Solving the structures of macromolecular complexes.

Cryo-electron microscopy (cryo-EM), the structural analysis of samples embedded in vitreous ice, is a powerful approach for determining three-dimensional (3D) structures of biological specimens. Over the past two decades, this technique has been used to successfully calculate subnanometer (<10 Å) resolution and, in some cases, near-atomic resolution structures of highly symmetrical and stable complexes such as icosahedral viruses and ribosomes, as well as samples that form ordered two-dimensional or helical arrays. However, determining high-resolution 3D structures of smaller, less symmetrical, and dynamic samples remains a significant challenge. The recent development of electron microscopes with automated data collection capabilities and robust direct electron detection cameras, as well as new powerful image processing algorithms, has dramatically expanded the number of biological macromolecules amenable for study using cryo-EM. In addition, these new technological and computational developments have been used to successfully determine <5 Å resolution 3D structures of samples, such as membrane proteins and complexes with either low or no symmetry, that traditionally were not considered promising candidates for high-resolution cryo-EM. With these exciting new advances, cryo-EM is now on pace to determine atomic resolution 3D structures.

The combined use of experimental viscosity ratios together with ab initio calculations for helium has driven significant improvements in the description of dilute gas transport properties. Here, we first use improvements made to ab initio helium calculations to update viscosity ratios measured for H2, Ar, CH4, and Xe by May et al. [Int. J. Thermophys. 28, 1085 (2007)] over the temperature range of 200–400 K, reducing the uncertainties of the data to 0.055%, 0.038%, 0.067%, and 0.084%, respectively. Separately, we extend the technique of combining viscosity ratios with ab initio calculations to develop new reference correlations for the dilute gas viscosity of 10 gases: helium, neon, argon, krypton, xenon, hydrogen, nitrogen, methane, ethane, and propane. This is achieved by combining the ratios of viscosities calculated ab initio at the target temperature and at 298.15 K with experimentally based reference viscosity values for each gas at 298.15 K. The new reference dilute gas viscosity correlations span temperature ranges from at least 150 K to 1200 K with relative uncertainties between 30% (krypton) and 85% (methane) lower than the original ab initio results. For the noble gases, ab initio calculations for the Prandtl number are used to develop reference correlations for thermal conductivity ranging from at least 100 K to 5000 K, with relative uncertainties ranging from 0.04% (argon) to 0.20% (xenon). The new reference correlations are compared with available experimental data at dilute gas conditions. In general, the data agree with the new correlations within the claimed experimental uncertainty.

Ab initio periodic calculations and classical molecular dynamics (MD) simulations were performed to investigate the adsorption mode of alanine and a number of short peptides, in particular two peptides, alanine-glutamic acid and alanine-lysine, taken as model systems for the ionic self-complementary oligopeptide EAK16-II, onto TiO(2) (110) rutile surface, and their conformational characteristics upon adsorption. The atomistic description of the rutile surface and its interactions with water and peptide molecules were based on ab initio calculations, the TIP3P water model, the AMBER force field, and available parameters. By comparison with ab initio calculations, it is shown that MD simulations of reasonable duration can describe the main characteristics of the peptide-TiO(2) surface interaction in solution, at least on a short time scale. Atom-atom radial distribution functions, atom-surface distances, backbone and side chain dihedral angle distributions, and peptide-surface interaction energies have been analyzed. Once adsorbed onto the TiO(2) rutile surface by a bidentate interaction of both carboxyl oxygens with two adjacent Ti atoms, the small peptide studied showed a clear propensity to remain there and undergo relatively limited hinge-bending motions.

