Protein NMR assignment by isotope pattern recognition.

The current standard method for amino acid signal identification in protein NMR spectra is sequential assignment using triple-resonance experiments. Good software and elaborate heuristics exist, but the process remains laboriously manual. Machine learning does help, but its training databases need millions of samples that cover all relevant physics and every kind of instrumental artifact. In this communication, we offer a solution to this problem. We propose polyadic decompositions to store millions of simulated three-dimensional NMR spectra, on-the-fly generation of artifacts during training, a probabilistic way to incorporate prior and posterior information, and integration with the industry standard CcpNmr software framework. The resulting neural nets take [1H,13C] slices of mixed pyruvate-labeled HNCA spectra (different CA signal shapes for different residue types) and return an amino acid probability table. In combination with primary sequence information, backbones of common proteins (GB1, MBP, and INMT) are rapidly assigned from just the HNCA spectrum.

Sensitive Detection of Structural Differences using a Statistical Framework for Comparative Crystallography.

Chemical and conformational changes underlie the functional cycles of proteins. Comparative crystallography can reveal these changes over time, over ligands, and over chemical and physical perturbations in atomic detail. A key difficulty, however, is that the resulting observations must be placed on the same scale by correcting for experimental factors. We recently introduced a Bayesian framework for correcting (scaling) X-ray diffraction data by combining deep learning with statistical priors informed by crystallographic theory. To scale comparative crystallography data, we here combine this framework with a multivariate statistical theory of comparative crystallography. By doing so, we find strong improvements in the detection of protein dynamics, element-specific anomalous signal, and the binding of drug fragments.

Scaling and Merging Time-Resolved Laue Data with Variational Inference.

Time-resolved X-ray crystallography (TR-X) at synchrotrons and free electron lasers is a promising technique for recording dynamics of molecules at atomic resolution. While experimental methods for TR-X have proliferated and matured, data analysis is often difficult. Extracting small, time-dependent changes in signal is frequently a bottleneck for practitioners. Recent work demonstrated this challenge can be addressed when merging redundant observations by a statistical technique known as variational inference (VI). However, the variational approach to time-resolved data analysis requires identification of successful hyperparameters in order to optimally extract signal. In this case study, we present a successful application of VI to time-resolved changes in an enzyme, DJ-1, upon mixing with a substrate molecule, methylglyoxal. We present a strategy to extract high signal-to-noise changes in electron density from these data. Furthermore, we conduct an ablation study, in which we systematically remove one hyperparameter at a time to demonstrate the impact of each hyperparameter choice on the success of our model. We expect this case study will serve as a practical example for how others may deploy VI in order to analyze their time-resolved diffraction data.

Laue-DIALS: open-source software for polychromatic X-ray diffraction data.

Most X-ray sources are inherently polychromatic. Polychromatic ("pink") X-rays provide an efficient way to conduct diffraction experiments as many more photons can be used and large regions of reciprocal space can be probed without sample rotation during exposure-ideal conditions for time-resolved applications. Analysis of such data is complicated, however, causing most X-ray facilities to discard >99% of X-ray photons to obtain monochromatic data. Key challenges in analyzing polychromatic diffraction data include lattice searching, indexing and wavelength assignment, correction of measured intensities for wavelength-dependent effects, and deconvolution of harmonics. We recently described an algorithm, Careless, that can perform harmonic deconvolution and correct measured intensities for variation in wavelength when presented with integrated diffraction intensities and assigned wavelengths. Here, we present Laue-DIALS, an open-source software pipeline that indexes and integrates polychromatic diffraction data. Laue-DIALS is based on the dxtbx toolbox, which supports the DIALS software commonly used to process monochromatic data. As such, Laue-DIALS provides many of the same advantages: an open-source, modular, and extensible architecture, providing a robust basis for future development. We present benchmark results showing that Laue-DIALS, together with Careless, provides a suitable approach to the analysis of polychromatic diffraction data, including for time-resolved applications.

