Structure Refinement Procedure Research Articles

Progressive assembly of multi-domain protein structures from cryo-EM density maps.

Progress in cryo-electron microscopy has provided the potential for large-size protein structure determination. However, the success rate for solving multi-domain proteins remains low because of the difficulty in modelling inter-domain orientations. Here we developed domain enhanced modeling using cryo-electron microscopy (DEMO-EM), an automatic method to assemble multi-domain structures from cryo-electron microscopy maps through a progressive structural refinement procedure combining rigid-body domain fitting and flexible assembly simulations with deep-neural-network inter-domain distance profiles. The method was tested on a large-scale benchmark set of proteins containing up to 12 continuous and discontinuous domains with medium- to low-resolution density maps, where DEMO-EM produced models with correct inter-domain orientations (template modeling score (TM-score) >0.5) for 97% of cases and outperformed state-of-the-art methods. DEMO-EM was applied to the severe acute respiratory syndrome coronavirus 2 genome and generated models with average TM-score and root-mean-square deviation of 0.97 and 1.3 Å, respectively, with respect to the deposited structures. These results demonstrate an efficient pipeline that enables automated and reliable large-scale multi-domain protein structure modelling from cryo-electron microscopy maps.

Determining structures in a native environment using single-particle cryoelectron microscopy images

Cryo-electron tomography is a powerful tool for structure determination in the native environment. However, this method requires the acquisition of tilt series, which is time-consuming and severely slows structure determination. By treating the densities of non-target protein as non-Gaussian noise, we developed a new target function that greatly improves the efficiency of recognizing the target protein in a single cryo-electron microscopy image. Moreover, we developed a sorting function that effectively eliminates the model dependence and improved the resolution during the subsequent structure refinement procedure. By eliminating model bias, our method allows using homolog proteins as models to recognize the target proteins in a complex context. Together, we developed an in situ single-particle analysis method. Our method was successfully applied to solve structures of glycoproteins on the surface of a non-icosahedral virus and Rubisco inside the carboxysome. Both data were collected within 24 h, thus allowing fast and simple structural determination.

Hydration mechanism in Ce-exchanged zeolites and heat release performances upon adsorption of water vapour in support of their potential use in thermochemical storage of energy under mild conditions of adsorbent regeneration and saturation

Abstract The potential use of commercially available 13X zeolite, modified by ion-exchange with cerium compensating cations possessing high charges and high hydration energies, has been tested in view of low-temperature storage of solar energy performed under mild operating conditions of low regeneration temperatures and low pressures of water vapour during the adsorption step. Structural and textural properties, sorption behaviour towards water vapour of three selected samples containing various proportions of Ce3+ and Ce4+ compensating cations and the pristine Na+-13X zeolite were studied by a variety of experimental techniques including Wavelength Dispersive X-Ray Fluorescence, Energy Dispersive X-ray Spectroscopy, X-ray diffraction, Thermogravimetric analysis, as well as measurements of the adsorption of gaseous nitrogen at 77 K and water vapour at 313 K. Based on the structure refinement procedure applied to the experimental XRD patterns, it was demonstrated that extra-framework cerium cations were preferentially located on sites I’ and II in dry and hydrated zeolites, showing relatively little dependence on the hydration level. Monte Carlo simulations were used to determine the limit values of the amount adsorbed and differential heat of adsorption, which could be obtained experimentally if the zeolite samples were completely dried. The potential of Ce-containing zeolites as adsorbents for the thermochemical energy storage was finally determined under flow conditions by firstly dehydrating samples at 353 K or 423 K and then saturating them at 296 K with water vapour at a mole fraction of 0.03. The choice of the operating conditions was decided so as to maintain the stability of the zeolite structure while taking the risk of reduced thermal performance of zeolite adsorbents undergoing incomplete regeneration-dehydration. Under such mild conditions, the modified 13X zeolites exhibited enhanced thermal performance in comparison with that of the pristine 13X, by releasing between 700 and 1100 kJ per kg of the adsorbent during a period of 6–8 h. Through a complementary study based on calorimetry measurements and molecular simulations, the understanding of the hydration-dehydration steps in Ce-exchanged zeolites and cation displacement upon hydration has allowed to establish the best compromise for the conditions of zeolite regeneration and saturation in the case of heat long-term storage applications.