Computational chemistry has led to the greater understanding of the molecular world, from the interaction of molecules, to the composition of molecular species and materials. Of the families of computational chemistry approaches available, the main families of electronic structure methods that are capable of accurate and/or reliable predictions of energetic, structural, and spectroscopic properties are ab initio methods and density functional theory (DFT). The focus of this dissertation is to improve the accuracy of predictions and computational efficiency (with respect to memory, disk space, and computer processing time) of some computational chemistry methods, which, in turn, can extend the size of molecule that can be addressed, and, for other methods, DFT, in particular, gain greater insight into which DFT methods are more reliable than others. Much, though not all, of the focus of this dissertation is upon transition metal species – species for which much less method development has been targeted or insight about method performance has been well established. The ab initio approach that has been targeted in this work is the correlation consistent composite approach (ccCA), which has proven to be a robust, ab initio computational method for main group and first row transition metal-containing molecules yielding, on average, accurate thermodynamic properties, i.e., within 1 kcal/mol of experiment for main group species and within 3 kcal/mol of experiment for first row transition metal molecules. In order to make ccCA applicable to systems containing any element from the periodic table, development of the method for second row transition metals and heavier elements, including lower p-block (5p and 6p) elements was pursued. The resulting method, the relativistic pseudopotential variant of ccCA (rp-ccCA), and its application are detailed for second row transition metals and lower p-block elements. Because of the computational cost of ab initio methods, DFT is a popular choice for the study of transition metals. Despite this, the most reliable density functionals for the prediction of energetic properties (e.g. enthalpy of formation, ionization potential, electron affinity, dissociation energy) of transition metal species, have not been clearly identified. The examination of DFT performance for first and second row transition metal thermochemistry (i.e., enthalpies of formation) was conducted and density functionals for the study of these species were identified. And, finally, to address the accuracy of spectroscopic and energetic properties, improvements for a series of density functionals have been established. In both DFT and ab initio methods, the harmonic approximation is typically employed. This neglect of anharmonic effects, such as those related to vibrational properties (e.g. zero-point vibrational energies, thermal contributions to enthalpy and entropy) of molecules, generally results in computational predictions that are not in agreement with experiment. To correct for the neglect of anharmonicity, scale factors can be applied to these vibrational properties, resulting in better alignment with experimental observations. Scale factors for DFT in conjunction with both the correlation and polarization consistent basis sets have been developed in this work.

In modern structural biology, there are three major methods for structural biologists to obtain structural information of macromolecules: cryo-electron microscopy (cryo-EM), nuclear magnetic resonance (NMR), and X-ray crystallography. Cryo-EM, in comparison with the other two methods, allows structural biologists to obtain the structures of various macromolecules in a more native and less perturbed system. Over the past decade, cryo-EM has enabled scientists to determine the structures of protein complexes at atomic resolution and made a profound impact in molecular bioscience and pharmaceutical sectors. Along with cryo-EM, another emerging technique called cryo-electron tomography (cryo-ET) has gained increasing importance in structural biology. It has the potential to visualize macromolecular complexes and assemblies in their native environments at high resolution, but there are still some challenges for small, sparse subjects and in approaching atomic resolution in situ. This chapter summarizes the major steps involved in structure determination using cryo-EM and cryo-ET and highlights the major challenges for in situ cryo-ET. We also present a few examples of near-atomic resolution structure determination of macromolecular assemblies both in purified systems in vitro and in native contexts in situ. Future perspectives are discussed as well.

BackgroundProteins in their majority act rarely as single entities. Multisubunit macromolecular complexes are the actors in most of the cellular processes. These nanomachines are hold together by weak protein-protein interactions and undergo functionally important conformational changes. TFIID is such a multiprotein complex acting in eukaryotic transcription initiation. This complex is first to be recruited to the promoter of the genes and triggers the formation of the transcription preinitiation complex involving RNA polymerase II which leads to gene transcription. The exact role of TFIID in this process is not yet understood.MethodsLast generation electron microscopes, improved data collection and new image analysis tools made it possible to obtain structural information of biological molecules at atomic resolution. Cryo-electron microscopy of vitrified samples visualizes proteins in a fully hydrated, close to native state. Molecular images are recorded at liquid nitrogen temperature in low electron dose conditions to reduce radiation damage. Digital image analysis of these noisy images aims at improving the signal-to-noise ratio, at separating distinct molecular views and at reconstructing a three-dimensional model of the biological particle.ResultsUsing these methods we showed the early events of an activated transcription initiation process. We explored the interaction of the TFIID coactivator with the yeast Rap1 activator, the transcription factor TFIIA and the promoter DNA. We demonstrated that TFIID serves as an assembly platform for transient protein-protein interactions, which are essential for transcription initiation.ConclusionsRecent developments in electron microscopy have provided new insights into the structural organization and the dynamic reorganization of large macromolecular complexes. Examples of near-atomic resolutions exist but the molecular flexibility of macromolecular complexes remains the limiting factor in most case. Electron microscopy has the potential to provide both structural and dynamic information of biological assemblies in order to understand the molecular mechanisms of their functions.

