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Blocking the 2-C-methyl-D-erythrithol-4-phosphate (MEP) pathway for isoprenoid biosynthesis offers interesting prospects for inhibiting Plasmodium or Mycobacterium spp. growth. Fosmidomycin (1) and its homologue FR900098 (2) potently inhibit 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in this pathway. Here we introduced aryl or aralkyl substituents at the β-position of the hydroxamate analogue of 2. While direct addition of a β-aryl moiety resulted in poor inhibition, longer linkers between the carbon backbone and the phenyl ring were generally associated with better binding to the enzymes. X-ray structures of the parasite Dxr-inhibitor complexes show that the "longer" compounds generate a substantially different flap structure, in which a key tryptophan residue is displaced, and the aromatic group of the ligand lies between the tryptophan and the hydroxamate's methyl group. Although the most promising new Dxr inhibitors lack activity against Escherichia coli and Mycobacterium smegmatis, they proved to be highly potent inhibitors of Plasmodium falciparum in vitro growth. (Figure Presented).

Blocking the 2-C-methyl-d-erythrithol-4-phosphate pathway for isoprenoid biosynthesis offers new ways to inhibit the growth of Plasmodium spp. Fosmidomycin [(3-(N-hydroxyformamido)propyl)phosphonic acid, 1] and its acetyl homologue FR-900098 [(3-(N-hydroxyacetamido)propyl)phosphonic acid, 2] potently inhibit 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in this biosynthetic pathway. Arylpropyl substituents were introduced at the -position of the hydroxamate analogue of 2 to study changes in lipophilicity, as well as electronic and steric properties. The potency of several new compounds on the P.falciparum enzyme approaches that of 1 and 2. Activities against the enzyme and parasite correlate well, supporting the mode of action. Seven X-ray structures show that all of the new arylpropyl substituents displace a key tryptophan residue of the active-site flap, which had made favorable interactions with 1 and 2. Plasticity of the flap allows substituents to be accommodated in many ways; in most cases, the flap is largely disordered. Compounds can be separated into two classes based on whether the substituent on the aromatic ring is at the meta or para position. Generally, meta-substituted compounds are better inhibitors, and in both classes, smaller size is linked to better potency. Sanjeewani Sooriyaarachchi, René Chofor, Serge Van Calenbergh and Sherry L. Mowbray contributed equally to the study.

The natural antibiotic fosmidomycin acts via inhibition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), an essential enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. Fosmidomycin is active on Mycobacterium tuberculosis DXR (MtDXR), but it lacks antibacterial activity probably because of poor uptake. alpha-Aryl substituted fosmidomycin analogues have more favorable physicochemical properties and are also more active in inhibiting malaria parasite growth. We have solved crystal structures of MtDXR in complex with 3,4-dichlorophenyl substituted fosmidomycin analogues; these show important differences compared to our previously described forsmidomycin-DXR complex. Our best inhibitor has an IC(50) = 0.15 mu M on MtDXR but still lacked activity in a mycobacterial growth assay (MIC > 32 mu g/mL). The combined results, however, provide insights into how DXR accommodates the new inhibitors and serve as an excellent starting point for the design of other novel and more potent inhibitors, particularly against pathogens where uptake is less of a problem, such as the malaria parasite.

Threonine biosynthesis is a general feature of prokaryotes, eukaryotic microorganisms, and higher plants. Since mammals lack the appropriate synthetic machinery, instead obtaining the amino acid through their diet, the pathway is a potential focus for the development of novel antibiotics, antifungal agents, and herbicides. Threonine synthase (TS), a pyridoxal-5-phosphate-dependent enzyme, catalyzes the final step in the pathway, in which L-homoserine phosphate and water are converted into threonine and inorganic phosphate. In the present publication, we report structural and functional studies of Mycobacterium tuberculosis TS, the product of the rv1295 (thrC) gene. The structure gives new insights into the catalytic mechanism of TSs in general, specifically by suggesting the direct involvement of the phosphate moiety of the cofactor, rather than the inorganic phosphate product, in transferring a proton from C4' to C-gamma in the formation of the alpha beta-unsaturated aldimine. It further provides a basis for understanding why this enzyme has a higher pH optimum than has been reported elsewhere for TSs and gives rise to the prediction that the equivalent enzyme from Thermus thermophilus will exhibit similar behavior. A deletion of the relevant gene generated a strain of M. tuberculosis that requires threonine for growth, such auxotrophic strains are frequently attenuated in vivo, indicating that TS is a potential drug target in this organism.

The Mycobacterium tuberculosis fatty acid synthase type II (FAS-II) system has the unique property of producing unusually long-chain fatty acids involved in the biosynthesis of mycolic acids, key molecules of the tubercle bacillus. The enzyme(s) responsible for dehydration of (3 R )-hydroxyacyl-ACP during the elongation cycles of the mycobacterial FAS-II remained unknown. This step is classically catalyzed by FabZ- and FabA-type enzymes in bacteria, but no such proteins are present in mycobacteria. Bioinformatic analyses and an essentiality study allowed the identification of a candidate protein cluster, Rv0635-Rv0636-Rv0637. Its expression in recombinant Escherichia coli strains leads to the formation of two heterodimers, Rv0635-Rv0636 (HadAB) and Rv0636-Rv0637 (HadBC), which also occurs in Mycobacterium smegmatis , as shown by split-Trp assays. Both heterodimers exhibit the enzymatic properties expected for mycobacterial FAS-II dehydratases: a marked specificity for both long-chain (≥C 12 ) and ACP-linked substrates. Furthermore, they function as 3-hydroxyacyl dehydratases when coupled with MabA and InhA enzymes from the M. tuberculosis FAS-II system. HadAB and HadBC are the long-sought (3 R )-hydroxyacyl-ACP dehydratases. The correlation between the substrate specificities of these enzymes, the organization of the orthologous gene cluster in different Corynebacterineae , and the structure of their mycolic acids suggests distinct roles for both heterodimers during the elongation process. This work describes bacterial monofunctional (3 R )-hydroxyacyl-ACP dehydratases belonging to the hydratase 2 family. Their original structure and the fact that they are essential for M. tuberculosis survival make these enzymes very good candidates for the development of antimycobacterial drugs.

Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to form bicarbonate, a reaction required for many functions, including carbon assimilation and pH homeostasis. Carbonic anhydrases are divided into at least three classes and are believed to share a zinc-hydroxide mechanism for carbon dioxide hydration. beta-carbonic anhydrases are broadly spread among the domains of life, and existing structures from different organisms show two distinct active site setups, one with three protein coordinations to the zinc (accessible) and the other with four (blocked). The latter is believed to be inconsistent with the zinc-hydroxide mechanism. The Mycobacterium tuberculosis Rv3588c gene, shown to be required for in vivo growth of the pathogen, encodes a beta-carbonic anhydrase with a steep pH dependence of its activity, being active at pH 8.4 but not at pH 7.5. We have recently solved the structure of this protein, which was a dimeric protein with a blocked active site. Here we present the structure of the thiocyanate complexed protein in a different crystal form. The protein now forms distinct tetramers and shows large structural changes, including a carboxylate shift yielding the accessible active site. This structure demonstrated for the first time that a beta-carbonic anhydrase can switch between the two states. A pH-dependent dimer to tetramer equilibrium was also demonstrated by dynamic light scattering measurements. The data presented here, therefore, suggest a carboxylate shift on/off switch for the enzyme, which may, in turn, be controlled by a dimer-to-tetramer equilibrium.

Endoglucanase Cel6A from Thermobifida fusca hydrolyzes the beta-1,4 linkages in cellulose at accessible points along the polymer. The structure of the catalytic domain of Cel6A from T. fusca in complex with a nonhydrolysable substrate analogue that acts as an inhibitor, methylcellobiosyl-4-thio-beta-cellobioside (Glc(2)-S-Glc(2)), has been determined to 1.5 A resolution. The glycosyl unit in subsite -1 was sterically hindered by Tyr73 and forced into a distorted (2)S(o) conformation. In the enzyme where Tyr73 was mutated to a serine residue, the hindrance was removed and the glycosyl unit in subsite -1 had a relaxed (4)C(1) chair conformation. The relaxed conformation was seen in two complex structures of the mutated enzyme, with cellotetrose (Glc(4)) at 1.64 A and Glc(2)-S-Glc(2) at 1.04 A resolution.

Glutamine synthetase catalyzes the ligation of glutamate and ammonia to form glutamine, with the resulting hydrolysis of ATP. The enzyme is a central component of bacterial nitrogen metabolism and is a potential drug target. Here, we report a high-yield recombinant expression system for glutamine synthetase of Mycobacterium tuberculosis together with a simple purification. The procedure allowed the structure of a complex with a phosphorylated form of the inhibitor methionine sulfoximine, magnesium, and ADP to be solved by molecular replacement and refined at 2.1-Å resolution. To our knowledge, this study provides the first reported structure for a taut form of the M. tuberculosis enzyme, similar to that observed for the Salmonella enzyme earlier. The phospho compound, generated in situ by an active enzyme, mimics the phosphorylated tetrahedral adduct at the transition state. Some differences in ligand interactions of the protein with both phosphorylated compound and nucleotide are observed compared with earlier structures; a third metal ion also is found. The importance of these differences in the catalytic mechanism is discussed; the results will help guide the search for specific inhibitors of potential therapeutic interest.

Xyloglucan endotransglycosylases (XETs) cleave and religate xyloglucan polymers in plant cell walls via a transglycosylation mechanism. Thus, XET is a key enzyme in all plant processes that require cell wall remodeling. To provide a basis for detailed structure-function studies, the crystal structure of Populus tremula x tremuloides XET16A (PttXET16A), heterologously expressed in Pichia pastoris, has been determined at 1.8-A resolution. Even though the overall structure of PttXET16A is a curved beta-sandwich similar to other enzymes in the glycoside hydrolase family GH16, parts of its substrate binding cleft are more reminiscent of the distantly related family GH7. In addition, XET has a C-terminal extension that packs against the conserved core, providing an additional beta-strand and a short alpha-helix. The structure of XET in complex with a xyloglucan nonasaccharide, XLLG, reveals a very favorable acceptor binding site, which is a necessary but not sufficient prerequisite for transglycosylation. Biochemical data imply that the enzyme requires sugar residues in both acceptor and donor sites to properly orient the glycosidic bond relative to the catalytic residues.

Ribose-5-phosphate isomerases (EC 5.3.1.6) inter-convert ribose-5-phosphate and ribulose-5-phosphate. This reaction allows the synthesis of ribose from other sugars, as well a means for salvage of carbohydrates after nucleotide breakdown. Two unrelated types of enzyme are known to catalyze the isomerization. The most common one, RpiA, is present in almost all organisms. The second type, RpiB, is found in many bacterial species.Here, we demonstrate that the RpiB from Mycobacterium tuberculosis (Rv2465c) has catalytic properties very similar to those previously reported for the Escherichia coli RpiB enzyme. Further, we report the structure of the mycobacterial enzyme, solved by molecular replacement and refined to 1.88A resolution. Comparison with the E.coli structure shows that there are important differences in the two active sites, including a change in the position and nature of the catalytic base. Sequence comparisons reveal that the M.tuberculosis and E.coli RpiB enzymes are in fact representative of two distinct sub-families. The mycobacterial enzyme represents a type found only in actinobacteria, while the enzyme from E.coli is typical of that seen in many other bacterial proteomes. Both RpiBs are very different from RpiA in structure as well as in the construction of the active site. Docking studies allow additional insights into the reactions of all three enzymes, and show that many features of the mechanism are preserved despite the different catalytic components.

Dextranase catalyzes the hydrolysis of the α-1,6-glycosidic linkage in dextran polymers. The structure of dextranase, Dex49A, from Penicillium minioluteum was solved in the apo-enzyme and product-bound forms. The main domain of the enzyme is a right-handed parallel β helix, which is connected to a β sandwich domain at the N terminus. In the structure of the product complex, isomaltose was found to bind in a crevice on the surface of the enzyme. The glycosidic oxygen of the glucose unit in subsite +1 forms a hydrogen bond to the suggested catalytic acid, Asp395. By NMR spectroscopy the reaction course was shown to occur with net inversion at the anomeric carbon, implying a single displacement mechanism. Both Asp376 and Asp396 are suitably positioned to activate the water molecule that performs the nucleophilic attack. A new clan that links glycoside hydrolase families 28 and 49 is suggested.

