scholarly journals OH cleavage from tyrosine: debunking a myth

2017 ◽  
Vol 24 (1) ◽  
pp. 7-18 ◽  
Author(s):  
Charles S. Bury ◽  
Ian Carmichael ◽  
Elspeth F Garman
Keyword(s):  
Radiation Damage ◽  
Electron Density ◽  
Active Sites ◽  
Solvent Accessibility ◽  
Protein Crystal ◽  
Protein Crystals ◽  
Tyrosyl Radical ◽  
Systematic Analysis ◽  
Oh Groups ◽  
The Impact

During macromolecular X-ray crystallography experiments, protein crystals held at 100 K have been widely reported to exhibit reproducible bond scission events at doses on the order of several MGy. With the objective to mitigate the impact of radiation damage events on valid structure determination, it is essential to correctly understand the radiation chemistry mechanisms at play. OH-cleavage from tyrosine residues is regularly cited as amongst the most available damage pathways in protein crystals at 100 K, despite a lack of widespread reports of this phenomenon in protein crystal radiation damage studies. Furthermore, no clear mechanism for phenolic C—O bond cleavage in tyrosine has been reported, with the tyrosyl radical known to be relatively robust and long-lived in both aqueous solutions and the solid state. Here, the initial findings of Tyr –OH group damage in a myrosinase protein crystal have been reviewed. Consistent with that study, at increasing doses, clear electron density loss was detectable local to Tyr –OH groups. A systematic investigation performed on a range of protein crystal damage series deposited in the Protein Data Bank has established that Tyr –OH electron density loss is not generally a dominant damage pathway in protein crystals at 100 K. Full Tyr aromatic ring displacement is here proposed to account for instances of observable Tyr –OH electron density loss, with the original myrosinase data shown to be consistent with such a damage model. Systematic analysis of the effects of other environmental factors, including solvent accessibility and proximity to disulfide bonds or hydrogen bond interactions, is also presented. Residues in known active sites showed enhanced sensitivity to radiation-induced disordering, as has previously been reported.

scholarly journals The early history of cryo-cooling for macromolecular crystallography

IUCrJ ◽  
2020 ◽  
Vol 7 (2) ◽  
pp. 148-157 ◽  
Author(s):  
David J. Haas
Keyword(s):  
Lactate Dehydrogenase ◽  
Radiation Damage ◽  
Analytical Data ◽  
Data Bank ◽  
Protein Crystal ◽  
Protein Crystals ◽  
X Ray ◽  
Royal Institution ◽  
History Of ◽  
Lysozyme Crystals

This paper recounts the first successful cryo-cooling of protein crystals that demonstrated the reduction in X-ray damage to macromolecular crystals. The project was suggested by David C. Phillips in 1965 at the Royal Institution of Great Britain and continued in 1967 at the Weizmann Institute of Science, where the first cryo-cooling experiments were performed on lysozyme crystals, and was completed in 1969 at Purdue University on lactate dehydrogenase crystals. A 1970 publication in Acta Crystallographica described the cryo-procedures, the use of cryo-protectants to prevent ice formation, the importance of fast, isotropic cryo-cooling and the collection of analytical data showing more than a tenfold decrease in radiation damage in cryo-cooled lactate dehydrogenase crystals. This was the first demonstration of any method that reduced radiation damage in protein crystals, which provided crystallographers with suitable means to employ synchrotron X-ray sources for protein-crystal analysis. Today, fifty years later, more than 90% of the crystal structures deposited in the Protein Data Bank have been cryo-cooled.

Low dose electron diffraction of wet protein crystals

1978 ◽  
Vol 36 (2) ◽  
pp. 6-7
Author(s):  
B.B. Chang ◽  
D.F. Parsons
Keyword(s):  
High Resolution ◽  
Electron Diffraction ◽  
Radiation Damage ◽  
Low Dose ◽  
Protein Crystal ◽  
Protein Crystals ◽  
Electron Dose ◽  
Resolution Electron ◽  
Diffraction Patterns ◽  
Iterative Procedures

High resolution electron diffraction patterns from wet unstained protein-crystals have been successfully obtained by using very low electron dose (∽10-2 e/Å2, much smaller dose than used by Unwin and Henderson (1975) for electron diffraction of their crystals). This enabled us to follow the radiation damage of the protein crystal due to increasing dosage by recording successive diffraction patterns given by the same crystal. Changes in intensities of both the high and the low order reflections can be studied. Another important consequence is that very low dose electron diffraction can be obtained from the same crystal and iterative procedures such as Gerchberg and Saxton's technique can be applied.The electron diffraction work with very low dose was performed at 200 kV using the environmental chamber in the Jeolco 200. To achieve best electron diffraction conditions with the specimen in the hydration chamber, the objective and intermediate lens currents have been changed.

scholarly journals Radiation damage in protein crystals examined under various conditions by different methods

