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Acta Cryst. (2014). A70, C211
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Members of eukaryotic nuclease I family are usually zinc, magnesium or calcium dependent, relatively small (about 300 amino acids) glycoproteins with important roles in various apoptotic processes, stress response, DNA repair machinery or sustenance scavenging. They produce 5'-mononucleotides, inorganic phosphate and mononucleosides as end products, have acidic pH optima and are able to cleave different homopolymers with usually no preference for DNA or RNA. P1/S1-like nucleases, a subgroup of nuclease I family, are zinc-dependent, with phospholipase C-like fold. They can be divided to single-strand specific (eg. S1 nuclease from Aspergillus oryzae) or unspecific. TBN1 from Solanum lycopersicum (tomato) is an unspecific P1/S1-like nuclease composed of 277 amino acids with a molecular mass of 37 kDa (when fully glycosylated). TBN1 plays an important role in specific apoptotic functions and cell senescence in plants and also exhibits anticancerogenic properties [1]. For our studies TBN1 was produced recombinantly in Nicotiana benthamiana leafs. Crystals were obtained using a combination of salt and polymer. Datasets for structural analysis were collected at BESSY II (Helmholtz-Zentrum Berlin) [2]. The final model was built and refined using data to 2.15 Å resolution. TBN1 is mainly α-helical with fold stabilized by four disulfide bridges and by the catalytic zinc cluster coordinated at the bottom of the active site cleft. Three oligosaccharides bonded on the surface significantly contribute to solubility of the enzyme. Oligomerization of TBN1 is mediated by binding of a peptide chain to the active site of a neighboring molecule and can be induced by inorganic phosphate. Based on the distribution of surface residues the possible binding sites for nucleic acids with secondary structure were identified. The newly discovered phospholipase activity significantly broadens the substrate promiscuity of TBN1 [3]. This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (grant No. EE2.3.30.0029), by the project ,,BIOCEV – Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University (CZ.1.05/1.1.00/02.0109), from the European Regional Development Fund, Grant Agency of the Czech Technical University in Prague, grant No. SGS13/219/OHK4/3T/14 and by Institute of Plant Molecular Biology, Biology Centre, AS CR, RVO:60077344.
Keywords: nuclease.

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Acta Cryst. (2014). A70, C249
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Natural killer (NK) cells, large granular lymphocytes, play an important role in the innate immune response against viruses, parasites and tumour cells. NK cells use a wide repertoire of surface receptors to modulate their activity [1]. The family of NKR-P1 surface receptors of NK cells belong to proteins with C-type lectin-like (CTL) fold. The overall architecture of other known CTL receptors (e.g. members of Ly49 family, NKG2D, CD94, mouse CLRg) is conserved [2]. The mechanism of ligand binding has been revealed by the crystal structure of Nkp65 bound to its keratinocyte ligand [3]. However, observation of domain swapping in crystal structure of mouse (m) NKR-P1A represents an unusual structural feature that might be involved in a new mechanism of ligand binding that would be specific for some members of NKR-P1 family. Nevertheless, our crystal structure of mNKR-P1A represents a unique structural observation that demands careful analysis. Even the latest structural studies do not answer the question of function or role of swapped domain of the receptor in potential ligand binding. We have generated new variants of mNKR-P1A of varied chain length that undergo biochemical and structural analysis including mass spectrometry.

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Acta Cryst. (2014). A70, C254
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Natural killer (NK) cells are large granular lymphocytes with innate immune reaction against tumor cells or cells affected by viral infection. They have a variety of receptors on their surface which mediate contact with cells tested by NK cells. Using X-ray crystallography, we determined a structure of an extracellular part of mouse C-type lectin related protein g (Clr-g, [1], PDB code 3RS1), a ligand for NK receptor NKR-P1F. The ligand and the receptor are both of C-type lectin like fold and this is the first determined structure of a CTL ligand of an NK receptor. The protein was produced in E. coli. The rod-like crystals appeared by spontaneous crystallization of the pure protein (2.5 mg/ml) on tube walls. Diffraction data of the vitrified crystal were measured at synchrotron BessyII of HZB in Berlin and were processed up to 1.95 Å. The protein was found in the form of dimers similar to that of CD69. The N-terminus of the chain (residues Met, Asn, Lys) is in the crystal bound to a neighbor dimer and shows thus that binding of a peptide to mouse Clr-g is possible, although not expected or confirmed by other experiments known to us. A model of interaction of Clr-g with NKR-P1F was designed based on electrostatic complementarity of both molecules. This work was supported by the Czech Science Foundation (grant No. P302/11/0855), Ministry of Education, Youth and Sports of the Czech Republic (grant No. LG14009) and by the project BIOCEV CZ.1.05/1.1.00/02.0109 from the ERDF.

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Acta Cryst. (2014). A70, C446
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Carnivorous pitcher plants of the genus Nepenthes secrete their own aspartic proteases, nepenthesins, to digest prey. Nepenthesins differ significantly in sequence from other plant aspartic proteases. This difference, which brings more cysteine residues into the structure of nepenthesins, in conjunction with putative N-glycosylation, can contribute to uniquely high temperature and pH stabilities of these proteases [1, 2]. In continuation of our previous study of the expression and biochemical and enzymatic characterization of a recombinant form of nepenthesin-1 (rNep-1) from Nepenthes gracilis [3], we report its crystallization and preliminary X-ray analysis. Crystals of rNep-1 in complex with the pepstatin A inhibitor have been grown using the hanging-drop vapour-diffusion technique. Diffraction data were collected to 2.9 Å resolution using synchrotron radiation at Bessy II of HZB, Berlin. The crystals belong to space group P21, with unit-cell parameters a = 54.4 Å, b = 86.6 Å, c = 95.8 Å, β = 1060. The self-rotation function combined with solvent-content calculations and Matthews coefficient suggest the presence of two molecules of rNep-1 in the asymmetric unit. This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (grants No. EE2.3.30.0029 and No. LG14009), by BIOCEV CZ.1.05/1.1.00/02.0109 from the European Regional Development Fund, and by the Grant Agency of the Czech Technical University in Prague, grant No. SGS13/219/OHK4/3T/14.

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Acta Cryst. (2014). A70, C613
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Development of X-ray diffraction technologies have made de novo phasing of protein structures by single-wavelength anomalous dispersion by sulphur (S-SAD) more common. As anomalous differences in the sulphur atomic factors are in the order of errors of measurement, careful intensity reading and data processing are crucial. S-SAD was used for de novo phasing of a small 12 kDa protein with 4 sulphur atoms per molecule at 2.3 Å, where the data did not enable a straightforward structure solution. Data processing was performed using XDS [1] and scaling using XSCALE. The sulphur substructure was determined by SHELXD [2] and phases were obtained from SHELXE [2]. Both algorithms strongly depend on input parameters and default values did not lead to the correct phases. Therefore a systematic search of optimal values of several parameters was used to find a solution. This method helped to confirm sulphur substructure and to differentiate the handedness of the solutions. Moreover, a script for comfortable conversion of SHELX outputs to MTZ format was developed, using programmes included in the CCP4 package [3]. The previously unsolvable protein structure was successfully resolved with the described procedure. This work was supported by the Grant Agency of the Czech Technical University in Prague, (SGS13/219/OHK4/3T/14), the Czech Science Foundation (P302/11/0855), project BIOCEV CZ.1.05/1.1.00/02.0109 from the ERDF.
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