Jan Stránský

Mori-Tanaka Based Estimates of Effective Thermal Conductivity of Various Engineering Materials

The purpose of this paper is to present a simple micromechanics-based model to estimate the effective thermal conductivity of macroscopically isotropic materials of matrix-inclusion type. The methodology is based on the well-established Mori-Tanaka method for composite media reinforced with ellipsoidal inclusions, extended to account for imperfect thermal contact at the matrix-inclusion interface, random orientation of particles and particle size distribution. Using simple ensemble averaging arguments, we show that the Mori-Tanaka relations are still applicable for these complex systems, provided that the inclusion conductivity is appropriately modified. Such conclusion is supported by the verification of the model against a detailed finite-element study as well as its validation against experimental data for a wide range of engineering material systems.

Structural and Catalytic Properties of S1 Nuclease from Aspergillus oryzae Responsible for Substrate Recognition, Cleavage, Non-Specificity, and Inhibition.

The single–strand–specific S1 nuclease from Aspergillus oryzae is an archetypal enzyme of the S1–P1 family of nucleases with a widespread use for biochemical analyses of nucleic acids. We present the first X–ray structure of this nuclease along with a thorough analysis of the reaction and inhibition mechanisms and of its properties responsible for identification and binding of ligands. Seven structures of S1 nuclease, six of which are complexes with products and inhibitors, and characterization of catalytic properties of a wild type and mutants reveal unknown attributes of the S1–P1 family. The active site can bind phosphate, nucleosides, and nucleotides in several distinguished ways. The nucleoside binding site accepts bases in two binding modes–shallow and deep. It can also undergo remodeling and so adapt to different ligands. The amino acid residue Asp65 is critical for activity while Asn154 secures interaction with the sugar moiety, and Lys68 is involved in interactions with the phosphate and sugar moieties of ligands. An additional nucleobase binding site was identified on the surface, which explains the absence of the Tyr site known from P1 nuclease. For the first time ternary complexes with ligands enable modeling of ssDNA binding in the active site cleft. Interpretation of the results in the context of the whole S1–P1 nuclease family significantly broadens our knowledge regarding ligand interaction modes and the strategies of adjustment of the enzyme surface and binding sites to achieve particular specificity.

Phosphate binding in the active centre of tomato multifunctional nuclease TBN1 and analysis of superhelix formation by the enzyme

Tomato multifunctional nuclease TBN1 belongs to the type I nuclease family, which plays an important role in apoptotic processes and cell senescence in plants. The newly solved structure of the N211D mutant is reported. Although the main crystal-packing motif (the formation of superhelices) is conserved, the details differ among the known structures. A phosphate ion was localized in the active site of the enzyme. The binding of the surface loop to the active centre is stabilized by the phosphate ion, which correlates with the observed aggregation of TBN1 in phosphate buffer. The conserved binding of the surface loop to the active centre suggests biological relevance of the contact in a regulatory function or in the formation of oligomers.

Best architecture of protein crystal. Database of protein-polymer interactions showing a unique role of PEG in protein crystallization

Protein molecules regularly ordered in crystal remain highly solvated and are continuously in dynamic equilibrium with solution. The stability of molecules in crystal is ensured by the 3D scaffold formed by intermolecular contacts between adhesive patches of neighbor molecules, whereas 30-80 % of crystal content remains dynamically disordered. That is why the stability of the 3D skeleton, i.e. protein crystal architecture (PCA) is so important. Large surface of protein molecules has usually many adhesive patches and thus we often observe different PCAs of the same protein (polymorphism). Each PCA has its own set of compatible adhesion modes and its own optimal solvent content in crystal. When incompatible adhesion modes are combined during crystal growth, one gets virtually non-diffracting solid phase. Principle of dominating adhesion mode plays a key role in the control of diffraction quality of crystal. It says that well diffracting crystals grow only when incompatible adhesion modes are suppressed. This can be achieved either by rational composition of crystallization solution using protein surface shielding agents (PSSA) [J.Synchr.Radiation (2011)18,50-52] or by chemical modification of the protein surface, or complexation with high affinity ligands. Analysis of already solved structures allows planned strengthening of dominant or weakening of non-compatible adhesion modes and a control of the diffraction quality of growing crystals. Theory of protein-protein adhesion modes shows why the poly(ethyleneglycol type polymers (PEGs) are the most successful precipitants [Z.Kristallogr.(2006)23,613-619]. Database of protein-polymers interactions (DPPI) [Z.Kristallogr.(2011)28,475-480] contains about 4000 of experimentally observed PEG-protein interfaces. It consists of a set of protein structures crystallizing with PEG and the script allowing easy visualization of PEG activities on protein surfaces. Seeing the PEG fragments interfering with protein-protein interfaces, with different types of salts, blocking competitive crystal contacts, protein oligomerization, crystallization, or biological activity, helps to understand the dynamic processes during crystal growth and allows rationalization of its performance on molecular basis. DPPI is available from hasekjh@seznam.cz. The project is supported by BIOCEV CZ.1.05/1.1.00/02.0109 from the ERDF, CSF project 15-15181S of the CSF, grant LG14009 of MSMT, BioStruct-X (EC FP7 project 283570) and Instruct of ESFRI. Figure 1. PEG induced adhesion mode as a leading motif for crystallization. The insert shows a typical crown ether conformation of polyether caused by interaction of five oxygens to lysine from one side. It induces strong hydrophobic interaction to the neighbor molecule on the opposite side. PDB code 3NBT.

