Acta Crystallographica Section D Biological Crystallography, 2001
The first observation of the unique environment for thyroxine (T(4)) binding in tetrameric rat tr... more The first observation of the unique environment for thyroxine (T(4)) binding in tetrameric rat transthyretin (rTTR) is reported as determined by X-ray diffraction. These data revealed different modes of hormone binding in the two unique hormone-binding sites in the rat TTR tetramer channel. Differences in the orientation of thyroxine and the position of water molecules in the two binding sites further suggest a mechanism for the docking pathway of the hormone into the channel of TTR. Crystals of the rat transthyretin-thyroxine complex are isomorphous with those reported for apo rTTR and crystallized in the tetragonal space group P4(3)2(1)2 with four independent TTR monomeric subunits in the asymmetric part of the crystal lattice. Data were collected to 2.5 A resolution and the structure was refined to R = 20.9% for 15 384 data in the resolution range 12-2.5 A. Similar to human TTR, the rat protein is also a 54 000 Da tetramer with four identical polypeptide chains of 127 amino-acid residues. Of the 22 amino-acid residues which differ between the human and rat sequences, none are in the thyroxine-binding domains. Analysis of these structural data reveals that the tertiary structure is similar to that of hTTR, with only small differences in the flexible loop regions on the surface of the structure. Conformational changes of the amino acids in the channel result in a hydrogen-bonded network that connects the two binding domains, in contrast to the hydrogen bonds formed along the tetramer interface in the apo transthyretin structure. These changes suggest a mechanism for the signal transmission between thyroxine-binding domains.
Acta Crystallographica Section D Biological Crystallography, 1996
The molecular structures of two human transthyretin (hTTR, prealbumin) complexes, co-crystallized... more The molecular structures of two human transthyretin (hTTR, prealbumin) complexes, co-crystallized with thyroxine (3,5,3',5'-tetraiodo-L-thyronine; T4) , and with 3',5'-dinitro-N-acetyl-L-thyronine (DNNAT), were determined by X-ray diffraction methods. Crystals of both structures are orthorhombic, space group P2~2~2, and have two independent monomers in the asymmetric unit of the crystal lattice. These structures have been refined to 17.0% for 8-2.0A resolution data for the T 4 complex (I), and to R= 18.4% for 8-2.2A resolution data for the DNNAT structure (II). This report provides a detailed description of T 4 binding to wild-type hTTR at 2.0A resolution, as well as DNNAT. In both structures, the two independent hormone-binding sites of the TTR tetramer are occupied by ligand. A 50% statistical disorder model was applied to account for the crystallographic twofold symmetry along the binding channel and the lack of such symmetry for the ligands. Results for the co-crystallized T 4 complex show that T 4 binds deep in the hormone-binding channel and displaces the bound water previously reported for T 4 soaked into a native transthyretin crystal . Nature (London), 268, 115-120]. DNNAT also binds deeper in the channel toward the tetramer center than T 4 with the nitro groups occupying the symmetrical innermost halogen pockets. The N-acetyl moiety does not form polar contacts with the protein side chains as it is oriented toward the center of the channel. The weak binding affinity of DNNAT results from the loss of hydrophobic interactions with the halogen binding pockets as observed in T 4 binding. These data suggest that the halogen-binding sites toward the tetramer center are of primary importance as they are occupied by analogues with weak affinity to TTR, and are therefore selected over the other halogen sites which contribute more strongly to the overall binding affinity.
The crystal and molecular structures of (Z)-4,4',6_triacetoxy aurone (I) and (Z)-3',5'-dibromo-2'... more The crystal and molecular structures of (Z)-4,4',6_triacetoxy aurone (I) and (Z)-3',5'-dibromo-2',4,4',6-tetrahydroxyaurone monohydrate (II) have been studied by X-ray analysis and AM1 molecular orbital methods. Aurones are characterized by a methine bridge between the benxofuran and phenolic rings and thus have only one flexible bond, the torsion about C (2) -C (21) -C ( 1' ) -C (6' ) ,8'. In the crystal both aurones have a (Z) -planar conformation defined by 8' values of 3.1(5) ' and 0.7(5)', for (I) and (II) respectively. Minimum energy conformations from AM1 molecular orbital calculations have 19' values of 12.5 ' for (I) and 3.3" and 31.3 ' for (II), depending on the orientation of the 2' -hydroxy hydrogen. The relative barrier to rotation about 8' for all AM1 conformations is less than 4 kcal mol-_'. When H2' 1 is in the plane of the phenolic ring cis to the methine bridge, it comes within 1.5 A of the methine hydrogen and causes the crystallographic conformation to be 2 kcal mol-' higher than the minimum energy structure at 31 O. When this hydrogen is placed trans to the methine bridge, the minimum energy conformation is 3.3 ', in agreement with the X-ray results. These observations suggest that caution should be used in the interpretation of AM1 results for such a sterically crowded system. Furthermore, these data indicate that, although a planar conformation is a minimum energy structure for aurones, less than 4 kcal mol-' is required to adopt a nonplanar conformation suggested as the active form for their action as inhibitors of the enzyme iodothyronine deiodinase. These aurones also act as competitive inhibitors for the binding of thyroxine to transthyretin, where similar conformational requirements are postulated.
