Using a SMALP platform to determine a sub-nm single particle cryo-EM membrane protein structure

The field of membrane protein structural biology has been revolutionized over the last few years with a number of high profile structures being solved using cryo-EM including Piezo, Ryanodine receptor, TRPV1 and the Glutamate receptor. Further developments in the EM field hold the promise of even greater progress in terms of greater resolution, which for membrane proteins is still typically within the 4-7 angstrom range. One advantage of a cryo-EM approach is the ability to study membrane proteins in more "native" like environments for example proteoliposomes, amphipols and nanodiscs. Recently, styrene maleic acid co-polymers (SMA) have been used to extract membrane proteins surrounded by native lipids (SMALPs) maintaining a more natural environment. We report here the structure of the Escherichia coli multidrug efflux transporter AcrB in a SMALP scaffold to sub-nm resolution, with the resulting map being consistent with high resolution crystal structures and other EM derived maps. However, both the C-terminal helix (TM12) and TM7 are poorly defined in the map. These helices are at the exterior of the helical bundle and form the greater interaction with the native lipids and SMA polymer and may represent a more dynamic region of the protein. This work shows the promise of using an SMA approach for single particle cryo-EM studies to provide sub-nm structures.Peer reviewe

Quantification of the unsharp masking technique of image enhancement

The technique of unsharp masking is described and its use as an image enhancement technique discussed. A mathematical model for the masking process is developed; experimental testing and MTF measurements of the masked and sharpened images are made to test the validity of the mathematical model as a predictor of the mask and final image characteristics. The effect of contrast, mask unsharpness, and source spread function size on the resulting MTF are presented. Subjective evaluations are used to determine the visually optimum image. It is shown that the visually best image is not necessarily the one with the largest MTF value or area; suggestions are made for adjusting existing image quality specifications to incorporate the results of unsharp masking techniques

The sensory features of a food cue influence its ability to act as an incentive stimulus and evoke dopamine release in the nucleus accumbens core

The sensory properties of a reward-paired cue (a Conditioned Stimulus; CS) may impact the motivational value attributed to the cue, and in turn influence the form of the conditioned response (CR) that develops. A cue with multiple sensory qualities, such as a moving lever-CS, may activate numerous neural pathways that process auditory and visual information, resulting in CRs that vary both within and between individuals. For example, CRs include approach to the lever-CS itself (rats that “sign-track;” ST), approach to the location of reward delivery (rats that “goal-track;” GT), or an “intermediate” combination of these behaviors. We found that the multimodal sensory features of the lever-CS were important to the development and expression of sign-tracking. When the lever-CS was covered, and thus could only be heard moving, STs continued to approach the lever location, but also started to approach the food cup during the CS period. While still predictive of reward, the auditory component of the lever-CS was a much weaker conditioned reinforcer than the visible lever-CS. This plasticity in behavioral responding observed in STs closely resembled behaviors normally seen in rats classified as “intermediates.” Furthermore, the ability of both the lever-CS and reward-delivery to evoke dopamine release in the nucleus accumbens was also altered by covering the lever – dopamine signaling in STs resembled neurotransmission observed in rats that normally only GT. These data suggest that while the visible lever-CS was attractive, wanted, and had incentive value for STs, when presented in isolation the auditory component of the cue was simply predictive of reward, lacking incentive salience. Therefore, the specific sensory features of cues may differentially contribute to responding and ensure behavioral flexibility

Investigating protein structure by means of mass spectrometry

The three-dimensional conformation of a protein is central to its biological function. Mass spectrometry (MS) has become an important tool for the study of various aspects of protein structure. This project investigates the use of MS for diagnosis of hemoglobinopathies, through primary structure identification, and for threedimensional protein structure analysis, through comparison to established methods and application to protein systems. Travelling-wave ion mobility mass spectrometry (TWIM-MS) was used to investigate the biological significance of gas-phase protein structure. Protein standards were analysed by TWIM-MS. Cross-sections were estimated for proteins studied, for charge states most indicative of native structure, and were found to be in good agreement with those calculated from published X-ray crystallography and nuclear magnetic resonance structures. These results illustrated that the TWIM-MS approach can provide biologically-relevant data on three-dimensional protein structure. TWIM-MS was then used to study the structural properties of the hemoglobin tetramer and its components. Results showed that globin monomers exist in similar conformations whether in apo- or holo- forms and that a heme-deficient dimer is unlikely to be a prerequisite for hemoglobin tetramer assembly. TWIM-MS was used to successfully differentiate between normal and sickle hemoglobin tetramers. The conformational changes occurring in VanS, a histidine kinase, upon autophosphorylation were investigated by TWIM-MS. Results provided insights into the mechanism of autophosphorylation. MS was used to follow the rate of the autophosphorylation and results obtained compared well with those from an established method. This demonstrated that MS offers a simple, reproducible alternative to conventional methods for the study of phosphorylation rates. MS was used to provide positive identification of a range of hemoglobinopathies caused by single point mutations. A high-throughput method was used to screen for hemoglobinopathies in South Asians with and without cardiovascular disease. Results showed a positive correlation between patients with hemoglobinopathies and those with cardiovascular disease

