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 4–7Å 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.
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•The first sub nm single particle reconstruction of a membrane protein using a SMALP scaffold•The ability to maintain a native like environment for structural studies holds great promise.•The resulting map is consistent with high resolution crystal structures and other EM derived maps.
Structure of the shutdown state of myosin-2 Scarff, Charlotte A; Carrington, Glenn; Casas-Mao, David ...
Nature (London),
12/2020, Letnik:
588, Številka:
7838
Journal Article
Recenzirano
Odprti dostop
Myosin-2 is essential for processes as diverse as cell division and muscle contraction. Dephosphorylation of its regulatory light chain promotes an inactive, 'shutdown' state with the ...filament-forming tail folded onto the two heads
, which prevents filament formation and inactivates the motors
. The mechanism by which this happens is unclear. Here we report a cryo-electron microscopy structure of shutdown smooth muscle myosin with a resolution of 6 Å in the head region. A pseudo-atomic model, obtained by flexible fitting of crystal structures into the density and molecular dynamics simulations, describes interaction interfaces at the atomic level. The N-terminal extension of one regulatory light chain interacts with the tail, and the other with the partner head, revealing how the regulatory light chains stabilize the shutdown state in different ways and how their phosphorylation would allow myosin activation. Additional interactions between the three segments of the coiled coil, the motor domains and the light chains stabilize the shutdown molecule. The structure of the lever in each head is competent to generate force upon activation. This shutdown structure is relevant to all isoforms of myosin-2 and provides a framework for understanding their disease-causing mutations.
Cryo‐electron microscopy (cryo‐EM) can now be used to determine high‐resolution structural information on a diverse range of biological specimens. Recent advances have been driven primarily by ...developments in microscopes and detectors, and through advances in image‐processing software. However, for many single‐particle cryo‐EM projects, major bottlenecks currently remain at the sample‐preparation stage; obtaining cryo‐EM grids of sufficient quality for high‐resolution single‐particle analysis can require the careful optimization of many variables. Common hurdles to overcome include problems associated with the sample itself (buffer components, labile complexes), sample distribution (obtaining the correct concentration, affinity for the support film), preferred orientation, and poor reproducibility of the grid‐making process within and between batches. This review outlines a number of methodologies used within the electron‐microscopy community to address these challenges, providing a range of approaches which may aid in obtaining optimal grids for high‐resolution data collection.
This paper describes different approaches that cryo‐EM users can take to improve the quality of their sample distribution and ice for high‐resolution single‐particle cryo‐EM.
Glycosylation is one of the most common post-translational modifications occurring in proteins. A detailed structural characterization of the involved carbohydrates, however, is still one of the ...greatest challenges in modern glycoproteomics, since multiple regio- and stereoisomers with an identical monosaccharide composition may exist. Recently, ion mobility-mass spectrometry (IM-MS), a technique in which ions are separated according to their mass, charge, and shape, has evolved as a promising technique for the separation and structural analysis of complex carbohydrates. This growing interest is based on the fact that the measured drift times can be converted into collision cross sections (CCSs), which can be compared, implemented into databases, and used as additional search criteria for structural identification. However, most of the currently used commercial IM-MS instruments utilize a nonuniform traveling wave field to propel the ions through the IM cell. As a result, CCS measurements cannot be performed directly and require calibration. Here, we present a calibration data set consisting of over 500 reference CCSs for negatively charged N-glycans and their fragments. Moreover, we show that dextran, already widely used as a calibrant in high performance liquid chromatography, is also a suitable calibrant for CCS estimations. Our data also indicate that a considerably increased error has to be taken into account when reference CCSs acquired in a different drift gas are used for calibration.
Initial performance of the COSINE-100 experiment Adhikari, G.; Adhikari, P.; de Souza, E. Barbosa ...