Proceedings of the National Academy of Sciences, 1992
The crystal structure of the complex of 3',5'dibromo-2',4,4',6-tetrahydroxyaurone, a flavone deri... more The crystal structure of the complex of 3',5'dibromo-2',4,4',6-tetrahydroxyaurone, a flavone derivative, with human transthyretin (TTR), a serum thyroid hormone transport protein, has been determined and refined to R = 17.9% for data to 2.3-A resolution and provides a detailed description of a protein-bound flavonoid structure. This bromoaurone is a potent competitor for thyroid hormone binding to TTR, a 54,980-dalton a4 tetrameric protein of 222 molecular
Proceedings of the National Academy of Sciences, 2007
Saccharomyces cerevisiae is an ideal host from which to obtain high levels of posttranslationally... more Saccharomyces cerevisiae is an ideal host from which to obtain high levels of posttranslationally modified eukaryotic proteins for x-ray crystallography. However, extensive replacement of methionine by selenomethionine for anomalous dispersion phasing has proven intractable in yeast. We report a general method to incorporate selenomethionine into proteins expressed in yeast based on manipulation of the appropriate metabolic pathways. sam1 ؊ sam2 ؊ mutants, in which the conversion of methionine to S-adenosylmethionine is blocked, exhibit reduced selenomethionine toxicity compared with wild-type yeast, increased production of protein during growth in selenomethionine, and efficient replacement of methionine by selenomethionine, based on quantitative mass spectrometry and x-ray crystallography. The structure of yeast tryptophanyl-tRNA synthetase was solved to 1.8 Å by using multiwavelength anomalous dispersion phasing with protein that was expressed and purified from the sam1 ؊ sam2 ؊ strain grown in selenomethionine. Six of eight selenium residues were located in the structure.
The crystal and molecular structures of morin (2', 3,4', 5,7-pentahydroxyflavone) hydrate (I), an... more The crystal and molecular structures of morin (2', 3,4', 5,7-pentahydroxyflavone) hydrate (I), and myricetin (3', 4', 5', 3,5,7-hexahydroxyflavone) triphenylphosphine oxide (TPPO) (1:2) co-crystal complex (II) have been studied by X-ray analysis and AM1 molecular orbital methods. The molecular conformation of the two flavones, described by the torsion angle 0 [C(3)-C(2)-C(l')-C(2')] between the benzopyrone and phenyl rings is -43.4" and 51.0" for molecules A and B of morin, respectively, and -37.0" for myricetin. Minimum energy conformations from AM1 molecular orbital calculations have 0 values of -38.2" for morin and -27.0" for myricetin. The energy profile for rotation about 0 for morin has a 28 kcal mol-' barrier at 0" due to steric interactions between the 2'hydroxy and the 3-hydroxy group. There are two local minima near 30 and 140", in good agreement with structural results. The profile for myricetin has two equivalent minima near 30 and 150" with a barrier of less than 2 kcal mol-' In the crystal both flavones form extensive networks of intra-and intermolecular hydrogen bonds. In (I), each morin conformer packs in alternating layers linked by water molecules, while in (II), TPPO stabilizes the crystal by formation of short hydrogen bonds (2.58-2.65 A) of the phosphoryl oxygen to the flavone. Myricetin also forms a two dimensional sheet-like packing in which myricetin molecules hydrogen bond to each other, as well as to TPPO. These conformational and hydrogen bonding patterns provide insight into specific types of ligandreceptor interactions and support structure activity data which suggest the importance of electronic and hydrogen bonding properties in the bioactivity of flavones.
The synthesis and biological activities of 14 6-substituted 2,4-diaminoquinazolines are reported.... more The synthesis and biological activities of 14 6-substituted 2,4-diaminoquinazolines are reported. These compounds were designed to improve the cell penetration of a previously reported series of 2,4-diamino-6-substituted-pyrido[2,3-d]pyrimidines which had shown significant potency and remarkable selectivity for Toxoplasma gondii dihydrofolate reductase (DHFR), but had much lower inhibitory effects on the growth of T. gondii cells in culture. The target N9-H analogues were obtained via regiospecific reductive amination of the appropriate benzaldehydes with 2,4,6-triaminoquinazoline, which, in turn, was synthesized from 2,4-diamino-6-nitroquinazoline. The N9-CH3 analogues were synthesized via a regiospecific reductive methylation of the corresponding N9-H precursors. The compounds were evaluated as inhibitors of DHFR from human, Pneumocystis carinii, T. gondii, rat liver, Lactobacillus casei, and Escherichia coli, and selected analogues were evaluated as inhibitors of the growth of tumor cells in culture. These analogues displayed potent T. gondii DHFR inhibition as well as inhibition of the growth of T. gondii cells in culture. Further, selected analogues were potent inhibitors of the growth of tumor cells in culture in the in vitro screening program of the National Cancer Institute with GI50s in the nanomolar and subnanomolar range. Crystallographic data for the ternary complex of hDHFR-NADPH and 2,4-diamino-6-[N-(2', 5'-dimethoxybenzyl)-N-methylamino]pyrido[2,3-d]pyrimidine, 1c, reveal the first structural details for a reversed N9-C10 folate bridge geometry as well as the first conformational details of a hybrid piritrexim-trimetrexate analogue.
