5-Aminopyrazole-4-carboxamide was used as an alternative scaffold to substitute for the pyrazolopyrimidine of a known “bumped kinase inhibitor” to create selective inhibitors of calcium-dependent protein kinase-1 from both Toxoplasma gondii and Cryptosporidium parvum. Compounds with low nanomolar inhibitory potencies against the target enzymes were obtained. The most selective inhibitors also exhibited submicromolar activities in T. gondii cell proliferation assays and were shown to be non-toxic to mammalian cells.
Toxoplasma gondii; Cryptosporidium parvum; Calcium-dependent protein kinase-1; Enzyme inhibitor; Selectivity
used as an alternative scaffold
to substitute for the pyrazolopyrimidine of a known “bumped
kinase inhibitor” to create selective inhibitors of calcium-dependent
protein kinase-1 from both Toxoplasma gondii and Cryptosporidium parvum. Compounds with low nanomolar inhibitory
potencies against the target enzymes were obtained. The most selective
inhibitors also exhibited submicromolar activities in T. gondii cell proliferation assays and were shown to be nontoxic to mammalian
Toxoplasma gondii; Cryptosporidium
parvum; calcium-dependent protein kinase-1; enzyme inhibitor; selectivity
Only a small percentage of protein kinases have been shown to adopt a distinct inactive ATP-binding site conformation, called the Asp-Phe-Gly-out (DFG-out) conformation. Given the high degree of homology within this enzyme family, we sought to understand the basis of this disparity on a sequence level. First, we identified two residue positions that sensitize mitogen-activated protein kinases (MAPKs) to inhibitors that stabilize the DFG-out inactive conformation. After characterizing the structure and dynamics of an inhibitor-sensitive MAPK mutant, we demonstrated the generality of this strategy by sensitizing a kinase (ASK1) not in the MAPK family to several DFG-out stabilizing ligands using the same residue positions. The use of specific inactive conformations may aid the study of noncatalytic roles of protein kinases, such as binding partner interactions and scaffolding effects.
Despite the enormous economic importance of Neospora caninum related veterinary diseases, the number of effective therapeutic agents is relatively small. Development of new therapeutic strategies to combat the economic impact of neosporosis remains an important scientific endeavor. This study demonstrates molecular, structural and phenotypic evidence that N. caninum calcium-dependent protein kinase 1 (NcCDPK1) is a promising molecular target for neosporosis drug development. Recombinant NcCDPK1 was expressed, purified and screened against a select group of bumped kinase inhibitors (BKIs) previously shown to have low IC50s against Toxoplasma gondii CDPK1 and T. gondii tachyzoites. NcCDPK1 was inhibited by low concentrations of BKIs. The three-dimensional structure of NcCDPK1 in complex with BKIs was studied crystallographically. The BKI-NcCDPK1 structures demonstrated the structural basis for potency and selectivity. Calcium-dependent conformational changes in solution as characterized by small-angle X-ray scattering are consistent with previous structures in low Calcium-state but different in the Calcium-bound active state than predicted by X-ray crystallography. BKIs effectively inhibited N. caninum tachyzoite proliferation in vitro. Electron microscopic analysis of N. caninum cells revealed ultra-structural changes in the presence of BKI compound 1294. BKI compound 1294 interfered with an early step in Neospora tachyzoite host cell invasion and egress. Prolonged incubation in the presence of 1294 interfered produced observable interference with viability and replication. Oral dosing of BKI compound 1294 at 50 mg/kg for 5 days in established murine neosporosis resulted in a 10-fold reduced cerebral parasite burden compared to untreated control. Further experiments are needed to determine the PK, optimal dosage, and duration for effective treatment in cattle and dogs, but these data demonstrate proof-of-concept for BKIs, and 1294 specifically, for therapy of bovine and canine neosporosis.
Protein kinases are key components of most mammalian signal transduction networks and are therapeutically relevant drug targets. Efforts to study protein kinase function would benefit from new technologies that are able to profile kinases in complex proteomes. Here, we describe active site-directed probes for profiling kinases in whole cell extracts and live cells. These probes contain general ligands that stabilize a specific inactive conformation of the ATP-binding sites of protein kinases, as well as trifluoromethylphenyl diazirine and alkyne moieties that allow covalent modification and enrichment of kinases, respectively. A diverse group of serine/threonine and tyrosine kinases were identified as specific targets of these probes in whole cell extracts. In addition, a number of kinase targets were selectively labeled in live cells. Our chemical proteomics approach should be valuable for interrogating protein kinase active sites in physiologically relevant environments.
