A NOESY-HSQC simulation program, SPIRIT

A program SPIRIT (Simulation Program considering Incomplete Recovery of z magnetization and INEPT Transfer efficiency) has been developed to simulate three-dimensional NOESY-HSQC spectra. This program takes into account (1) different transfer efficiency during INEPT and reverse INEPT durations due to differential relaxation rates and¹ J coupling constants; (2) the different effect of the sensitivity-enhancement scheme on CH, CH₂ and CH₃ systems; and (3) incomplete recovery of longitudinal magnetization between scans. The simulation program incorporates anisotropic tumbling mode for symmetric tops, and allows for differential external relaxation rates for protons. Some well-defined internal motions, such as the fast rotation of methyl groups, are taken into account. The simulation program also allows for input of multiple conformations and their relative populations to calculate the average relaxation matrix to account for slow internal motions. With the SPIRIT program, the sensitivity-enhanced NOESY-HSQC experiment can be used directly in the evaluation of the accuracy of structures, which can potentially be improved by direct refinement against the primary data. Abbreviations: NOESY, nuclear Overhauser enhancement spectroscopy; HSQC, heteronuclear single quantum correlation; INEPT, insensitive nuclei enhanced by polarization transfer.     

Crystal structure of the phospholipase A and acyltransferase 4 (PLAAT4) catalytic domain

Phospholipase A and Acyltransferase 4 (PLAAT4) is a class II tumor suppressor, that also plays a role as a restrictor of intracellular Toxoplasma gondii infection through restriction of parasitic vacuole size. The catalytic N-terminal domain (NTD) interacts with the C-terminal domain (CTD), which is important for sub-cellular targeting and enzymatic function. The dynamics of the NTD main (L1) loop and the L2(B6) loop adjacent to the active site, have been shown to be important regulators of enzymatic activity. Here, we present the crystal structure of PLAAT4 NTD, determined from severely intergrown crystals using automated, laser-based crystal harvesting and data reduction technologies. The structure showed the L1 loop in two distinct conformations, highlighting a complex network of interactions likely influencing its conformational flexibility. Ensemble refinement of the crystal structure recapitulates the major correlated motions observed in solution by NMR. Our analysis offers useful insights on millisecond dynamics based on the crystal structure, complementing NMR studies which preclude structural information at this time scale.     

Crystallographic Refinement and Structure-Factor Time-Averaging by Molecular Dynamics in the Absence of a Physical Force Field

Abstract

The application of Molecular-Dynamics simulation in protein-crystallographic structure refinement has become common practice. In this paper, structure optimizations are described where the driving force is derived only from the crystallographic data and not from any physical potential energy function. Under this extreme condition ab initio structure refinement and the application of structure-factor time averaging was investigated using a small 9 atom test system. Success in ab initio refinement, where the starting atomic positions are randomly distributed, depends on the resolution of the crystallographic data used in the optimization. The presence of high resolution data introduces false minima in the X-ray energy profile, enhancing the search problem significantly. On the same system, we also tested the method of time-averaged crystallographically restrained Molecular Dynamics, again in the absence of a physical force field. In this method, the diffraction data is modelled by an ensemble of structures instead of one single structure. In comparison to conventional single-structure refinement, more reflections were required to determine a correct atomic distribution. A time-averaging simulation at 0.2 nm resolution (40 reflections) yielded an incorrect distribution, although a low R-factor was obtained. Simulations at 0.1 nm resolution (248 reflections) gave both low R-factors, 3 to 4%, and correct atomic distributions. The scale factor between the observed and time-averaged calculated structure factor amplitudes appeared to be unstable, when optimized during a time-averaging simulation. Tests of time-averaged restrained simulations with noise added to the observed structure-factor amplitudes, indicated that noise is modelled when no information in the form of constraints or restraints is available to distinguish it from real data.     

Protein-protein recognition analyzed by docking simulation.

Antibody-lysozyme and protease-inhibitor complexes are reconstituted by docking lysozyme as a rigid body onto the combining site of the antibodies and the inhibitors onto the active site of the proteases. Simplified protein models with one sphere per residue are subjected to simulated annealing using a crude energy function where the attractive component is proportional to the interface area. The procedure finds clusters of orientations in which a steric fit between the two protein components is achieved over a large contact surface. With five out of six complexes, the native structure of the complexes determined by X-ray crystallography is among those retained. Docked complexes are then subjected to conformational energy refinement with full atomic detail. With Fab HyHEL 5 and lysozyme, a native-like complex has the lowest refined energy. It can also be retrieved when starting with the X-ray structure of free lysozyme. However, some non-native complexes cannot be rejected: they form large interfaces, have a large number of H-bonds, and few unpaired polar groups. While these are necessary features of protein-protein recognition, they are not sufficient in determining specificity.     

