Sampling of the NMR time domain along concentric rings

Abstract We present a novel approach to sampling the NMR time domain, whereby the sampling points are aligned on concentric rings, which we term concentric ring sampling (CRS). Radial sampling constitutes a special case of CRS where each ring has the same number of points and the same relative orientation. We derive theoretically that the most efficient CRS approach is to place progressively more points on rings of larger radius, with the number of points growing linearly with the radius, a method that we call linearly increasing CRS (LCRS). For cases of significant undersampling to reduce measurement time, a randomized LCRS (RLCRS) is also described. A theoretical treatment of these approaches is provided, including an assessment of artifacts and sensitivity. The analytical treatment of sensitivity also addresses the sensitivity of radially sampled data processed by Fourier transform. Optimized CRS approaches are found to produce artifact-free spectra of the same resolution as Cartesian sampling, for the same measurement time. Additionally, optimized approaches consistently yield fewer and smaller artifacts than radial sampling, and have a sensitivity equal to Cartesian and better than radial sampling. We demonstrate the method using numerical simulations, as well as a 3D HNCO experiment on protein G B1 domain

mAb therapy controls CNS-resident lyssavirus infection via a CD4 T cell-dependent mechanism

DATA AVAILABILITY : This study includes no data deposited in external repositories.Infections with rabies virus (RABV) and related lyssaviruses are uniformly fatal once virus accesses the central nervous system (CNS) and causes disease signs. Current immunotherapies are thus focused on the early, pre-symptomatic stage of disease, with the goal of peripheral neutralization of virus to prevent CNS infection. Here, we evaluated the therapeutic efficacy of F11, an antilyssavirus human monoclonal antibody (mAb), on established lyssavirus infections. We show that a single dose of F11 limits viral load in the brain and reverses disease signs following infection with a lethal dose of lyssavirus, even when administered after initiation of robust virus replication in the CNS. Importantly, we found that F11-dependent neutralization is not sufficient to protect animals from mortality, and a CD4 T cell-dependent adaptive immune response is required for successful control of infection. F11 significantly changes the spectrum of leukocyte populations in the brain, and the FcRc-binding function of F11 contributes to therapeutic efficacy. Thus, mAb therapy can drive potent neutralization-independent T cell-mediated effects, even against an established CNS infection by a lethal neurotropic virus.https://www.embopress.org/journal/17574684am2024Medical VirologySDG-03:Good heatlh and well-bein

Development of new approaches to NMR data collection for protein structure determination

Multidimensional nuclear magnetic resonance (NMR) spectroscopy has become one of the most important techniques available for studying the structure and function of biological macromolecules at atomic resolution. The conventional approach to multidimensional NMR involves the sampling of the time domain on a Cartesian grid followed by a multidimensional Fourier transform (FT). While this approach yields high quality spectra, as the number of dimensions is increased the time needed for sampling on a Cartesian grid increases exponentially, making it impractical to record 4-D spectra at high resolution and impossible to record 5-D spectra at all. This thesis describes new approaches to data collection and processing that make it possible to obtain spectra at higher resolution and/or with a higher dimensionality than was previously feasible with the conventional method. The central focus of this work has been the sampling of the time domain along radial spokes, which was recently introduced into the NMR community. If each radial spoke is processed by an FT with respect to radius, a set of projections of the higher-dimensional spectrum are obtained. Full spectra at high resolution can be generated from these projections via tomographic reconstruction. We generalized the lower-value reconstruction algorithm from the literature, and later integrated it with the backprojection algorithm in a hybrid reconstruction method. These methods permit the reconstruction of accurate 4-D and 5- D spectra at very high resolution, from only a small number of projections, as we demonstrated in the reconstruction of 4-D and 5-D sequential assignment spectra on small and large proteins. For nuclear Overhauser spectroscopy (NOESY), used to measure interproton distances in proteins, one requires quantitative reconstructions. We have successfully obtained these using filtered backprojection, which we found was equivalent to processing the radially sampled data by a polar FT. All of these methods represent significant gains in data collection efficiency over conventional approaches. The polar FT interpretation suggested that the problem could be analyzed using FT theory, to design even more efficient methods. We have developed a new approach to sampling, using concentric rings of sampling points, which represents a further improvement in efficiency and sensitivity over radial sampling.Dissertatio

Rapid Protein Global Fold Determination Using Ultrasparse Sampling, High-Dynamic Range Artifact Suppression, and Time-Shared NOESY

