scholarly journals SAXS studies of X-ray induced disulfide bond damage: Engineering high-resolution insight from a low-resolution technique

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0239702
Author(s):  
Timothy R. Stachowski ◽  
Mary E. Snell ◽  
Edward H. Snell
Keyword(s):  
Radiation Damage ◽  
Disulfide Bond ◽  
Large Scale ◽  
Specific Interaction ◽  
X Rays ◽  
Low Resolution ◽  
Damage Site ◽  
Site Specific ◽  
X Ray ◽  
X Ray Crystallography

A significant problem in biological X-ray crystallography is the radiation chemistry caused by the incident X-ray beam. This produces both global and site-specific damage. Site specific damage can misdirect the biological interpretation of the structural models produced. Cryo-cooling crystals has been successful in mitigating damage but not eliminating it altogether; however, cryo-cooling can be difficult in some cases and has also been shown to limit functionally relevant protein conformations. The doses used for X-ray crystallography are typically in the kilo-gray to mega-gray range. While disulfide bonds are among the most significantly affected species in proteins in the crystalline state at both cryogenic and higher temperatures, there is limited information on their response to low X-ray doses in solution, the details of which might inform biomedical applications of X-rays. In this work we engineered a protein that dimerizes through a susceptible disulfide bond to relate the radiation damage processes seen in cryo-cooled crystals to those closer to physiologic conditions. This approach enables a low-resolution technique, small angle X-ray scattering (SAXS), to detect and monitor a residue specific process. A dose dependent fragmentation of the engineered protein was seen that can be explained by a dimer to monomer transition through disulfide bond cleavage. This supports the crystallographically derived mechanism and demonstrates that results obtained crystallographically can be usefully extrapolated to physiologic conditions. Fragmentation was influenced by pH and the conformation of the dimer, providing information on mechanism and pointing to future routes for investigation and potential mitigation. The novel engineered protein approach to generate a large-scale change through a site-specific interaction represents a promising tool for advancing radiation damage studies under solution conditions.

scholarly journals xMDFF: molecular dynamics flexible fitting of low-resolution X-ray structures

2014 ◽  
Vol 70 (9) ◽  
pp. 2344-2355 ◽  
Author(s):  
Ryan McGreevy ◽  
Abhishek Singharoy ◽  
Qufei Li ◽  
Jingfen Zhang ◽  
Dong Xu ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Large Scale ◽  
Protein Structures ◽  
Data Bank ◽  
Real Space ◽  
Low Resolution ◽  
X Ray ◽  
X Ray Crystallography ◽  
Atomic Structures ◽  
Electron Density Map

X-ray crystallography remains the most dominant method for solving atomic structures. However, for relatively large systems, the availability of only medium-to-low-resolution diffraction data often limits the determination of all-atom details. A new molecular dynamics flexible fitting (MDFF)-based approach, xMDFF, for determining structures from such low-resolution crystallographic data is reported. xMDFF employs a real-space refinement scheme that flexibly fits atomic models into an iteratively updating electron-density map. It addresses significant large-scale deformations of the initial model to fit the low-resolution density, as tested with synthetic low-resolution maps of D-ribose-binding protein. xMDFF has been successfully applied to re-refine six low-resolution protein structures of varying sizes that had already been submitted to the Protein Data Bank. Finally,viasystematic refinement of a series of data from 3.6 to 7 Å resolution, xMDFF refinements together with electrophysiology experiments were used to validate the first all-atom structure of the voltage-sensing protein Ci-VSP.

scholarly journals Sub-atomic resolution X-ray crystallography and neutron crystallography: promise, challenges and potential

IUCrJ ◽  
2015 ◽  
Vol 2 (4) ◽  
pp. 464-474 ◽  
Author(s):  
Matthew P. Blakeley ◽  
Samar S. Hasnain ◽  
Svetlana V. Antonyuk
Keyword(s):  
Radiation Damage ◽  
Diffraction Data ◽  
Atomic Resolution ◽  
Current Status ◽  
X Rays ◽  
Macromolecular Crystallography ◽  
X Ray ◽  
X Ray Crystallography ◽  
Small Proteins ◽  
Neutron Crystallography