The Cytoplasmic Domain of the SARS-CoV-2 Envelope Protein Assembles into a ß-Sheet Bundle in Lipid Bilayers.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope (E) protein forms a pentameric ion channel in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment (ERGIC) of the infected cell. The cytoplasmic domain of E interacts with host proteins to cause virus pathogenicity and may also mediate virus assembly and budding. To understand the structural basis of these functions, here we investigate the conformation and dynamics of an E protein construct (residues 8-65) that encompasses the transmembrane domain and the majority of the cytoplasmic domain using solid-state NMR. 13C and 15N chemical shifts indicate that the cytoplasmic domain adopts a ß-sheet-rich conformation that contains three ß-strands separated by turns. The five subunits associate into an umbrella-shaped bundle that is attached to the transmembrane helices by a disordered loop. Water-edited NMR spectra indicate that the third ß-strand at the C terminus of the protein is well hydrated, indicating that it is at the surface of the ß-bundle. The structure of the cytoplasmic domain cannot be uniquely determined from the inter-residue correlations obtained here due to ambiguities in distinguishing intermolecular and intramolecular contacts for a compact pentameric assembly of this small domain. Instead, we present four structural topologies that are consistent with the measured inter-residue contacts. These data indicate that the cytoplasmic domain of the SARS-CoV-2 E protein has a strong propensity to adopt ß-sheet conformations when the protein is present at high concentrations in lipid bilayers. The equilibrium between the ß-strand conformation and the previously reported α-helical conformation may underlie the multiple functions of E in the host cell and in the virion.

Microtubule-binding core of the tau protein.

The protein tau associates with microtubules to maintain neuronal health. Posttranslational modifications of tau interfere with this binding, leading to tau aggregation in neurodegenerative disorders. Here, we use solid-state nuclear magnetic resonance (NMR) to investigate the structure of the microtubule-binding domain of tau. Wild-type tau that contains four microtubule-binding repeats and a pseudorepeat R' is studied. Complexed with taxol-stabilized microtubules, the immobilized residues exhibit well-resolved two-dimensional spectra that can be assigned to the amino-terminal region of R4 and the R' domain. When tau coassembles with tubulin to form unstable microtubules, the R' signals remain, whereas the R4 signals disappear, indicating that R' remains immobilized, whereas R4 becomes more mobile. Therefore, R' outcompetes the other four repeats to associate with microtubules. These NMR data, together with previous cryo-electron microscopy densities, indicate an extended conformation for microtubule-bound R'. R' contains the largest number of charged residues among all repeats, suggesting that charge-charge interaction drives tau-microtubule association.

pH- and Calcium-Dependent Aromatic Network in the SARS-CoV-2 Envelope Protein.

The envelope (E) protein of the SARS-CoV-2 virus is a membrane-bound viroporin that conducts cations across the endoplasmic reticulum Golgi intermediate compartment (ERGIC) membrane of the host cell to cause virus pathogenicity. The structure of the closed state of the E transmembrane (TM) domain, ETM, was recently determined using solid-state NMR spectroscopy. However, how the channel pore opens to mediate cation transport is unclear. Here, we use 13C and 19F solid-state NMR spectroscopy to investigate the conformation and dynamics of ETM at acidic pH and in the presence of calcium ions, which mimic the ERGIC and lysosomal environment experienced by the E protein in the cell. Acidic pH and calcium ions increased the conformational disorder of the N- and C-terminal residues and also increased the water accessibility of the protein, indicating that the pore lumen has become more spacious. ETM contains three regularly spaced phenylalanine (Phe) residues in the center of the peptide. 19F NMR spectra of para-fluorinated Phe20 and Phe26 indicate that both residues exhibit two sidechain conformations, which coexist within each channel. These two Phe conformations differ in their water accessibility, lipid contact, and dynamics. Channel opening by acidic pH and Ca2+ increases the population of the dynamic lipid-facing conformation. These results suggest an intricate aromatic network that regulates the opening of the ETM channel pore. This aromatic network may be a target for E inhibitors against SARS-CoV-2 and related coronaviruses.

Binding Sites of a Positron Emission Tomography Imaging Agent in Alzheimer's ß-Amyloid Fibrils Studied Using 19F Solid-State NMR.