The European physical journal. C, Particles and fields,
02/2018, Letnik:
78, Številka:
2
Journal Article
Recenzirano
Odprti dostop
COSINE is a dark matter search experiment based on an array of low background NaI(Tl) crystals located at the Yangyang underground laboratory. The assembly of COSINE-100 was completed in the summer ...of 2016 and the detector is currently collecting physics quality data aimed at reproducing the DAMA/LIBRA experiment that reported an annual modulation signal. Stable operation has been achieved and will continue for at least 2 years. Here, we describe the design of COSINE-100, including the shielding arrangement, the configuration of the NaI(Tl) crystal detection elements, the veto systems, and the associated operational systems, and we show the current performance of the experiment.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Structural biology generally provides static snapshots of protein conformations that can provide information on the functional mechanisms of biological systems. Time-resolved structural biology ...provides a means to visualize, at near-atomic resolution, the dynamic conformational changes that macromolecules undergo as they function. X-ray free-electron-laser technology has provided a powerful tool to study enzyme mechanisms at atomic resolution, typically in the femtosecond to picosecond timeframe. Complementary to this, recent advances in the resolution obtainable by electron microscopy and the broad range of samples that can be studied make it ideally suited to time-resolved approaches in the microsecond to millisecond timeframe to study large loop and domain motions in biomolecules. Here we describe a cryo-EM grid preparation device that permits rapid mixing, voltage-assisted spraying and vitrification of samples. It is shown that the device produces grids of sufficient ice quality to enable data collection from single grids that results in a sub-4 Å reconstruction. Rapid mixing can be achieved by blot-and-spray or mix-and-spray approaches with a delay of ∼10 ms, providing greater temporal resolution than previously reported mix-and-spray approaches.
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.
We present results from a 54.7 live-day shielded run of the DRIFT-IId detector, the world's most sensitive, directional, dark matter detector. Several improvements were made relative to our previous ...work including a lower threshold for detection, a more robust analysis and a tenfold improvement in our gamma rejection factor. After analysis, no events remain in our fiducial region leading to an exclusion curve for spin-dependent WIMP-proton interactions which reaches 0.28pb at 100GeV/c2, a fourfold improvement on our previous work. We also present results from a 45.4 live-day unshielded run of the DRIFT-IId detector during which 14 nuclear recoil-like events were observed. We demonstrate that the observed nuclear recoil rate of 0.31 ± 0.08 events per day is consistent with detection of ambient, fast neutrons emanating from the walls of the Boulby Underground Science Facility.
The Negative Ion Drift (NID) gas SF6 has favourable properties for track reconstruction in directional Dark Matter (DM) searches utilising low pressure gaseous Time Projection Chambers (TPCs). ...However, the electronegative nature of the gas means that it is more difficult to achieve significant gas gains with regular Thick Gaseous Electron Multipliers (ThGEMs). Typically, the maximum attainable gas gain in SF6 and other Negative Ion (NI) gas mixtures, previously achieved with an 55Fe X-ray source or electron beam, is on the order of 103 1,2,3,4; whereas electron drift gases like CF4 and similar mixtures are readily capable of reaching gas gains on the order of 104 or greater 5,9,7,8,6. In this paper, a novel two stage Multi-Mesh ThGEM (MMThGEM) structure is presented. The MMThGEM was used to amplify charge liberated by an 55Fe X-ray source in 40 Torr of SF6. By expanding on previously demonstrated results 10, the device was pushed to its sparking limit and stable gas gains up to ˜50000 were observed. The device was further optimised by varying the field strengths of both the collection and transfer regions in isolation. Following this optimisation procedure, the device was able to produce a maximum stable gas gain of ˜90000. These results demonstrate an order of magnitude improvement in gain with the NID gas over previously reported values and ultimately benefits the sensitivity of a NITPC to low energy recoils in the context of a directional DM search.
Abstract The PocketWATCH facility is a unique multi-purpose test bed designed to replicate the conditions of large water Cherenkov detectors. Housed at the University of Sheffield, the facility ...consists of a light-tight 2000 L ultrapure water tank with purification and temperature control systems. Water temperature, resistivity, and UV attenuation in the tank are monitored and shown to be stable over time. The system is also shown to be compatible with a solution of 0.2% gadolinium sulfate, allowing further utility in testing equipment bound for the next generation neutrino and nucleon decay water Cherenkov particle detectors. The relevant water quality parameters are shown to be stable whilst running in Gd-mode, thereby providing a suitable test bed for hardware development in a realistic, ex situ environment.
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