Crystal structure prediction by ionic network analysis: the example of (p–T–X)-structure relationships in olivines

The method of Ionic Network Analysis (INA) is defined by reference to the known crystal structures of olivine minerals. It is based on a reversible transformation between two alternative representations of ionic crystal structures: (a) the crystallographic and (b) the interactional. Whereas the former encompasses unit-cell parameters and atomic coordinates, the latter consists of selected interaction vectors between ions. Since the lengths and orientations of these vary only slightly between crystal structures obtained under systematically varying (p, T, X) conditions, they may be used to predict the crystal structures at intermediate (p, T, X) values by interpolation. Two interactional networks are constructed, one for the anions and the other for cations. As both networks lead to independent calculated values of the unit-cell parameters, it is possible to exploit the known, continuous (p, T, X) variations of cell parameters as normative constraints for the prediction of atomic coordinates within a predictive structural refinement procedure. Continuously varying structurally based parameters such as the volumes of cation coordination polyhedra may likewise be used. The choice of olivines for developing the method has been guided by the availability of pressure, temperature and compositional structural data for them. However, the ideas are expounded sufficiently generally for the method to be applied to other minerals.

Why direct and post-refinement determinations of absolute structure may give different results.

Direct determination of the Flack parameter as part of the structure refinement procedure usually gives different, though similar, values to post-refinement methods. The source of this discrepancy has been probed by analysing a range of data sets taken from the recent literature. Most significantly, it was observed that the directly refined Flack (x) parameter and its standard uncertainty are usually not much influenced by changes in the refinement weighting schemes, but if they are then there are probably problems with the data or model. Post-refinement analyses give Flack parameters strongly influenced by the choice of weights. Weights derived from those used in the main least squares lead to post-refinement estimates of the Flack parameters and their standard uncertainties very similar to those obtained by direct refinement. Weights derived from the variances of the observed structure amplitudes are more appropriate and often yield post-refinement Flack parameters similar to those from direct refinement, but always with lower standard uncertainties. Substantial disagreement between direct and post-refinement determinations are strongly indicative of problems with the data, which may be difficult to identify. Examples drawn from 28 structure determinations are provided showing a range of different underlying problems. It seems likely that post-refinement methods taking into account the slope of the normal probability plot are currently the most robust estimators of absolute structure and should be reported along with the directly refined values.

Accurate equilibrium structures of fluoro- and chloroderivatives of methane

This work is a systematic study of molecular structure of fluoro-, chloro-, and fluorochloromethanes. For the first time, the accurate ab initio structure is computed for 10 molecules (CF4, CClF3, CCl2F2, CCl3F, CHClF2, CHCl2F, CH2F2, CH2ClF, CH2Cl2, and CCl4) at the coupled cluster level of electronic structure theory including single and double excitations augmented by a perturbational estimate of the effects of connected triple excitations [CCSD(T)] with all electrons being correlated and Gaussian basis sets of at least quadruple-ζ quality. Furthermore, when possible, namely for the molecules CH2F2, CH2Cl2, CH2ClF, CHClF2, and CCl2F2, accurate semi-experimental equilibrium (rSEe) structure has also been determined. This is achieved through a least-squares structural refinement procedure based on the equilibrium rotational constants of all available isotopomers, determined by correcting the experimental ground-state rotational constants with computed ab initio vibration–rotation interaction constants and electronic g-factors. The computed and semi-experimental equilibrium structures are in excellent agreement with each other, but the rSEe structure is generally more accurate, in particular for the CF and CCl bond lengths. The carbon–halogen bond length is discussed within the framework of the ligand close-packing model as a function of the atomic charges. For this purpose, the accurate equilibrium structures of some other molecules with alternative ligands, such as CH3Li, CF3CCH, and CF3CN, are also computed.

The Shape of TiO2-B Nanoparticles

The shape of nanoparticles can be important in defining their properties. Establishing the exact shape of particles is a challenging task when the particles tend to agglomerate and their size is just a few nanometers. Here we report a structure refinement procedure for establishing the shape of nanoparticles using powder diffraction data. The method utilizes the fundamental formula of Debye coupled with a Monte Carlo-based optimization and has been successfully applied to TiO2-B nanoparticles. Atomistic modeling and molecular dynamics simulations of ensembles of all the ions in the nanoparticle reveal surface hydroxylation as the underlying reason for the established shape and structural features.