We have simulated exchange of inner-sphere and bulk water molecules for different sizes of Al3+(aq) clusters, Al(H2O)63+ + nH2O for n = 0, 1, 6, or 12, with ab initio and molecular dynamics simulations, in order to understand how robust the ab initio method is for identifying hydrolytic reaction pathways of particular importance to geochemistry. In contrast to many interfacial reactions, this particular elementary reaction is particularly simple and well-constrained by experiment. Nevertheless, we find that a rich array of parallel reaction pathways depend sensitively on the details of the solvation sphere and structure and that larger clusters are not necessarily better. Inner-sphere water exchange in Al3+(aq) may occur through two Langford-Gray dissociative pathways, one in which the incoming and outgoing waters are cis, the other in which they are trans to one another. A large majority of exchanges in the molecular dynamics simulations occurred via the trans mechanism, in contrast to the predictions of the ab initio method. In Al(H2O)63+ + H2O, the cis mechanism has a transition state of 84.3 kJ/mol, which is in good agreement with previous experimental and ab initio results, while the trans mechanism has only a saddle point with two negative frequencies, not a transition state, at 89.7 kJ/mol. In addition to the exchange mechanisms, dissociation pathways could be identified that were considerably lower in energy than experiment and varied considerably between 60 and 100 kJ/mol, depending on the particular geometry and cluster size, with no clear relation between the two. Ab initio calculations using large clusters with full second coordination spheres (n = 12) were unable to find dissociation or exchange transition states because the network of hydrogen bonds in the second coordination sphere was too rigid to accommodate the outgoing inner-sphere water. Our results indicate that caution should surround ab initio simulation of complicated dynamic processes such as hydrolysis, ion exchange, and interfacial reactions that involve several steps. Dynamic methods of simulation need to accompany static methods such as ab initio calculation, and it is best to consider simulated pathways as hypotheses to be tested experimentally rather than definitive properties of the reaction.

Infrared and Raman spectra, ab initio calculations, barriers to internal rotation, and vibrational assignment of 4-fluoro-2-pentyne

Stable isomers, transition states, and their photoabsorption spectra for the Na4+ cluster are determined by ab initio MRCI calculations. Three stable geometries of symmetry C2v, D2h, and D2d and two transition states of symmetry Cs and C2 are identified. The D2d configuration is a new geometry not previously obtained in ab initio calculations. The photoabsorption spectra of the three isomers and the transition states are used to interpret experimental absorption data. Dissociation dynamics and possible laser control of isomer populations are discussed in terms of the electronic states of the cluster.

The formation of Mn–C short-range ordering (SRO) has a great influence on the mechanical properties of high-Mn steels. In the present work, the formation of Mn–C SRO during recrystallization of an X60Mn18 steel was investigated by means of a combined study employing small angle neutron scattering (SANS) and ab initio ground-state energy calculations based on density-functional theory. The SANS measurements prove the presence of Mn–C SRO in the recrystallization annealed X60Mn18 steel and indicate the evolution of the SRO during recrystallization. The results show that with the increase in annealing time, the mean size of the Mn–C SRO decreases, whereas the number density increases. The ab initio calculations well describe the energetically favored condition of Mn–C SRO and provide the theoretical explanation of the clustering formation and evolution in the X60Mn18 steel. The stress-strain curve of the X60Mn18 steel exhibits a high strain-hardening rate and the plastic deformation is characterized with a series of serrations during a uniaxial tensile test. In the end, the correlation between Mn–C SRO and the serrated flow of high-Mn steels is further discussed.