The structure of Giardia prolyl-tRNA synthetase cocrystallized with proline and ATP shows evidence for half-of-the-sites activity, leading to a corresponding mixture of reaction substrates and product (prolyl-AMP) in the two active sites of the dimer.
The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolution crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine.
aminoacyl-tRNA synthetases; protozoa; structural genomics; Giardia lamblia
Calcium-dependent protein kinase-1 (CDPK1) from Cryptosporidium parvum (CpCDPK1) and Toxoplasma gondii (TgCDPK1) have become attractive targets for discovering selective inhibitors to combat infections caused by these protozoa. We used structure-based design to improve a series of benzoylbenzimidazole-based compounds in terms of solubility, selectivity, and potency against CpCDPK1 and TgCDPK1. The best inhibitors show inhibitory potencies below 50 nM and selectivity well above 200-fold over two human kinases with small gatekeeper residues.
Cryptosporidium parvum; Toxoplasma gondii; calcium-dependent protein kinase 1; enzyme inhibitor; selectivity
Protein kinases, key regulators of intracellular signal transduction, have emerged as an important class of drug targets. Chemical proteomic tools that facilitate the functional interrogation of protein kinase active sites are powerful reagents for studying the regulation of this large enzyme family and for performing inhibitor selectivity screens. Here we describe a new crosslinking strategy that enables rapid and quantitative profiling of protein kinase active sites in lysates and live cells. Applying this methodology to the SRC-family kinases (SFKs) SRC and HCK led to the identification of a series of conformation-specific, ATP-competitive inhibitors that display a distinct preference for autoinhibited forms of these kinases. Furthermore, we show that ligands that demonstrate this selectivity are able to modulate the ability of the regulatory domains of SRC and HCK to engage in intermolecular binding interactions. These studies provide insight into the regulation of this important family of tyrosine kinases.
Diseases caused by the apicomplexan protozoans Toxoplasma gondii and Cryptosporidium parvum are a major health concern. The life cycle of these parasites is regulated by a family of calcium-dependent protein kinases (CDPKs) that have no direct homologs in the human host. Fortuitously, CDPK1 from both parasites contains a rare glycine gatekeeper residue adjacent to the ATP-binding pocket. This has allowed creation of a series of C3-substituted pyrazolopyrimidine compounds that are potent inhibitors selective for CDPK1 over a panel of human kinases. Here we demonstrate that selectivity is further enhanced by modification of the scaffold at the C1 position. The explanation for this unexpected result is provided by crystal structures of the inhibitors bound to CDPK1 and the human kinase c-SRC. Furthermore, the insight gained from these studies was applied to transform an alternative ATP-competitive scaffold lacking potency and selectivity for CDPK1 into a low nanomolar inhibitor of this enzyme with no activity against SRC.
Toxoplasmosis is a disease of prominent health concern that is caused by the protozoan parasite, Toxoplasma gondii. Proliferation of T. gondii is dependent on its ability to invade host cells, which is mediated, in part, by calcium-dependent protein kinase 1 (CDPK1). We have developed ATP competitive inhibitors of TgCDPK1 that block invasion of parasites into host cells, preventing their proliferation. The presence of a unique glycine gatekeeper residue in TgCDPK1 permits selective inhibition of the parasite enzyme over human kinases. These potent TgCDPK1 inhibitors do not inhibit the growth of human cell lines and represent promising candidates as toxoplasmosis therapeutics.
eq is a well defined entity that represents one property, mean-square displacement, of the anisotropic atomic displacement parameter tensor from which it is derived. B
eq is not, however, the best estimate of the B factor that would result from isotropic model refinement. A new entity B
est is proposed to serve this purpose.