Crystallography is a multidisciplinary field that links divergent areas of mathematics, science a... more Crystallography is a multidisciplinary field that links divergent areas of mathematics, science and engineering to provide knowledge of life on an atomic scale. Crystal growth, a key component of the field, is an ideal vehicle for education. Crystallization has been used with a 'grocery store chemistry' approach and linked to high-throughput remote-access screening technologies. This approach provides an educational opportunity that can effectively teach the scientific method, readily accommodate different levels of educational experience, and reach any student with access to a grocery store, a post office and the internet. This paper describes the formation of the program through the students who helped develop and prototype the procedures. A summary is presented of the analysis and preliminary results and a description given of how the program could be linked with other aspects of crystallography. This approach has the potential to bridge the gap between students in remote locations and with limited funding, and access to scientific resources, providing students with an international-level research experience. teaching and education 1200 Joseph R. Luft et al. Grocery store crystallography
Methods in molecular biology (Clifton, N.J.), 2008
The Structural Genomics of Pathogenic Protozoa (SGPP) Consortium aimed to determine crystal struc... more The Structural Genomics of Pathogenic Protozoa (SGPP) Consortium aimed to determine crystal structures of proteins from trypanosomatid and malaria parasites in a high throughput manner. The pipeline of target selection, protein production, crystallization, and structure determination, is sketched. Special emphasis is given to a number of technology developments including domain prediction, the use of "co-crystallants," and capillary crystallization.…
Acta Crystallographica Section D Biological Crystallography, 2003
The results of the crystal structure determination of human dihydrofolate reductase (hDHFR) as a ... more The results of the crystal structure determination of human dihydrofolate reductase (hDHFR) as a binary complex with the potent N9ÐC10 reversed-bridge antifolate inhibitor 2,4-diamino-6-[N-(3 H ,4 H ,5 H -trimethoxybenzyl)-N-methylamino]pyrido[2,3-d]pyrimidine (1) are reported for two independent polymorphic rhombohedral R3 lattices [R3(1) and R3(2)]. Data from these two crystal forms were re®ned to 1.90 A Ê resolution for complex R3(1), with R = 0.186 for 9689 data, and to 1.80 A Ê resolution for complex R3(2), with R = 0.194 for 13 305 data. Changes in the loop geometry between the two structures re¯ects contact differences in the packing environments in the two R3 lattices. The largest changes (between 0.5 and 1.7 A Ê ) are observed for the loop regions encompassing residues 16±25, 40±48, 81±89, 99±108, 143±148 and 161±169. Comparison of the intermolecular contacts of these loops reveals that the R3(2) lattice is more tightly packed, as re¯ected in its smaller V M value and smaller solvent content. The conformation of inhibitor (1) is similar in both structures and the N9ÐC10 bridge geometry is more similar to that observed for the normal C9ÐN10 bridge of trimetrexate (TMQ) than to the other N9ÐC10 reversed-bridge antifolates previously reported. The effect of the N9ÐC10 reversedbridge geometry is to distort the bridge from coplanarity with the pyrido[2,3-d]pyrimidine ring system and to twist the C10 methylene conformation towards a gauche conformation. This also in¯uences the conformation of the methoxybenzyl ring, moving it away from a trans position and placing the 5 H -methoxy group deeper within the hydrophobic pocket made by Leu60, Pro61 and Asn64 of the hDHFR active site.
Acta Crystallographica Section D Biological Crystallography, 2003
A technique for automatically evaluating microbatch (400 nl) protein-crystallization trials is de... more A technique for automatically evaluating microbatch (400 nl) protein-crystallization trials is described. This method addresses analysis problems introduced at the sub-microlitre scale, including non-uniform lighting and irregular droplet boundaries. The droplet is segmented from the well using a loopy probabilistic graphical model with a two-layered grid topology. A vector of 23 features is extracted from the droplet image using the Radon transform for straight-edge features and a bank of correlation ®lters for microcrystalline features. Image classi®cation is achieved by linear discriminant analysis of its feature vector. The results of the automatic method are compared with those of a human expert on 32 1536-well plates. Using the human-labeled images as ground truth, this method classi®es images with 85% accuracy and a ROC score of 0.84. This result compares well with the experimental repeatability rate, assessed at 87%. Images falsely classi®ed as crystalpositive variously contain speckled precipitate resembling microcrystals, skin effects or genuine crystals falsely labeled by the human expert. Many images falsely classi®ed as crystalnegative variously contain very ®ne crystal features or dendrites lacking straight edges. Characterization of these misclassi®cations suggests directions for improving the method.
