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

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-mm3 crystals 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 cytochrome c′, 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

Lectin-Functionalized Polyethylene Glycol for Relief of Mucosal Dryness

The importance of lubrication between oral surfaces provided by the salivary film is most acutely apparent when it is disrupted, a prevalent consequence of salivary gland hypofunction experienced with aging, a symptom of certain diseases, or a side effect of some medical interventions. Sufferers report difficulty with speech and oral food processing and collectively is detrimental to quality of life. Polyethylene glycol (PEG) is widely employed as a successful biocompatible boundary lubricant in engineering and biomedical applications. It is hypothesized that the immobilization of PEG to biological materials such as oral epithelial cells and tissue can mimic the salivary film and provide durable relief from the symptoms of mucosal dryness. To do so, PEG is functionalized with a sugar binding lectin (wheat germ agglutinin) to enhance epithelial adhesion through lectin-sugar interactions. Retention and lubricity are characterized on an ex vivo oral tissue tribology rig. WGA-PEG coats and retains on mucin films, oral epithelial cells, and porcine tongue tissue, and offers sustained reduction in coefficient of friction (COF). WGA-PEG could be developed into a useful topical treatment for reducing oral friction and the perception of dry mouth

Perdeuterated GbpA Enables Neutron Scattering Experiments of a Lytic Polysaccharide Monooxygenase

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Direct visualization of a Fe(IV)-OH intermediate in a heme enzyme

Catalytic heme enzymes carry out a wide range of oxidations in biology. They have in common a mechanism that requires formation of highly oxidized ferryl intermediates. It is these ferryl intermediates that provide the catalytic engine to drive the biological activity. Unravelling the nature of the ferryl species is of fundamental and widespread importance. The essential question is whether the ferryl is best described as a Fe(IV)=O or a Fe(IV)–OH species, but previous spectroscopic and X-ray crystallographic studies have not been able to unambiguously differentiate between the two species. Here we use a different approach. We report a neutron crystal structure of the ferryl intermediate in Compound II of a heme peroxidase; the structure allows the protonation states of the ferryl heme to be directly observed. This, together with pre-steady state kinetic analyses, electron paramagnetic resonance spectroscopy and single crystal X-ray fluorescence, identifies a Fe(IV)–OH species as the reactive intermediate. The structure establishes a precedent for the formation of Fe(IV)–OH in a peroxidase

Depression, Somatization and Anxiety in Female Patients with Temporomandibular Disorders (TMD)

The aim of this research was to determine the possible differences in degrees of depression, somatization and anxiety between the acute and chronic female patients with temporomandibular disorders (TMD), and whether these differences exist in healthy female patients. Ninety female patients were involved in this research; 60 of them were TMD patients of the Dental Polyclinic, while other 30 females came for a rutine recall visit and had no problem related to TMD. Patients were aged 22 to 67 years, the average age being 38.5±12 years. All patients were asked to fill in the RDC/TMD protocol and three psychological tests (Emotions Profile Index, Somatization Scale and life Events Scale). Following the analysis of the RDC/TMD protocol and psychological tests, it was determined that the chronic female patients had higher depression and somatization scores in comparison with the acute patients (p<0.01); the acute patients self-perceive higher levels of anxiety in relation to the control group; furthermore, the patients reporting higher levels of depression were more inclined to somatization and had experienced a greater number of stress events in the past six months. It is beyond doubt that patients suffering from the TMD’s exhibit higher levels of depression, somatization and anxiety compared to the healthy ones, which proves that physiological factors may play a predisposing role in combination with reduced level of body tolerance to pain, and a decreased tolerance to stress

Brief Report: Testing the Psychometric Properties of the Spence Children’s Anxiety Scale (SCAS) and the Screen for Child Anxiety Related Emotional Disorders (SCARED) in Autism Spectrum Disorder

Anxiety is a prevalent and impairing co-morbidity among individuals with autism spectrum disorder (ASD), yet assessment measures, including screening tools, are seldom validated with autism samples. We explored the psychometric properties of the child and parent reports of the Spence Children’s Anxiety Scale (SCAS) and the Screen for Anxiety Related Disorder-71 (SCARED-71) with 49 males with ASD (10–16 years, 63% co-occurring anxiety). Both measures had excellent internal consistency and fair-good parent–child agreement. The SCAS has a higher proportion of items evaluating observable behaviors. Predictive power of the measures did not differ. Higher cut-points in the parent reports (SCARED only) and lower cut-points in the child reports may enhance prediction in this sample. Choice of measure and cut-points should be considered alongside intended purpose