Improved cross validation of a static ubiquitin structure derived from high precision residual dipolar couplings measured in a drug-based liquid crystalline phase.

The antibiotic squalamine forms a lyotropic liquid crystal at very low concentrations in water (0.3-3.5% w/v), which remains stable over a wide range of temperature (1-40 °C) and pH (4-8). Squalamine is positively charged, and comparison of the alignment of ubiquitin relative to 36 previously reported alignment conditions shows that it differs substantially from most of these, but is closest to liquid crystalline cetyl pyridinium bromide. High precision residual dipolar couplings (RDCs) measured for the backbone 1H-15N, 15N-13C′, 1Hα-13Cα, and 13C′-13Cα one-bond interactions in the squalamine medium fit well to the static structural model previously derived from NMR data. Inclusion into the structure refinement procedure of these RDCs, together with 1H-15N and 1Hα-13Cα RDCs newly measured in Pf1, results in improved agreement between alignment-induced changes in 13C′ chemical shift, 3JHNHα values, and 13Cα-13Cβ RDCs and corresponding values predicted by the structure, thereby validating the high quality of the single-conformer structural model. This result indicates that fitting of a single model to experimental data provides a better description of the average conformation than does averaging over previously reported NMR-derived ensemble representations. The latter can capture dynamic aspects of a protein, thus making the two representations valuable complements to one another.

Deformation of the benzene ring upon fluorination: equilibrium structures of all fluorobenzenes

Born−Oppenheimer equilibrium structure (r BO e) estimates are reported for benzene and all 12 possible fluorobenzenes, based on geometry optimizations performed at the coupled cluster level of electronic structure theory including single and double excitations augmented by a perturbational estimate of the effects of connected triple excitations [CCSD(T)] and Gaussian basis sets of at least triple zeta quality. Furthermore, accurate semiexperimental equilibrium (r SE e) structures are determined for C6H6, C6H5F, and 1,2- and 1,3-difluorobenzene. They are obtained through a least-squares structural refinement procedure based on equilibrium rotational constants of as many isotopologues as feasible, determined by correcting experimental vibrationally averaged ground-state rotational constants with computed ab initio vibration–rotation interaction constants and electronic g-factors, and using a few structural constraints based on the best r BO e estimates. The r BO e and r SE e equilibrium structures are in excellent agreement with each other for the four semirigid molecules but in almost all cases they differ significantly from previously determined equilibrium structure estimates based on rotational spectroscopy or gas electron diffraction. The nature of deformations of the benzene ring induced by a single fluorine substitution can be characterized as follows: (a) the strongest effect is the pushing of the ipso carbon atom toward the ring center resulting in a deformation at the ipso [by +2.7(1)°] and ortho [−1.7(1)°] CCC angles, (b) a simultaneous decrease in the ortho CC bond length of the benzene ring by 0.009 Å and (c) a decrease of all the CH bond lengths. Additivity relations concerning the F substitution effects are obtained based on the equilibrium structures of all possible fluorobenzenes.

Microstructural evolution of nanostructured Ti0.7Ni0.3N prepared by reactive ball-milling

Nanocrystalline stoichiometric Ti0.7Ni0.3N powder has been synthesized by ball-milling the α-Ti (hcp) and Ni (fcc) powders under N2 gas at room temperature. The α-Ti phase partially transforms to the transient (-Ti phase after 1h of milling. After 5.5h of milling, very broad reflections of Ti0.7Ni0.3N phase is noticed. Complete formation of Ti0.7Ni0.3N phase is observed after 9h of milling. Microstructure in terms of lattice imperfections of unmilled and all ball-milled powder mixtures are primarily characterized by analyzing the X-ray powder diffraction patterns employing the Rietveld structure refinement procedure. It clearly reveals the presence of Ti0.7Ni0.3N phase and inclusion of nitrogen atoms into the α-Ti–Ni matrix on the way to formation of nitride phase. Microstructure of the ball milled nitride powders is also characterized by HRTEM. Particle size of Ti0.7Ni0.3N phase obtained from XRD method of characterization is ∼5nm which is very close to that obtained from HRTEM.

