Charles E. Bugg

Structure of ubiquitin refined at 1.8 A resolution.

The crystal structure of human erythrocytic ubiquitin has been refined at 1.8 A resolution using a restrained least-squares procedure. The crystallographic R-factor for the final model is 0.176. Bond lengths and bond angles in the molecule have root-mean-square deviations from ideal values of 0.016 A and 1.5 degrees, respectively. A total of 58 water molecules per molecule of ubiquitin are included in the final model. The last four residues in the molecule appear to have partial occupancy or large thermal motion. The overall structure of ubiquitin is extremely compact and tightly hydrogen-bonded; approximately 87% of the polypeptide chain is involved in hydrogen-bonded secondary structure. Prominent secondary structural features include three and one-half turns of alpha-helix, a short piece of 3(10)-helix, a mixed beta-sheet that contains five strands, and seven reverse turns. There is a marked hydrophobic core formed between the beta-sheet and alpha-helix. The molecule features a number of unusual secondary structural features, including a parallel G1 beta-bulge, two reverse Asx turns, and a symmetrical hydrogen-bonding region that involves the two helices and two of the reverse turns.

Structure of calmodulin refined at 2.2 A resolution.

The crystal structure of mammalian calmodulin has been refined at 2.2 A (1 A = 0.1 nm) resolution using a restrained least-squares method. The final crystallographic R-factor, based on 6685 reflections in the range 2.2 A less than or equal to d less than or equal to 5.0 A with intensities exceeding 2.5 sigma, is 0.175. Bond lengths and bond angles in the molecule have root-mean-square deviations from ideal values of 0.016 A and 1.7 degrees, respectively. The refined model includes residues 5 to 147, four Ca2+ and 69 water molecules per molecule of calmodulin. The electron density for residues 1 to 4 and 148 is poorly defined, and they are not included in the model. The molecule is shaped somewhat like a dumbbell, with an overall length of 65 A; the two lobes are connected by a seven-turn alpha-helix. Prominent secondary structural features include seven alpha-helices, four Ca2+-binding loops, and two short, double-stranded antiparallel beta-sheets between pairs of adjacent Ca2+-binding loops. The four Ca2+-binding domains in calmodulin have a typical EF hand conformation (helix-loop-helix) and are similar to those described in other Ca2+-binding proteins. The X-ray structure determination of calmodulin shows a large hydrophobic cleft in each half of the molecule. These hydrophobic regions probably represent the sites of interaction with many of the pharmacological agents known to bind to calmodulin.

Algorithm for ribbon models of proteins

Abstract A method is presented to draw smooth, 3D ribbon models of proteins. The procedure calculates closely-spaced guide coordinates based on the peptide plane and passes regular, nearly parallel B-spline curves through them. This becomes a simple process with a graphics device having built-in B-spline generating capabilities such as the Evans and Sutherland PS300. Examination of ribbons such as these provides a useful tool for the crystallographer. Any irregularity in the ribbon is a strong visual cue, suggestive of potential problem areas during the refinement process.

Three-dimensional structure of recombinant human granulocyte-macrophage colony-stimulating factor.

The crystal structure of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) has been determined at 2.8 A resolution using multiple isomorphous replacement techniques. There are two molecules in the crystallographic asymmetric unit, which are related by an approximate non-crystallographic 2-fold axis. The overall structure is highly compact and globular with a predominantly hydrophobic core. The main structural feature of rhGM-CSF is a four alpha-helix bundle, which represents approximately 42% of the structure. The helices are arranged in a left-handed antiparallel bundle with two overhand connections. Within the connections is a two-stranded antiparallel beta-sheet. The tertiary structure of rhGM-CSF has a topology similar to that of porcine growth factor and interferon-beta. Most of the proposed critical regions for receptor binding are located on a continuous surface at one end of the molecule that includes the C terminus.

Crystallographic and Modeling Methods in Molecular Design

This up-to-date review of forefront approaches used in structure-based drug design and protein engineering is intended for chemists, molecular biologists, pharmacologists, crystallographers, computer modelers, and graduate students interested in drug design and protein engineering. Leaders in protein crystallography, molecular dynamics, computer graphics, and molecular modeling report on their major contributions to this rapidly evolving field. Readers can expect to obtain an overall introduction to the major approaches that are now being pursued in structure-based drug design and protein engineering.

Preliminary investigations of protein crystal growth using the Space Shuttle

Abstract Protein crystal growth in space is of interest because of the potential applications for unique studies of crystallization processes. Theoretical and experimental research indicates that gravitational fields produce density-driven convective flow patterns which can influence crystal growth, and these convective effects can be controlled under microgravity conditions. Microgravity can also be used to control sedimentation effects. As part of a program to investigate the influence of gravity on protein crystal growth, ground and shuttle-based experiments are in progress, and suitable techniques and equipment for protein crystal growth in space are being developed. The research program includes several phases of hardware development, beginning with a simple prototype system, and evolving to an automated protein crystal growth unit that will permit the major variables in protein crystallization to be monitored and controlled during the crystal growth processes. As part of the first step in hardware development, protein crystal growth experiments have been performed on four different shuttle flight missions.

