Deterministic schedules for robust and reproducible non-uniform sampling in multidimensional NMR

We show that a simple, general, and easily reproducible method for generating non-uniform sampling (NUS) schedules preserves the benefits of random sampling, including inherently reduced sampling artifacts, while removing the pitfalls associated with choosing an arbitrary seed. Sampling schedules are generated from a discrete cumulative distribution function (CDF) that closely fits the continuous CDF of the desired probability density function. We compare random and deterministic sampling using a Gaussian probability density function applied to 2D HSQC spectra. Data are processed using the previously published method of Spectroscopy by Integration of Frequency and Time domain data (SIFT). NUS spectra from deterministic sampling schedules were found to be at least as good as those from random schedules at the SIFT critical sampling density, and significantly better at half that sampling density. The method can be applied to any probability density function and generalized to greater than two dimensions.National Institutes of Health (U.S.) (Grant EB001035)National Institutes of Health (U.S.) (Grant EB003151)National Institutes of Health (U.S.) (Grant EB001960)National Institutes of Health (U.S.) (Grant EB002026

Efficient resonance assignment of proteins in MAS NMR by simultaneous intra- and inter-residue 3D correlation spectroscopy

Resonance assignment is the first step in NMR structure determination. For magic angle spinning NMR, this is typically achieved with a set of heteronuclear correlation experiments (NCaCX, NCOCX, CONCa) that utilize SPECIFIC-CP [superscript 15]N–[superscript 13]C transfers. However, the SPECIFIC-CP transfer efficiency is often compromised by molecular dynamics and probe performance. Here we show that one-bond ZF-TEDOR [superscript 15]N– [superscript 13]C transfers provide simultaneous NCO and NCa correlations with at least as much sensitivity as SPECIFIC-CP for some non-crystalline samples. Furthermore, a 3D ZF-TEDOR-CC experiment provides heteronuclear sidechain correlations and robustness with respect to proton decoupling and radiofrequency power instabilities. We demonstrate transfer efficiencies and connectivities by application of 3D ZF-TEDOR-DARR to a model microcrystalline protein, GB1, and a less ideal system, GvpA in intact gas vesicles.National Institutes of Health. National Institute for Biomedical Imaging and Bioengineering (Grant EB-001960)National Institutes of Health. National Institute for Biomedical Imaging and Bioengineering (Grant EB-002926)National Institutes of Health. National Institute for Biomedical Imaging and Bioengineering (Grant EB-001035

Selectively dispersed isotope labeling for protein structure determination by magic angle spinning NMR

The power of nuclear magnetic resonance spectroscopy derives from its site-specific access to chemical, structural and dynamic information. However, the corresponding multiplicity of interactions can be difficult to tease apart. Complimentary approaches involve spectral editing on the one hand and selective isotope substitution on the other. Here we present a new “redox” approach to the latter: acetate is chosen as the sole carbon source for the extreme oxidation numbers of its two carbons. Consistent with conventional anabolic pathways for the amino acids, [1-[superscript 13]C] acetate does not label α carbons, labels other aliphatic carbons and the aromatic carbons very selectively, and labels the carboxyl carbons heavily. The benefits of this labeling scheme are exemplified by magic angle spinning spectra of microcrystalline immunoglobulin binding protein G (GB1): the elimination of most J-couplings and one- and two-bond dipolar couplings provides narrow signals and long-range, intra- and inter-residue, recoupling essential for distance constraints. Inverse redox labeling, from [2-[superscript 13]C] acetate, is also expected to be useful: although it retains one-bond couplings in the sidechains, the removal of CA–CO coupling in the backbone should improve the resolution of NCACX spectra.National Institutes of Health. National Institute for Biomedical Imaging and Bioengineering (Grant EB-001035)National Institutes of Health. National Institute for Biomedical Imaging and Bioengineering (Grant EB-001960)National Institutes of Health. National Institute for Biomedical Imaging and Bioengineering (Grant EB-002026

Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR

The human voltage dependent anion channel 1 (VDAC) is a 32 kDa β-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms two-dimensional lipid crystals, showing remarkable spectral resolution (0.5–0.3 ppm for [superscript 13]C line widths and <0.5 ppm [superscript 15]N line widths at 750 MHz). In addition to the benefits of working in a lipid bilayer, several distinct advantages are observed with the lipid crystalline preparation. First, the strong signals and sharp line widths facilitated extensive NMR resonance assignments for an integral membrane β-barrel protein in lipid bilayers by MAS NMR. Second, a large number of residues in loop regions were readily observed and assigned, which can be challenging in detergent-solubilized membrane proteins where loop regions are often not detected due to line broadening from conformational exchange. Third, complete backbone and side chain chemical shift assignments could be obtained for the first 25 residues, which comprise the functionally important N-terminus. The reported assignments allow us to compare predicted torsion angles for VDAC prepared in DMPC 2D lipid crystals, DMPC liposomes, and LDAO-solubilized samples to address the possible effects of the membrane mimetic environment on the conformation of the protein. Concluding, we discuss the strengths and weaknesses of the reported assignment approach and the great potential for even more complete assignment studies and de novo structure determination via [superscript 1]H detected MAS NMR.National Institutes of Health (U.S.) (Grant EB001960)National Institutes of Health (U.S.) (Grant EB002026

Magic Angle Spinning Nuclear Magnetic Resonance Characterization of Voltage-Dependent Anion Channel Gating in Two-Dimensional Lipid Crystalline Bilayers

National Institutes of Health (U.S.) (EB001960)National Institutes of Health (EB002026

Intermolecular Structure Determination of Amyloid Fibrils with 2 Magic-Angle Spinning and Dynamic Nuclear Polarization NMR

We describe magic-angle spinning NMR experiments designed to elucidate the interstrand architecture of amyloid fibrils. Three methods are introduced for this purpose, two being based on the analysis of long-range [superscript 13]C–[superscript 13]C correlation spectra and the third based on the identification of intermolecular interactions in [superscript 13]C–[superscript 15]N spectra. We show, in studies of fibrils formed by the 86-residue SH3 domain of PI3 kinase (PI3-SH3 or PI3K-SH3), that efficient [superscript 13]C–[superscript 13]C correlation spectra display a resonance degeneracy that establishes a parallel, in-register alignment of the proteins in the amyloid fibrils. In addition, this degeneracy can be circumvented to yield direct intermolecular constraints. The [superscript 13]C–[superscript 13]C experiments are corroborated by [superscript 15]N–[superscript 13]C correlation spectra obtained from a mixed [[superscript 15]N,[superscript 12]C]/[[superscript 14]N,[superscript 13]C] sample which directly quantify interstrand distances. Furthermore, when the spectra are recorded with signal enhancement provided by dynamic nuclear polarization (DNP) at 100 K, we demonstrate a dramatic increase (from 23 to 52) in the number of intermolecular [superscript 15]N–[superscript 13]C constraints detectable in the spectra. The increase in the information content is due to the enhanced signal intensities and to the fact that dynamic processes, leading to spectral intensity losses, are quenched at low temperatures. Thus, acquisition of low temperature spectra addresses a problem that is frequently encountered in MAS spectra of proteins. In total, the experiments provide 111 intermolecular [superscript 13]C–[superscript 13]C and [superscript 15]N–[superscript 13]C constraints that establish that the PI3-SH3 protein strands are aligned in a parallel, in-register arrangement within the amyloid fibril.National Institutes of Health (U.S.) (Grant EB-003151)National Institutes of Health (U.S.) (Grant EB-002804)National Institutes of Health (U.S.) (Grant EB-002026

Microchannel cooling for the LHCb VELO Upgrade I

The LHCb VELO Upgrade I, currently being installed for the 2022 start of LHC Run 3, uses silicon microchannel coolers with internally circulating bi-phase \cotwo for thermal control of hybrid pixel modules operating in vacuum. This is the largest scale application of this technology to date. Production of the microchannel coolers was completed in July 2019 and the assembly into cooling structures was completed in September 2021. This paper describes the R\&D path supporting the microchannel production and assembly and the motivation for the design choices. The microchannel coolers have excellent thermal peformance, low and uniform mass, no thermal expansion mismatch with the ASICs and are radiation hard. The fluidic and thermal performance is presented.Comment: 31 pages, 27 figure

The Genetic Links to anxiety and depression (GLAD) study: Online recruitment into the largest recontactable study of depression and anxiety