Segment assembly, structure alignment and iterative simulation in protein structure prediction

Segment assembly, structure alignment and iterative simulation in protein structure prediction

New Methods to Improve Protein Structure Prediction and Refinement

We have developed and combined several novel methods to improve protein structure prediction from the amino acid sequence, and the structural refinement of protein models. One of the most promising developments in protein structure prediction are many-body potentials that take into account dense packing, and cooperativity of interactions in protein cores. We developed a method that uses whole protein information filtered through machine learners to score protein models based on their likeness to native structures. Testing on CASP 9 targets showed that our method is superior to the common DFIRE and its derivatives as well as to the current version of RWPlus, both of which are considered a standard in the field. By combing statistical contact potentials with entropies from the elastic network models of proteins we can compute free energy and improve coarse-grained modeling of protein structure and dynamics. The consideration of protein flexibility and its fluctuational dynamics improves protein structure prediction, and leads to a better refinement of computational models of proteins. We proposed a novel protein structural refinement procedure based on Anisotropic Network Model (ANM) of protein fluctuational dynamics and Go-like model of energy score. The starting structures were models from past CASP experiments. We changed positions of C-alpha atoms using ANM, creating a new set of 250 structures from the initial model, and computed energies of these structures using Go-like energy score. The top 5 coarse-grained structures were fully rebuilt with BBQ and Scrwl4. To remove bond stretches and the excluded volume clashes, short Molecular Mechanics simulations (up to 10,000 steps) were performed with OPLS-AA force field and implicit solvent GBSA-OBC. The whole structural refinement process was performed iteratively leading to the improvement of average RMSD from 3.8A to 2.6A in 50 iterations.

Multi‐LZerD: Multiple protein docking for asymmetric complexes

The tertiary structures of protein complexes provide a crucial insight about the molecular mechanisms that regulate their functions and assembly. However, solving protein complex structures by experimental methods is often more difficult than single protein structures. Here, we have developed a novel computational multiple protein docking algorithm, Multi-LZerD, that builds models of multimeric complexes by effectively reusing pairwise docking predictions of component proteins. A genetic algorithm is applied to explore the conformational space followed by a structure refinement procedure. Benchmark on eleven hetero-multimeric complexes resulted in near-native conformations for all but one of them (a root mean square deviation smaller than 2.5Å). We also show that our method copes with unbound docking cases well, outperforming the methodology that can be directly compared with our approach. Multi-LZerD was able to predict near-native structures for multimeric complexes of various topologies.

Computational Methods for Multiple Protein Docking for Asymmetric Complexes

Protein complexes are involved in many biological processes mediating diverse important cellular functions. The tertiary structures of protein complexes provide a crucial insight about the molecular mechanisms that regulate their functions and assembly. However, solving protein complex structures by experimental methods is often more difficult than single protein structures. Computational prediction methods are expected to make significant contributions in this area. Until now most of the protein docking prediction methods focus only on pairwise docking. There exist a handful of methods for the prediction of multimeric complexes, but almost all of them assume specific properties such as homomericity or symmetry. Considering a substantial number of multimeric complexes of diverse kinds exist in a cell, there is an urgent need for the development of a multiple protein docking prediction method that does not target a specific type of complexes.We have developed a novel multiple protein docking algorithm, Multi-LZerD, that builds models of multimeric complexes by effectively reusing pairwise docking predictions of component proteins. A genetic algorithm is applied to explore the conformational space followed by a structure refinement procedure. Benchmark on nine hetero-multimeric complexes resulted in near native conformations for all of them (a root mean square deviation smaller than 2.5A). We also show that our method handles unbound docking cases well, outperforming the only methodology developed so far that can be directly compared to our approach. Multi-LZerD was able to predict near native structures for multimeric complexes of various topologies.

Surface structure refinement including anomalous crystal truncation rods

A surface structure refinement procedure is introduced, which uses both anomalous and non-anomalous crystal truncation rod data simultaneously. It is shown how a single structural model can be refined against data sets measured at different wavelengths and how this can greatly reduce correlations between fit parameters. The structure factors are computed taking into account the anomalous dispersion corrections and are scaled to the data, whereby each data set is assigned its own scale factor. The procedure is implemented in the widely used surface diffraction programROD. The structure of a one unit cell thin LaAlO3film on an SrTiO3(001) substrate is refined by making use of a non-anomalous data set and one taken at the LaL1-edge.

