Vladimir Y. Lunin

On macromolecular refinement at subatomic resolution with interatomic scatterers

Modelling deformation electron density using interatomic scatters is simpler than multipolar methods, produces comparable results at subatomic resolution and can easily be applied to macromolecules.

Atomic structure of the Serratia marcescens endonuclease at 1.1 A resolution and the enzyme reaction mechanism.

The three-dimensional crystal structure of Serratia marcescens endonuclease has been refined at 1.1 A resolution to an R factor of 12.9% and an R(free) of 15.6% with the use of anisotropic temperature factors. The model contains 3694 non-H atoms, 715 water molecules, four sulfate ions and two Mg(2+)-binding sites at the active sites of the homodimeric protein. It is shown that the magnesium ion linked to the active-site Asn119 of each monomer is surrounded by five water molecules and shows an octahedral coordination geometry. The temperature factors for the bound Mg(2+) ions in the A and B subunits are 7.08 and 4.60 A(2), respectively, and the average temperature factors for the surrounding water molecules are 12.13 and 10.3 A(2), respectively. In comparison with earlier structures, alternative side-chain conformations are defined for 51 residues of the dimer, including the essential active-site residue Arg57. A plausible mechanism of enzyme function is proposed based on the high-resolution S. marcescens nuclease structure, the functional characteristics of the natural and mutational forms of the enzyme and consideration of its structural analogy with homing endo-nuclease I-PpoI.

A procedure compatible with X-PLOR for the calculation of electron-density maps weighted using an R-free-likelihood approach

A program has been developed that uses an R-free-likelihood-based technique to estimate the errors of phases calculated from atomic models. This technique allows one to obtain realistic estimates when it is applied to refined models. The program reads a file of structure factors in the X-PLOR format and creates a new one that contains information necessary to calculate weighted maps. The output file can be used directly by X-PLOR.

Low-resolution data analysis for low-density lipoprotein particle

The knowledge of the molecular structure of LDL, a large lipoprotein complex, is of great interest for medical investigations. Currently available LDL crystals do not diffract to high resolution and do not allow the application of standard crystallographic techniques. Additional difficulties arise because of a very dense crystal packing and the presence of several components with quite different mean densities. Several ab initio phasing methods previously reported by the authors have been successfully applied to find a crystallographic image of LDL at a resolution of 27 A. The most promising results have been obtained using direct phasing with a connectivity analysis of the electron-density maps. The current image makes it possible to discern a single particle covered by a layer of relatively high density that is asymmetrically distributed on the particle surface. It shows a partition of high and low densities inside the particle and, in particular, strips of varying density in the lipid core.

Metrics for comparison of crystallographic maps

Rank scaling of Fourier syntheses leads to new tools for the comparison of crystallographic contour maps. The new metrics are in better agreement with a visual map analysis than the conventional map correlation coefficient.

X-ray-induced deterioration of disulfide bridges at atomic resolution.

Overall and site-specific X-ray-induced damage to porcine pancreatic elastase was studied at atomic resolution at temperatures of 100 and 15 K. The experiments confirmed that irradiation causes small movements of protein domains and bound water molecules in protein crystals. These structural changes occur not only at 100 K but also at temperatures as low as 15 K. An investigation of the deterioration of disulfide bridges demonstrated the following. (i) A decrease in the occupancy of S(γ) atoms and the appearance of new cysteine rotamers occur simultaneously. (ii) The occupancy decrease is observed for all S(γ) atoms, while new rotamers arise for some of the cysteine residues; the appearance of new conformations correlates with the accessibility to solvent. (iii) The sum of the occupancies of the initial and new conformations of a cysteine residue is approximately equal to the occupancy of the second cysteine residue in the bridge. (iv) The most pronounced changes occur at doses below 1.4 × 10(7) Gy, with only small changes occurring at higher doses. Comparison of the radiation-induced changes in an elastase crystal at 100 and 15 K suggested that the dose needed to induce a similar level of deterioration of the disulfide bonds and atomic displacements at 15 K to those seen at 100 K is more than two times higher.

