent the stresses that damage crystals when flash-cooling are described. A method of using isopentane to assess whether cryogenic temperatures have been preserved when dismounting screened crystals is also presented.

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.

Design of a novel Peltier-based cooling device and its use in neutron diffraction data collection of perdeuterated yeast pyrophosphatase

2010 ◽  
Vol 43 (5) ◽  
pp. 1113-1120 ◽  
Author(s):  
Esko Oksanen ◽  
François Dauvergne ◽  
Adrian Goldman ◽  
Monika Budayova-Spano
Keyword(s):  
Data Collection ◽  
Neutron Diffraction ◽  
Diffraction Data ◽  
Catalytic Mechanism ◽  
Inorganic Pyrophosphatase ◽  
Neutron Diffraction Data ◽  
Cooling Device ◽  
Data Set ◽  
X Ray ◽  
X Ray Crystallography

H atoms play a central role in enzymatic mechanisms, but H-atom positions cannot generally be determined by X-ray crystallography. Neutron crystallography, on the other hand, can be used to determine H-atom positions but it is experimentally very challenging. Yeast inorganic pyrophosphatase (PPase) is an essential enzyme that has been studied extensively by X-ray crystallography, yet the details of the catalytic mechanism remain incompletely understood. The temperature instability of PPase crystals has in the past prevented the collection of a neutron diffraction data set. This paper reports how the crystal growth has been optimized in temperature-controlled conditions. To stabilize the crystals during neutron data collection a Peltier cooling device that minimizes the temperature gradient along the capillary has been developed. This device allowed the collection of a full neutron diffraction data set.

scholarly journals Protein microcrystallography using synchrotron radiation

IUCrJ ◽  
2017 ◽  
Vol 4 (5) ◽  
pp. 529-539 ◽  
Author(s):  
Masaki Yamamoto ◽  
Kunio Hirata ◽  
Keitaro Yamashita ◽  
Kazuya Hasegawa ◽  
Go Ueno ◽  
...  
Keyword(s):  
Synchrotron Radiation ◽  
Data Collection ◽  
Diffraction Data ◽  
Structure Determination ◽  
Structural Features ◽  
X Ray ◽  
Data Collection Strategy ◽  
Technological Developments ◽  
Novel Method

The progress in X-ray microbeam applications using synchrotron radiation is beneficial to structure determination from macromolecular microcrystals such as smallin mesocrystals. However, the high intensity of microbeams causes severe radiation damage, which worsens both the statistical quality of diffraction data and their resolution, and in the worst cases results in the failure of structure determination. Even in the event of successful structure determination, site-specific damage can lead to the misinterpretation of structural features. In order to overcome this issue, technological developments in sample handling and delivery, data-collection strategy and data processing have been made. For a few crystals with dimensions of the order of 10 µm, an elegant two-step scanning strategy works well. For smaller samples, the development of a novel method to analyze multiple isomorphous microcrystals was motivated by the success of serial femtosecond crystallography with X-ray free-electron lasers. This method overcame the radiation-dose limit in diffraction data collection by using a sufficient number of crystals. Here, important technologies and the future prospects for microcrystallography are discussed.

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