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SOLSA is the first automated expert system for on-site cores analysis. The scope is to provide a prototype to be an innovative and necessary tool for geo-metallurgy, in order to optimize the valorization of the ore. The Expert System consists in the combination of an integrated drilling rig providing cores of high quality, an automated scanner and phase identification software, developed for nickel laterites and bauxites but usable as well in other sectors. SOLSA combines non-destructive sensors and the whole system is driven by an innovative, user-friendly and intelligent software. SOLSA provides more complete information while optimizing the exploration stage, with a significant reduction of costs and return time. Such objective involves, in the first place, to fast, cheaper and systematic acquisition of the data needed for optimizing the process. The adding value takes place first at the exploration or grade control stage, furnishing systematic characterization and regionalization of the different types of ore. Then such information can be used for improving the ore scheduling at the mining and processing stages, toward improving the recovery and efficiency of the processing

Background and objectives: Vascular calcification (VC) is one of the factors associated with mortality in hemodialysis (HD) patients. The purpose of the study was to assess associations between prevalent VC and disturbances of calcium-phosphate metabolism as well as changes in vitamin D (25(OH)D), FGF 23 and MGP levels and to evaluate the possible impact of VC and changes of these biomarkers on survival in HD patients. Methods: The study population consisted of 81 prevalent patients in the hemodialysis unit of Hospital of Lithuanian University of Health Sciences Kaunas Clinics. A simple vascular calcification score (SVCS) was evaluated as it is described by Adragao et al. 25(OH)D (nmol/L), FGF 23 (ng/L) and MGP (ng/mL) were measured and analysed. Results: Patients were divided into two groups: SVCS Conclusion: VC in HD patients is highly influenced by age and presence of diabetes and associated with higher risk of death. No significant association was found between MGP and FGF 23 and VC as well as between these two biomarkers and risk of death. Lower 25(OH)D levels were associated with mortality in this dialysis patients cohort.

Rational design of compounds that would bind specific pockets of the target proteins is a difficult task in drug design. The 12 isoforms of catalytically active human carbonic anhydrases (CAs) have highly similar active sites that make it difficult to design inhibitors selective for one or several CA isoforms. A series of CA inhibitors based on 2-chloro/bromo-benzenesulfonamide that is largely fixed in the CA active site together with one or two tails yielded compounds that were synthesized and evaluated as inhibitors of CA isoforms. Introduction of a second tail had significant influence on the binding affinity and two-tailed compounds in most cases provided high affinity and selectivity for CA IX and CA XIV. The contacts between several compounds and CA amino acids were determined by X-ray crystallography. Together with the intrinsic enthalpy and entropy of binding they provided the structure-thermodynamics correlations for this series of compounds with the insight how to rationally build compounds with desired CA isoform as a target.

International audience; The Material Properties Open Database (MPOD, http//mpod.cimav.edu.mx) is a functional element of the web-based open databases system linked with crystallography. MPOD delivers single-crystal tensor properties in several representations, ranging from numerical matrices to 3D printing. Longitudinal moduli surfaces can be displayed in computers as well as in smart cell phones. Properties are stored as '.mpod' files. IUCr formatting standards (CIF) are followed. The original published paper containing the data is cited. Structural and experimental information is also registered and linked. 'Coupling properties', say piezo-effects and magnetoelectricity, represent interactions linking different subsystems in a material. Currently, piezoelectricity occupies a significant fraction of cases in MPOD. The implications of crystal symmetry in piezoelectricity are systematically taken into account. Matrices' elements and longitudinal moduli surfaces are checked for consistency with the Neumann principle. The inclusion of magnetoelectric axial tensors introduces exciting features into MPOD.