Acta Crystallographica Section D Biological Crystallography, 2005
Structural data are reported to 2.5 A resolution for the first full analysis of the methotrexate-... more Structural data are reported to 2.5 A resolution for the first full analysis of the methotrexate-resistant Leu22Arg (L22R) variant of mouse dihydrofolate reductase (mDHFR) crystallized as a ternary complex with methotrexate (MTX) and the cofactor NADPH. These results are compared with the MTX and NADPH ternary complexes of L22R human DHFR (hDHFR) and those of mouse and human wild-type DHFR enzymes. The conformation of mDHFR Arg22 is such that it makes hydrogen-bonding contacts with Asp21, Trp24 and a structural water molecule, observations which were not made in the L22R hDHFR ternary complex. These data show that there is little difference between the structures of the wild type and L22R variant for either mouse or human DHFR; however, there are significant differences between the species. Comparison of these structures reveals that the active site of mDHFR is larger than that in the hDHFR structure. In mDHFR, the position of MTX is shifted 0.6 A toward helix C (residues 59-65), which in turn is shifted 1.2 A away from the active site relative to that observed in the hDHFR ternary complexes. In the L22R variant mDHFR structure, MTX makes shorter contacts to the conserved residues Ile7, Val115 and Tyr121 than in the L22R variant human DHFR structure. These contacts are comparable in both wild-type enzymes. The unexpected results from this comparison of the mouse and human DHFR complexes bound with the same ligand and cofactor illustrate the importance of detailed study of several species of enzyme, even when there is a high sequence homology between them. These data suggest that the differences in binding interactions of the L22R variant are in agreement with the weaker binding affinity for MTX in the variant enzymes; the larger size of the binding site in mDHFR supports the observation that the binding affinity of MTX for L22R mDHFR is significantly weaker than that of the L22R hDHFR enzyme.
Acta Crystallographica Section D Biological Crystallography, 2004
Structural data are reported for the first examples of the tetrahydroquinazoline antifolate (6R,6... more Structural data are reported for the first examples of the tetrahydroquinazoline antifolate (6R,6S)-2,4-diamino-6-(1-indolinomethyl)-5,6,7,8-tetrahydroquinazoline (1) and its trimethoxy analogue (6R,6S)-2,4-diamino-6-(3',4',5'-trimethoxybenzyl)-5,6,7,8-tetrahydroquinazoline (2) as inhibitor complexes with dihydrofolate reductase (DHFR) from human (hDHFR) and Pneumocystis carinii (pcDHFR) sources. The indoline analogue (1) was crystallized as ternary complexes with NADPH and hDHFR (1.9 A resolution) and pcDHFR (2.3 A resolution), while the trimethoxy quinazoline analogue (2) was crystallized as a binary complex with hDHFR in two polymorphic rhombohedral R3 lattices: R3(1) to 1.8 A resolution and R3(2) to 2.0 A resolution. Structural analysis of these potent and selective DHFR-inhibitor complexes revealed preferential binding of the 6S-equatorial isomer in each structure. This configuration is similar to that of the natural tetrahydrofolate substrate; that is, 6S. These data also show that in both the hDHFR and pcDHFR ternary complexes with (1) the indoline ring is partially disordered, with two static conformations that differ between structures. These conformers also differ from that observed for the trimethoxybenzyl ring of tetrahydroquinazoline (2). There is also a correlation between the disorder of the flexible loop 23 and the disorder of the cofactor nicotinamide ribose ring in the pcDHFR-NADPH-(1) ternary complex. Comparison of the Toxoplasma gondii DHFR (tgDHFR) sequence with those of other DHFRs provides insight into the role of sequence and conformation in inhibitor-binding preferences which may aid in the design of novel antifolates with specific DHFR selectivity.
X-ray crystallography typically requires the mounting of crystals, which can make the sample diff... more X-ray crystallography typically requires the mounting of crystals, which can make the sample difficult to manipulate when it is small and the microscope objective is close to the crystallization plate. By simply moving the objective to the bottom of a clear crystallization plate (inverting the normal view), crystals were able to be manipulated and harvested from wells having a 0.9 mm diameter and 5.0 mm depth. The mounting system enabled the structural solution of the 187 amino acid N-terminal domain of Saccharomyces cerevisiae glutaminyl-tRNA synthetase from crystals that appeared during high-throughput screening but proved recalcitrant to scale-up and optimization. While not a general mounting solution, the simple expedient of removing the objective lens from the area where manipulation and harvesting occur greatly facilitates the manual, or even automated, process.