Background: Anxiety and depression are common, debilitating and costly. These disorders are influenced by multiple risk factors, from genes to psychological vulnerabilities and environmental stressors, but research is hampered by a lack of sufficiently large comprehensive studies. We are recruiting 40,000 individuals with lifetime depression or anxiety and broad assessment of risks to facilitate future research. Methods: The Genetic Links to Anxiety and Depression (GLAD) Study (www.gladstudy.org.uk) recruits individuals with depression or anxiety into the NIHR Mental Health BioResource. Participants invited to join the study (via media campaigns) provide demographic, environmental and genetic data, and consent for medical record linkage and recontact. Results: Online recruitment was effective; 42,531 participants consented and 27,776 completed the questionnaire by end of July 2019. Participants’ questionnaire data identified very high rates of recurrent depression, severe anxiety, and comorbidity. Participants reported high rates of treatment receipt. The age profile of the sample is biased toward young adults, with higher recruitment of females and the more educated, especially at younger ages. Discussion: This paper describes the study methodology and descriptive data for GLAD, which represents a large, recontactable resource that will enable future research into risks, outcomes, and treatment for anxiety and depression

Evaluating the performance of land surface model ORCHIDEE-CAN v1.0 on water and energy flux estimation with a single- and multi-layer energy budget scheme

Canopy structure is one of the most important vegetation characteristics for land-atmosphere interactions, as it determines the energy and scalar exchanges between the land surface and the overlying air mass. In this study we evaluated the performance of a newly developed multilayer energy budget in the ORCHIDEE-CAN v1.0 land surface model (Organising Carbon and Hydrology In Dynamic Ecosystems - CANopy), which simulates canopy structure and can be coupled to an atmospheric model using an implicit coupling procedure. We aim to provide a set of accept-able parameter values for a range of forest types. Top-canopy and sub-canopy flux observations from eight sites were collected in order to conduct this evaluation. The sites crossed climate zones from temperate to boreal and the vegetation types included deciduous, evergreen broad-leaved and evergreen needle-leaved forest with a maximum leaf area index (LAI; all-sided) ranging from 3.5 to 7.0. The parametrization approach proposed in this study was based on three selected physical processes - namely the diffusion, advection, and turbulent mixing within the canopy. Short-term sub-canopy observations and long-term surface fluxes were used to calibrate the parameters in the sub-canopy radiation, turbulence, and resistance modules with an automatic tuning process. The multi-layer model was found to capture the dynamics of sub-canopy turbulence, temperature, and energy fluxes. The performance of the new multi-layer model was further compared against the existing single-layer model. Although the multi-layer model simulation results showed few or no improvements to both the nighttime energy balance and energy partitioning during winter compared with a single-layer model simulation, the increased model complexity does provide a more detailed description of the canopy micrometeorology of various forest types. The multi-layer model links to potential future environmental and ecological studies such as the assessment of in-canopy species vulnerability to climate change, the climate effects of disturbance intensities and frequencies, and the consequences of biogenic volatile organic compound (BVOC) emissions from the terrestrial ecosystem.Peer reviewe

The Maunder minimum (1645-1715) was indeed a grand minimum: a reassessment of multiple datasets

Aims. Although the time of the Maunder minimum (1645–1715) is widely known as a period of extremely low solar activity, it is still being debated whether solar activity during that period might have been moderate or even higher than the current solar cycle (number 24). We have revisited all existing evidence and datasets, both direct and indirect, to assess the level of solar activity during the Maunder minimum. Methods. We discuss the East Asian naked-eye sunspot observations, the telescopic solar observations, the fraction of sunspot active days, the latitudinal extent of sunspot positions, auroral sightings at high latitudes, cosmogenic radionuclide data as well as solar eclipse observations for that period. We also consider peculiar features of the Sun (very strong hemispheric asymmetry of the sunspot location, unusual differential rotation and the lack of the K-corona) that imply a special mode of solar activity during the Maunder minimum. Results. The level of solar activity during the Maunder minimum is reassessed on the basis of all available datasets. Conclusions. We conclude that solar activity was indeed at an exceptionally low level during the Maunder minimum. Although the exact level is still unclear, it was definitely lower than during the Dalton minimum of around 1800 and significantly below that of the current solar cycle #24. Claims of a moderate-to-high level of solar activity during the Maunder minimum are rejected with a high confidence level