Vascular calcification (VC) is one of the factors associated with cardiovascular mortality in hemodialysis (HD) patients. Recommendations concerning screening for VC differ. Possible ability to prevent and reversibility of VC are major subjects on debate whether screening for VC could improve outcomes of renal patients. The objective of the study was to evaluate the significance of simple vascular calcification score (SVCS) based on plane radiographic films and to test its association with non-fatal cardiovascular events in patients on chronic HD.A study population consisted of 95 prevalent HD patients in the HD unit of Hospital of Lithuanian University of Health sciences Kaunas Clinics. Clinical data and laboratory tests information were collected from medical records. SVCS was evaluated as it is described by Adragao et al. After measurement of VC, HD patients were observed for novel non-fatal cardiovascular events.Patients were divided into two groups: SVCS≥3 (57 patients [60%]) and3 (38 patients [40%]). The Kaplan-Meier survival curves show a significant difference in non-fatal cardiovascular events in the group with SVCS≥3 vs.3 group (26.3% vs. 7.8%; log rank 5,49; P=0.018). Multivariate Cox regression analysis confirmed a negative impact of VC, hyperphosphatemia, and lower ejection fraction on cardiovascular events. No statistically significant differences were observed comparing parameters of Ca-P metabolism disorders between groups with different SVCS. On separate analysis, the presence of VC in hands was also associated with higher rate of novel cardiovascular events (score 0 goup-5 events [10.6%] vs. score≥1 group-13 events [27%], log rank P=0.035).VC assessed by simple and inexpensive radiological method was an independent predictor of novel non-fatal cardiovascular events in HD patients.

International audience; The objective of the SOLSA project (EU-H2020) is to develop an analytical expert system for on-line-on-mine-real-time mineralogical and geochemical analyses on sonic drill cores. As one aspect of the system, this paper presents the building of the hyperspectral library and its incorporation into sparse unmixing techniques for mineral identification. Twenty seven spectra representing 14 minerals have been collected for the library. Three sparse unmixing techniques have been investigated and evaluated using simulated data generated from our hyperspectral library, and real hyperspectral data acquired from a serpentinized harzburgite sample. Among the three techniques, the collaborative sparse unmixing by variable splitting and augmented Lagrangian (CLSUnSAL) method provided the best accurate results on the simulated data. In addition, the results of the CLSUnSAL method show high correlation with that of the QEMSCAN® analysis on the harzburgite hyperspectral data.

DNA cytosine methylation is a widespread epigenetic mark. Biological effects of DNA methylation are mediated by the proteins that preferentially bind to 5-methylcytosine (5mC) in different sequence contexts. Until now two different structural mechanisms have been established for 5mC recognition in eukaryotes; however, it is still unknown how discrimination of the 5mC modification is achieved in prokaryotes. Here we report the crystal structure of the N-terminal DNA-binding domain (McrB-N) of the methyl-specific endonuclease McrBC from Escherichia coli. The McrB-N protein shows a novel DNA-binding fold adapted for 5mC-recognition. In the McrB-N structure in complex with methylated DNA, the 5mC base is flipped out from the DNA duplex and positioned within a binding pocket. Base flipping elegantly explains why McrBC system restricts only T4-even phages impaired in glycosylation [Luria, S.E. and Human, M.L. (1952) A nonhereditary, host-induced variation of bacterial viruses. J. Bacteriol., 64, 557–569]: flipped out 5-hydroxymethylcytosine is accommodated in the binding pocket but there is no room for the glycosylated base. The mechanism for 5mC recognition employed by McrB-N is highly reminiscent of that for eukaryotic SRA domains, despite the differences in their protein folds.

Type IIS restriction endonucleases recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. The restriction endonuclease BpuJI recognizes the asymmetric sequence 5′-CCCGT; however, it cuts at multiple sites in the vicinity of the target sequence. BpuJI consists of two physically separate domains, with catalytic and dimerization functions in the C-terminal domain and DNA recognition functions in the N-terminal domain. Here we report the crystal structure of the BpuJI recognition domain bound to cognate DNA at 1.3-A resolution. This region folds into two winged-helix subdomains, D1 and D2, interspaced by the DL subdomain. The D1 and D2 subdomains of BpuJI share structural similarity with the similar subdomains of the FokI DNA-binding domain; however, their orientations in protein–DNA complexes are different. Recognition of the 5′-CCCGT target sequence is achieved by BpuJI through the major groove contacts of amino acid residues located on both the helix–turn–helix motifs and the N-terminal arm. The role of these interactions in DNA recognition is also corroborated by mutational analysis.