As part of a program to discover improved glycoside hydrolase family 12 (GH 12) endoglucanases, we have studied the biochemical diversity of several GH 12 homologs. The H. schweinitzii Cel12A enzyme differs from the T. reesei Cel12A enzyme by only 14 amino acids (93% sequence identity), but is much less thermally stable. The bacterial Cel12A enzyme from S. sp. 11AG8 shares only 28% sequence identity to the T. reesei enzyme, and is much more thermally stable. Each of the 14 sequence differences from H. schweinitzii Cel12A were introduced in T. reesei Cel12A to determine the effect of these amino acid substitutions on enzyme stability. Several of the T. reesei Cel12A variants were found to have increased stability, and the differences in apparent midpoint of thermal denaturation (T(m)) ranged from a 2.5 degrees C increase to a 4.0 degrees C decrease. The least stable recruitment from H. schweinitzii Cel12A was A35S. Consequently, the A35V substitution was recruited from the more stable S. sp. 11AG8 Cel12A and this T. reesei Cel12A variant was found to have a T(m) 7.7 degrees C higher than wild type. Thus, the buried residue at position 35 was shown to be of critical importance for thermal stability in this structural family. There was a ninefold range in the specific activities of the Cel12 homologs on o-NPC. The most and least stable T. reesei Cel12A variants, A35V and A35S, respectively, were fully active. Because of their thermal tolerance, S. sp. 11AG8 Cel12A and T. reesei Cel12A variant A35V showed a continual increase in activity over the temperature range of 25 degrees C to 60 degrees C, whereas the less stable enzymes T. reesei Cel12A wild type and the destabilized A35S variant, and H. schweinitzii Cel12A showed a decrease in activity at the highest temperatures. The crystal structures of the H. schweinitzii, S. sp. 11AG8, and T. reesei A35V Cel12A enzymes have been determined and compared with the wild-type T. reesei Cel12A enzyme. All of the structures have similar Calpha traces, but provide detailed insight into the nature of the stability differences. These results are an example of the power of homolog recruitment as a method for identifying residues important for stability.

As macromolecular crystal structures are determined and refined in an increasingly automated fashion, careful assessment of the reliability and quality of the resulting models becomes increasingly important. Here, we analyze various issues related to the reliability and quality of macromolecular crystal structures deposited between 1991 and 2000. We find that the average resolution at which these structures are determined is essentially constant. In line with this observation, the average quality as measured by Ramachandran analysis does not improve as a function of time. On the other hand, an observed decrease of the average discrepancy between free and conventional R values suggests that the fit of model and data is improving. Finally, we present a surprising correlation between the tendency of crystallographers to deposit their experimental data and the free R values of their models.

Conformational changes of periplasmic binding proteins are essential for their function in chemotaxis and transport. The allose-binding protein from Escherichia coli is, like other receptors in its family, composed of two α/β domains joined by a three-stranded hinge. In the previously determined structure of the closed, ligand-bound form (Chaudhuri, B. N., Ko, J., Park, C., Jones, T. A., and Mowbray, S. L. (1999) J. Mol. Biol. 286, 1519–1531), the ligand-binding site is buried between the two domains. We report here the structures of three distinct open, ligand-free forms of this receptor, one solved at 3.1-A resolution and two others at 1.7-A resolution. Together, these allow a description of the conformational changes associated with ligand binding. A few large, coupled torsional changes in the hinge strands are sufficient to generate the overall bending motion, with only minor disruption of the individual domains. Integral water molecules appear to act as structural “ball bearings” in this process. The conformational changes of the related ribose-binding protein follow a distinct pattern. The observed differences between the two proteins can be interpreted in the context of changes in sequence and in crystal packing and provide new insights into the nature of hinge bending motion in this class of periplasmic binding proteins.

Pantothenate kinase (PanK) catalyzes the phosphorylation of pantothenate, the first committed and rate-limiting step toward coenzyme A (CoA) biosynthesis. In our earlier reports, we had established that the type I isoform encoded by the coaA gene is an essential pantothenate kinase in Mycobacterium tuberculosis , and this vital information was then exploited to screen large libraries for identification of mechanistically different classes of PanK inhibitors. The present report summarizes the synthesis and expansion efforts to understand the structure-activity relationships leading to the optimization of enzyme inhibition along with antimycobacterial activity. Additionally, we report the progression of two distinct classes of inhibitors, the triazoles, which are ATP competitors, and the biaryl acetic acids, with a mixed mode of inhibition. Cocrystallization studies provided evidence of these inhibitors binding to the enzyme. This was further substantiated with the biaryl acids having MIC against the wild-type M. tuberculosis strain and the subsequent establishment of a target link with an upshift in MIC in a strain overexpressing PanK. On the other hand, the ATP competitors had cellular activity only in a M. tuberculosis knockdown strain with reduced PanK expression levels. Additionally, in vitro and in vivo survival kinetic studies performed with a M. tuberculosis PanK ( Mt PanK) knockdown strain indicated that the target levels have to be significantly reduced to bring in growth inhibition. The dual approaches employed here thus established the poor vulnerability of PanK in M. tuberculosis .

Methionyl-tRNA synthetase (MetRS) from Mycobacterium smegmatis was recombinantly expressed in Escherichia coli and purified using Ni(2+)-affinity and size-exclusion chromatography. Crystals formed readily in the presence of the ligands methionine and adenosine. These two ligands are components of an intermediate in the two-step catalytic mechanism of MetRS. The crystals were produced using the vapour-diffusion method and a full data set to 2.1 A resolution was collected from a single crystal. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 155.9, b = 138.9, c = 123.3 A, beta = 124.8 degrees . The presence of three molecules in the asymmetric unit corresponded to a solvent content of 60% and a Matthews coefficient of 3.1 A(3) Da(-1). Structure determination is in progress.

