Protein structure determination by proton-detected magic-angle spinning (MAS) NMR has focused on highly deuterated samples, in which only a small number of protons are introduced and observation of ...signals from side chains is extremely limited. Here, we show in two fully protonated proteins that, at 100-kHz MAS and above, spectral resolution is high enough to detect resolved correlations from amide and side-chain protons of all residue types, and to reliably measure a dense network of ¹H-¹H proximities that define a protein structure. The high data quality allowed the correct identification of internuclear distance restraints encoded in 3D spectra with automated data analysis, resulting in accurate, unbiased, and fast structure determination. Additionally, we find that narrower proton resonance lines, longer coherence lifetimes, and improved magnetization transfer offset the reduced sample size at 100-kHz spinning and above. Less than 2 weeks of experiment time and a single 0.5-mg sample was sufficient for the acquisition of all data necessary for backbone and side-chain resonance assignment and unsupervised structure determination. We expect the technique to pave the way for atomic-resolution structure analysis applicable to a wide range of proteins.
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Around half of all protein structures solved nowadays using solution-state nuclear magnetic resonance (NMR) spectroscopy have been because of automated data analysis. The pervasiveness of ...computational approaches in general hides, however, a more nuanced view in which the full variety and richness of the field appears. This review is structured around a comparison of methods associated with three NMR observables: classical nuclear Overhauser effect (NOE) constraint gathering in contrast with more recent chemical shift and residual dipole coupling (RDC) based protocols. In each case, the emphasis is placed on the latest research, covering mainly the past 5 years. By describing both general concepts and representative programs, the objective is to map out a field in which – through the very profusion of approaches – it is all too easy to lose one's bearings.
Among bacterial toxin-antitoxin systems, to date no antitoxin has been identified that functions by cleaving toxin mRNA. Here we show that YjdO (renamed GhoT) is a membrane lytic peptide that causes ...ghost cell formation (lysed cells with damaged membranes) and increases persistence (persister cells are tolerant to antibiotics without undergoing genetic change). GhoT is part of a new toxin-antitoxin system with YjdK (renamed GhoS) because in vitro RNA degradation studies, quantitative real-time reverse-transcription PCR and whole-transcriptome studies revealed that GhoS masks GhoT toxicity by cleaving specifically yjdO (ghoT) mRNA. Alanine substitutions showed that Arg28 is important for GhoS activity, and RNA sequencing indicated that the GhoS cleavage site is rich in U and A. The NMR structure of GhoS indicates it is related to the CRISPR-associated-2 RNase, and GhoS is a monomer. Hence, GhoT-GhoS is to our knowledge the first type V toxin-antitoxin system where a protein antitoxin inhibits the toxin by cleaving specifically its mRNA.
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MATCH (Memetic Algorithm and Combinatorial Optimization Heuristics) is a new memetic algorithm for automated sequence-specific polypeptide backbone NMR assignment of proteins. MATCH employs local ...optimization for tracing partial sequence-specific assignments within a global, population-based search environment, where the simultaneous application of local and global optimization heuristics guarantees high efficiency and robustness. MATCH thus makes combined use of the two predominant concepts in use for automated NMR assignment of proteins. Dynamic transition and inherent mutation are new techniques that enable automatic adaptation to variable quality of the experimental input data. The concept of dynamic transition is incorporated in all major building blocks of the algorithm, where it enables switching between local and global optimization heuristics at any time during the assignment process. Inherent mutation restricts the intrinsically required randomness of the evolutionary algorithm to those regions of the conformation space that are compatible with the experimental input data. Using intact and artificially deteriorated APSY-NMR input data of proteins, MATCH performed sequence-specific resonance assignment with high efficiency and robustness.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Re‐protonation is key: A combination of a high magnetic field (1 GHz) and ultra‐fast magic‐angle spinning (60 kHz) allows easy detection of NMR spectra revealing details of secondary and tertiary ...structures of medium‐sized proteins. The technique was applied to the 153‐residue microcrystalline ZnII‐loaded superoxide dismutase (ZnII‐SOD) fully 2H,13C,15N‐labeled and 100 % re‐protonated at the exchangeable sites.
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As methods for analysis of biomolecular structure and dynamics using nuclear magnetic resonance spectroscopy (NMR) continue to advance, the resulting 3D structures, chemical shifts, and other NMR ...data are broadly impacting biology, chemistry, and medicine. Structure model assessment is a critical area of NMR methods development, and is an essential component of the process of making these structures accessible and useful to the wider scientific community. For these reasons, the Worldwide Protein Data Bank (wwPDB) has convened an NMR Validation Task Force (NMR-VTF) to work with wwPDB partners in developing metrics and policies for biomolecular NMR data harvesting, structure representation, and structure quality assessment. This paper summarizes the recommendations of the NMR-VTF, and lays the groundwork for future work in developing standards and metrics for biomolecular NMR structure quality assessment.
•Well-developed methods can form a basis for standardized NMR structure assessment•Tools for validation of X-ray crystal structures are appropriate for NMR structures•NMR structure validation is generally applicable only to “well-defined” regions•Further research is required to address key issues of NMR structure validation
As analyses of biomolecular structure and dynamics by nuclear magnetic resonance (NMR) spectroscopy continue to advance, the resulting structures have a broad impact. Montelione et al. summarize recommendations for developing metrics and policies for biomolecular NMR structure representation and validation.
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The J-UNIO (JCSG protocol using the software UNIO) procedure for automated protein structure determination by NMR in solution is introduced. In the present implementation, J-UNIO makes use of ...APSY-NMR spectroscopy, 3D heteronuclear-resolved
1
H,
1
H-NOESY experiments, and the software UNIO. Applications with proteins from the JCSG target list with sizes up to 150 residues showed that the procedure is highly robust and efficient. In all instances the correct polypeptide fold was obtained in the first round of automated data analysis and structure calculation. After interactive validation of the data obtained from the automated routine, the quality of the final structures was comparable to results from interactive structure determination. Special advantages are that the NMR data have been recorded with 6–10 days of instrument time per protein, that there is only a single step of chemical shift adjustments to relate the backbone signals in the APSY-NMR spectra with the corresponding backbone signals in the NOESY spectra, and that the NOE-based amino acid side chain chemical shift assignments are automatically focused on those residues that are heavily weighted in the structure calculation. The individual working steps of J-UNIO are illustrated with the structure determination of the protein YP_926445.1 from
Shewanella amazonensis
, and the results obtained with 17 JCSG targets are critically evaluated.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Pseudocontact shifts (PCSs) arise in paramagnetic systems in which the susceptibility tensor is anisotropic. PCSs depend upon the distance from the paramagnetic center and the position relative to ...the susceptibility tensor, and they can be used as structural restraints in protein structure determination. We show that the use of 1H-detected solid-state correlations provides facile and rapid detection and assignment of site-specific PCSs, including resolved 1H PCSs, in a large metalloprotein, Co2+-substituted superoxide dismutase (Co2+-SOD). With only 3 mg of sample and a small set of experiments, several hundred PCSs were measured and assigned, and these PCSs were subsequently used in combination with 1H–1H distance and dihedral angle restraints to determine the protein backbone geometry with a precision paralleling those of state-of-the-art liquid-state determinations of diamagnetic proteins, including a well-defined active site.
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