Nucleic acids symposium series 43(16), 8100 - 8110(2015). doi:10.1093/nar/gkv768, Type II restriction endonuclease BsaWI recognizes a degenerated sequence 5′-W/CCGGW-3′ (W stands for A or T, ‘/’ denotes the cleavage site). It belongs to a large family of restriction enzymes that contain a conserved CCGG tetranucleotide in their target sites. These enzymes are arranged as dimers or tetramers, and require binding of one, two or three DNA targets for their optimal catalytic activity. Here, we present a crystal structure and biochemical characterization of the restriction endonuclease BsaWI. BsaWI is arranged as an ‘open’ configuration dimer and binds a single DNA copy through a minor groove contacts. In the crystal primary BsaWI dimers form an indefinite linear chain via the C-terminal domain contacts implying possible higher order aggregates. We show that in solution BsaWI protein exists in a dimer-tetramer-oligomer equilibrium, but in the presence of specific DNA forms a tetramer bound to two target sites. Site-directed mutagenesis and kinetic experiments show that BsaWI is active as a tetramer and requires two target sites for optimal activity. We propose BsaWI mechanism that shares common features both with dimeric Ecl18kI/SgrAI and bona fide tetrameric NgoMIV/SfiI enzymes., Published by Oxford Univ. Press, Oxford

DNA base flipping is an important mechanism in molecular enzymology, but its study is limited by the lack of an accessible and reliable diagnostic technique. A series of crystalline complexes of a DNA methyltransferase, M.HhaI, and its cognate DNA, in which a fluorescent nucleobase analogue, 2-aminopurine (AP), occupies defined positions with respect the target flipped base, have been prepared and their structures determined at higher than 2 angstrom resolution. From time-resolved fluorescence measurements of these single crystals, we have established that the fluorescence decay function of AP shows a pronounced, characteristic response to base flipping: the loss of the very short (similar to 100 ps) decay component and the large increase in the amplitude of the long (similar to 10 ns) component. When AP is positioned at sites other than the target site, this response is not seen. Most significantly, we have shown that the same clear response is apparent when M.HhaI complexes with DNA in solution, giving an unambiguous signal of base flipping. Analysis of the AP fluorescence decay function reveals conformational heterogeneity in the DNA-enzyme complexes that cannot be discerned from the present X-ray structures.

Restriction endonuclease Eco57I from Escherichia coli recognizes asymmetric DNA sequence 5′-CTGAAG and has both restriction (DNA cleavage a short distance away from the recognition site) and modification (methylation) activities residing in a single polypeptide chain. Single crystals of wild-type Eco57I ternary complexes with double-stranded DNA and sinefungin, a stimulator of endonuclease activity, were obtained by the vapor diffusion technique and characterized crystallographically for different variants of the DNA component. The best data for the complex with 25-mer DNA were collected to 4.2-A resolution at 100 K using synchrotron radiation. The crystals are orthorhombic, space group P 2 1 2 1 2, with a =164.3, b =293.0, c =71.1 A, and contain two to four copies of the protein in the asymmetric unit.

Human carbonic anhydrase IX (CA IX) is highly expressed in tumor tissues, and its selective inhibition provides a potential target for the treatment of numerous cancers. Development of potent, highly selective inhibitors against this target remains an unmet need in anticancer therapeutics. A series of fluorinated benzenesulfonamides with substituents on the benzene ring was designed and synthesized. Several of these exhibited a highly potent and selective inhibition profile against CA IX. Three fluorine atoms significantly increased the affinity by withdrawing electrons and lowering the pKa of the benzenesulfonamide group. The bulky ortho substituents, such as cyclooctyl or even cyclododecyl groups, fit into the hydrophobic pocket in the active site of CA IX but not CA II, as shown by the compound's co-crystal structure with chimeric CA IX. The strongest inhibitor of recombinant human CA IX's catalytic domain in human cells achieved an affinity of 50 pM. However, the high affinity diminished the selectivity. The most selective compound for CA IX exhibited 10 nM affinity. The compound that showed the best balance between affinity and selectivity bound with 1 nM affinity. The inhibitors described in this work provide the basis for novel anticancer therapeutics targeting CA IX.

The crystal structure of the NgoMIV restriction endonuclease in complex with cleaved DNA has been determined at 1.6 A resolution. The crystallographic asymmetric unit contains a protein tetramer and two DNA molecules cleaved at their recognition sites. This is the first structure of a tetrameric restriction enzyme–DNA complex. In the tetramer, two primary dimers are arranged back to back with two oligonucleotides bound in clefts on opposite sides of the tetramer. The DNA molecules retain a B-type conformation and have an enclosed angle between their helical axes of 60°. Sequence-specific interactions occur in both the major and minor grooves. Two Mg2+ ions are located close to the cleaved phosphate at the active site of NgoMIV. Biochemical experiments show that interactions between the recognition sites within the tetramer greatly increase DNA cleavage efficiency.