Background: Cel6A is one of the two cellobiohydrolases produced by Trichoderma reesei . The catalytic core has a structure that is a variation of the classic TIM barrel. The active site is located inside a tunnel, the roof of which is formed mainly by a pair of loops. Results: We describe three new ligand complexes. One is the structure of the wild-type enzyme in complex with a nonhydrolysable cello-oligosaccharide, methyl 4-S- β -cellobiosyl-4-thio- β -cellobioside (Glc) 2 -S-(Glc) 2 , which differs from a cellotetraose in the nature of the central glycosidic linkage where a sulphur atom replaces an oxygen atom. The second structure is a mutant, Y169F, in complex with the same ligand, and the third is the wild-type enzyme in complex with m-iodobenzyl β -D-glucopyranosyl- β (1,4)-D-xylopyranoside (IBXG). Conclusions: The (Glc) 2 -S-(Glc) 2 ligand binds in the -2 to +2 sites in both the wild-type and mutant enzymes. The glucosyl unit in the -1 site is distorted from the usual chair conformation in both structures. The IBXG ligand binds in the -2 to +1 sites, with the xylosyl unit in the -1 site where it adopts the energetically favourable chair conformation. The -1 site glucosyl of the (Glc) 2 -S-(Glc) 2 ligand is unable to take on this conformation because of steric clashes with the protein. The crystallographic results show that one of the tunnel-forming loops in Cel6A is sensitive to modifications at the active site, and is able to take on a number of different conformations. One of the conformational changes disrupts a set of interactions at the active site that we propose is an integral part of the reaction mechanism.

We report our evaluation of the CASP3 comparative modelling competition. Our analysis covers the accuracy of the over-all fold, the bridging of insertions and deletions, and the adding of side-chains. We describe our attempts at automating aspects of the evaluation.

The antimalarial compound fosmidomycin targets DXR, the enzyme that catalyzes the first committed step in the MEP pathway producing the universally essential isoprenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate. The MEP pathway is used by a number of pathogens, including Mycobacterium tuberculosis and apicomplexan parasites, and differs from the classical mevalonate pathway that is essential in humans. Using a structure-based approach, we designed a number of analogues of fosmidomycin, including a series that are substituted in both the Cα and the hydroxamate positions. The latter proved to be a stable framework for the design of inhibitors that extend from the cramped substrate-binding site and can, for the first time, bridge the substrate and cofactor binding sites. A number of these compounds are more potent than fosmidomycin in terms of killing Plasmodium falciparum in an in vitro assay; the best has an IC50 of 40 nM. De tre (3) första författarna delar förstaförfattarskapet.

Mycobacterium tuberculosis, the bacterial causative agent of tuberculosis, currently affects millions of people. The emergence of drug-resistant strains makes development of new antibiotics targeting the bacterium a global health priority. Pantothenate kinase, a key enzyme in the universal biosynthesis of the essential cofactor CoA, was targeted in this study to find new tuberculosis drugs. The biochemical characterizations of two new classes of compounds that inhibit pantothenate kinase from M. tuberculosis are described, along with crystal structures of their enzyme-inhibitor complexes. These represent the first crystal structures of this enzyme with engineered inhibitors. Both classes of compounds bind in the active site of the enzyme, overlapping with the binding sites of the natural substrate and product, pantothenate and phosphopantothenate, respectively. One class of compounds also interferes with binding of the cofactor ATP. The complexes were crystallized in two crystal forms, one of which is in a new space group for this enzyme and diffracts to the highest resolution reported for any pantothenate kinase structure. These two crystal forms allowed, for the first time, modeling of the cofactor-binding loop in both open and closed conformations. The structures also show a binding mode of ATP different from that previously reported for the M. tuberculosis enzyme but similar to that in the pantothenate kinases of other organisms.

The errors that can be introduced into a protein model during model building and refinement vary tremendously in their importance and severity [1,2]. At one extreme, the mainchain may be totally incorrectly traced in the experimental map, or the molecular replacement solution may be wrong. Minor errors may include an incorrect peptide orientation, or misplaced or excessive water molecules. The reasons why errors creep into models are many, but for a structure built into an experimental map, the main ones are limited resolution and poorly phased diffraction data. Other things being equal, the resolution of the diffraction data should be the ultimate variable that determines the accuracy of a structural investigation. Inevitably, life is more complicated, and a successful structural investigation is often a learning experience for the people involved.

We thank the Editors for allowing us to publish this diatribe, and Dr Alex Cameron for providing us with the coordinates of his unpublished glutathione transferase structure. We are preparing a considerably longer and more technical paper concerning rebuilding and refinement for a forthcoming volume of Methods in Enzymology, a preprint of which will be made available via the World Wide Web (http://onyx.bmc.uu.se/).

Arg15 is a conserved active-site residue in class Alpha glutathione transferases. X-ray diffraction studies of human glutathione transferase A1-1 have shown that N o of this amino acid residue is adjacent to the sulfur atom of a glutathione derivative bound to the active site, suggesting the presence of a hydrogen bond. The phenolic hydroxyl group of Tyr9 also forms a hydrogen bond to the sulfur atom of glutathione, and removal of this hydroxyl group causes partial inactivation of the enzyme. The present study demonstrates by use of site-directed mutagenesis the functional significance of Arg15 for catalysis. Mutation of Arg15 into Leu reduced the catalytic activity by 25-fold, whereas substitution by Lys caused only a threefold decrease, indicating the significance of a positively charged residue at position 15. Mutation of Arg15 into Ala or His caused a substantial reduction of the specific activity (200 or 400-fold, respectively), one order of magnitude more pronounced than the effect of the Tyr9→Phe mutation. Double mutations involving residues 9 and 15 demonstrated that the effects of mutations at the two positions were additive except for the substitution of His for Arg15, which appeared to cause secondary structural effects. The p K a value of the phenolic hydroxyl of Tyr9 was determined by UV absorption difference spectroscopy and was found to be 8.1 in the wild-type enzyme. The corresponding p K a values of mutants R15K, R15H and R15L were 8.5, 8.7 and 8.8, respectively, demonstrating the contribution of the guanidinium group of Arg15 to the electrostatic field in the active site. Addition of glutathione caused an increased p K a value of Tyr9; this effect was not obtained with S -methylglutathione. These results show that Tyr9 is protonated when glutathione is bound to the enzyme at physiological pH values. The involvement of an Arg residue in the binding and activation of glutathione is a feature that distinguishes class Alpha glutathione transferases from members in other glutathione transferase classes.