2009 ◽  
Vol 16 (2) ◽  
pp. 129-132 ◽  
Author(s):  
Elspeth F. Garman ◽  
Colin Nave
Keyword(s):  
Radiation Damage ◽  
Electronic State ◽  
Room Temperature ◽  
Protein Crystal ◽  
Protein Crystals ◽  
X Rays ◽  
X Ray Diffraction ◽  
Radiation Physics ◽  
X Ray ◽  
The Past

Investigation of radiation damage in protein crystals has progressed in several directions over the past couple of years. There have been improvements in the basic procedures such as calibration of the incident X-ray intensity and calculation of the dose likely to be deposited in a crystal of known size and composition with this intensity. There has been increased emphasis on using additional techniques such as optical, Raman or X-ray spectroscopy to complement X-ray diffraction. Apparent discrepancies between the results of different techniques can be explained by the fact that they are sensitive to different length scales or to changes in the electronic state rather than to movement of atoms. Investigations have been carried out at room temperature as well as cryo-temperatures and, in both cases, with the introduction of potential scavenger molecules. These and other studies are leading to an overall description of the changes which can occur when a protein crystal is irradiated with X-rays at both cryo- and room temperatures. Results from crystallographic and spectroscopic radiation-damage experiments can be reconciled with other studies in the field of radiation physics and chemistry.

Exile, Diaspora, and Return

2017 ◽  
Author(s):  
Luis Roniger ◽  
Leonardo Senkman ◽  
Saúl Sosnowski ◽  
Mario Sznajder
Keyword(s):  
Systematic Analysis ◽  
The Arts ◽  
Scientific Disciplines ◽  
Early Twenty ◽  
Transnational Connections ◽  
Late Twentieth ◽  
Institutional Impact ◽  
Global Arena ◽  
Education Science ◽  
The Impact

This book explores how Argentina, Chile, Paraguay, and Uruguay have been affected by postexilic relocations, transnational migrant displacements, and diasporas. It provides a systematic analysis of the formation of exile communities and diaspora politics, the politics of return, and the agenda of democratization in the late twentieth and early twenty-first centuries, focusing on the impact of intellectuals, academics, activists, and public figures who had experienced exile on the reconstitution and transformation of their societies following democratization. Readers are offered a kaleidoscope of intellectual itineraries, debates, and contributions held in the public domain by individuals who confronted and fought authoritarian rule. The book covers their contributions to the restructuring and transformation of scientific disciplines and of the humanities and the arts, as well as their collective institutional impact on higher education, science and technology, and public institutions. Bringing together sociopolitical, cultural, and policy analysis with the testimonies of dozens of intellectuals, academics, political activists, and policymakers, the book addresses the impact of exile on people’s lives and on their fractured experiences, the debates and prospects of return, the challenges of dis-exile and postexilic trends, and, finally, the ways in which those who experienced exile impacted democratized institutions, public culture, and discourse. It also follows some crucial shifts in the frontiers of citizenship, moving analysis to transnational connections and permanent diasporas, including the diasporas of knowledge that increasingly changed the very meaning of being national and transnational, while connecting those countries to the global arena.

Resolution of a protein sequence ambiguity by X-ray crystallographic and mass spectrometric methods

1992 ◽  
Vol 25 (2) ◽  
pp. 205-210 ◽  
Author(s):  
L. J. Keefe ◽  
E. E. Lattman ◽  
C. Wolkow ◽  
A. Woods ◽  
M. Chevrier ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Electron Density ◽  
Mass Spectrometric ◽  
Amino Acid Sequences ◽  
Data Matrix ◽  
Protein Crystal ◽  
Insertion Mutant ◽  
Laser Desorption Mass Spectrometry ◽  
X Ray

Ambiguities in amino acid sequences are a potential problem in X-ray crystallographic studies of proteins. Amino acid side chains often cannot be reliably identified from the electron density. Many protein crystal structures that are now being solved are simple variants of a known wild-type structure. Thus, cloning artifacts or other untoward events can readily lead to cases in which the proposed sequence is not correct. An example is presented showing that mass spectrometry provides an excellent tool for analyzing suspected errors. The X-ray crystal structure of an insertion mutant of Staphylococcal nuclease has been solved to 1.67 Å resolution and refined to a crystallographic R value of 0.170 [Keefe & Lattman (1992). In preparation]. A single residue has been inserted in the C-terminal α helix. The inserted amino acid was believed to be an alanine residue, but the final electron density maps strongly indicated that a glycine had been inserted instead. To confirm the observations from the X-ray data, matrix-assisted laser desorption mass spectrometry was employed to verify the glycine insertion. This mass spectrometric technique has sufficient mass accuracy to detect the methyl group that distinguishes glycine from alanine and can be extended to the more common situation in which crystallographic measurements suggest a problem with the sequence, but cannot pinpoint its location or nature.