Crystallographic structural models for macromolecules have typically included an isotropic displacement parameter B
iso for each atom. In cases where the structural model instead includes anisotropic displacement parameters U
ij, the derived quantity B
eq can be substituted for B
iso for many purposes. B
eq is not, however, the best predictor of the value B
iso that would hypothetically have been obtained by direct refinement of an isotropic model. A new entity B
est is proposed that represents an estimate for B
iso that minimizes the Kullback–Leibler divergence from a paired anisotropic model. In general B
eq < 1, with the difference between the two values becoming larger for atoms that are more anisotropic. Although this difference does not affect direct refinement of either isotropic or anisotropic models, it is relevant to any analysis that compares isotropic and anisotropic models of the same underlying structure. In particular, it may lead to improved selection of multi-group TLS models based on analysis of an initial isotropic refinement.
macromolecular refinement; TLS; anisotropy
Ultrafiltration provides a generic method to discover ligands for protein drug targets with millimolar to micromolar Kd, the typical range of fragment-based drug discovery. This method was tailored to a 96-well format, and cocktails of fragment-sized molecules, with molecular masses between 150 and 300 Da, were screened against medical structural genomics target proteins. The validity of the method was confirmed through competitive binding assays in the presence of ligands known to bind the target proteins.
Ultrafiltration; Screening; Fragment-based drug discovery; Compound library
The single tyrosyl tRNA-synthetase (TyrRS) gene in trypanosomatid genomes codes for a protein that is twice the length of TyrRS from virtually all other organisms. Each half of the double-length TyrRS contains a catalytic domain and an anticodon-binding domain, however the two halves retain only 17% sequence identity to each other. The structural and functional consequences of this duplication and divergence are unclear. TyrRS normally forms a homodimer in which the active site of one monomer pairs with the anticodon-binding domain from the other. However, crystal structures of Leishmania major TyrRS show that instead the two halves of a single molecule form a pseudo-dimer resembling the canonical TyrRS dimer. Curiously, the C-terminal copy of the catalytic domain has lost the catalytically important HIGH and KMSKS motifs characteristic of Class I aminoacyl-tRNA synthetases. Thus the pseudo-dimer contains only one functional active site, contributed by the N-terminal half, and only one functional anticodon recognition site, contributed by the C-terminal half. Despite biochemical evidence for negative cooperativity between the two active sites of the usual TyrRS homodimer, previous structures have captured a crystallographically-imposed symmetric state. As the L. major TyrRS pseudo-dimer is inherently asymmetric, conformational variations observed near the active site may be relevant to understanding how the state of a single active site is communicated across the dimer interface. Furthermore, substantial differences between trypanosomal TyrRS and human homologs are promising for the design of inhibitors that selectively target the parasite enzyme.
aminoacyl-tRNA synthetase; protozoa; drug target; tropical disease
The PROSPERO server analyzes sequence and experimental protein characterization results, then uses that analysis to predict crystallization outcome and suggest priorities for futher work on difficult targets. The server allows users to upload data from six types of experiment, to organize those data by sample and project, and to share those data with collaborators.
The growth of diffracting crystals from purified proteins is often a major bottleneck in determining structures of biological and medical interest. The PROSPERO web server, http://skuld.bmsc.washington.edu/prospero, is intended both to provide a means of organizing the potentially large numbers of experimental characterizations measured from such proteins, and to provide useful guidance for structural biologists who have succeeded in purifying their target protein but have reached an impasse in the difficult and poorly understood process of turning purified protein into well diffracting crystals. These researchers need to decide which of many possible rescue options are worth pursuing, given finite resources. This choice is even more crucial when attempting to solve high-priority but relatively difficult structures of eukaryotic proteins. The site currently uses the HyGX1 predictor, which was trained and validated on protein samples from pathogenic protozoa (eukaryotes) using results from six types of experiment. PROSPERO allows users to store, analyze and display multiple results for each sample, to group samples into projects, and to share results and predictions with collaborators.
protein crystallography; protein characterization; PROSPERO; computer programs
Effective control and eradication of malaria will require new tools to prevent transmission. Current antimalarial therapies targeting the asexual stage of Plasmodium do not prevent transmission of circulating gametocytes from infected humans to mosquitoes. Here, we describe a new class of transmission-blocking compounds, bumped kinase inhibitors (BKIs), which inhibit microgametocyte exflagellation. Oocyst formation and sporozoite production, necessary for transmission to mammals, were inhibited in mosquitoes fed on either BKI-1–treated human blood or mice treated with BKI-1. BKIs are hypothesized to act via inhibition of Plasmodium calcium-dependent protein kinase 4 and predicted to have little activity against mammalian kinases. Our data show that BKIs do not inhibit proliferation of mammalian cell lines and are well tolerated in mice. Used in combination with drugs active against asexual stages of Plasmodium, BKIs could prove an important tool for malaria control and eradication.
