T. Petrova

Neutron structure of type III Antifreeze Protein allows the reconstruction of AFP-ice interface

Antifreeze proteins (AFPs) inhibit ice growth at sub‐zero temperatures. The prototypical type‐III AFPs have been extensively studied, notably by X‐ray crystallography, solid‐state and solution NMR, and mutagenesis, leading to the identification of a compound ice‐binding surface (IBS) composed of two adjacent ice‐binding sections, each which binds to particular lattice planes of ice crystals, poisoning their growth. This surface, including many hydrophobic and some hydrophilic residues, has been extensively used to model the interaction of AFP with ice. Experimentally observed water molecules facing the IBS have been used in an attempt to validate these models. However, these trials have been hindered by the limited capability of X‐ray crystallography to reliably identify all water molecules of the hydration layer. Due to the strong diffraction signal from both the oxygen and deuterium atoms, neutron diffraction provides a more effective way to determine the water molecule positions (as D2O). Here we report the successful structure determination at 293 K of fully perdeuterated type‐III AFP by joint X‐ray and neutron diffraction providing a very detailed description of the protein and its solvent structure. X‐ray data were collected to a resolution of 1.05 Å, and neutron Laue data to a resolution of 1.85 Å with a “radically small” crystal volume of 0.13 mm3. The identification of a tetrahedral water cluster in nuclear scattering density maps has allowed the reconstruction of the IBS‐bound ice crystal primary prismatic face. Analysis of the interactions between the IBS and the bound ice crystal primary prismatic face indicates the role of the hydrophobic residues, which are found to bind inside the holes of the ice surface, thus explaining the specificity of AFPs for ice versus water. Copyright

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.

High-resolution neutron and X-ray diffraction room-temperature studies of an H-FABP-oleic acid complex: study of the internal water cluster and ligand binding by a transferred multipolar electron-density distribution

Neutron and high-resolution X-ray crystallography were used to determine fully the structure of the internal water cluster in H-FABP. Analysis of the orientation and electrostatic properties of the water molecules showed significant alignment of the permanent dipoles of the water molecules with the protein electrostatic field.

A likelihood-based search for the macromolecular position in the crystalline unit cell

The goal of this work is to analyse whether the generalized likelihood criterion can be used to find the best spherical envelope of a macromolecule in a unit cell. A family of spherical envelopes is ranged in accordance with their likelihood values calculated by means of a Monte Carlo-type computer procedure. Two kinds of envelope families were tested. The first one was composed of spherical envelopes of fixed radius but different positions in the unit cell. In the second case, the sphere radii were linked to their centre position so that the total volume occupied by all symmetry-related spheres was roughly equal to the total volume occupied by the real molecule. The experiments showed that when using the first type of envelope the level of the signal for the right solution is higher than the one obtained with the straightforward R-factor-based single-Gaussian-atom search, but spurious maxima (usually placed on the symmetry axes) may still exist. The use of the second type of envelope family reduces the level of the spurious maxima.

Perdeuteration: improved visualization of solvent structure in neutron macromolecular crystallography

The 1.8 Å resolution neutron structure of deuterated type III antifreeze protein in which the methyl groups of leucine and valine residues are selectively protonated is presented. Comparison between this and the 1.85 Å resolution neutron structure of perdeuterated type III antifreeze protein indicates that perdeuteration improves the visibility of solvent molecules located in close vicinity to hydrophobic residues, as cancellation effects between H atoms of the methyl groups and nearby heavy-water molecules (D2O) are avoided.