Acta Crystallographica Section D Biological Crystallography, 2001
The first observation of the unique environment for thyroxine (T(4)) binding in tetrameric rat tr... more The first observation of the unique environment for thyroxine (T(4)) binding in tetrameric rat transthyretin (rTTR) is reported as determined by X-ray diffraction. These data revealed different modes of hormone binding in the two unique hormone-binding sites in the rat TTR tetramer channel. Differences in the orientation of thyroxine and the position of water molecules in the two binding sites further suggest a mechanism for the docking pathway of the hormone into the channel of TTR. Crystals of the rat transthyretin-thyroxine complex are isomorphous with those reported for apo rTTR and crystallized in the tetragonal space group P4(3)2(1)2 with four independent TTR monomeric subunits in the asymmetric part of the crystal lattice. Data were collected to 2.5 A resolution and the structure was refined to R = 20.9% for 15 384 data in the resolution range 12-2.5 A. Similar to human TTR, the rat protein is also a 54 000 Da tetramer with four identical polypeptide chains of 127 amino-acid residues. Of the 22 amino-acid residues which differ between the human and rat sequences, none are in the thyroxine-binding domains. Analysis of these structural data reveals that the tertiary structure is similar to that of hTTR, with only small differences in the flexible loop regions on the surface of the structure. Conformational changes of the amino acids in the channel result in a hydrogen-bonded network that connects the two binding domains, in contrast to the hydrogen bonds formed along the tetramer interface in the apo transthyretin structure. These changes suggest a mechanism for the signal transmission between thyroxine-binding domains.
Acta Crystallographica Section D Biological Crystallography, 1996
The molecular structures of two human transthyretin (hTTR, prealbumin) complexes, co-crystallized... more The molecular structures of two human transthyretin (hTTR, prealbumin) complexes, co-crystallized with thyroxine (3,5,3',5'-tetraiodo-L-thyronine; T4) , and with 3',5'-dinitro-N-acetyl-L-thyronine (DNNAT), were determined by X-ray diffraction methods. Crystals of both structures are orthorhombic, space group P2~2~2, and have two independent monomers in the asymmetric unit of the crystal lattice. These structures have been refined to 17.0% for 8-2.0A resolution data for the T 4 complex (I), and to R= 18.4% for 8-2.2A resolution data for the DNNAT structure (II). This report provides a detailed description of T 4 binding to wild-type hTTR at 2.0A resolution, as well as DNNAT. In both structures, the two independent hormone-binding sites of the TTR tetramer are occupied by ligand. A 50% statistical disorder model was applied to account for the crystallographic twofold symmetry along the binding channel and the lack of such symmetry for the ligands. Results for the co-crystallized T 4 complex show that T 4 binds deep in the hormone-binding channel and displaces the bound water previously reported for T 4 soaked into a native transthyretin crystal . Nature (London), 268, 115-120]. DNNAT also binds deeper in the channel toward the tetramer center than T 4 with the nitro groups occupying the symmetrical innermost halogen pockets. The N-acetyl moiety does not form polar contacts with the protein side chains as it is oriented toward the center of the channel. The weak binding affinity of DNNAT results from the loss of hydrophobic interactions with the halogen binding pockets as observed in T 4 binding. These data suggest that the halogen-binding sites toward the tetramer center are of primary importance as they are occupied by analogues with weak affinity to TTR, and are therefore selected over the other halogen sites which contribute more strongly to the overall binding affinity.
The crystal and molecular structures of (Z)-4,4',6_triacetoxy aurone (I) and (Z)-3',5'-dibromo-2'... more The crystal and molecular structures of (Z)-4,4',6_triacetoxy aurone (I) and (Z)-3',5'-dibromo-2',4,4',6-tetrahydroxyaurone monohydrate (II) have been studied by X-ray analysis and AM1 molecular orbital methods. Aurones are characterized by a methine bridge between the benxofuran and phenolic rings and thus have only one flexible bond, the torsion about C (2) -C (21) -C ( 1' ) -C (6' ) ,8'. In the crystal both aurones have a (Z) -planar conformation defined by 8' values of 3.1(5) ' and 0.7(5)', for (I) and (II) respectively. Minimum energy conformations from AM1 molecular orbital calculations have 19' values of 12.5 ' for (I) and 3.3" and 31.3 ' for (II), depending on the orientation of the 2' -hydroxy hydrogen. The relative barrier to rotation about 8' for all AM1 conformations is less than 4 kcal mol-_'. When H2' 1 is in the plane of the phenolic ring cis to the methine bridge, it comes within 1.5 A of the methine hydrogen and causes the crystallographic conformation to be 2 kcal mol-' higher than the minimum energy structure at 31 O. When this hydrogen is placed trans to the methine bridge, the minimum energy conformation is 3.3 ', in agreement with the X-ray results. These observations suggest that caution should be used in the interpretation of AM1 results for such a sterically crowded system. Furthermore, these data indicate that, although a planar conformation is a minimum energy structure for aurones, less than 4 kcal mol-' is required to adopt a nonplanar conformation suggested as the active form for their action as inhibitors of the enzyme iodothyronine deiodinase. These aurones also act as competitive inhibitors for the binding of thyroxine to transthyretin, where similar conformational requirements are postulated.