Comparison of DEM-Based Wang Tilings and PUC

This contribution presents a statistical evaluation of microstructures assembled by means of2D Wang tiles, morphology of which is designed employing an algorithm based on Discrete ElementMethod. Comparison between realization of Wang tiling and Periodic Unit Cell representation is providedin terms of spatial descriptors. Namely, radial distribution function g2 and two-point probabilityfunction S2 are used to assess the artefacts related to periodicity in synthesized microstructures. Wedemonstrate significant reduction in periodicity achieved with Wang tiling (compared to the periodicsynthesis) in both the microstructures and results of numerical simulations.

Stochastic Wang Tiles Generation Using the Discrete Element Method

An algorithm generating morphology of 2D and 3D stochastic Wang tiles is presented in thiscontribution. The algorithm is based on the discrete element method (DEM) and is therefore applicableprimarily for matrix-based microstructures with separate inclusions with emphasis on producingmaximally random dense packings. An open-source free DEM code YADE was used for all the computations.The method is illustrated with a 2D exemplary realization of Wang tiling and comparisonagainst Periodic Unit Cell representation in terms of spatial statistics is provided.

Changes of LLT1, a ligand for human NKR-P1, with varied glycosylation and crystallization conditions

Natural killer (NK) cells are a type of lymphocytes which kill tumor, virally infected or stressed cells. Decision to kill a cell is made as a result of balance of signals from plenty of activating and inhibitory receptors on surface of the natural killer cells. LLT1 is a ligand expressed primarily on activated lymphocytes (including NK cells itself). It is a binding partner for NKR-P1, receptor on surface of NK cells. Both NKR-P1 and LLT1 have an extracellular part of C-type lectin like (CTL) fold. Receptors and ligands with CTL fold have not been yet excessively studied and their interactions are not still understood. Here we would like to present four crystal structures of LLT1 which we have recently published [1]. LLT1 with homogenous GlcNAc2Man5 glycosylation was expressed in HEK293S GnTIcells [2]. The four LLT1 structures differ by its oligomeric state (monomeric, dimeric and hexameric [three dimers in compact packing]) under various pH. Monomeric and dimeric LLT1 crystal structures originate from protein deglycosylated after the first GlcNAc, while the hexameric form corresponds to LLT1 with the original GlcNAc2Man5 glycosylation. The poster will present types of crystal interactions leading to formation of the four crystal structures. This study was supported by BIOCEV CZ.1.05/1.1.00/02.0109 from the ERDF, by the Czech Science Foundation (project 15-15181S), by the Ministry of Education, Youth and Sports of the Czech Republic (grant LG14009), by Charles University (UNCE 204025/2012, SVV 260079/2014, GA UK 161216), BioStruct-X (EC FP7 project 283570) and Instruct, part of the European Strategy Forum on Research Infrastructures (ESFRI) supported by national member subscriptions. [1] Skálová et al., Acta Cryst., 2015, D71, 578-591. (Open access) [2] Bláha et al., Protein Expres. Purif. 2015, 109, 7-13. Figure 1. Structure of dimeric LLT1 with denoted positions of glycosylation sites.

Optimization in S-SAD phasing - difference between solved and unsolved structure

J. Stránský, T. Kovaľ, L. Østergaard, J. Dušková, T. Skálová, J. Hašek, P. Kolenko, K. Fejfarová, J. Dohnálek Academy of Sciences of the Czech Republic, Institute of Biotechnology, Prague, Czech Republic, Czech Technical University in Prague, Faculty of Nuclear Sciences and Physical Engineering, Prague, Czech Republic, Academy of the Sciences of the Czech Republic, Institute of Macromolecular Chemistry, Prague , Czech Republic, Novozymes A/S, Bagsvaerd, Denmark

Correction: Sejnoha, M. et al. Mori-Tanaka Based Estimates of Effective Thermal Conductivity of Various Engineering Materials. Micromachines 2011, 2, 129–149

We have discovered a mistake in our original derivation related to the definition of the apparent conductivity due to orientation averaging. [...]