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 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.

scholarly journals Synchrotron microcrystal native-SAD phasing at a low energy

IUCrJ ◽  
2019 ◽  
Vol 6 (4) ◽  
pp. 532-542 ◽  
Author(s):  
Gongrui Guo ◽  
Ping Zhu ◽  
Martin R. Fuchs ◽  
Wuxian Shi ◽  
Babak Andi ◽  
...  
Keyword(s):  
Data Analysis ◽  
Data Collection ◽  
Diffraction Data ◽  
De Novo ◽  
Low Energy ◽  
Anomalous Scattering ◽  
Free Electron Lasers ◽  
X Ray ◽  
Structural Evaluation ◽  
Sad Phasing

De novo structural evaluation of native biomolecules from single-wavelength anomalous diffraction (SAD) is a challenge because of the weakness of the anomalous scattering. The anomalous scattering from relevant native elements – primarily sulfur in proteins and phosphorus in nucleic acids – increases as the X-ray energy decreases toward their K-edge transitions. Thus, measurements at a lowered X-ray energy are promising for making native SAD routine and robust. For microcrystals with sizes less than 10 µm, native-SAD phasing at synchrotron microdiffraction beamlines is even more challenging because of difficulties in sample manipulation, diffraction data collection and data analysis. Native-SAD analysis from microcrystals by using X-ray free-electron lasers has been demonstrated but has required use of thousands of thousands of microcrystals to achieve the necessary accuracy. Here it is shown that by exploitation of anomalous microdiffraction signals obtained at 5 keV, by the use of polyimide wellmounts, and by an iterative crystal and frame-rejection method, microcrystal native-SAD phasing is possible from as few as about 1 200 crystals. Our results show the utility of low-energy native-SAD phasing with microcrystals at synchrotron microdiffraction beamlines.

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.

Global indicators of X-ray data quality

2001 ◽  
Vol 34 (2) ◽  
pp. 130-135 ◽  
Author(s):  
Manfred S. Weiss
Keyword(s):  
Data Quality ◽  
Diffraction Data ◽  
Optical Resolution ◽  
Data Set ◽  
X Ray ◽  
Global Indicators ◽  
Nominal Resolution

Global indicators of the quality of diffraction data are presented and discussed, and are evaluated in terms of their performance with respect to various tasks. Based on the results obtained, it is suggested that some of the conventional indicators still in use in the crystallographic community should be abandoned, such as the nominal resolutiondminor the mergingRfactorRmerge, and replaced by more objective and more meaningful numbers, such as the effective optical resolutiondeff,optand the redundancy-independent mergingRfactorRr.i.m.. Furthermore, it is recommended that the precision-indicating mergingRfactorRp.i.m.should be reported with every diffraction data set published, because it describes the precision of the averaged measurements, which are the quantities normally used in crystallography as observables.

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 Data Exploration Toolkitfor serial diffraction experiments

2015 ◽  
Vol 71 (2) ◽  
pp. 352-356 ◽  
Author(s):  
Oliver B. Zeldin ◽  
Aaron S. Brewster ◽  
Johan Hattne ◽  
Monarin Uervirojnangkoorn ◽  
Artem Y. Lyubimov ◽  
...  
Keyword(s):  
Data Collection ◽  
Diffraction Data ◽  
Data Exploration ◽  
Data Sets ◽  
Significant Heterogeneity ◽  
Free Electron Lasers ◽  
X Ray ◽  
Experimental Strategy ◽  
Serial Crystallography

Ultrafast diffraction at X-ray free-electron lasers (XFELs) has the potential to yield new insights into important biological systems that produce radiation-sensitive crystals. An unavoidable feature of the `diffraction before destruction' nature of these experiments is that images are obtained from many distinct crystals and/or different regions of the same crystal. Combined with other sources of XFEL shot-to-shot variation, this introduces significant heterogeneity into the diffraction data, complicating processing and interpretation. To enable researchers to get the most from their collected data, a toolkit is presented that provides insights into the quality of, and the variation present in, serial crystallography data sets. These tools operate on the unmerged, partial intensity integration results from many individual crystals, and can be used on two levels: firstly to guide the experimental strategy during data collection, and secondly to help users make informed choices during data processing.

Fine-needle capillary mounting for protein microcrystals

2012 ◽  
Vol 45 (4) ◽  
pp. 785-788 ◽  
Author(s):  
Masatomo Makino ◽  
Izumi Wada ◽  
Nobuhiro Mizuno ◽  
Kunio Hirata ◽  
Nobutaka Shimizu ◽  
...  
Keyword(s):  
Data Quality ◽  
Optical Tweezers ◽  
Diffraction Data ◽  
Cell Biology ◽  
Capillary Action ◽  
Fine Needle ◽  
X Ray

Protein microcrystals of less than 10 µm in size are now applicable to X-ray studies by synchrotron microbeam technology. However, because of their small size, they are difficult to handle and mount. In addition, the deterioration of data quality by scattering from the mounting apparatus and crystallization solvent is not negligible. To address these issues, a simple mounting method is proposed using a fine-needle capillary similar to that used for microinjection in cell biology. In this method, microcrystals are pulled up by capillary action or pipetting, and are held at the tip together with a small amount of cryoprotectant. The quality of the diffraction data using this method is comparable to that of data from conventional cryoloops. This solid apparatus is hopefully suitable for automation of microcrystal handling coupled with optical tweezers.

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 A step towards long-wavelength protein crystallography: subjecting protein crystals to a vacuum

2015 ◽  
Vol 48 (3) ◽  
pp. 913-916 ◽  
Author(s):  
Santosh Panjikar ◽  
Lars Thomsen ◽  
Kane Michael O'Donnell ◽  
Alan Riboldi-Tunnicliffe
Keyword(s):  
Data Collection ◽  
Data Quality ◽  
Crystal Lattice ◽  
Liquid Nitrogen ◽  
Proteinase K ◽  
Protein Crystals ◽  
X Ray ◽  
Long Wavelength ◽  
Flash Cooling

Using the UHV experimental endstation on the soft X-ray beamline at the Australian Synchrotron, lysozyme and proteinase K crystals have been exposed to a vacuum of 10−5 mbar, prior to flash-cooling in a bath of liquid nitrogen. Subsequent data collection on the MX2 beamline at the Australian Synchrotron demonstrated that, for lysozyme and proteinase K, it is possible to subject these mounted crystals to a vacuum pressure of 10−5 mbar without destroying the crystal lattice. Despite the lower data quality of the vacuum-pumped crystals compared with control crystals, it is demonstrated that the protein crystals can survive in a vacuum under suitable conditions.

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