Demonstration of radon removal from SF6 using molecular sieves

The gas SF6 has become of interest as a negative ion drift gas for use in directional dark matter searches. However, as for other targets in such searches, it is important that radon contamination can be removed as this provides a source of unwanted background events. In this work we demonstrate for the first time filtration of radon from SF6 gas by using a molecular sieve. Four types of sieves from Sigma-Aldrich were investigated, namely 3Å, 4Å, 5Å and 13X. A manufactured radon source was used for the tests. This was attached to a closed loop system in which gas was flowed through the filters and a specially adapted Durridge RAD7 radon detector. In these measurements, it was found that only the 5Å type was able to significantly reduce the radon concentration without absorbing the SF6 gas. The sieve was able to reduce the initial radon concentration of 3875 ± 13 Bqm−3 in SF6 gas by 87% when cooled with dry ice. The ability of the cooled 5Å molecular sieve filter to significantly reduce radon concentration from SF6 provides a promising foundation for the construction of a radon filtration setup for future ultra-sensitive SF6 gas rare-event physics experiments

Characterisation of large area THGEMs and experimental measurement of the Townsend coefficients for CF4

Whilst the performance of small THGEMs is well known, here we consider the challenges in scaling these up to large area charge readouts. We first verify the expected gain of larger THGEMs by reporting experimental Townsend coefficients for a 10 cm diameter THGEM in low-pressure CF$_4$. Large area 50 cm by 50 cm THGEMs were sourced from a commercial PCB supplier and geometrical imperfections were observed which we quantified using an optical camera setup. The large area THGEMs were experimentally characterised at Boulby Underground Laboratory through a series of gain calibrations and alpha spectrum measurements. ANSYS, Magboltz and Garfield++ simulations of the design of a TPC based on the large area THGEMs are presented. We also consider their implications for directional dark matter research and potential applications within nuclear security

Lowering the energy threshold in COSINE-100 dark matter searches

COSINE-100 is a dark matter detection experiment that uses NaI(Tl) crystal detectors operating at the Yangyang underground laboratory in Korea since September 2016. Its main goal is to test the annual modulation observed by the DAMA/LIBRA experiment with the same target medium. Recently DAMA/LIBRA has released data with an energy threshold lowered to 1 keV, and the persistent annual modulation behavior is still observed at 9.5$\sigma$. By lowering the energy threshold for electron recoils to 1 keV, COSINE-100 annual modulation results can be compared to those of DAMA/LIBRA in a model-independent way. Additionally, the event selection methods provide an access to a few to sub-GeV dark matter particles using constant rate studies. In this article, we discuss the COSINE-100 event selection algorithm, its validation, and efficiencies near the threshold

Measurement of directional range components of nuclear recoil tracks in a fiducialised dark matter detector

We present results from the first measurement of axial range components of fiducialized neutron induced nuclear recoil tracks using the DRIFT directional dark matter detector. Nuclear recoil events are fiducialized in the DRIFT experiment using temporal charge carrier separations between different species of anions in 30:10:1 Torr of CS$_2$:CF$_4$:O$_2$ gas mixture. For this measurement, neutron-induced nuclear recoil tracks were generated by exposing the detector to $^{252}$Cf source from different directions. Using these events, the sensitivity of the detector to the expected axial directional signatures were investigated as the neutron source was moved from one detector axis to another. Results obtained from these measurements show clear sensitivity of the DRIFT detector to the axial directional signatures in this fiducialization gas mode

Examination of ataxin-3 (atx-3) aggregation by structural mass spectrometry techniques: A rationale for expedited aggregation upon polyglutamine (polyQ) expansion

Expansion of polyglutamine stretches leads to the formation of polyglutamine-containing neuronal aggregates and neuronal death in nine diseases for which there currently are no treatments or cures. This is largely due to a lack in understanding of the mechanisms by which expanded polyglutamine regions contribute to aggregation and disease. To complicate matters further, several of the polyglutamine-disease related proteins, including ataxin-3, have a multistage aggregation mechanism in which flanking domain self-assembly precedes polyglutamine aggregation yet is influenced by polyglutamine expansion. How polyglutamine expansion influences flanking domain aggregation is poorly understood. Here, we use a combination of mass spectrometry and biophysical approaches to investigate this issue for ataxin-3. We show that the conformational dynamics of the flanking Josephin domain in ataxin-3 with an expanded polyglutamine tract are altered in comparison to those exhibited by its nonexpanded counterpart, specifically within the aggregation-prone region of the Josephin domain (amino acid residues 73-96). Expansion thus exposes this region more frequently in ataxin-3 containing an expanded polyglutamine tract, providing a molecular explanation of why aggregation is accelerated upon polyglutamine expansion. Here, harnessing the power of ion mobility spectrometry-mass spectrometry, oligomeric species formed during aggregation are characterized and a model for oligomer growth proposed. The results suggest that a conformational change occurs at the dimer level that initiates self-assembly. New insights into ataxin-3 fibril architecture are also described, revealing the region of the Josephin domain involved in protofibril formation and demonstrating that polyglutamine aggregation proceeds as a distinct second step after protofibril formation without requiring structural rearrangement of the protofibril core. Overall, the results enable the effect of polyglutamine expansion on every stage of ataxin-3 self-assembly, from monomer through to fibril, to be described and a rationale for expedited aggregation upon polyglutamine expansion to be provided