The X-ray crystal structure of Citrobacter freundii restriction endonuclease Cfr 10I has been determined at a resolution of 2.15 A by multiple isomorphous replacement methods and refined to an R -factor of 19.64%. The structure of Cfr 10I represents the first structure of a restriction endonuclease recognizing a degenerated nucleotide sequence. Structural comparison of Cfr 10I with previously solved structures of other restriction enzymes suggests that recognition of specific sequence occurs through contacts in the major and the minor grooves of DNA. The arrangement of the putative active site residues shows some striking differences from previously described restriction endonucleases and supports a two-metal- ion mechanism of catalysis.

S-Alkylation of pyrimidine derivatives (I) with ω-bromoacetylbenzenesulfonamides (II) and (IV) affords the title compounds (III) and (V), respectively.

EcoRII restriction endonuclease is specific for the 5′-CCWGG sequence (W stands for A or T); however, it shows no activity on a single recognition site. To activate cleavage it requires binding of an additional target site as an allosteric effector. EcoRII dimer consists of three structural units: a central catalytic core, made from two copies of the C-terminal domain (EcoRII-C), and two N-terminal effector DNA binding domains (EcoRII-N). Here, we report DNA-bound EcoRII-N and EcoRII-C structures, which show that EcoRII combines two radically different structural mechanisms to interact with the effector and substrate DNA. The catalytic EcoRII-C dimer flips out the central T:A base pair and makes symmetric interactions with the CC:GG half-sites. The EcoRII-N effector domain monomer binds to the target site asymmetrically in a single defined orientation which is determined by specific hydrogen bonding and van der Waals interactions with the central T:A pair in the major groove. The EcoRII-N mode of the target site recognition is shared by the large class of higher plant transcription factors of the B3 superfamily.

Fabry's disease is an X-linked inborn error of glycosphingolipid metabolism caused by a deficiency of the lysosomal hydrolase alpha-galactosidase A. Due to deficiency of this enzyme activity, a progressive lysosomal accumulation of glycosphingolipids, in particular globotriaosylceramide, takes place within endothelial cells and cells of the vascular and nervous systems, myocardial cells, endothelial, and mesangial and epithelial cells of the kidney, eventually leading to organ dysfunction. The degree of renal involvement generally correlates with the progression of glycosphingolipid accumulation and may lead to renal insufficiency and failure. Renal dysfunction can progress to end-stage renal failure, which usually occurs in the third to fifth decade of life. The prevalence of this disease among males on chronic hemodialysis is different in various countries. Screening for alpha-galactosidase A deficiency by blood spot tests was performed among 536 male dialysis patients in all 42 hemodialysis centers in Lithuania in the period of April-June, 2005. All tests, showed normal galactosidase A enzymatic activity.No patient with suspicion of Fabry's disease was found by this screening method.