Seminvariant density decomposition and connectivity analysis and their application to very low resolution macromolecular phasing.

A low-resolution Fourier synthesis is thought to show a molecule as a compact region of a high electron density. As a consequence, the number of such regions, chosen at a proper cut-off level, should be equal to the number of molecules in the unit cell. This hypothesis may be used as a basis for selection criteria in multisolution ab initio phasing procedures. However, when working with a small number of reflections, this hypothesis may break down. The suggested Fourier-synthesis decomposition explains some reasons for failure and provides a connectivity-based procedure for the determination of macromolecular position in the crystal unit cell and the phasing of several low-resolution reflections. The simplest decomposition consists in separating the reflections into two sets according to whether their phases do or do not depend on a permitted origin shift. It is shown that the partial Fourier syntheses corresponding to these subsets are simply a half-sum and a half-difference of the initial electron-density distribution with its shifted copy. Therefore, they display the true images overlapped with the shifted ones (or with shifted and additionally flipped copies for the latter synthesis). The paper generalizes the decomposition for the case of a finite subgroup of the group of permitted origin shifts and reveals the role of one-phase sem-invariants.

On the Ab Initio Solution of the Phase Problem for Macromolecules at Very Low Resolution. II. Generalized Likelihood Based Approach to Cluster Discrimination

The multisolution strategies for direct phasing at very low resolution, such as the few atoms model technique, result in a number of alternative phase sets, each of them arising from a cluster of closely related models. Use of a Monte-Carlo type computer procedure is suggested to choose between the possible phase sets. It consists of generating a large number of pseudo-atom models inside the mask defined by a trial phase set and the use of histograms of magnitude correlation to evaluate the masks. It is shown that the procedure may be considered as a generalization of the statistical maximum-likelihood principle and may be used as a powerful supplementary tool in the likelihood-based approaches to the phase problem solution.

Direct phasing by binary integer programming

In the absence of phase information, a variety of electron-density distributions is consistent with the observed magnitudes. This ambiguity may be reduced significantly if the distribution values are restricted to 0 or 1, i.e. when the object of search is an envelope rather than a continuous electron-density distribution. The binarizing in both real (the grid-point density values) and reciprocal (the phases) spaces allows the usual structure-factor equations to be replaced by a system of linear inequalities with binary unknowns. A special computer procedure is applied to obtain several sets of values, which satisfy or almost satisfy these inequalities. The averaging of the found phase sets allows the final map to be calculated. The approach was tested with calculated and experimental data for a known protein structure. The size of the grid for the envelope calculation is at the moment the major limitation of the approach. Nevertheless, even for a very small grid, some structure information can be extracted and used as a starting point for further phase improvement or as a way to solve the molecular replacement problem.

X-Ray-Radiation-Induced Cooperative Atomic Movements in Protein

X-rays interact with biological matter and cause damage. Proteins and other macromolecules are damaged primarily by ionizing X-ray photons and secondarily by reactive radiolytic chemical species. In particular, protein molecules are damaged during X-ray diffraction experiments with protein crystals, which is, in many cases, a serious hindrance to structure solution. The local X-ray-induced structural changes of the protein molecule have been studied using a number of model systems. However, it is still not well understood whether these local chemical changes lead to global structural changes in protein and what the mechanism is. We present experimental evidence at atomic resolution indicating the movement of large parts of the protein globule together with bound water molecules in the early stages of radiation damage to the protein crystal. The data were obtained from a crystal cryocooled to approximately 100 K and diffracting to 1 A. The movement of the protein structural elements occurs simultaneously with the decarboxylation of several glutamate and aspartate residues that mediate contacts between moving protein structural elements and with the rearrangement of the water network. The analysis of the anisotropy of atomic displacement parameters reveals that the observed atomic movements occur at different rates in different unit cells of the crystal. Thus, the examination of the cooperative atomic movement enables us to better understand how radiation-induced local chemical and structural changes of the protein molecule eventually lead to disorder in protein crystals.