Xyloglucan endotransglycosylases (XETs) cleave and religate xyloglucan polymers in plant cell walls. Recombinant XET from poplar has been purified from a Pichia pastoris expression system and crystallized. Two different crystal forms were obtained by vapour diffusion from potassium sodium tartrate and from an imidazole buffer using sodium acetate as a precipitant. Data were collected from these crystal forms to 3.5 and 2.1 A resolution, respectively. The first crystal form was found to belong to space group P3(1)21 or P3(2)21 (unit-cell parameters a = 98.6, b = 98.6, c = 98.5 A) and the second crystal form to space group P6(3) (unit-cell parameters a = 188.7, b = 188.7, c = 46.1 A).

The transketolase (TKT) enzyme in Mycobacterium tuberculosis represents a novel drug target for tuberculosis treatment and has low homology with the orthologous human enzyme. Here, we report on the structural and kinetic characterization of the transketolase from M. tuberculosis (TBTKT), a homodimer whose monomers each comprise 700 amino acids. We show that TBTKT catalyses the oxidation of donor sugars xylulose-5-phosphate and fructose-6-phosphate as well as the reduction of the acceptor sugar ribose-5-phosphate. An invariant residue of the TKT consensus sequence required for thiamine cofactor binding is mutated in TBTKT; yet its catalytic activities are unaffected, and the 2.5 Å resolution structure of full-length TBTKT provides an explanation for this. Key structural differences between the human and mycobacterial TKT enzymes that impact both substrate and cofactor recognition and binding were uncovered. These changes explain the kinetic differences between TBTKT and its human counterpart, and their differential inhibition by small molecules. The availability of a detailed structural model of TBTKT will enable differences between human and M. tuberculosis TKT structures to be exploited to design selective inhibitors with potential antitubercular activity.

Background Lipases constitute a family of enzymes that hydrolyze triglycerides. They occur in many organisms and display a wide variety of substrate specificities. In recent years, much progress has been made towards explaining the mechanism of these enzymes and their ability to hydrolyze their substrates at an oil–water interface. Results We have determined the DNA and amino acid sequences for lipase B from the yeast Candida antarctica . The primary sequence has no significant homology to any other known lipase and deviates from the consensus sequence around the active site serine that is found in other lipases. We have determined the crystal structure of this enzyme using multiple isomorphous replacement methods for two crystal forms. Models for the orthorhombic and monoclinic crystal forms of the enzyme have been refined to 1.55 a and 2.1 a resolution, respectively. Lipase B is an α / β type protein that has many features in common with previously determined lipase structures and other related enzymes. In the monoclinic crystal form, lipid-like molecules, most likely β -octyl glucoside, can be seen close to the active site. The behaviour of these lipid molecules in the crystal structure has been studied at different pH values. Conclusions The structure of Candida antarctica lipase B shows that the enzyme has a Ser–His–Asp catalytic triad in its active site. The structure appears to be in an ‘open' conformation with a rather restricted entrance to the active site. We believe that this accounts for the substrate specificity and high degree of stereospecificity of this lipase.

A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize isopentenyl diphosphate via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway found in humans. As part of a structure-based drug-discovery program against tuberculosis, IspD, the enzyme that carries out the third step in the MEP pathway, was targeted. Constructs of both the Mycobacterium smegmatis and the Mycobacterium tuberculosis enzymes that were suitable for structural and inhibitor-screening studies were engineered. Two crystal structures of the M. smegmatis enzyme were produced, one in complex with CTP and the other in complex with CMP. In addition, the M. tuberculosis enzyme was crystallized in complex with CTP. Here, the structure determination and crystallographic refinement of these crystal forms and the enzymatic characterization of the M. tuberculosis enzyme construct are reported. A comparison with known IspD structures allowed the definition of the structurally conserved core of the enzyme. It indicates potential flexibility in the enzyme and in particular in areas close to the active site. These well behaved constructs provide tools for future target-based screening of potential inhibitors. The conserved nature of the extended active site suggests that any new inhibitor will potentially exhibit broad-spectrum activity.

This review focuses on bioinformatics technologies employed by the EU-sponsored multidisciplinary VIZIER consortium (Comparative Structural Genomics of Viral Enzymes Involved in Replication, FP6 Project: 2004-511960, active from 1 November 2004 to 30 April 2009), to achieve its goals. From the management of the information flow of the project, to bioinformatics-mediated selection of RNA viruses and prediction of protein targets, to the analysis of 3D protein structures and antiviral compounds, these technologies provided a communication framework and integrated solutions for steady and timely advancement of the project. RNA viruses form a large class of major pathogens that affect humans and domestic animals. Such RNA viruses as HIV, Influenza virus and Hepatitis C virus are of prime medical concern today, but the identities of viruses that will threaten human population tomorrow are far from certain. To contain outbreaks of common or newly emerging infections, prototype drugs against viruses representing the Virus Universe must be developed. This concept was championed by the VIZIER project which brought together experts in diverse fields to produce a concerted and sustained effort for identifying and validating targets for antivirus therapy in dozens of RNA virus lineages. (C) 2010 Elsevier B.V. All rights reserved.

The filamentous fungus, Trichoderma reesei produces all of the enzymatic activities required for efficient hydrolysis of highly ordered crystalline cellulose. All the principal enzymes involved in cellulose hydrolysis possess two functional domains, one directly involved in catalysis and the other in substrate recognition and binding. The availability of high-resolution three dimensional structures of the two domains allows genetic engineering to be used for detailed structure-function studies of these important enzymes.

The Rv3588c gene product of Mycobacterium tuberculosis, a beta-carbonic anhydrase (CA, EC 4.2.1.1) denominated here mtCA 2, shows the highest catalytic activity for CO(2) hydration (k(cat) of 9.8 x 10(5)s(-1), and k(cat)/K(m) of 9.3 x 10(7)M(-1)s(-1)) among the three beta-CAs encoded in the genome of this pathogen. A series of sulfonamides/sulfamates was assayed for their interaction with mtCA 2, and some diazenylbenzenesulfonamides were synthesized from sulfanilamide/metanilamide by diazotization followed by coupling with amines or phenols. Several low nanomolar mtCA 2 inhibitors have been detected among which acetazolamide, ethoxzolamide and some 4-diazenylbenzenesulfonamides (K(I)s of 9-59 nM). As the Rv3588c gene was shown to be essential to the growth of M. tuberculosis, inhibition of this enzyme may be relevant for the design of antituberculosis drugs possessing a novel mechanism of action.

