Benoit Guillot

Advances in protein and small-molecule charge-density refinement methods using MoPro

With an increasing number of biological macromolecule structures solved at ultra-high resolution and with the advances of supramolecular chemistry, it becomes necessary to extend to large systems experimental charge-density study methods that are usually applied to small molecules. The latest developments in the refinement program MoPro (Molecular Properties), dedicated to the charge-density refinement at (sub)atomic resolution of structures ranging from small molecules to biological macromolecules, are presented. MoPro uses the Hansen & Coppens [Acta Cryst. (1978), A34, 909–921] multipolar pseudo-atom model for the electron-density refinement. Alternative methods are also proposed, such as modelling bonding and lone-pair electron density by virtual spherical atoms. For proteins at atomic resolution, a charge-density database developed in the laboratory enables the transfer of multipolar parameters. The program allows complex refinement strategies to be written and has numerous restraints, constraints and analysis tools for use in the structure and electron-density analysis. New kappa and multipolar parameter restraints/constraints are also implemented and discussed. Furthermore, constraints on the electron density, such as local symmetry and atom equivalence, are easily defined. Some examples of applications, from small molecules to large unit cells (including the enzyme aldose reductase), are given in order to guide the MoPro user and to show the large field of applicability of this code.

Refinement of proteins at subatomic resolution with MOPRO

Crystallography at subatomic resolution permits the observation and measurement of the non-spherical character of the atomic electron density. Charge density studies are being performed on molecules of increasing size. The MOPRO least-squares refinement software has thus been developed, by extensive modifications of the program MOLLY, for protein and supramolecular chemistry applications. The computation times are long because of the large number of reflections and the complexity of the multipolar model of the atomic electron density; the structure factor and derivative calculations have thus been parallelized. Stereochemical and dynamical restraints as well as the conjugate gradient algorithm have been implemented. A large number of the normal matrix off-diagonal terms turn out to be very small and the block diagonal approximation is thus particularly efficient in the case of large structures at very high resolution.

On the application of an experimental multipolar pseudo-atom library for accurate refinement of small-molecule and protein crystal structures

With an increasing number of biomacromolecular crystal structures being measured to ultra-high resolution, it has become possible to extend to large systems experimental charge-density methods that are usually applied to small molecules. A library has been built of average multipole populations describing the electron density of chemical groups in all 20 amino acids found in proteins. The library uses the Hansen & Coppens multipolar pseudo-atom model to derive molecular electron density and electrostatic potential distributions. The library values are obtained from several small peptide or amino acid crystal structures refined against ultra-high-resolution X-ray diffraction data. The library transfer is applied automatically in the MoPro software suite to peptide and protein structures measured at atomic resolution. The transferred multipolar parameters are kept fixed while the positional and thermal parameters are refined. This enables a proper deconvolution of thermal motion and valence-electron-density redistributions, even when the diffraction data do not extend to subatomic resolution. The use of the experimental library multipolar atom model (ELMAM) also has a major impact on crystallographic structure modelling in the case of small-molecule crystals at atomic resolution. Compared to a spherical-atom model, the library transfer results in a more accurate crystal structure, notably in terms of thermal displacement parameters and bond distances involving H atoms. Upon transfer, crystallographic statistics of fit are improved, particularly free R factors, and residual electron-density maps are cleaner.

A comparison between experimental and theoretical aspherical-atom scattering factors for charge-density refinement of large molecules

The differences between two databases describing the polypeptide main chain in terms of charge-density parameters, directly usable in protein structure refinements, are discussed. These databases contain averaged multipole populations of peptide pseudo-atoms obtained from refinement against theoretical simulated data and against high-resolution experimental data on small peptide or amino acid molecules. The main discrepancy becomes apparent when electrostatic properties are calculated.

Elucidation of the phosphate binding mode of DING proteins revealed by subangstrom X-ray crystallography.

PfluDING is a bacterial protein isolated from Pseudomonas fluorescens that belongs to the DING protein family, which is ubiquitous in eukaryotes and extends to prokaryotes. DING proteins and PfluDING have very similar topologies to phosphate Solute Binding Proteins (SBPs). The three-dimensional structure of PfluDING was obtained at subangstrom resolution (0.88 and 0.98 A) at two different pHs (4.5 and 8.5), allowing us to discuss the hydrogen bond network that sequesters the phosphate ion in the binding site. From this high resolution data, we experimentally elucidated the molecular basis of phosphate binding in phosphate SBPs. The phosphate ion is tightly bound to the protein via 12 hydrogen bonds between phosphate oxygen atoms and OH and NH groups of the protein. The proton on one oxygen atom of the phosphate dianion forms a 2.5 A low barrier hydrogen bond with an aspartate, with the energy released by forming this strong bond ensuring the specificity for the dianion even at pH 4.5. In particular, contrary to previous theories on phosphate SBPs, accurate electrostatic potential calculations show that the binding cleft is positively charged. PfluDING structures reveal that only dibasic phosphate binds to the protein at both acidic and basic phosphate, suggesting that the protein binding site environment stabilizes the HPO(4)(2-) form of phosphate.