The aim of the study was to determine the prevalence and quality of control of disorders of calcium and phosphorus metabolism among patients on hemodialysis in Lithuania during the period of 2004-2005 and to assess rarely used methods of treatment such as parathyroidectomy and administration of calcimimetics.All Lithuanian hemodialysis centers were visited, and data on disorders of calcium-phosphorus metabolism were collected in December 2004 and 2005. The quality of control was evaluated according to Kidney Disease Outcome Quality Initiative recommendations.According to Kidney Disease Outcome Quality Initiative guidelines, normal parathyroid hormone levels were found in 20.4% of hemodialysis patients in 2004 and 18.8% of hemodialysis patients in 2005; normal levels of phosphate were in 41.9% and 39.4%, respectively; normal levels of calcium were observed in 44.7% of patients in 2004 and in 42.3% of patients in 2005. In 2005 as compared to 2004, there were statistically significantly more patients with low parathyroid hormone level (39.9% and 45.8%, respectively, P0.05). Only in 5.6% of patients in 2004 and 3.9% of patients in 2005, all four parameters of calcium-phosphate metabolism (calcium, phosphate, and of parathyroid hormone levels and calcium-phosphate product) were within the normal range. No parameters in the normal range were found in 17-20% of patients. The use of alfacalcidol significantly increased: 316 (30.8%) patients in 2004 and 388 (35.7%) patients in 2005 were treated with alfacalcidol (P0.05). Alfacalcidol was prescribed for 16.5% of patients in 2004 and for 17% of patients in 2005, in whom parathyroid hormone level was below the normal range in the presence of hypercalcemia and hyperphosphatemia. The use of calcimimetics was considered rational in 142 (13.8%) patients in 2004 and 119 (10.9%) patients in 2005. According to the data of our study, parathyroidectomy was indicated in 19 (1.85%) patients in 2004 and 17 (1.56%) patients in 2005.According to Kidney Disease Outcome Quality Initiative recommendations, the control of disorders of calcium-phosphate metabolism in Lithuanian hemodialysis patients was insufficient in 2004 and 2005. One-third of the patients were treated with alfacalcidol when parathyroid hormone level was low and hypercalcemia and hyperphosphatemia persisted. Calcimimetics for the treatment of secondary hyperparathyroidism were administered in about 10% of patients.

Restricion endonuclease Ecl18kI is specific for the sequence /CCNGG and cleaves it before the outer C to generate 5 nt 5′-overhangs. It has been suggested that Ecl18kI is evolutionarily related to NgoMIV, a 6-bp cutter that cleaves the sequence G/CCGGC and leaves 4 nt 5′-overhangs. Here, we report the crystal structure of the Ecl18kI–DNA complex at 1.7 A resolution and compare it with the known structure of the NgoMIV–DNA complex. We find that Ecl18kI flips both central nucleotides within the CCNGG sequence and buries the extruded bases in pockets within the protein. Nucleotide flipping disrupts Watson–Crick base pairing, induces a kink in the DNA and shifts the DNA register by 1 bp, making the distances between scissile phosphates in the Ecl18kI and NgoMIV cocrystal structures nearly identical. Therefore, the two enzymes can use a conserved DNA recognition module, yet recognize different sequences, and form superimposable dimers, yet generate different cleavage patterns. Hence, Ecl18kI is the first example of a restriction endonuclease that flips nucleotides to achieve specificity for its recognition site.

Among all restriction endonucleases known to date, BfiI is unique in cleaving DNA in the absence of metal ions. BfiI represents a different evolutionary lineage of restriction enzymes, as shown by its crystal structure at 1.9-Å resolution. The protein consists of two structural domains. The N-terminal catalytic domain is similar to Nuc, an EDTA-resistant nuclease from the phospholipase D superfamily. The C-terminal DNA-binding domain of BfiI exhibits a β-barrel-like structure very similar to the effector DNA-binding domain of the Mg 2+ -dependent restriction enzyme EcoRII and to the B3-like DNA-binding domain of plant transcription factors. BfiI presumably evolved through domain fusion of a DNA-recognition element to a nonspecific nuclease akin to Nuc and elaborated a mechanism to limit DNA cleavage to a single double-strand break near the specific recognition sequence. The crystal structure suggests that the interdomain linker may act as an autoinhibitor controlling BfiI catalytic activity in the absence of a specific DNA sequence. A psi-blast search identified a BfiI homologue in a Mesorhizobium sp. BNC1 bacteria strain, a plant symbiont isolated from an EDTA-rich environment.

We analyzed data of 383 hemodialysis patients admitted to the hemodialysis center of Kaunas University of Medicine Hospital between 1 January 1994 and 31 December 2004. The aim of the study was to estimate their survival and identify it influencing factors. Demographic data (gender, birth date), cause of renal insufficiency, date of hemodialysis (HD) initiation, blood analyses at the start of HD (hemoglobin, C-reactive protein, serum albumin, creatinine, calcium, phosphate), how HD were started (through central venous catheter or permanent vascular access), time of the first nephrologist consultation before initiating of HD were recorded for each patient in a special form. The total survival rate was estimated using the Kaplan-Maier method. Mean survival of HD patients was only 21.93 months (95% confidence intervals (CI) 18.7-25.16 months). It was influenced by a high early mortality (17.23% of patients died within the first three months from the beginning of HD (36.5% of all dead patients)). The lowest survival rate was of those patients who started HD immediately after the first consultation with a nephrologist. Early referral to nephrologist, normal serum albumin and C-reactive protein concentrations had a positive impact on survival in hemodialysis patients.