Crystal packing modifies ligand binding affinity: The case of aldose reductase

The relationship between the structures of protein–ligand complexes existing in the crystal and in solution, essential in the case of fragment‐based screening by X‐ray crystallography (FBS‐X), has been often an object of controversy. To address this question, simultaneous co‐crystallization and soaking of two inhibitors with different ratios, Fidarestat (FID; Kd = 6.5 nM) and IDD594 (594; Kd = 61 nM), which bind to h‐aldose reductase (AR), have been performed. The subatomic resolution of the crystal structures allows the differentiation of both inhibitors, even when the structures are almost superposed. We have determined the occupation ratio in solution by mass spectrometry (MS) Occ(FID)/Occ(594) = 2.7 and by X‐ray crystallography Occ(FID)/Occ(594) = 0.6. The occupancies in the crystal and in solution differ 4.6 times, implying that ligand binding potency is influenced by crystal contacts. A structural analysis shows that the Loop A (residues 122–130), which is exposed to the solvent, is flexible in solution, and is involved in packing contacts within the crystal. Furthermore, inhibitor 594 contacts the base of Loop A, stabilizing it, while inhibitor FID does not. This is shown by the difference in B‐factors of the Loop A between the AR–594 and AR–FID complexes. A stable loop diminishes the entropic energy barrier to binding, favoring 594 versus FID. Therefore, the effect of the crystal environment should be taken into consideration in the X‐ray diffraction analysis of ligand binding to proteins. This conclusion highlights the need for additional methodologies in the case of FBS‐X to validate this powerful screening technique, which is widely used. Proteins 2012.

New possibilities of X-ray nanocrystallography of biological macromolecules based on X-ray free-electron lasers

X-ray serial nanocrystallography is a new technique for determining the three-dimensional structure of biological macromolecules from data on the diffraction of ultrashort pulses generated by X-ray free-electron lasers. The maximum achievable resolution for a set of experimental data as a function of the sample sizes and parameters of the equipment is estimated based on simulations of the diffraction process with allowance for changes in the electronic structure of the atoms of the sample under the influence of X-rays. Estimates show that nanocrystallography greatly enhances the possibilities of X-ray analysis, reducing the requirements for the minimum permitted size of the crystals and enabling to explore poorly crystallizable molecular objects, such as many membrane proteins and complexes of macromolecules.

Mask-based approach to phasing of single-particle diffraction data

A Monte Carlo-type approach for low- and medium-resolution phasing of single-particle diffraction data is suggested. Firstly, the single-particle phase problem is substituted with the phase problem for an imaginary crystal. A unit cell of this crystal contains a single isolated particle surrounded by a large volume of bulk solvent. The developed phasing procedure then generates a large number of connected and finite molecular masks, calculates their Fourier coefficients, selects the sets with magnitudes that are highly correlated with the experimental values and finally aligns the selected phase sets and calculates the averaged phase values. A test with the known structure of monomeric photosystem II resulted in phases that have 97% correlation with the exact phases in the full 25 Å resolution shell (1054 structure factors) and correlations of 99, 94, 81 and 79% for the resolution shells ∞-60, 60-40, 40-30 and 30-25 Å, respectively. The same procedure may be used for crystallographic ab initio phasing.

Femtosecond X-ray free-electron lasers: A new tool for studying nanocrystals and single macromolecules

A brief overview of the design of femtosecond X-ray free-electron lasers (XFEL), characteristics of the emitted X-ray pulses, and potentialities of XFEL are presented. A concise analysis of the problems in modeling X-ray scattering patterns produced by ultraintense radiation sources is given.

X-rays-Induced Cooperative Atomic Movement in a Protein Crystal

Protein molecules are damaged during X-ray diffraction experiments with protein crystals, which is in many cases a serious hindrance to structure solution. It is still not well understood whether radiation-induced local chemical changes lead to global structural changes in protein and what the mechanism is. We present experimental evidence at atomic resolution that irradiation causes the displacement of big parts of the protein molecule and water network. Radiation-induced structural changes in a protein molecule were studied in a series of diffraction experiments in which multiple data sets corresponding to increasing absorbed doses were collected from the same crystals of human aldose reductase (h-AR) and elastase at atomic resolution. There is a pronounced correlation between collective atomic movements and local and global damage to the crystal. Radiation-induced atomic shifts start at places with the pronounced local damage and are the largest for the damaged residues and structure fragments connected to damaged residues. An analysis of atomic displacement parameters (ADPs) revealed a distinct increase in the anisotropic character of ADP’s for the atoms of some segments of the structures. This effect was pronounced for those atoms that initially had approximately isotropic ADPs and shifted over relatively large distances during irradiation. Because their displacements in different cells of the crystal occur not exactly at the same moment, this leads to an additional static disorder component.