Amyloid imaging by positron emission tomography (PET) is an important method for diagnosing neurodegenerative disorders such as Alzheimer's disease. Many 11C- and 18F-labeled PET tracers show varying binding capacities, specificities, and affinities for their target proteins. The structural basis of these variations is poorly understood. Here we employ 19F and 13C solid-state NMR to investigate the binding sites of a PET ligand, flutemetamol, to the 40-residue Alzheimer's ß-amyloid peptide (Aß40). Analytical high-performance liquid chromatography and 19F NMR spectra show that flutemetamol binds the current Aß40 fibril polymorph with a stoichiometry of one ligand per four to five peptides. Half of the ligands are tightly bound while the other half are loosely bound. 13C and 15N chemical shifts indicate that this Aß40 polymorph has an immobilized N-terminus, a non-ß-sheet His14, and a non-ß-sheet C-terminus. We measured the proximity of the ligand fluorine to peptide residues using 19F-13C and 19F-1H rotational-echo double-resonance (REDOR) experiments. The spectra show that three segments in the peptide, 12VHH14, 18VFF20, and 39VV40, lie the closest to the ligand. REDOR-constrained docking simulations indicate that these three segments form multiple binding sites, and the ligand orientations and positions at these sites are similar across different Aß polymorphs. Comparison of the flutemetamol-interacting residues in Aß40 with the small-molecule binding sites in other amyloid proteins suggest that conjugated aromatic compounds preferentially bind ß-sheet surface grooves lined by aromatic, polar, and charged residues. These motifs may explain the specificity of different PET tracers to different amyloid proteins.

Inclusion of the C-Terminal Domain in the ß-Sheet Core of Heparin-Fibrillized Three-Repeat Tau Protein Revealed by Solid-State Nuclear Magnetic Resonance Spectroscopy.

Many neurodegenerative diseases such as Alzheimer's disease are characterized by pathological ß-sheet filaments of the tau protein, which spread in a prion-like manner in patient brains. To date, high-resolution structures of tau filaments obtained from patient brains show that the ß-sheet core only includes portions of the microtubule-binding repeat domains and excludes the C-terminal residues, indicating that the C-terminus is dynamically disordered. Here, we use solid-state NMR spectroscopy to identify the ß-sheet core of full-length 0N3R tau fibrillized using heparin. Assignment of 13C and 15N chemical shifts of the rigid core of the protein revealed a single predominant ß-sheet conformation, which spans not only the R3, R4, R' repeats but also the entire C-terminal domain (CT) of the protein. This massive ß-sheet core qualitatively differs from all other tau fibril structures known to date. Using long-range correlation NMR experiments, we found that the R3 and R4 repeats form a ß-arch, similar to that seen in some of the brain-derived tau fibrils, but the R1 and R3 domains additionally stack against the CT, reminiscent of previously reported transient interactions of the CT with the microtubule-binding repeats. This expanded ß-sheet core structure suggests that the CT may have a protective effect against the formation of pathological tau fibrils by shielding the amyloidogenic R3 and R4 domains, preventing side-on nucleation. Truncation and post-translational modification of the CT in vivo may thus play an important role in the progression of tauopathies.

In vitro 0N4R tau fibrils contain a monomorphic ß-sheet core enclosed by dynamically heterogeneous fuzzy coat segments.

Misfolding of the microtubule-binding protein tau into filamentous aggregates is characteristic of many neurodegenerative diseases such as Alzheimer's disease and progressive supranuclear palsy. Determining the structures and dynamics of these tau fibrils is important for designing inhibitors against tau aggregation. Tau fibrils obtained from patient brains have been found by cryo-electron microscopy to adopt disease-specific molecular conformations. However, in vitro heparin-fibrillized 2N4R tau, which contains all four microtubule-binding repeats (4R), was recently found to adopt polymorphic structures. Here we use solid-state NMR spectroscopy to investigate the global fold and dynamics of heparin-fibrillized 0N4R tau. A single set of 13C and 15N chemical shifts was observed for residues in the four repeats, indicating a single ß-sheet conformation for the fibril core. This rigid core spans the R2 and R3 repeats and adopts a hairpin-like fold that has similarities to but also clear differences from any of the polymorphic 2N4R folds. Obtaining a homogeneous fibril sample required careful purification of the protein and removal of any proteolytic fragments. A variety of experiments and polarization transfer from water and mobile side chains indicate that 0N4R tau fibrils exhibit heterogeneous dynamics: Outside the rigid R2-R3 core, the R1 and R4 repeats are semirigid even though they exhibit ß-strand character and the proline-rich domains undergo large-amplitude anisotropic motions, whereas the two termini are nearly isotropically flexible. These results have significant implications for the structure and dynamics of 4R tau fibrils in vivo.