The 2.9A resolution crystal structure of apo wild-type GroEL was determined for the first time and represents the reference structure, facilitating the study of structural and functional differences observed in GroEL variants. Until now the crystal structure of the mutant Arg13Gly, Ala126Val GroEL was used for this purpose. We show that, due to the mutations as well as to the presence of a crystallographic symmetry, the ring-ring interface was inaccurately described. Analysis of the present structure allowed the definition of structural elements at this interface, essential for understanding the inter-ring allosteric signal transmission. We also show unambiguously that there is no ATP-induced 102 degrees rotation of the apical domain helix I around its helical axis, as previously assumed in the crystal structure of the (GroEL-KMgATP)(14) complex, and analyze the apical domain movements. These results enabled us to compare our structure with other GroEL crystal structures already published, allowing us to suggest a new route through which the allosteric signal for negative cooperativity propagates within the molecule. The proposed mechanism, supported by known mutagenesis data, underlines the importance of the switching of salt bridges.

Villous adenoma comprises 5.6% of colon tumors. Adenomas of the colon could be divided into tubular, villous, and tubulovillous adenomas. Villous adenomas are not as common as tubular neoplasms; however, usually they are larger and could cause a depleting syndrome characterized by dehydration, hyponatremia, hypokalemia, hypochloremia, and metabolic acidosis. In severe cases, shock and renal failure in older patients could cause death. Case of villous adenoma is presented in this article. Seventy-eight-year-old male who suffered permanent diarrhea for few years followed with acute renal failure. Villous adenoma was diagnosed. The conservative treatment had short renal function improvement. Hemodialysis had to be performed. Renal function was completely recovered after radical tumor surgery.

Crystal structures of Type II restriction endonucleases demonstrate a conserved common core and active site residues but diverse structural elements involved in DNA sequence discrimination. Comparative structural analysis of restriction enzymes recognizing the same nucleotide sequence might therefore contribute to our understanding of the structural diversity of specificity determinants within restriction enzymes. We have solved the crystal structure of the Bacillus stearothermophilus restriction endonuclease Bse634I by the multiple isomorphous replacement technique to 2.17 A resolution. Bse634I is an isoschisomer of the Cfr10I restriction enzyme whose crystal structure has been reported previously. Comparative structural analysis of the first pair of isoschisomeric enzymes revealed conserved structural determinants of sequence recognition and catalysis. However, conformations of the N-terminal subdomains differed between Bse634I/Cfr10I, suggesting a rigid body movement that might couple DNA recognition and catalysis. Structural similarities extend to the quaternary structure level: crystal contacts suggest that Bse634I similarly to Cfr10I is arranged as a tetramer. Kinetic analysis reveals that Bse634I is able to interact simultaneously with two recognition sites supporting the tetrameric architecture of the protein. Thus, restriction enzymes Bse634I, Cfr10I and NgoMIV, recognizing overlapping nucleotide sequences, exhibit a conserved tetrameric architecture that is of functional importance.

The use of prior chemical knowledge such as bond lengths, bond angles about constituent blocks of macromolecules and ligands is an essential part of macromolecular crystal structure analysis. One of the reliable sources of such chemical knowledge is small molecule database where small molecule crystal structures have been analysed against high-resolution, high-quality experimental data. Furthermore, vast amount of data in small molecule database provide comprehensive coverage of flexible chemical environment and enable proper statistical analysis to avoid biased representation of those chemical properties. This presentation describes our work on organization of the data from open-access and daily-updated small molecule database, Crystallography Open Database(COD) [1], into a new generation of CCP4 monomer library (Dictionary), a container of prior chemical knowledge [2]. In order to describe specific environment atoms are in, they are classified into different atomic types based on local graphs and some basic chemical properties of atoms. This scheme can be applied to any small molecule databases. The atom types, and values of bond lengths and bond associated with them, are further clustered into a hierarchical tree and an isomorphism-mapping algorithm is implemented to facilitate fast search among a large number of atom types (typically several millions). This also provides a mechanism to derive reliable values for bond lengths and angles of novel ligands. Metal and non-organic atoms are treated differently with organic ones. The original data in COD are curated using several criteria and further statistical analysis on derived values of bond lengths and angles are allow to extract reliable chemical information from such databanks as COD. There are several software tools associated with new dictionary including 1) generate "ideal" bond lengths and angles for unknown ligand; 2) generate starting coordinates to represent one of the conformation of the ligand under consideration.