The International Year of Crystallography saw the number of macromolecular structures deposited in the Protein Data Bank cross the 100000 mark, with more than 90000 of these provided by X-ray crystallography. The number of X-ray structures determined to sub-atomic resolution (i.e.≤1 Å) has passed 600 and this is likely to continue to grow rapidly with diffraction-limited synchrotron radiation sources such as MAX-IV (Sweden) and Sirius (Brazil) under construction. A dozen X-ray structures have been deposited to ultra-high resolution (i.e.≤0.7 Å), for which precise electron density can be exploited to obtain charge density and provide information on the bonding character of catalytic or electron transfer sites. Although the development of neutron macromolecular crystallography over the years has been far less pronounced, and its application much less widespread, the availability of new and improved instrumentation, combined with dedicated deuteration facilities, are beginning to transform the field. Of the 83 macromolecular structures deposited with neutron diffraction data, more than half (49/83, 59%) were released since 2010. Sub-mm3crystals are now regularly being used for data collection, structures have been determined to atomic resolution for a few small proteins, and much larger unit-cell systems (cell edges >100 Å) are being successfully studied. While some details relating to H-atom positions are tractable with X-ray crystallography at sub-atomic resolution, the mobility of certain H atoms precludes them from being located. In addition, highly polarized H atoms and protons (H+) remain invisible with X-rays. Moreover, the majority of X-ray structures are determined from cryo-cooled crystals at 100 K, and, although radiation damage can be strongly controlled, especially since the advent of shutterless fast detectors, and by using limited doses and crystal translation at micro-focus beams, radiation damage can still take place. Neutron crystallography therefore remains the only approach where diffraction data can be collected at room temperature without radiation damage issues and the only approach to locate mobile or highly polarized H atoms and protons. Here a review of the current status of sub-atomic X-ray and neutron macromolecular crystallography is given and future prospects for combined approaches are outlined. New results from two metalloproteins, copper nitrite reductase and cytochromec′, are also included, which illustrate the type of information that can be obtained from sub-atomic-resolution (∼0.8 Å) X-ray structures, while also highlighting the need for complementary neutron studies that can provide details of H atoms not provided by X-ray crystallography.

scholarly journals Analysis of global and site-specific radiation damage in cryo-EM

2018 ◽  
Author(s):  
Johan Hattne ◽  
Dan Shi ◽  
Calina Glynn ◽  
Chih-Te Zee ◽  
Marcus Gallagher-Jones ◽  
...  
Keyword(s):  
Electron Beam ◽  
Radiation Damage ◽  
Disulfide Bonds ◽  
Atomic Resolution ◽  
Proteinase K ◽  
Electron Radiation ◽  
Site Specific ◽  
X Ray ◽  
X Ray Crystallography ◽  
Crystal Electron

SummaryMicro-crystal electron diffraction (MicroED) is an emerging method in cryo-EM for structure determination using nanocrystals. It has been used to solve structures of a diverse set of biomolecules and materials, in some cases to sub-atomic resolution. However, little is known about the damaging effects of the electron beam on samples during such measurements. We assess global and site-specific damage from electron radiation on nanocrystals of proteinase K and of a prion hepta-peptide and find that the dynamics of electron-induced damage follow well-established trends observed in X-ray crystallography. Metal ions are perturbed, disulfide bonds are broken, and acidic side chains are decarboxylated while the diffracted intensities decay exponentially with increasing exposure. A better understanding of radiation damage in MicroED improves our assessment and processing of all types of cryo-EM data.

scholarly journals A SAXS-based approach to rationally evaluate radical scavengers – toward eliminating radiation damage in solution and crystallographic studies

2021 ◽  
Vol 28 (5) ◽  
Author(s):  
Timothy R. Stachowski ◽  
Mary E. Snell ◽  
Edward H. Snell
Keyword(s):  
Ascorbic Acid ◽  
Radiation Damage ◽  
Disulfide Bond ◽  
Sodium Nitrate ◽  
Large Scale ◽  
Relative Effectiveness ◽  
Mitigation Strategies ◽  
Chemical Additives ◽  
X Ray ◽  
Solution Studies