Tryptophanyl-tRNA synthetase (TrpRS) is an essential enzyme that is recognizably conserved across all forms of life. It is responsible for activating and attaching tryptophan to a cognate tRNATrp molecule for use in protein synthesis. In some eukaryotes this original core function has been supplemented or modified through the addition of extra domains or the expression of variant TrpRS isoforms. The three TrpRS structures from pathogenic protozoa described here represent three illustrations of this malleability in eukaryotes. The Cryptosporidium parvum genome contains a single TrpRS gene, which codes for an N-terminal domain of uncertain function in addition to the conserved core TrpRS domains. Sequence analysis indicates that this extra domain, conserved among several apicomplexans, is related to the editing domain of some AlaRS and ThrRS. The C. parvum enzyme remains fully active in charging tRNATrp after truncation of this extra domain. The crystal structure of the active, truncated enzyme is presented here at 2.4 Å resolution. The Trypanosoma brucei genome contains separate cytosolic and mitochondrial isoforms of TrpRS that have diverged in their respective tRNA recognition domains. The crystal structure of the T. brucei cytosolic isoform is presented here at 2.8 Å resolution. The Entamoeba histolytica genome contains three sequences that appear to be TrpRS homologs. However one of these, whose structure is presented here at 3.0 Å resolution, has lost the active site motifs characteristic of the Class I aminoacyl-tRNA synthetase catalytic domain while retaining the conserved features of a fully formed tRNATrp recognition domain. The biological function of this variant E. histolytica TrpRS remains unknown, but, on the basis of a completely conserved tRNA recognition region and evidence for ATP but not tryptophan binding, it is tempting to speculate that it may perform an editing function. Together with a previously reported structure of an unusual TrpRS from Giardia, these protozoan structures broaden our perspective on the extent of structural variation found in eukaryotic TrpRS homologs.
Glycogen synthase kinase-3 (GSK-3) is a drug target under intense investigation in pharmaceutical companies and constitutes an attractive piggyback target for eukaryotic pathogens. Two different GSKs are found in trypanosomatids, one about 150 residues shorter than the other. GSK-3 short (GeneDB: Tb927.10.13780) has previously been validated genetically as a drug target in Trypanosoma brucei by RNAi induced growth retardation; and chemically by correlation between enzyme and in vitro growth inhibition. Here, we report investigation of the equivalent GSK-3 short enzymes of L. major (LmjF18.0270) and L. infantum (LinJ18_V3.0270, identical in amino acid sequences to LdonGSK-3 short) and a crystal structure of LmajGSK-3 short at 2 Å resolution. The inhibitor structure-activity relationships (SARs) of L. major and L. infantum are virtually identical, suggesting that inhibitors could be useful for both cutaneous and visceral leishmaniasis. Leishmania spp. GSK-3 short has different inhibitor SARs than TbruGSK-3 short, which can be explained mostly by two variant residues in the ATP-binding pocket. Indeed, mutating these residues in the ATP-binding site of LmajGSK-3 short to the TbruGSK-3 short equivalents results in a mutant LmajGSK-3 short enzyme with SAR more similar to that of TbruGSK-3 short. The differences between human GSK-3β (HsGSK-3β) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found.
Glycogen synthase kinase-3; Leishmania spp.; Trypanosoma brucei; African sleeping sickness; drug development; protein kinase
A simple rule of thumb based on resolution is not adequate to identify the best treatment of atomic displacements in macromolecular structural models. The choice to use isotropic B factors, anisotropic B factors, TLS models or some combination of the three should be validated through statistical analysis of the model refinement.