Proceedings of the National Academy of Sciences, 1992
The crystal structure of the complex of 3',5'dibromo-2',4,4',6-tetrahydroxyaurone, a flavone deri... more The crystal structure of the complex of 3',5'dibromo-2',4,4',6-tetrahydroxyaurone, a flavone derivative, with human transthyretin (TTR), a serum thyroid hormone transport protein, has been determined and refined to R = 17.9% for data to 2.3-A resolution and provides a detailed description of a protein-bound flavonoid structure. This bromoaurone is a potent competitor for thyroid hormone binding to TTR, a 54,980-dalton a4 tetrameric protein of 222 molecular
Proceedings of the National Academy of Sciences, 2007
Saccharomyces cerevisiae is an ideal host from which to obtain high levels of posttranslationally... more Saccharomyces cerevisiae is an ideal host from which to obtain high levels of posttranslationally modified eukaryotic proteins for x-ray crystallography. However, extensive replacement of methionine by selenomethionine for anomalous dispersion phasing has proven intractable in yeast. We report a general method to incorporate selenomethionine into proteins expressed in yeast based on manipulation of the appropriate metabolic pathways. sam1 ؊ sam2 ؊ mutants, in which the conversion of methionine to S-adenosylmethionine is blocked, exhibit reduced selenomethionine toxicity compared with wild-type yeast, increased production of protein during growth in selenomethionine, and efficient replacement of methionine by selenomethionine, based on quantitative mass spectrometry and x-ray crystallography. The structure of yeast tryptophanyl-tRNA synthetase was solved to 1.8 Å by using multiwavelength anomalous dispersion phasing with protein that was expressed and purified from the sam1 ؊ sam2 ؊ strain grown in selenomethionine. Six of eight selenium residues were located in the structure.
The crystal and molecular structures of morin (2', 3,4', 5,7-pentahydroxyflavone) hydrate (I), an... more The crystal and molecular structures of morin (2', 3,4', 5,7-pentahydroxyflavone) hydrate (I), and myricetin (3', 4', 5', 3,5,7-hexahydroxyflavone) triphenylphosphine oxide (TPPO) (1:2) co-crystal complex (II) have been studied by X-ray analysis and AM1 molecular orbital methods. The molecular conformation of the two flavones, described by the torsion angle 0 [C(3)-C(2)-C(l')-C(2')] between the benzopyrone and phenyl rings is -43.4" and 51.0" for molecules A and B of morin, respectively, and -37.0" for myricetin. Minimum energy conformations from AM1 molecular orbital calculations have 0 values of -38.2" for morin and -27.0" for myricetin. The energy profile for rotation about 0 for morin has a 28 kcal mol-' barrier at 0" due to steric interactions between the 2'hydroxy and the 3-hydroxy group. There are two local minima near 30 and 140", in good agreement with structural results. The profile for myricetin has two equivalent minima near 30 and 150" with a barrier of less than 2 kcal mol-' In the crystal both flavones form extensive networks of intra-and intermolecular hydrogen bonds. In (I), each morin conformer packs in alternating layers linked by water molecules, while in (II), TPPO stabilizes the crystal by formation of short hydrogen bonds (2.58-2.65 A) of the phosphoryl oxygen to the flavone. Myricetin also forms a two dimensional sheet-like packing in which myricetin molecules hydrogen bond to each other, as well as to TPPO. These conformational and hydrogen bonding patterns provide insight into specific types of ligandreceptor interactions and support structure activity data which suggest the importance of electronic and hydrogen bonding properties in the bioactivity of flavones.
The synthesis and biological activities of 14 6-substituted 2,4-diaminoquinazolines are reported.... more The synthesis and biological activities of 14 6-substituted 2,4-diaminoquinazolines are reported. These compounds were designed to improve the cell penetration of a previously reported series of 2,4-diamino-6-substituted-pyrido[2,3-d]pyrimidines which had shown significant potency and remarkable selectivity for Toxoplasma gondii dihydrofolate reductase (DHFR), but had much lower inhibitory effects on the growth of T. gondii cells in culture. The target N9-H analogues were obtained via regiospecific reductive amination of the appropriate benzaldehydes with 2,4,6-triaminoquinazoline, which, in turn, was synthesized from 2,4-diamino-6-nitroquinazoline. The N9-CH3 analogues were synthesized via a regiospecific reductive methylation of the corresponding N9-H precursors. The compounds were evaluated as inhibitors of DHFR from human, Pneumocystis carinii, T. gondii, rat liver, Lactobacillus casei, and Escherichia coli, and selected analogues were evaluated as inhibitors of the growth of tumor cells in culture. These analogues displayed potent T. gondii DHFR inhibition as well as inhibition of the growth of T. gondii cells in culture. Further, selected analogues were potent inhibitors of the growth of tumor cells in culture in the in vitro screening program of the National Cancer Institute with GI50s in the nanomolar and subnanomolar range. Crystallographic data for the ternary complex of hDHFR-NADPH and 2,4-diamino-6-[N-(2', 5'-dimethoxybenzyl)-N-methylamino]pyrido[2,3-d]pyrimidine, 1c, reveal the first structural details for a reversed N9-C10 folate bridge geometry as well as the first conformational details of a hybrid piritrexim-trimetrexate analogue.