Glutamine synthetase (GS, EC 6.3.1.2; also known as gamma-glutamyl:ammonia ligase) catalyzes the ATP-dependent condensation of glutamate and ammonia to form glutamine. The enzyme has essential roles in different tissues and species, which have led to its consideration as a drug or an herbicide target. In this article, we describe studies aimed at the discovery of new antimicrobial agents targeting Mycobacterium tuberculosis, the causative pathogen of tuberculosis. A number of distinct classes of GS inhibitors with an IC(50) of micromolar value or better were identified via high-throughput screening. A commercially available purine analogue similar to one of the clusters identified (the diketopurines), 1-[(3,4-dichlorophenyl)methyl]-3,7-dimethyl-8-morpholin-4-yl-purine-2,6-dione, was also shown to inhibit the enzyme, with a measured IC(50) of 2.5+/-0.4 microM. Two X-ray structures are presented: one is a complex of the enzyme with the purine analogue alone (2.55-A resolution), and the other includes the compound together with methionine sulfoximine phosphate, magnesium and phosphate (2.2-A resolution). The former represents a relaxed, inactive conformation of the enzyme, while the latter is a taut, active one. These structures show that the compound binds at the same position in the nucleotide site, regardless of the conformational state. The ATP-binding site of the human enzyme differs substantially, explaining why it has an approximately 60-fold lower affinity for this compound than the bacterial GS. As part of this work, we devised a new synthetic procedure for generating l-(SR)-methionine sulfoximine phosphate from l-(SR)-methionine sulfoximine, which will facilitate future investigations of novel GS inhibitors.

The Modoc virus (MODV) is a flavivirus with no known vector (NKV). Evolutionary studies have shown that the viruses in the MODV group have evolved in association with mammals (bats, rodents) without transmission by an arthropod vector. MODV methyltransferase is the first enzyme from this evolutionary branch to be structurally characterized. The high-resolution structure of the methyltransferase domain of the MODV NS5 protein (MTase(MODV)) was determined. The protein structure was solved in the apo form and in complex with its cofactor S-adenosyl-L-methionine (SAM). Although it belongs to a separate evolutionary branch, MTase(MODV) shares structural characteristics with flaviviral MTases from the other branches. Its capping machinery is a relatively new target in flaviviral drug development and the observed structural conservation between the three flaviviral branches indicates that it may be possible to identify a drug that targets a range of flaviviruses. The structural conservation also supports the choice of MODV as a possible model for flavivirus studies.

The complex of fatty-acid-binding protein (FABP) from bovine heart (cFABP, pI4.9) with endogenous lipid was crystallized in the presence of ammonium sulfate as precipitant. The needle-shaped crystals belong to the monoclinic space group C2, with unit-cell constants a = 5.262(6) nm, b = 7.631(8) nm, c = 3.945(5) nm and beta = 94.47(9) degrees. A native data set to 0.35 nm resolution was collected using synchrotron radiation and film methods. An initial model for the three-dimensional structure of the protein was constructed based on the crystal structure of the related bovine P2 myelin protein [Jones, T.A., Bergfors, T., Sedzik, J.Unge, T. (1988) EMBO J. 7, 1597-1604] to which the amino acid sequence of bovine cFABP was adapted. Energy minimizations were carried out under different conditions using both an all-atom and a united-atom force field. The optimized models were used to determine the crystal structure of cFABP by molecular-replacement techniques. The structure was refined by simulated annealing to R = 0.267. As the bound lipid is heterogeneous, it could not be located in the electron-density map and/or the attained resolution was not sufficient. Bovine cFABP is composed of ten antiparallel beta strands forming a beta barrel, and by two alpha helices. The structural features are similar to those of other members of the superfamily of hydrophobic molecule transporters.

Glutamine synthetase (GS) catalyzes the ligation of glutamate and ammonia to form glutamine, with concomitant hydrolysis of ATP. In mammals, the activity eliminates cytotoxic ammonia, at the same time converting neurotoxic glutamate to harmless glutamine; there are a number of links between changes in GS activity and neurodegenerative disorders, such as Alzheimer's disease. In plants, because of its importance in the assimilation and re-assimilation of ammonia, the enzyme is a target of some herbicides. GS is also a central component of bacterial nitrogen metabolism and a potential drug target. Previous studies had investigated the structures of bacterial and plant GSs. In the present publication, we report the first structures of mammalian GSs. The apo form of the canine enzyme was solved by molecular replacement and refined at a resolution of 3 A. Two structures of human glutamine synthetase represent complexes with: a) phosphate, ADP, and manganese, and b) a phosphorylated form of the inhibitor methionine sulfoximine, ADP and manganese; these structures were refined to resolutions of 2.05 A and 2.6 A, respectively. Loop movements near the active site generate more closed forms of the eukaryotic enzymes when substrates are bound; the largest changes are associated with the binding of the nucleotide. Comparisons with earlier structures provide a basis for the design of drugs that are specifically directed at either human or bacterial enzymes. The site of binding the amino acid substrate is highly conserved in bacterial and eukaryotic GSs, whereas the nucleotide binding site varies to a much larger degree. Thus, the latter site offers the best target for specific drug design. Differences between mammalian and plant enzymes are much more subtle, suggesting that herbicides targeting GS must be designed with caution.

Selenomethionine incorporation is a standard method for determining the phases in protein crystallography by single- or multiwavelength anomalous dispersion. Recombinant expression of selenomethionine-containing protein in non-auxotrophic Pichia pastoris strains yield an incorporation of about 50%. The expression of a mutated variant of Penicillium minioluteum dextranase in P. pastoris is used to illustrate the method utilized to obtain selenomethionyl-substituted protein and to show the phasing power of the acquired anomalous signal. The dextranase structure was solved using the anomalous signal achieved from 50% selenomethionine incorporation.