As computational chemistry methods enjoy unprecedented growth, computer power increases and price/performance ratio drops, a large number of crystal structures can today be refined and their properties computed using modern theoretical approaches, such as DFT, post-HF, QM/MM, MCMM methods. Availability of several open source codes for computational and quantum chemistry and open-access crystallographic databases enables large scale computations of material structures and properties. We thus increasingly feel that an open collection of theoretically computed chemical structures would be a valuable resource for the scientific community. To address this need, we have launched a Theoretical Crystallography Open Database (TCOD, http://www.crystallography.net/tcod/). The TCOD sets a goal to collect a comprehensive set of computed crystal structures that would be made available under an Open Data license and invites all scientists to deposit their published results or pre-publication data. Accompanied with a large set of experimental structures in the COD database [1], the TCOD opens immediate possibilities for experimental and theoretical data cross-validation. To ensure high quality of deposited data, TCOD offers ontologies in a form of CIF [2] dictionaries that describe parameters of computed chemical and crystal structures, and an automated pipeline that checks each submitted structure against a set of community-specified criteria for convergence, computation quality and reproducibility. The scope of TCOD and validation tools make TCOD a high-quality, comprehensive theoretical structure database, useful in a broad range of disciplines. First-principle calculations are also of huge interest to simulate physical properties, either prospectively or for materials that do not grow as sufficiently large crystals. The property results can now be tested against the Material Properties Open Database [3] (http://www.materialproperties.org/) to ameliorate the used models.

It is thought that most of the type II restriction endonucleases interact with DNA as homodimers. Cfr10I is a typical type II restriction enzyme that recognises the 5'-Pu decreases CCGGPy sequence and cleaves it as indicated by the arrow. Gel-filtration and analytical ultracentrifugation data presented here indicate that Cfr10I is a homotetramer in isolation. The only SfiI restriction enzyme that recognises the long interrupted recognition sequence 5'-GGCCNNNNNGGCC has been previously reported to operate as a tetramer however, its structure is unknown. Analysis of Cfr10I crystals revealed that a single molecule in the asymmetric unit is repeated by D2 symmetry to form a tetramer. To determine whether the packing of the Cfr10I in the crystal reflects the quaternary structure of the protein in solution, the tryptophan W220 residue located at the putative dimer-dimer interface was mutated to alanine, and the structural and functional consequences of the substitution were analysed. Equilibrium sedimentation experiments revealed that, in contrast to the wild-type Cfr10I, the W220A mutant exists in solution predominantly as a dimer. In addition, the tetramer seems to be a catalytically important form of Cfr10I, since the DNA cleavage activity of the W220A mutant is < 0.1% of that of the wild-type enzyme. Further, analysis of plasmid DNA cleavage suggests that the Cfr10I tetramer is able to interact with two copies of the recognition sequence, located on the same DNA molecule. Indeed, electron microscopy studies demonstrated that two distant recognition sites are brought together through the DNA looping induced by the simultaneous binding of the Cfr10I tetramer to both sites. These data are consistent with the tetramer being a functionally important form of Cfr10I.