An analytical function representing the energy of interaction in the acetone dimer was constructed on the basis of ab initio (MP2/6-31+G*) calculations for 327 configurations. The inclusion of electron correlation in the ab initio calculations was essential for satisfactory reproduction of experimental data, as was the avoidance of basis set superposition error by means of the counterpoise method. The analytical potential function was fitted to the ab initio data using a weighting scheme favoring good fit in low-energy regions. The minimum of the function, −4.83 kcal/mol, corresponds to a C2h symmetry in which the monomers are antiparallel and are linked by four hydrogen bonds. Three low-energy stationary points corresponding to transition states with differing numbers of hydrogen bonds and energy were also located. The quality of the function was corroborated by inspection of derived isoenergetic contour maps and of its prediction for the temperature dependence of the second virial coefficient. As a further test, a Monte Carlo simulation of liquid acetone was performed in the canonical ensemble, and energetic properties and radial distribution functions were computed.

The molecular structures of trans-1,2-dichloro-1,2-disilylethene and 1-bromo-1-silylethene have been determined by gas-phase electron diffraction (GED) and ab initio molecular orbital calculations (MP2/6-311G). Both compounds were found to have highly asymmetric coordination around the carbon atoms with [ab initio (r(e))/GED (r(a))] C=C-Cl [117.0/117.0(2) degrees] and C=C-Si [126.2/128.1(1) degrees] in the C(2)(h) structure of trans-1,2-dichloro-1,2-disilylethene and C=C-Br [119.2/120.7(4) degrees] and C=C-Si [125.0/125.0(4) degrees] in the C(s) structure of 1-bromo-1-silylethene. Other important structural parameters for trans-1,2-dichloro-1,2-disilylethene are C=C [135.2/134.5(3) pm], C-Si [189.4/187.9(2) pm], and C-Cl [175.1/174.9(1) pm], and C=C [134.2/133.4(2) pm], C-Si [187.8/187.2(3) pm], and C-Br [191.3/191.0(3) pm] for 1-bromo-1-silylethene. Further ab initio calculations were carried out on CH(2)CRX and trans-(CRX)(2) (R = SiH(3), CH(3), or H; X = H, F, Cl, or Br) to gauge the effects of electron-withdrawing and electron-donating groups on the structures. They reveal some even more distorted structures. The asymmetric appearance of these molecules can largely be accounted for by valence shell electron pair repulsion theory.

Structural and conformational analysis of 1-monofluorosilacyclobutane and 1-monochlorosilacyclobutane. A gas-phase electron diffraction and ab initio investigation

The distribution of deformation density of 2,5‐dimethyl thiathiophthene and 2,4‐diphenyl thiathiophthene are calculated via density functional method. The results are in good agreement with the corresponding experimental and theoretical distributions from an ab initio method. The ionization potentials obtained in this calculation on 2,5‐dimethyl thiathiophthene are in good agreement with those obtained experimentally from photoelectron spectroscopy, it is in better agreement than those VIPs based on ab initio calculations. Net atomic charges are compared with the experimental multipole refinement and with those calculated with ab initio and DFT methods for various atomic partitions.

In this study we compare the binding energies of polycoordinated complexes of Zn2+ within cavities composed of model “hard” (H2O, OH−) or “soft” (CH3SH, CH3S−) ligands. Ab initio supermolecule computations are performed at the HF and MP2 levels using extended basis sets to determine the binding energies and their components as a function of: the number of ligands, ranging from three to six; the net charge of the cavity; and the “hard” versus “soft” character of the ligands. These ab initio computations are used to test the reliability of the SIBFA molecular mechanics procedure, originally formulated and calibrated on the basis of ab initio computations, for such charged systems. The SIBFA intermolecular interaction energies match the corresponding ab initio values using a coreless effective potential split-valence basis set with a relative error of ≤3%. Extensions to binuclear Zn2+ complexes, such as those that occur in the Zn-binding sites of Gal4 and β-lactamase proteins, are performed to test the applicability of the methodology for such systems. © 2000 John Wiley & Sons, Inc. J Comput Chem 21: 1011–1039, 2000