X-ray-based techniques are a powerful tool in structural biology but the radiation-induced chemistry that results can be detrimental and may mask an accurate structural understanding. In the crystallographic case, cryocooling has been employed as a successful mitigation strategy but also has its limitations including the trapping of non-biological structural states. Crystallographic and solution studies performed at physiological temperatures can reveal otherwise hidden but relevant conformations, but are limited by their increased susceptibility to radiation damage. In this case, chemical additives that scavenge the species generated by radiation can mitigate damage but are not always successful and the mechanisms are often unclear. Using a protein designed to undergo a large-scale structural change from breakage of a disulfide bond, radiation damage can be monitored with small-angle X-ray scattering. Using this, we have quantitatively evaluated how three scavengers commonly used in crystallographic experiments – sodium nitrate, cysteine, and ascorbic acid – perform in solution at 10°C. Sodium nitrate was the most effective scavenger and completely inhibited fragmentation of the disulfide bond at a lower concentration (500 µM) compared with cysteine (∼5 mM) while ascorbic acid performed best at 5 mM but could only reduce fragmentation by ∼75% after a total accumulated dose of 792 Gy. The relative effectiveness of each scavenger matches their reported affinities for solvated electrons. Saturating concentrations of each scavenger shifted fragmentation from first order to a zeroth-order process, perhaps indicating the direct contribution of photoabsorption. The SAXS-based method can detect damage at X-ray doses far lower than those accessible crystallographically, thereby providing a detailed picture of scavenger processes. The solution results are also in close agreement with what is known about scavenger performance and mechanism in a crystallographic setting and suggest that a link can be made between the damage phenomenon in the two scenarios. Therefore, our engineered approach might provide a platform for more systematic and comprehensive screening of radioprotectants that can directly inform mitigation strategies for both solution and crystallographic experiments, while also clarifying fundamental radiation damage mechanisms.

Studies of metaphase chromosomes in the scanning transmission x-ray microscope: Image fidelity, radiation damage and specimen preparation

1992 ◽  
Vol 50 (2) ◽  
pp. 1038-1039
Author(s):  
Shawn Williams ◽  
Xiaodong Zhang ◽  
Susan Lamm ◽  
Jack Van’t Hof
Keyword(s):  
Visible Light ◽  
Radiation Damage ◽  
Cross Section ◽  
Imaging Techniques ◽  
Dose Range ◽  
Specimen Preparation ◽  
X Rays ◽  
Metaphase Chromosomes ◽  
X Ray ◽  
Scanning Transmission

The Scanning Transmission X-ray Microscope (STXM) is well suited for investigating metaphase chromosome structure. The absorption cross-section of soft x-rays having energies between the carbon and oxygen K edges (284 - 531 eV) is 6 - 9.5 times greater for organic specimens than for water, which permits one to examine unstained, wet biological specimens with resolution superior to that attainable using visible light. The attenuation length of the x-rays is suitable for imaging micron thick specimens without sectioning. This large difference in cross-section yields good specimen contrast, so that fewer soft x-rays than electrons are required to image wet biological specimens at a given resolution. But most imaging techniques delivering better resolution than visible light produce radiation damage. Soft x-rays are known to be very effective in damaging biological specimens. The STXM is constructed to minimize specimen dose, but it is important to measure the actual damage induced as a function of dose in order to determine the dose range within which radiation damage does not compromise image quality.

scholarly journals Lower resolution Laue neutron data yield correct nanocluster formulations

2014 ◽  
Vol 70 (a1) ◽  
pp. C187-C187
Author(s):  
Alison Edwards
Keyword(s):  
Neutron Diffraction ◽  
Single Crystal ◽  
Diffraction Pattern ◽  
Precise Determination ◽  
Data Sets ◽  
X Rays ◽  
Low Resolution ◽  
Neutron Data ◽  
X Ray ◽  
Product Formulation

"The renaissance in Laue studies - at neutron sources - provides us with access to single crystal neutron diffraction data for synthetic compounds without requiring synthesis of prohibitively large amounts of compound or improbably large crystals. Such neutron diffraction studies provide vital data where proof of the presence or absence of hydrogen in particular locations is required and which cannot validly be proved by X-ray studies. Since the commissioning of KOALA at OPAL in 2009[1] we have obtained numerous data sets which demonstrate the vital importance of measuring data even where the extent of the diffraction pattern is at relatively low resolution - especially when compared to that obtainable for the same compound with X-rays. In the Laue experiment performed with a fixed radius detector, data reduction is only feasible for crystals in the ""goldilocks"" zone – where the unit cell is relatively large for the detector, a correspondingly low resolution diffraction pattern in which adjacent spots are less affected by overlap will yield more data against which a structure can be refined than a pattern of higher resolution – one where neighbouring spots overlap rendering both unusable (in our current methodology). Analogous application of powder neutron diffraction in such determinations is also considered. Single crystal neutron diffraction studies of several important compounds (up to 5KDa see figure below)[2] in which precise determination of hydride content by neutron diffraction was pivotal to the final formulation will be presented. The neutron data sets typically possess 20% or fewer unique data at substantially "lower resolution" than the corresponding X-ray data sets. Careful refinement clearly reveals chemical detail which is typically unexplored in related X-ray diffraction studies reporting high profile chemistry despite the synthetic route being one which hydride ought to be considered/excluded in product formulation."