According to the crystal structure of Cfr 10I restriction endonuclease the acidic residues D134, E71 and E204 are clustered together and presumably chelate metal ion(s) at the active site. Indeed, investigation of the DNA cleavage properties of substitutional mutants of Cfr 10I D134A, E71Q, E71A and E204Q reveals that D134, E71 and E204 residues are essential for cleavage activity, supporting their active site function. Structural comparison indicates that the D134 residue of Cfr 10I spatially overlaps with aspartate residues D91 and D74, from the invariant active site motifs 90PDX19EAK and 73PDX15DIK of Eco RI and Eco RV, respectively. However, structural studies in conjunction with mutational analyses suggest that the sequence motif 133PDX55KX13E corresponds to the active site of Cfr 10I, but differs from canonical active site motifs of Eco RI and Eco RV. According to the crystal structure of Cfr 10I the serine S188 residue from the 188SVK sequence motif is a spatial equivalent of the acidic residue from the (E/D)XK-part of the active site motif, which is conserved between Eco RI and Eco RV. Site-directed mutagenesis experiments of Cfr 10I, however, revealed that the S188 was not so important for catalysis while the E204 residue located 2.8 A away indeed was essential for cleavage, suggesting that the glutamate E204 rather than the S188 residue contributes to the metal binding site in Cfr 10I. In addition, model-building studies suggest that mutual interchange of the E204 and S188 residues should lead only to minor positional differences of the carboxylate residues of glutamate side-chains. The double mutant S188E/E204S was therefore prepared by site-directed mutagenesis where the active site motif 133PDX55KX13E of Cfr 10I was changed to a canonical motif 133PDX53EVK, which is similar to that of Eco RI and Eco RV. Interestingly, the double mutant S188E/E204S of Cfr 10I with redesigned active site structure, exhibited 10% of Wt cleavage activity in a λ DNA cleavage assay. Thus, structure guided redesign of the catalytic/metal binding site of Cfr 10I, provides novel experimental evidence to suggest that spatial rather than sequence conservation plays the dominant role in the formation of restriction enzyme active sites.

Gel shift analysis reveals [Lagunavicius, A.,Siksnys, V. (1997) Biochemistry 36 (preceding paper in this issue)] that at pH 8.3 in the absence of Mg2+, MunI restriction endonuclease exhibits little DNA binding specificity, as compared with the D83A and E98A mutants of MunI. This suggests that charged carboxylate residue(s) influence the DNA binding specificity of MunI. In our efforts to establish the determinants of MunI binding specificity, we investigated the possible role of the ionic milieu, and we found that lowering pH or elevating Ca2+ levels per se induces specific DNA recognition by WT MunI. In contrast to the binding experiments at pH 8.3, gel shift analysis at pH 6.5 indicated tight sequence-specific binding of WT MunI in the absence of Mg2+, suggesting that protonation of active site carboxylate residue(s) which manifest anomalously high pKa value(s) control binding specificity. Interestingly, Ca2+ ion concentrations, which did not support DNA cleavage by MunI also induced DNA binding specificity in WT MunI at pH 8.3. To explore possible structural changes upon DNA binding, we then used a limited proteolysis technique. Trypsin cleavage of MunI-DNA complexes indicated that in the presence of cognate DNA the MunI restriction endonuclease became resistant to proteolytic cleavage, suggesting that binding of specific DNA induced a structural change. CD measurements confirmed this observation, suggesting minor secondary structural differences between complexes of MunI with cognate and noncognate DNA. These results therefore suggest that binding of MunI to its recognition sequence triggers a conformational transition that correctly juxtaposes active site carboxylate residues, which then chelate Mg2+ ions. In the absence of Mg2+ ions, at pH 8.3, conditions in which carboxylate groups would be expected to be completely ionized, electrostatic repulsion between charged carboxylates and phosphate oxygens is enhanced such as to interfere with specific DNA binding. Elimination of such repulsive constraints by replacement of carboxylate residues, by lowering pH, or by metal ion binding, then promotes MunI binding specificity.

Structural investigations into quadruplexes, formed from guanine rich sequences found in the human chromosome, and development of small molecule ligands that can stabilize these structures have been the main focus of our research. The targeting of the telomere, a G-rich hexanucleotide repeat sequence TAGGGT, found at the end of all mammalian chromosomes has lead to the development of a series of ligands that can effectively inhibit the enzyme telomerase. Critically a 3’ single stranded overhang, located at the end of the chromosome, is the substrate for the ribonucleoprotein telomerase. Folding of these telomeric repeats into higher order DNA structures inhibits access of telomerase and prevents telomere maintenance. Additionally, the binding of other associated proteins that form part of the telosome, are also disrupted leading rapidly to chromosomal damage, such as chromosomal fusions and apoptosis.Wewill discuss our research into the folding topologies that are available to these telomeric sequences and other target sequence found in the chromosome, and the design of selective ligands that target these novel topologies. This is of particular importance with the identification of quanine rich sequences that have been shown to form stable quadruplex structures within promotor regions of onogenes. The structural diversity of the quadruplex structures formed from these G-rich DNA sequences will also be reviewed. m04.o03