Structural analysis and multipole modelling of quercetin monohydrate – a quantitative and comparative study

The multipolar atom model, constructed by transferring the charge-density parameters from an experimental or theoretical database, is considered to be an easy replacement of the widely used independent atom model. The present study on a new crystal structure of quercetin monohydrate [2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one monohydrate], a plant flavonoid, determined by X-ray diffraction, demonstrates that the transferred multipolar atom model approach greatly improves several factors: the accuracy of atomic positions and the magnitudes of atomic displacement parameters, the residual electron densities and the crystallographic figures of merit. The charge-density features, topological analysis and electrostatic interaction energies obtained from the multipole models based on experimental database transfer and periodic quantum mechanical calculations are found to compare well. This quantitative and comparative study shows that in the absence of high-resolution diffraction data, the database transfer approach can be applied to the multipolar electron density features very accurately.

Charge-density analysis of a protein structure at subatomic resolution: the human aldose reductase case

The valence electron density of the protein human aldose reductase was analyzed at 0.66 angstroms resolution. The methodological developments in the software MoPro to adapt standard charge-density techniques from small molecules to macromolecular structures are described. The deformation electron density visible in initial residual Fourier difference maps was significantly enhanced after high-order refinement. The protein structure was refined after transfer of the experimental library multipolar atom model (ELMAM). The effects on the crystallographic statistics, on the atomic thermal displacement parameters and on the structure stereochemistry are analyzed. Constrained refinements of the transferred valence populations Pval and multipoles Plm were performed against the X-ray diffraction data on a selected substructure of the protein with low thermal motion. The resulting charge densities are of good quality, especially for chemical groups with many copies present in the polypeptide chain. To check the effect of the starting point on the result of the constrained multipolar refinement, the same charge-density refinement strategy was applied but using an initial neutral spherical atom model, i.e. without transfer from the ELMAM library. The best starting point for a protein multipolar refinement is the structure with the electron density transferred from the database. This can be assessed by the crystallographic statistical indices, including Rfree, and the quality of the static deformation electron-density maps, notably on the oxygen electron lone pairs. The analysis of the main-chain bond lengths suggests that stereochemical dictionaries would benefit from a revision based on recently determined unrestrained atomic resolution protein structures.

Ultra-high-resolution X-ray structure of proteins

The constant advances in synchrotron radiation sources and crystallogenesis methods and the impulse of structural genomics projects have brought biocrystallography to a context favorable to subatomic resolution protein and nucleic acid structures. Thus, as soon as such precision can be frequently obtained, the amount of information available in the precise electron density should also be easily and naturally exploited, similarly to the field of small molecule charge density studies. Indeed, the use of a nonspherical model for the atomic electron density in the refinement of subatomic resolution protein structures allows the experimental description of their electrostatic properties. Some methods we have developed and implemented in our multipolar refinement program MoPro for this purpose are presented. Examples of successful applications to several subatomic resolution protein structures, including the 0.66Å resolution human aldose reductase, are described.

Topological analysis of hydrogen bonds and weak interactions in protein helices via transferred experimental charge density parameters.

Helices represent the most abundant secondary structure motif in proteins and are often involved in various functional roles. They are stabilized by a network of hydrogen bonds between main chain carbonyl and amide groups. Several surveys scrutinized these hydrogen bonds, mostly based on the geometry of the interaction. Alternatively, the topological analysis of the electron density provides a powerful technique to investigate hydrogen bonds. For the first time, transferred experimental charge density parameters (ELMAM database) were used to carry out a topological analysis of the electron density in protein helices. New parameters for the description of the hydrogen bond geometry are proposed. Bonding contacts between the amide N and carbonyl O atoms (N···O) of helices, poorly addressed in the literature so far, were characterized from topological, geometrical, and local energetic analyses. Particularly, a geometrical criterion allowing for the discrimination between hydrogen bonds and N···O contacts is proposed.

Charge-density analysis and electrostatic properties of 2-carboxy-4-methylanilinium chloride monohydrate obtained using a multipolar and a spherical-charges model

The new crystal structure of 2-carboxy-4-methylanilinium chloride monohydrate was determined by X-ray diffraction and refined using three different electron-density models. In the first model, the ELMAM2 multipolar electron-density database was transferred to the molecule. Theoretical structure factors were also computed from periodic density functional theory calculations and yielded, after multipolar-atoms refinement, the second charge-density model. An alternative electron-density modelling, based on spherical atoms and additional charges on the covalent bonds and electron lone-pair sites, was used in the third model in the refinement versus the theoretical data. The crystallographic refinements, structural properties, electron-density distributions and molecular electrostatic potentials obtained from the different charge-density models were compared. As the number of variables refined in the different models is the same, the R factor is a good indicator of refinement quality. The R factor is best for multipolar modelling, presumably because of the greater flexibility and larger number of parameters to model the electron density compared to the spherical-charges model. The electrostatic potentials around the molecule show a high correlation coefficient between the three models.