In structural studies of large proteins by NMR, global fold determination plays an increasingly important role in providing a first look at a target’s topology and reducing assignment ambiguity in NOESY spectra of fully protonated samples. In this work, we demonstrate the use of ultrasparse sampling, a new data processing algorithm, and a 4-D time-shared NOESY experiment (1) to collect all NOEs in ²H/¹³C/¹⁵N-labeled protein samples with selectively protonated amide and ILV methyl groups at high resolution in only four days, and (2) to calculate global folds from this data using fully automated resonance assignment. The new algorithm, SCRUB, incorporates the CLEAN method for iterative artifact removal but applies an additional level of iteration, permitting real signals to be distinguished from noise and allowing nearly all artifacts generated by real signals to be eliminated. In simulations with 1.2% of the data required by Nyquist sampling, SCRUB achieves a dynamic range over 10000:1 (250× better artifact suppression than CLEAN) and completely quantitative reproduction of signal intensities, volumes, and line shapes. Applied to 4-D time-shared NOESY data, SCRUB processing dramatically reduces aliasing noise from strong diagonal signals, enabling the identification of weak NOE crosspeaks with intensities 100× less than those of diagonal signals. Nearly all of the expected peaks for interproton distances under 5 Å were observed. The practical benefit of this method is demonstrated with structure calculations for 23 kDa and 29 kDa test proteins using the automated assignment protocol of CYANA, in which unassigned 4-D time-shared NOESY peak lists produce accurate and well-converged global fold ensembles, whereas 3-D peak lists either fail to converge or produce significantly less accurate folds. The approach presented here succeeds with an order of magnitude less sampling than required by alternative methods for processing sparse 4-D data

Rapid Protein Global Fold Determination Using Ultrasparse Sampling, High-Dynamic Range Artifact Suppression, and Time-Shared NOESY

In structural studies of large proteins by NMR, global fold determination plays an increasingly important role in providing a first look at a target’s topology and reducing assignment ambiguity in NOESY spectra of fully protonated samples. In this work, we demonstrate the use of ultrasparse sampling, a new data processing algorithm, and a 4-D time-shared NOESY experiment (1) to collect all NOEs in ²H/¹³C/¹⁵N-labeled protein samples with selectively protonated amide and ILV methyl groups at high resolution in only four days, and (2) to calculate global folds from this data using fully automated resonance assignment. The new algorithm, SCRUB, incorporates the CLEAN method for iterative artifact removal but applies an additional level of iteration, permitting real signals to be distinguished from noise and allowing nearly all artifacts generated by real signals to be eliminated. In simulations with 1.2% of the data required by Nyquist sampling, SCRUB achieves a dynamic range over 10000:1 (250× better artifact suppression than CLEAN) and completely quantitative reproduction of signal intensities, volumes, and line shapes. Applied to 4-D time-shared NOESY data, SCRUB processing dramatically reduces aliasing noise from strong diagonal signals, enabling the identification of weak NOE crosspeaks with intensities 100× less than those of diagonal signals. Nearly all of the expected peaks for interproton distances under 5 Å were observed. The practical benefit of this method is demonstrated with structure calculations for 23 kDa and 29 kDa test proteins using the automated assignment protocol of CYANA, in which unassigned 4-D time-shared NOESY peak lists produce accurate and well-converged global fold ensembles, whereas 3-D peak lists either fail to converge or produce significantly less accurate folds. The approach presented here succeeds with an order of magnitude less sampling than required by alternative methods for processing sparse 4-D data

Rapid Protein Global Fold Determination Using Ultrasparse Sampling, High-Dynamic Range Artifact Suppression, and Time-Shared NOESY

In structural studies of large proteins by NMR, global fold determination plays an increasingly important role in providing a first look at a target’s topology and reducing assignment ambiguity in NOESY spectra of fully protonated samples. In this work, we demonstrate the use of ultrasparse sampling, a new data processing algorithm, and a 4-D time-shared NOESY experiment (1) to collect all NOEs in ²H/¹³C/¹⁵N-labeled protein samples with selectively protonated amide and ILV methyl groups at high resolution in only four days, and (2) to calculate global folds from this data using fully automated resonance assignment. The new algorithm, SCRUB, incorporates the CLEAN method for iterative artifact removal but applies an additional level of iteration, permitting real signals to be distinguished from noise and allowing nearly all artifacts generated by real signals to be eliminated. In simulations with 1.2% of the data required by Nyquist sampling, SCRUB achieves a dynamic range over 10000:1 (250× better artifact suppression than CLEAN) and completely quantitative reproduction of signal intensities, volumes, and line shapes. Applied to 4-D time-shared NOESY data, SCRUB processing dramatically reduces aliasing noise from strong diagonal signals, enabling the identification of weak NOE crosspeaks with intensities 100× less than those of diagonal signals. Nearly all of the expected peaks for interproton distances under 5 Å were observed. The practical benefit of this method is demonstrated with structure calculations for 23 kDa and 29 kDa test proteins using the automated assignment protocol of CYANA, in which unassigned 4-D time-shared NOESY peak lists produce accurate and well-converged global fold ensembles, whereas 3-D peak lists either fail to converge or produce significantly less accurate folds. The approach presented here succeeds with an order of magnitude less sampling than required by alternative methods for processing sparse 4-D data