Isopentenyl diphosphate is the precursor of various isoprenoids that are essential to all living organisms. It is produced by the mevalonate pathway in humans but by an alternate route in plants, protozoa, and many bacteria. 1-deoxy-D-xylulose-5-phosphate reductoisomerase catalyzes the second step of this non-mevalonate pathway, which involves an NADPH-dependent rearrangement and reduction of 1-deoxy-D-xylulose 5-phosphate to form 2-C-methyl-D-erythritol 4-phosphate. The use of different pathways, combined with the reported essentiality of the enzyme makes the reductoisomerase a highly promising target for drug design. Here we present several high resolution structures of the Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase, representing both wild type and mutant enzyme in various complexes with Mn(2+), NADPH, and the known inhibitor fosmidomycin. The asymmetric unit corresponds to the biological homodimer. Although crystal contacts stabilize an open active site in the B molecule, the A molecule displays a closed conformation, with some differences depending on the ligands bound. An inhibition study with fosmidomycin resulted in an estimated IC(50) value of 80 nm. The double mutant enzyme (D151N/E222Q) has lost its ability to bind the metal and, thereby, also its activity. Our structural information complemented with molecular dynamics simulations and free energy calculations provides the framework for the design of new inhibitors and gives new insights into the reaction mechanism. The conformation of fosmidomycin bound to the metal ion is different from that reported in a previously published structure and indicates that a rearrangement of the intermediate is not required during catalysis.

Epoxide hydrolases are vital to many organisms by virtue of their roles in detoxification, metabolism and processing of signaling molecules. The Mycobacterium tuberculosis genome encodes an unusually large number of epoxide hydrolases, suggesting that they might be of particular importance to these bacteria. We report here the first structure of an epoxide hydrolase from M.tuberculosis, solved to a resolution of 2.5 A using single-wavelength anomalous dispersion (SAD) from a selenomethionine-substituted protein. The enzyme features a deep active-site pocket created by the packing of three helices onto a curved six-stranded beta-sheet. This structure is similar to a previously described limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis and unlike the alpha/beta-hydrolase fold typical of mammalian epoxide hydrolases (EH). A number of changes in the mycobacterial enzyme create a wider and deeper substrate-binding pocket than is found in its Rhodococcus homologue. Interestingly, each structure contains a different type of endogenous ligand of unknown origin bound in its active site. As a consequence of its wider substrate-binding pocket, the mycobacterial EH is capable of hydrolyzing long or bulky lipophilic epoxides such as 10,11-epoxystearic acid and cholesterol 5,6-oxide at appreciable rates, suggesting that similar compound(s) will serve as its physiological substrate(s).

The Mycobacterium tuberculosis genome contains about 4000 genes, of which approximately a third code for proteins of unknown function or are classified as conserved hypothetical proteins. We have determined the three-dimensional structure of one of these, the rv0216 gene product, which has been shown to be essential for M. tuberculosis growth in vivo. The structure exhibits the greatest similarity to bacterial and eukaryotic hydratases that catalyse the R-specific hydration of 2-enoyl coenzyme A. However, only part of the catalytic machinery is conserved in Rv0216 and it showed no activity for the substrate crotonyl-CoA. The structure of Rv0216 allows us to assign new functional annotations to a family of seven other M. tuberculosis proteins, a number if which are essential for bacterial survival during infection and growth.

Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to form bicarbonate. This activity is universally required for fatty acid biosynthesis as well as for the production of a number of small molecules, pH homeostasis, and other functions. At least three different carbonic anhydrase families are known to exist, of which the alpha-class found in humans has been studied in most detail. In the present work, we describe the structures of two of the three beta-class carbonic anhydrases that have been identified in Mycobacterium tuberculosis, i.e. Rv1284 and Rv3588c. Both structures were solved by molecular replacement and then refined to resolutions of 2.0 and 1.75 A, respectively. The active site of Rv1284 is small and almost completely shielded from solvent, whereas that of Rv3588c is larger and quite open to solution. Differences in coordination of the active site metal are also observed. In Rv3588c, an aspartic acid side chain displaces a water molecule and coordinates directly to the zinc ion, thereby closing the zinc coordination sphere and breaking the salt link to a nearby arginine that is a feature of Rv1284. The two carbonic anhydrases thus exhibit both of the metal coordination geometries that have previously been observed for structures in this family. Activity studies demonstrate that Rv3588c is a completely functional carbonic anhydrase. The apparent lack of activity of Rv1284 in the present assay system is likely exacerbated by the observed depletion of zinc in the preparation.

A short review of the author's computer-graphics developments since 1976 is presented. Some of the major developments in these programs are reviewed and a description is provided of some of the more recent tools that can be used for electron-density map interpretation. These tools include a secondary-structure template-building system that works in conjunction with a new sequence-decoration system.

The Uppsala Electron Density Server (EDS; http://eds.bmc.uu.se/) is a web-based facility that provides access to electron-density maps and statistics concerning the fit of crystal structures and their maps. Maps are available for approximately 87% of the crystallographic Protein Data Bank (PDB) entries for which structure factors have been deposited and for which straightforward map calculations succeed in reproducing the published R value to within five percentage points. Here, an account is provided of the methods that are used to generate the information contained in the server. Some of the problems that are encountered in the map-generation process as well as some spin-offs of the project are also discussed.

Traditionally, epoxide hydrolases (EH) have been regarded as xenobiotic-metabolizing enzymes implicated in the detoxification of foreign compounds. They are known to play a key role in the control of potentially genotoxic epoxides that arise during metabolism of many lipophilic compounds. Although this is apparently the main function for the mammalian microsomal epoxide hydrolase (mEH), evidence is now accumulating that the mammalian soluble epoxide hydrolase (sEH), despite its proven role in xenobiotic metabolism, also has a central role in the formation and breakdown of physiological signaling molecules. In addition, a certain class of microbial epoxide hydrolases has recently been identified that is an integral part of a catabolic pathway, allowing the use of specific terpens as sole carbon sources. The recently available x-ray structures of a number of EHs mirror their respective functions: the microbial terpen EH differs in its fold from the canonical alpha/beta hydrolase fold of the xenobiotic-metabolizing mammalian EHs. It appears that the latter fold is the perfect solution for the efficient detoxification of a large variety of structurally different epoxides by a single enzyme, whereas the smaller microbial EH, which has a particularly high turnover number with its prefered substrate, seems to be the better solution for the hydrolysis of one specific substrate. The structure of the sEH also includes an additional catalytic domain that has recently been shown to possess phosphatase activity. Although the physiological substrate for this second active site has not been identified so far, the majority of known phosphatases are involved in signaling processes, suggesting that the sEH phosphatase domain also has a role in the regulation of physiological functions.