Crystallography is a multidisciplinary field that links divergent areas of mathematics, science a... more Crystallography is a multidisciplinary field that links divergent areas of mathematics, science and engineering to provide knowledge of life on an atomic scale. Crystal growth, a key component of the field, is an ideal vehicle for education. Crystallization has been used with a 'grocery store chemistry' approach and linked to high-throughput remote-access screening technologies. This approach provides an educational opportunity that can effectively teach the scientific method, readily accommodate different levels of educational experience, and reach any student with access to a grocery store, a post office and the internet. This paper describes the formation of the program through the students who helped develop and prototype the procedures. A summary is presented of the analysis and preliminary results and a description given of how the program could be linked with other aspects of crystallography. This approach has the potential to bridge the gap between students in remote locations and with limited funding, and access to scientific resources, providing students with an international-level research experience. teaching and education 1200 Joseph R. Luft et al. Grocery store crystallography
Methods in molecular biology (Clifton, N.J.), 2008
The Structural Genomics of Pathogenic Protozoa (SGPP) Consortium aimed to determine crystal struc... more The Structural Genomics of Pathogenic Protozoa (SGPP) Consortium aimed to determine crystal structures of proteins from trypanosomatid and malaria parasites in a high throughput manner. The pipeline of target selection, protein production, crystallization, and structure determination, is sketched. Special emphasis is given to a number of technology developments including domain prediction, the use of "co-crystallants," and capillary crystallization.…
Acta Crystallographica Section D Biological Crystallography, 2003
The results of the crystal structure determination of human dihydrofolate reductase (hDHFR) as a ... more The results of the crystal structure determination of human dihydrofolate reductase (hDHFR) as a binary complex with the potent N9ÐC10 reversed-bridge antifolate inhibitor 2,4-diamino-6-[N-(3 H ,4 H ,5 H -trimethoxybenzyl)-N-methylamino]pyrido[2,3-d]pyrimidine (1) are reported for two independent polymorphic rhombohedral R3 lattices [R3(1) and R3(2)]. Data from these two crystal forms were re®ned to 1.90 A Ê resolution for complex R3(1), with R = 0.186 for 9689 data, and to 1.80 A Ê resolution for complex R3(2), with R = 0.194 for 13 305 data. Changes in the loop geometry between the two structures re¯ects contact differences in the packing environments in the two R3 lattices. The largest changes (between 0.5 and 1.7 A Ê ) are observed for the loop regions encompassing residues 16±25, 40±48, 81±89, 99±108, 143±148 and 161±169. Comparison of the intermolecular contacts of these loops reveals that the R3(2) lattice is more tightly packed, as re¯ected in its smaller V M value and smaller solvent content. The conformation of inhibitor (1) is similar in both structures and the N9ÐC10 bridge geometry is more similar to that observed for the normal C9ÐN10 bridge of trimetrexate (TMQ) than to the other N9ÐC10 reversed-bridge antifolates previously reported. The effect of the N9ÐC10 reversedbridge geometry is to distort the bridge from coplanarity with the pyrido[2,3-d]pyrimidine ring system and to twist the C10 methylene conformation towards a gauche conformation. This also in¯uences the conformation of the methoxybenzyl ring, moving it away from a trans position and placing the 5 H -methoxy group deeper within the hydrophobic pocket made by Leu60, Pro61 and Asn64 of the hDHFR active site.
Acta Crystallographica Section D Biological Crystallography, 2003
A technique for automatically evaluating microbatch (400 nl) protein-crystallization trials is de... more A technique for automatically evaluating microbatch (400 nl) protein-crystallization trials is described. This method addresses analysis problems introduced at the sub-microlitre scale, including non-uniform lighting and irregular droplet boundaries. The droplet is segmented from the well using a loopy probabilistic graphical model with a two-layered grid topology. A vector of 23 features is extracted from the droplet image using the Radon transform for straight-edge features and a bank of correlation ®lters for microcrystalline features. Image classi®cation is achieved by linear discriminant analysis of its feature vector. The results of the automatic method are compared with those of a human expert on 32 1536-well plates. Using the human-labeled images as ground truth, this method classi®es images with 85% accuracy and a ROC score of 0.84. This result compares well with the experimental repeatability rate, assessed at 87%. Images falsely classi®ed as crystalpositive variously contain speckled precipitate resembling microcrystals, skin effects or genuine crystals falsely labeled by the human expert. Many images falsely classi®ed as crystalnegative variously contain very ®ne crystal features or dendrites lacking straight edges. Characterization of these misclassi®cations suggests directions for improving the method.