scholarly journals Quantitative radiation damage studies in macromolecular X-ray crystallography

2013 ◽  
Vol 69 (a1) ◽  
pp. s407-s408
Author(s):  
Markus Gerstel ◽  
Natalya Olekhnovich ◽  
Jonathan Brooks-Bartlett ◽  
Zygmunt S. Derewenda ◽  
Charlotte M. Deane ◽  
...  
Keyword(s):  
Radiation Damage ◽  
X Ray ◽  
X Ray Crystallography

scholarly journals A young and obscured AGN embedded in the giant radio galaxy Mrk 1498

2019 ◽  
Author(s):  
L Hernández-García ◽  
F Panessa ◽  
L Bassani ◽  
G Bruni ◽  
F Ursini ◽  
...  
Keyword(s):  
Large Scale ◽  
Radio Galaxy ◽  
Conclusive Evidence ◽  
X Rays ◽  
Nuclear Activity ◽  
X Ray ◽  
Multi Wavelength ◽  
Tidal Streams ◽  
A Minor ◽  
Extended Emission

Abstract Mrk 1498 is part of a sample of galaxies with extended emission line regions (extended outwards up to a distance of ∼7 kpc) suggested to be photo-ionized by an AGN that has faded away or that is still active but heavily absorbed. Interestingly, the nucleus of Mrk 1498 is at the center of two giant radio lobes with a projected linear size of 1.1 Mpc. Our multi-wavelength analysis reveals a complex nuclear structure, with a young radio source (Giga-hertz Peaked Spectrum) surrounded by a strong X-ray nuclear absorption, a mid-infrared spectrum that is dominated by the torus emission, plus a circum-nuclear extended emission in the [OIII] image (with radius of ∼ 1 kpc), most likely related to the ionization of the AGN, aligned with the small and large scale radio jet and extended also at X-rays. In addition a large-scale extended emission (up to ∼ 10 kpc) is only visible in [OIII]. These data show conclusive evidence of a heavily absorbed nucleus and has recently restarted its nuclear activity. To explain its complexity, we propose that Mrk 1498 is the result of a merging event or secular processes, such as a minor interaction, that has triggered the nuclear activity and produced tidal streams. The large-scale extended emission that gives place to the actual morphology could either be explained by star formation or outflowing material from the AGN.

scholarly journals Optical control, selection and analysis of population dynamics in ultrafast protein X-ray crystallography

2019 ◽  
Vol 377 (2145) ◽  
pp. 20170474 ◽  
Author(s):  
Christopher D. M. Hutchison ◽  
Jasper J. van Thor
Keyword(s):  
Structural Dynamics ◽  
Time Domain ◽  
Optical Pumping ◽  
High Repetition Rate ◽  
X Rays ◽  
X Ray Diffraction ◽  
X Ray ◽  
Vibrational Coherence ◽  
X Ray Crystallography ◽  
Ultrafast Optical

Ultrafast pump-probe X-ray crystallography has now been established at X-ray free electron lasers that operate at hard X-ray energies. We discuss the performance and development of current applications in terms of the available data quality and sensitivity to detect and analyse structural dynamics. A discussion of technical capabilities expected at future high repetition rate applications as well as future non-collinear multi-pulse schemes focuses on the possibility to advance the technique to the practical application of the X-ray crystallographic equivalent of an impulse time-domain Raman measurement of vibrational coherence. Furthermore, we present calculations of the magnitude of population differences and distributions prepared with ultrafast optical pumping of single crystals in the typical serial femtosecond crystallography geometry, which are developed for the general uniaxial and biaxial cases. The results present opportunities for polarization resolved anisotropic X-ray diffraction analysis of photochemical populations for the ultrafast time domain. This article is part of the theme issue ‘Measurement of ultrafast electronic and structural dynamics with X-rays’.

scholarly journals Closing the gap between electron and X-ray crystallography

2015 ◽  
Vol 71 (6) ◽  
pp. 737-739 ◽  
Author(s):  
Enrico Mugnaioli
Keyword(s):  
Electron Diffraction ◽  
Structure Characterization ◽  
Diffraction Tomography ◽  
X Rays ◽  
Electron Crystallography ◽  
X Ray ◽  
X Ray Crystallography ◽  
Closing The Gap ◽  
Refinement Algorithm

The development of a proper refinement algorithm that takes into account dynamical scattering guarantees, for electron crystallography, results approaching X-rays in terms of precision, accuracy and reliability. The combination of such dynamical refinement and electron diffraction tomography establishes a complete pathway for the structure characterization of single sub-micrometric crystals.

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