Crystal structure and disorder in Poly( l-lactic acid) δ form ( α′ form) and the phase transition mechanism to the ordered α form

Crystal structure of poly( l-lactic acid) (PLLA) α′ form (or the newly-termed “ δ” form) has been analyzed on the basis of the X-ray diffraction data. The oriented δ form was obtained by stretching and annealing the melt-quenched sample at ca. 100 °C. The unit cell parameters were a = 10.80 Å, b = 6.20 Å, c (fiber axis) = 28.80 Å and α = β = γ = 90°. As seen from the lack of even-numbered 00l reflections (002, 004, ..) except the 0010 reflection, the chain conformation of the δ form was found to be more remarkably disordered than that of the regular α form. As already reported, the α form takes the (10/3) helix with approximately 2 1 screw symmetry although, more strictly speaking, this screw symmetry is not actually existent. The δ form does not possess any symmetry along the chain axis although the local torsional angles are not very much different from each other. As for the chain packing structure, the two different possibilities were assumed in the structural refinement procedure: (i) the corner and center chains direct upward and downward along the chain axis, respectively, just like the case of regular α form (alternate packing mode) and (ii) the upward and downward chains are located at the same lattice site at 50% probability (statistically-disordered packing mode). The structure analysis using the Bragg reflections suggested the preferability of the model (i). The diffuse and streaky lines were observed along the layer lines, originating from the relative-height disorder between the neighboring chains in the δ form. The X-ray diffuse scatterings coming from the various types of the disordered structure have been simulated, among which the disordered multi-domain structure model was found to be the most plausible model, where the domains constructed by the alternate arrangement of upward and downward chains are gathered together with the mismatch in the relative height between the neighboring domains. In this way the disorder-to-order phase transition from the δ to α form was found to occur through the conformational ordering of the chains accompanied with the chain packing regularization as well as the matching of the neighboring domain height to give a large single domain of the α form.

An Improved Gas Electron Diffractometer – The Instrument, Data Collection, Reduction and Structure Refinement Procedures

Graphical Abstract An Improved Gas Electron Diffractometer – The Instrument, Data Collection, Reduction and Structure Refinement Procedures

Use of Relaxation Enhancements in a Paramagnetic Environment for the Structure Determination of Proteins Using NMR Spectroscopy

More information: Relaxation enhancements induced by an inert paramagnetic environment (PREs) are used together with a limited NOE data set in a model-free alternative protein structure refinement procedure. Structures of two model systems (8 kDa ubiquitin and the 42 kDa maltodextrin-binding protein in complex with β-cyclodextrin) were obtained employing PREs and NOEs of exchangeable protons.

Generalized X-ray and neutron crystallographic analysis: more accurate and complete structures for biological macromolecules

X-ray and neutron crystallographic techniques provide complementary information on the structure and function of biological macromolecules. X-ray and neutron (XN) crystallographic data have been combined in a joint structure-refinement procedure that has been developed using recent advances in modern computational methodologies, including cross-validated maximum-likelihood target functions with gradient-based optimization and simulated annealing. The XN approach for complete (including hydrogen) macromolecular structure analysis provides more accurate and complete structures, as demonstrated for diisopropyl fluorophosphatase, photoactive yellow protein and human aldose reductase. Furthermore, this method has several practical advantages, including the easier determination of the orientation of water molecules, hydroxyl groups and some amino-acid side chains.

Structural and photoacoustic studies of Zn4Sb3 and ZnSb phases prepared by mechanical alloying

The structural and photoacoustic properties of nanostructured zinc antimonides Zn4Sb3 and ZnSb prepared by mechanical alloying were studied by x-ray diffraction (XRD) and photoacoustic absorption spectroscopy (PAS). The XRD patterns revealed the existence of Zn4Sb3 and elemental Zn for 10 h of milling, Zn4Sb3 and ZnSb for 20 h of milling, and ZnSb and elemental Zn for 30 h of milling. Using the Rietveld structural refinement procedure, the experimental XRD patterns were simulated and the relative amounts of each phase for each of the milling times above were calculated. The thermal diffusivity values corresponding to each milling time were measured from the PAS signal amplitude and phase. Using the thermal diffusivity value and the relative amounts of ZnSb and elemental Zn, we estimated the thermal diffusivity for the ZnSb phase in nanometric form. The result seems to corroborate the suggestion of other researchers that the thermal conductivity tends to be smaller in materials with nanometric crystallites.