Acta Crystallographica Section D Biological Crystallography, 2005
Structural data are reported to 2.5 A resolution for the first full analysis of the methotrexate-... more Structural data are reported to 2.5 A resolution for the first full analysis of the methotrexate-resistant Leu22Arg (L22R) variant of mouse dihydrofolate reductase (mDHFR) crystallized as a ternary complex with methotrexate (MTX) and the cofactor NADPH. These results are compared with the MTX and NADPH ternary complexes of L22R human DHFR (hDHFR) and those of mouse and human wild-type DHFR enzymes. The conformation of mDHFR Arg22 is such that it makes hydrogen-bonding contacts with Asp21, Trp24 and a structural water molecule, observations which were not made in the L22R hDHFR ternary complex. These data show that there is little difference between the structures of the wild type and L22R variant for either mouse or human DHFR; however, there are significant differences between the species. Comparison of these structures reveals that the active site of mDHFR is larger than that in the hDHFR structure. In mDHFR, the position of MTX is shifted 0.6 A toward helix C (residues 59-65), which in turn is shifted 1.2 A away from the active site relative to that observed in the hDHFR ternary complexes. In the L22R variant mDHFR structure, MTX makes shorter contacts to the conserved residues Ile7, Val115 and Tyr121 than in the L22R variant human DHFR structure. These contacts are comparable in both wild-type enzymes. The unexpected results from this comparison of the mouse and human DHFR complexes bound with the same ligand and cofactor illustrate the importance of detailed study of several species of enzyme, even when there is a high sequence homology between them. These data suggest that the differences in binding interactions of the L22R variant are in agreement with the weaker binding affinity for MTX in the variant enzymes; the larger size of the binding site in mDHFR supports the observation that the binding affinity of MTX for L22R mDHFR is significantly weaker than that of the L22R hDHFR enzyme.
Acta Crystallographica Section D Biological Crystallography, 2004
Structural data are reported for the first examples of the tetrahydroquinazoline antifolate (6R,6... more Structural data are reported for the first examples of the tetrahydroquinazoline antifolate (6R,6S)-2,4-diamino-6-(1-indolinomethyl)-5,6,7,8-tetrahydroquinazoline (1) and its trimethoxy analogue (6R,6S)-2,4-diamino-6-(3',4',5'-trimethoxybenzyl)-5,6,7,8-tetrahydroquinazoline (2) as inhibitor complexes with dihydrofolate reductase (DHFR) from human (hDHFR) and Pneumocystis carinii (pcDHFR) sources. The indoline analogue (1) was crystallized as ternary complexes with NADPH and hDHFR (1.9 A resolution) and pcDHFR (2.3 A resolution), while the trimethoxy quinazoline analogue (2) was crystallized as a binary complex with hDHFR in two polymorphic rhombohedral R3 lattices: R3(1) to 1.8 A resolution and R3(2) to 2.0 A resolution. Structural analysis of these potent and selective DHFR-inhibitor complexes revealed preferential binding of the 6S-equatorial isomer in each structure. This configuration is similar to that of the natural tetrahydrofolate substrate; that is, 6S. These data also show that in both the hDHFR and pcDHFR ternary complexes with (1) the indoline ring is partially disordered, with two static conformations that differ between structures. These conformers also differ from that observed for the trimethoxybenzyl ring of tetrahydroquinazoline (2). There is also a correlation between the disorder of the flexible loop 23 and the disorder of the cofactor nicotinamide ribose ring in the pcDHFR-NADPH-(1) ternary complex. Comparison of the Toxoplasma gondii DHFR (tgDHFR) sequence with those of other DHFRs provides insight into the role of sequence and conformation in inhibitor-binding preferences which may aid in the design of novel antifolates with specific DHFR selectivity.
X-ray crystallography typically requires the mounting of crystals, which can make the sample diff... more X-ray crystallography typically requires the mounting of crystals, which can make the sample difficult to manipulate when it is small and the microscope objective is close to the crystallization plate. By simply moving the objective to the bottom of a clear crystallization plate (inverting the normal view), crystals were able to be manipulated and harvested from wells having a 0.9 mm diameter and 5.0 mm depth. The mounting system enabled the structural solution of the 187 amino acid N-terminal domain of Saccharomyces cerevisiae glutaminyl-tRNA synthetase from crystals that appeared during high-throughput screening but proved recalcitrant to scale-up and optimization. While not a general mounting solution, the simple expedient of removing the objective lens from the area where manipulation and harvesting occur greatly facilitates the manual, or even automated, process.
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Papers by Joseph Luft