In choosing and refining any crystallographic structural model, there is tension between the desire to extract the most detailed information possible and the necessity to describe no more than what is justified by the observed data. A more complex model is not necessarily a better model. Thus, it is important to validate the choice of parameters as well as validating their refined values. One recurring task is to choose the best model for describing the displacement of each atom about its mean position. At atomic resolution one has the option of devoting six model parameters (a ‘thermal ellipsoid’) to describe the displacement of each atom. At medium resolution one typically devotes at most one model parameter per atom to describe the same thing (a ‘B factor’). At very low resolution one cannot justify the use of even one parameter per atom. Furthermore, this aspect of the structure may be described better by an explicit model of bulk displacements, the most common of which is the translation/libration/screw (TLS) formalism, rather than by assigning some number of parameters to each atom individually. One can sidestep this choice between atomic displacement parameters and TLS descriptions by including both treatments in the same model, but this is not always statistically justifiable. The choice of which treatment is best for a particular structure refinement at a particular resolution can be guided by general considerations of the ratio of model parameters to the number of observations and by specific statistics such as the Hamilton R-factor ratio test.
atomic displacements; B factors; TLS models; model parameters
Leishmania parasites cause two million new cases of leishmaniasis each year with several hundreds of millions people at risk. Due to the paucity and shortcomings of available drugs, we have undertaken the crystal structure determination of a key enzyme from Leishmania major in hopes of creating a platform for the rational design of new therapeutics. Crystals of the catalytic core of methionyl-tRNA synthetase from L. major (LmMetRS) were obtained with the substrates MgATP and methionine present in the crystallization medium. These crystals yielded the 2.0 Å resolution structure of LmMetRS in complex with two products, methionyladenylate and pyrophosphate, along with a Mg2+ ion that bridges them. This is the first class I aminoacyl-tRNA synthetase (aaRS) structure with pyrophosphate bound. The residues of the class I aaRS signature sequence motifs, KISKS and HIGH, make numerous contacts with the pyrophosphate. Substantial differences between the LmMetRS structure and previously reported complexes of E. coli MetRS (EcMetRS) with analogs of the methionyladenylate intermediate product are observed, even though one of these analogs only differs by one atom from the intermediate. The source of these structural differences is attributed to the presence of the product pyrophosphate in LmMetRS. Analysis of the LmMetRS structure in light of the Aquifex aeolicus MetRS-tRNAMet complex shows that major rearrangements of multiple structural elements of enzyme and/or tRNA are required to allow the CCA acceptor triplet to reach the methionyladenylate intermediate in the active site. Comparison with sequences of human cytosolic and mitochondrial MetRS reveals interesting differences near the ATP- and methionine-binding regions of LmMetRS, suggesting that it should be possible to obtain compounds that selectively inhibit the parasite enzyme.
aminoacyl-tRNA synthetase; protozoa; drug target; leishmaniasis; trypanosomiasis; enzyme product complex
Human African trypanosomiasis continues to be an important public health threat in extensive regions of sub-Saharan Africa. Treatment options for infected patients are unsatisfactory due to toxicity, difficult administration regimes, and poor efficacy of available drugs. The aminoacyl-tRNA synthetases were selected as attractive drug targets due to their essential roles in protein synthesis and cell survival. Comparative sequence analysis disclosed differences between the trypanosome and mammalian methionyl-tRNA synthetases (MetRSs) that suggested opportunities for selective inhibition using drug-like molecules. Experiments using RNA interference on the single MetRS of Trypanosoma brucei demonstrated that this gene product was essential for normal cell growth. Small molecules (diaryl diamines) similar to those shown to have potent activity on prokaryotic MetRS enzymes were synthesized and observed to have inhibitory activity on the T. brucei MetRS (50% inhibitory concentration, <50 nM) and on bloodstream forms of T. brucei cultures (50% effective concentration, as low as 4 nM). Twenty-one compounds had a close correlation between enzyme binding/inhibition and T. brucei growth inhibition, indicating that they were likely to be acting on the intended target. The compounds had minimal effects on mammalian cell growth at 20 μM, demonstrating a wide therapeutic index. The most potent compound was tested in the murine model of trypanosomiasis and demonstrated profound parasite suppression and delayed mortality. A homology model of the T. brucei MetRS based on other MetRS structures was used to model binding of the lead diaryl diamine compounds. Future studies will focus on improving the pharmacological properties of the MetRS inhibitors.