As part of a program to discover improved glycoside hydrolase family 12 (GH 12) endoglucanases, we have extended our previous work on the structural and biochemical diversity of GH 12 homologs to include the most stable fungal GH 12 found, Humicola grisea Cel12A. The H. grisea enzyme was much more stable to irreversible thermal denaturation than the Trichoderma reesei enzyme. It had an apparent denaturation midpoint (T(m)) of 68.7 degrees C, 14.3 degrees C higher than the T. reesei enzyme. There are an additional three cysteines found in the H. grisea Cel12A enzyme. To determine their importance for thermal stability, we constructed three H. grisea Cel12A single mutants in which these cysteines were exchanged with the corresponding residues in the T. reesei enzyme. We also introduced these cysteine residues into the T. reesei enzyme. The thermal stability of these variants was determined. Substitutions at any of the three positions affected stability, with the largest effect seen in H. grisea C206P, which has a T(m) 9.1 degrees C lower than that of the wild type. The T. reesei cysteine variant that gave the largest increase in stability, with a T(m) 3.9 degrees C higher than wild type, was the P201C mutation, the converse of the destabilizing C206P mutation in H. grisea. To help rationalize the results, we have determined the crystal structure of the H. grisea enzyme and of the most stable T. reesei cysteine variant, P201C. The three cysteines in H. grisea Cel12A play an important role in the thermal stability of this protein, although they are not involved in a disulfide bond.

Chemosensory proteins (CSPs) have been proposed to transport hydrophobic chemicals from air to olfactory or taste receptors. They have been isolated from several sensory organs of a wide range of insect species. The x-ray structure of CSPMbraA6, a 112-aa antennal protein from the moth Mamestra brassicae (Mbra ), was shown to exhibit a novel type of α-helical fold. We have performed a structural and binding study of CSPMbraA6 to get some insights into its possible molecular function. Tryptophan fluorescence quenching demonstrates the ability of CSPMbraA6 to bind several types of semio-chemicals or surrogate ligands with μM K d . Its crystal structure in complex with one of these compounds, 12-bromo-dodecanol, reveals extensive conformational changes on binding, resulting in the formation of a large cavity filled by three ligand molecules. Furthermore, binding cooperativity was demonstrated for some ligands, suggesting a stepwise binding. The peculiar rearrangement of CSPMbraA6 conformation and the cooperativity phenomenon might trigger the recognition of chemicals by receptors and induce subsequent signal transduction.

Epoxide hydrolases are essential for the processing of epoxide-containing compounds in detoxification or metabolism. The classic epoxide hydrolases have an alpha/beta hydrolase fold and act via a two-step reaction mechanism including an enzyme-substrate intermediate. We report here the structure of the limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis, solved using single-wavelength anomalous dispersion from a selenomethionine-substituted protein and refined at 1.2 A resolution. This enzyme represents a completely different structure and a novel one-step mechanism. The fold features a highly curved six-stranded mixed beta-sheet, with four alpha-helices packed onto it to create a deep pocket. Although most residues lining this pocket are hydrophobic, a cluster of polar groups, including an Asp-Arg-Asp triad, interact at its deepest point. Site-directed mutagenesis supports the conclusion that this is the active site. Further, a 1.7 A resolution structure shows the inhibitor valpromide bound at this position, with its polar atoms interacting directly with the residues of the triad. We suggest that several bacterial proteins of currently unknown function will share this structure and, in some cases, catalytic properties.

Chemosensory proteins (CSPs) are believed to be involved in chemical communication and perception. Such proteins, of M(r) 13,000, have been isolated from several sensory organs of a wide range of insect species. Several CSPs have been identified in the antennae and proboscis of the moth Mamestra brassicae. One of them, CSPMbraA6, a 112-amino acid antennal protein, has been expressed in large quantities and is soluble in the Escherichia coli periplasm. X-ray structure determination has been performed in parallel with ligand binding assays using tryptophan fluorescence quenching. The protein has overall dimensions of 25 x 30 x 32 A and exhibits a novel type of alpha-helical fold with six helices connected by alpha-alpha loops. A narrow channel extends within the protein hydrophobic core. Fluorescence quenching with brominated alkyl alcohols or fatty acids and modeling studies indicates that CSPMbraA6 is able to bind such compounds with C12-18 alkyl chains. These ubiquitous proteins might have the role of extracting hydrophobic linear compounds (pheromones, odors, or fatty acids) dispersed in the phospholipid membrane and transporting them to their receptor.

Conformational changes of periplasmic binding proteins are essential for their function in chemotaxis and transport. The allose-binding protein from Escherichia coli is, like other receptors in its family, composed of two alpha/beta domains joined by a three-stranded hinge. In the previously determined structure of the closed, ligand-bound form (Chaudhuri, B. N., Ko, J., Park, C., Jones, T. A., and Mowbray, S. L. (1999) J. Mol. Biol. 286, 1519-1531), the ligand-binding site is buried between the two domains. We report here the structures of three distinct open, ligand-free forms of this receptor, one solved at 3.1-A resolution and two others at 1.7-A resolution. Together, these allow a description of the conformational changes associated with ligand binding. A few large, coupled torsional changes in the hinge strands are sufficient to generate the overall bending motion, with only minor disruption of the individual domains. Integral water molecules appear to act as structural "ball bearings" in this process. The conformational changes of the related ribose-binding protein follow a distinct pattern. The observed differences between the two proteins can be interpreted in the context of changes in sequence and in crystal packing and provide new insights into the nature of hinge bending motion in this class of periplasmic binding proteins.

Dextranase from the fungus Penicillium minioluteum hydrolyses alpha-1,6-glycosidic bonds in dextran polymers. The enzyme has been expressed in Pichia pastoris in the presence of selenomethionine (SeMet). The level of SeMet incorporation was estimated by amino-acid analysis to be 50%. The protein has been crystallized in space group P2(1)2(1)2, with unit-cell parameters a = 103.6, b = 115.3, c = 49.8 A and one molecule per asymmetric unit. The crystals diffract to 2.0 A and the presence of SeMet in the crystals has been confirmed by an X-ray absorption spectrum.

Protein crystallography is an exacting trade, and the results may contain errors that are difficult to identify. It is the crystallographer's responsibility to make sure that incorrect protein structures do not reach the literature.