The hydration shell of myoglobin

The space in the unit cell of a metmyoglobin crystal not occupied by myoglobin atoms was filled with water using Monte Carlo calculations. Independent calculations with different amounts of water have been performed. Structure factors were calculated using the water coordinates thus obtained and the known coordinates of the myoglobin atoms. A comparison with experimental structure factors showed that both the low and the high resolution regime could be well reproduced with 814 Monte Carlo water molecules per unit cell with a B-value of 50 Ų. The Monte Carlo water molecules yield a smaller standard R-value (0.166) than using a homogeneous electron density for the simulation of the crystal water (R = 0.212). A reciprocal space refinement of the water and the protein coordinates has been performed. Monte Carlo calculations can be used to obtain information for crystallographically invisible parts of the unit cell and yield better coordinates for the visible part in the refinement. Correspondence to: F. Parak     

Torsion angle dynamics: Reduced variable conformational sampling enhances crystallographic structure refinement

A reduced variable conformational sampling strategy for macromolecules based on molecular dynamics in torsion angle space is evaluated using crystallographic refinement as a prototypical search problem. Bae and Haug's algorithm for constrained dynamics [Bae, D. S., Haug, E. J. A recursive formulation for constrained mechanical system dynamics. Mech. Struct. Mach. 15:359–382, 1987], originally developed for robotics, was used. Their formulation solves the equations of motion exactly for arbitrary holonomic constraints, and hence differs from commonly used approximation algorithms. It uses gradients calculated in Cartesian coordinates, and thus also differs from internal coordinate formulations. Molecular dynamics can be carried out at significantly higher temperatures due to the elimination of the high frequency bond and angle vibrations. The sampling strategy presented here combines high temperature torsion angle dynamics with repeated trajectories using different initial velocities. The best solutions can be identified by the free R value, or the R value if experimental phase information is appropriately included in the refinement. Applications to crystallographic refinement show a significantly increased radius of convergence over conventional techniques. For a test system with diffraction data to 2 Å resolution, slow-cooling protocols fail to converge if the backbone atom root mean square (rms) coordinate deviation from the crystal structure is greater than 1.25 Å, but torsion angle refinement can correct backbone atom rms coordinate deviations up to approximately 1.7 Å. © 1994 Wiley-Liss, Inc.     

Protein–protein structure prediction by scoring molecular dynamics trajectories of putative poses

The prediction of protein–protein interactions and their structural configuration remains a largely unsolved problem. Most of the algorithms aimed at finding the native conformation of a protein complex starting from the structure of its monomers are based on searching the structure corresponding to the global minimum of a suitable scoring function. However, protein complexes are often highly flexible, with mobile side chains and transient contacts due to thermal fluctuations. Flexibility can be neglected if one aims at finding quickly the approximate structure of the native complex, but may play a role in structure refinement, and in discriminating solutions characterized by similar scores. We here benchmark the capability of some state‐of‐the‐art scoring functions (BACH‐SixthSense, PIE/PISA and Rosetta) in discriminating finite‐temperature ensembles of structures corresponding to the native state and to non‐native configurations. We produce the ensembles by running thousands of molecular dynamics simulations in explicit solvent starting from poses generated by rigid docking and optimized in vacuum. We find that while Rosetta outperformed the other two scoring functions in scoring the structures in vacuum, BACH‐SixthSense and PIE/PISA perform better in distinguishing near‐native ensembles of structures generated by molecular dynamics in explicit solvent. Proteins 2016; 84:1312–1320. © 2016 Wiley Periodicals, Inc.     

CMM—An enhanced platform for interactive validation of metal binding sites

Metal ions bound to macromolecules play an integral role in many cellular processes. They can directly participate in catalytic mechanisms or be essential for the structural integrity of proteins and nucleic acids. However, their unique nature in macromolecules can make them difficult to model and refine, and a substantial portion of metal ions in the PDB are misidentified or poorly refined. CheckMyMetal (CMM) is a validation tool that has gained widespread acceptance as an essential tool for researchers working on metal-macromolecule complexes. CMM can be used during structure determination or to validate metal binding sites in structural models within the PDB. The functionalities of CMM have recently been greatly enhanced and provide researchers with additional information that can guide modeling decisions. The new version of CMM shows metals in the context of electron density maps and allows for on-the-fly refinement of metal binding sites. The improvements should increase the reproducibility of biomedical research. The web server is available at https://cmm.minorlab.org .