The protozoans Cryptosporidium parvum and Toxoplasma gondii are parasites of major health concern to humans. Both parasites contain a group of calcium-dependent protein kinases (CDPKs) which are found in plants and ciliates but not in humans or fungi. Here, we describe a series of potent inhibitors that target CDPK1 in C. parvum (CpCDPK1) and T. gondii (TgCDPK1). These inhibitors are highly selective for CpCDPK1 and TgCDPK1 over the mammalian kinases SRC and ABL. Furthermore, they are able to block an early stage of C. parvum invasion of HCT-8 host cells, which is similar to their effects on T. gondii invasion of human fibroblasts.
Phosphorylation; protozoans; calcium-dependent protein kinases; Cryptosporidium parvum; Toxoplasma gondii; apicomplexan
The protozoans Cryptosporidium parvum and Toxoplasma gondii are parasites of major health concern to humans. Both parasites contain a group of calcium-dependent protein kinases (CDPKs), which are found in plants and ciliates but not in humans or fungi. Here we describe a series of potent inhibitors that target CDPK1 in C. parvum (CpCDPK1) and T. gondii (TgCDPK1). These inhibitors are highly selective for CpCDPK1 and TgCDPK1 over the mammalian kinases SRC and ABL. Furthermore, they are able to block an early stage of C. parvum invasion of HCT-8 host cells, which is similar to their effects on T. gondii invasion of human fibroblasts.
Phosphorylation; protozoans; calcium-dependent protein kinases; Cryptosporidium parvum; Toxoplasma gondii; apicomplexan
The great power of protein crystallography to reveal biological structure is often limited by the tremendous effort required to produce suitable crystals. A hybrid crystal growth predictive model is presented that combines both experimental and sequence-derived data from target proteins, including novel variables derived from physico-chemical characterization such as R30, the ratio between a protein‘s DSF intensity at 30 °C and at Tm. This hybrid model is shown to be more powerful than sequence-based prediction alone – and more likely to be useful for prioritizing and directing the efforts of structural genomics and individual structural biology laboratories.
Crystal growth; protein characterization; thermal shift assay; dynamic light scattering; limited proteolysis; regression partition tree
The 2.1 Å crystal structure of tryptophanyl-tRNA synthetase (TrpRS) from the diplomonad Giardia lamblia reveals that the N-terminus of this class I aminoacyl-tRNA synthetase forms a 16-residue α-helix. This helix replaces a β-hairpin that is required by human TrpRS for normal activity and has been inferred to play a similar role in all eukaryotic TrpRS. The primary sequences of TrpRS homologs from several basal eukaryotes including Giardia lack a set of three residues observed to stabilize interactions with this β-hairpin in the human TrpRS. Thus the present structure suggests that the activation reaction mechanism of TrpRS from the basal eukaryote G. lamblia differs from that of higher eukaryotes. Furthermore, the protein as observed in the crystal forms an (α2)2 homotetramer. The canonical dimer interface observed in all previous structures of tryptophanyl-tRNA synthetases is maintained, but in addition each N-terminal α-helix reciprocally interlocks with the equivalent helix from a second dimer to form a dimer of dimers. Although we have no evidence for tetramer formation in vivo, modeling indicates that the crystallographically observed tetrameric structure would be compatible with the tRNA binding mode used by dimeric TrpRS and TyrRS.
aminoacyl-tRNA synthetase; protozoa; structural genomics
Guidelines and specific tests for validating macromolecular crystal structures that include TLS models are introduced. Validation may used to troubleshoot problems during refinement, to confirm the internal consistency of the model as part of deposition into the Protein Data Bank or to assess the plausibility of interpretating the boundary between two TLS groups as indicating a hinge point between structural domains.
The use of TLS (translation/libration/screw) models to describe anisotropic displacement of atoms within a protein crystal structure has become increasingly common. These models may be used purely as an improved methodology for crystallographic refinement or as the basis for analyzing inter-domain and other large-scale motions implied by the crystal structure. In either case it is desirable to validate that the crystallographic model, including the TLS description of anisotropy, conforms to our best understanding of protein structures and their modes of flexibility. A set of validation tests has been implemented that can be integrated into ongoing crystallographic refinement or run afterwards to evaluate a previously refined structure. In either case validation can serve to increase confidence that the model is correct, to highlight aspects of the model that may be improved or to strengthen the evidence supporting specific modes of flexibility inferred from the refined TLS model. Automated validation checks have been added to the PARVATI and TLSMD web servers and incorporated into the CCP4i user interface.
validation; TLS models; anisotropy