Assessing the Impact of Secondary Structure and Solvent Accessibility on Protein Evolution

Genetics ◽  
1998 ◽  
Vol 149 (1) ◽  
pp. 445-458 ◽  
Author(s):  
Nick Goldman ◽  
Jeffrey L Thorne ◽  
David T Jones
Keyword(s):  
Amino Acid ◽  
Secondary Structure ◽  
Protein Evolution ◽  
Solvent Accessibility ◽  
Strong Association ◽  
Length Distribution ◽  
Parametric Bootstrap ◽  
Amino Acid Replacement ◽  
Physical Constraints ◽  
The Impact

Abstract Empirically derived models of amino acid replacement are employed to study the association between various physical features of proteins and evolution. The strengths of these associations are statistically evaluated by applying the models of protein evolution to 11 diverse sets of protein sequences. Parametric bootstrap tests indicate that the solvent accessibility status of a site has a particularly strong association with the process of amino acid replacement that it experiences. Significant association between secondary structure environment and the amino acid replacement process is also observed. Careful description of the length distribution of secondary structure elements and of the organization of secondary structure and solvent accessibility along a protein did not always significantly improve the fit of the evolutionary models to the data sets that were analyzed. As indicated by the strength of the association of both solvent accessibility and secondary structure with amino acid replacement, the process of protein evolution—both above and below the species level—will not be well understood until the physical constraints that affect protein evolution are identified and characterized.

scholarly journals University students’ representations of Europe and self-identification as Europeans: a synthesis of qualitative evidence for future policy formulation

2020 ◽  
Vol 8 (1) ◽  
Author(s):  
Esther Cores-Bilbao ◽  
María del Carmen Méndez-García ◽  
M. Carmen Fonseca-Mora
Keyword(s):  
University Students ◽  
Evidence Synthesis ◽  
Foreign Language Learning ◽  
Educational Interventions ◽  
Policy Formulation ◽  
Qualitative Evidence Synthesis ◽  
The European Union ◽  
Systematic Analysis ◽  
Qualitative Evidence ◽  
The Impact

AbstractThe current European context is characterised by the emergence of socio-political tensions that threaten to derail the cohesion objectives traditionally promoted by the authorities of the European Union. With EU citizenship in the shadow of Brexit, the fear of dismemberment of the current Europe of the 28 looms over a renewed debate on concepts like European identity, European citizenship or EU legitimacy and the involvement of its constituents in European affairs, as well as the role of education for promoting democratic awareness among young Europeans. This work aims to collect, appraise and synthesise qualitative evidence obtained in primary research exploring the perceptions of European university students about their civic and cultural identity. This systematic analysis sets out to identify predictors of positive self-identification with the EU and its institutions, focusing on the impact that different educational interventions have had on the attitudes and perceptions expressed by university students, and the importance of foreign language learning in the results obtained. The authors report their assessment of quality of the findings in a Cochrane-style qualitative evidence synthesis (QES), based on the GRADE-CERQual (Confidence in Evidence from Reviews of Qualitative research) method. The 12 informed findings described in this study support decision-making in future education policy formulation.

Abstract P163: Global and Regional Consumption of Specific Dietary Fats and Dietary Cholesterol in 1990 and 2005: Systematic Analysis of Country-Specific Nutrition Surveys Worldwide

Circulation ◽  
2012 ◽  
Vol 125 (suppl_10) ◽  
Author(s):  
Renata Micha ◽  
Shahab Khatibzadeh ◽  
Edward Giovannucci ◽  
John Powles ◽  
Peilin Shi ◽  
...  
Keyword(s):  
Chronic Diseases ◽  
Dietary Habits ◽  
Dietary Cholesterol ◽  
Assessment Method ◽  
Dietary Fats ◽  
Systematic Analysis ◽  
Diet Surveys ◽  
Nationally Representative ◽  
The Impact ◽  
Age And Sex

Background. Assessing the impact of diet on chronic diseases worldwide has been limited by availability only of food disappearance data rather than reliable and systematically assessed consumption data on dietary habits globally. Objective. To review and access published and unpublished national diet surveys worldwide in a systematic and consistent way to produce comprehensive intake data of specific dietary fats and their uncertainties by country, region, age, and sex in 1990 and 2005. Methods. We developed methods to identify, assess, and obtain exposure data (mean, SD) from nationally representative diet surveys worldwide on saturated, n-6, n-3 and trans fats, and dietary cholesterol. To address missing data and estimate mean intake, we developed and applied a multi-level hierarchical Bayesian model that accounted for country- and region-level data, measurement comparability, study representativeness, and diet assessment method. Time-varying country-level covariates were used to inform the estimates, including FAO food availability data, population, GDP, latitude, metabolic risks, and other diet covariates. Uncertainty of the estimates accounted for uncertainty from sampling and statistical modeling. Results. We obtained relevant data (85% by direct author contact) from 76 nationally representative and 15 large regional surveys from 49 countries in 15 regions, covering 75% of the world’s population. Several countries and regions lacked representative data. Data were most frequently available for saturated fat and dietary cholesterol (Figure). Results for other fats will be presented at the meeting. Conclusions. These new methods developed to systematically assess, compile and estimate the exposure distribution of specific dietary fats and cholesterol in a uniform fashion globally allow, for the first time, characterization of consumption habits and trends by country, region, age and sex. Such global assessment is imperative for estimating the impact of dietary fats on chronic diseases worldwide.

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