Blind Testing of Routine, Fully Automated Determination of Protein Structures from NMR Data

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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 1 H-1 H 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.NMR spectroscopy | magic-angle spinning | protein structures | proton detection | viral nucleocapsids D espite tremendous progress in the analysis of biomolecular samples over the last two decades (1-7), routine application of magic-angle spinning (MAS) NMR in biology is still limited by the inherently low sensitivity. The direct detection of proton resonances is a straightforward way to counter this problem, but entails a trade-off with resolution due to the strong homonuclear dipolar interactions among proton nuclei. High-resolution proton-detected methods were first demonstrated with modest spinning frequencies by today's standards (∼10 kHz) and relied on a reduction of 1 H-1 H couplings by high levels of dilution with deuterium, typically perdeuteration, and complete (8, 9) or partial (10-12) protonation at exchangeable sites. The need for narrow proton resonances without such extreme levels of deuteration has motivated a continuous technological development, resulting in a dramatic increase in the available spinning frequency (13)(14)(15)(16)(17)(18)(19)(20).At MAS frequencies of 40-60 kHz, deuteration and 100% reprotonation at exchangeable sites, primarily amide protons, result in resolved and sensitive spectra, similar in quality to the case of higher dilution levels and lower spinning frequencies (21-23). This opens the way to rapid sequential assignment of backbone resonances (24-27), as well as to the unambiguous measurement of detailed structural and dynamical parameters (28-32). A further increase in the MAS frequency to 100 kHz allows resonance assignment (20), a structure determination of a model protein (16), and interaction studies (15) with as little as 0.5 mg of sample. However, a high deuteration level severely limits observation of side-chain s...

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confidence: 99%

Structure of fully protonated proteins by proton-detected magic-angle spinning NMR

Andreas

Jaudzems

Staněk

et al. 2016

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“…This information has been used to characterize protein secondary structures (17,18) and to determine the allowed ranges of dihedral angles (19). More recently it has been demonstrated that by encoding the chemical shift information into molecular fragments it is possible to accurately determine the structures of smallto medium-sized globular proteins (14)(15)(16)(20)(21)(22)(23).…”

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confidence: 99%

“…A further problem is that the discrete fragment libraries may not necessarily cover the entire relevant space of protein conformations. Although fragment replacement methods are thus efficient in finding a structure compatible with the available data, it is not straightforward to demonstrate that such a structure actually represents the native state, even if, for practical purposes, it does so in most cases (22). Likewise, it is difficult to use fragment-based approaches to generate alternative conformers that represent the less densely populated regions of the native free energy landscape.…”

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confidence: 99%

Equilibrium simulations of proteins using molecular fragment replacement and NMR chemical shifts

Boomsma

Tian

Frellsen

et al. 2014

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Methods of protein structure determination based on NMR chemical shifts are becoming increasingly common. The most widely used approaches adopt the molecular fragment replacement strategy, in which structural fragments are repeatedly reassembled into different complete conformations in molecular simulations. Although these approaches are effective in generating individual structures consistent with the chemical shift data, they do not enable the sampling of the conformational space of proteins with correct statistical weights. Here, we present a method of molecular fragment replacement that makes it possible to perform equilibrium simulations of proteins, and hence to determine their free energy landscapes. This strategy is based on the encoding of the chemical shift information in a probabilistic model in Markov chain Monte Carlo simulations. First, we demonstrate that with this approach it is possible to fold proteins to their native states starting from extended structures. Second, we show that the method satisfies the detailed balance condition and hence it can be used to carry out an equilibrium sampling from the Boltzmann distribution corresponding to the force field used in the simulations. Third, by comparing the results of simulations carried out with and without chemical shift restraints we describe quantitatively the effects that these restraints have on the free energy landscapes of proteins. Taken together, these results demonstrate that the molecular fragment replacement strategy can be used in combination with chemical shift information to characterize not only the native structures of proteins but also their conformational fluctuations.

“…Despite this, the use of chemical shifts for determination and refinement of protein structures (14)(15)(16)(17) is not the aim of this work, although a simple refinement routine is used here only as a tool to assess the reliability of a proposed methodology to repair flaws in proteins.…”

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confidence: 99%

Physics-based method to validate and repair flaws in protein structures

Martin

Arnautova

Icazatti

et al. 2013

A method that makes use of information provided by the combination of 13 C α and 13 C β chemical shifts, computed at the density functional level of theory, enables one to (i) validate, at the residue level, conformations of proteins and detect backbone or side-chain flaws by taking into account an ensemble average of chemical shifts over all of the conformations used to represent a protein, with a sensitivity of ∼90%; and (ii) provide a set of (χ1/χ2) torsional angles that leads to optimal agreement between the observed and computed 13 C α and 13 C β chemical shifts. The method has been incorporated into the CheShift-2 protein validation Web server. To test the reliability of the provided set of (χ1/ χ2) torsional angles, the side chains of all reported conformations of five NMR-determined protein models were refined by a simple routine, without using NOE-based distance restraints. The refinement of each of these five proteins leads to optimal agreement between the observed and computed 13 C α and 13 C β chemical shifts for ∼94% of the flaws, on average, without introducing a significantly large number of violations of the NOE-based distance restraints for a distance range ≤ 0.5 Å, in which the largest number of distance violations occurs. The results of this work suggest that use of the provided set of (χ1/χ2) torsional angles together with other observables, such as NOEs, should lead to a fast and accurate refinement of the side-chain conformations of protein models.S ince the seminal observation by Kendrew (1) that "it is the spatial relations between the side-chains which determine the chemical behavior and biological specificity of the protein molecule as a whole and these relations cannot be determined, except in a fragmentary manner, by purely chemical techniques" interest has been focused on the development of accurate methods to validate and determine side-chain conformations in proteins (ref. 2 and references therein). Side-chain chemical shifts have also been used for protein structure validation because these observables are highly sensitive to protein structural packing (3). The latter interest arises because it is largely accepted that a proper protein structure determination requires validation methods in which the observable values used to validate the structures are not used in their determination (4, 5), and it will assure spectroscopists and other users that a given protein model is a good representation of the native structure in solution. However, the validation process involves two crucial steps: (i) detecting flaws in the structure at the residue level and (ii) providing details as to how these flaws can be repaired. Existing validation methods are mainly concerned with determining the quality of the whole structure but only sometimes with highlighting where the flaws are located at the residue level (3,(6)(7)(8)(9)(10)(11)(12). To the best of our knowledge, these validation methods do not provide detailed information as to how such flaws can be eliminated. Therefore, it is left to the sp...