The crystal structure of guanosine dihydrate and inosine dihydrate

Crystals of the dihydrates of guanosine (C_(10)H_(13)N_5O_5) and inosine (C_(10)H_(12)N_4O_5) are nearly isostructural. They are monoclinic, space group P2_1, with cell dimensions ɑ = 17·518, b = 11 ·502, c = 6·658 A, β = 98·17° (guanosine) and ɑ = 17·573, b =11·278, c=6-654 A, β = 98·23° (inosine). There are two nucleoside molecules and four water molecules per asymmetric unit. Data were collected on an automated diffractometer; the structures were solved by Patterson and trial-and-error methods and refined to R indices of about 0·035. The structure features hydrogen bonding between purine bases to form ribbons parallel to b and parallel stacking of purine bases along c; the separation between adjacent rings within a stack is 3·3 A. The conformations about the glycosidic C-N bond and the puckerings of the sugar rings arc quite different for the two molecules in the asymmetric unit.

Structure of variant-3 scorpion neurotoxin from Centruroides sculpturatus Ewing, refined at 1.8 A resolution.

The three-dimensional structure of the variant-3 protein neurotoxin from the scorpion Centruroides sculpturatus Ewing has been determined by X-ray diffraction data. The initial model for the 65-residue protein was obtained at 3 A resolution by multiple-isomorphous-replacement methods. The structure was refined at 1.8 A resolution by restrained difference-Fourier methods, and by free-atom, block-diagonal least-squares. Considering the 4900 reflections for which d = 1.8-7 A and Fo greater than 2.5 sigma (Fo), the final R-index is 0.16 for the restrained model, and 0.14 for the free-atom model. Average estimated errors in atomic co-ordinates are about 0.1 A. The refined structure includes 492 protein atoms; one molecule of 2-methyl-2,4-pentanediol, which is tightly bound in a hydrophobic pocket on the surface of the protein; and 72 additional solvent sites. The major secondary structural features are two and a half turns of alpha-helix and a three-strand stretch of antiparallel beta-sheet. The helix is connected to the middle strand of the beta-sheet by two disulfide bridges, and a third disulfide bridge is located nearby. Several loops extend out of this dense core of secondary structure. The protein displays several reverse turns and a highly contorted proline-rich, COOH-terminal segment. One of the proline residues (Pro59) assumes a cis-conformation. The structure involves 44 intramolecular hydrogen bonds. The crystallographic results suggest two major corrections in the published primary structure; one of these has been confirmed by new chemical sequence data. The protein displays a large flattened surface that contains a high concentration of hydrophobic residues, along with most of the conserved amino acids that are found in the scorpion neurotoxins.

The three-dimensional structure of scorpion neurotoxins

The crystal and molecular structure of a toxin from the scorpion Centruroides sculpturatus has been solved by standard x-ray crystallographic methods at 3 A resolution. Subsequently the 3 A model has been refined and the resolution has been extended to 1.8 A using the gradient-curvature method. The final reliability index of 0.17 The structure has two and a half turns of alpha-helix, a three-strand stretch of antiparallel beta-sheet and several beta-turns. Three of the four disulfide bridges are found in close interaction with the alpha-helix and beta-sheet structures in what constitutes a very rigid part of the molecule. Examination of available scorpion toxin sequences reveals several sections containing invariant and/or semiinvariant amino acids. Many of these residues are found clustered on a rather large flat surface which is also clearly more hydrophobic than other areas on the molecule. These observations suggest that this surface may play a role in the biological action of scorpion toxins. Secondary structure predictions calculated using the method of Dufton and Hider agree well with the x-ray structure. This is also true for other scorpion toxins and reinforces the idea that scorpion toxins are a family of structurally related proteins.

Structure of scorpion toxin variant-3 at 1·2 Å resolution

The crystal structure of the variant-3 protein neurotoxin from the scorpion Centruroides sculpturatus Ewing has been refined at 1.2 A resolution using restrained least-squares. The final model includes 492 non-hydrogen protein atoms, 453 protein hydrogen atoms, eight 2-methyl-2,4-pentanediol (MPD) solvent atoms, and 125 water oxygen atoms. The variant-3 protein model geometry deviates from ideal bond lengths by 0.024 A and from ideal angles by 3.6 degrees. The crystallographic R-factor for structure factors calculated from the final model is 0.192 for 17,706 unique reflections between 10.0 to 1.2 A. A comparison between the models of the initial 1.8 A and the 1.2 A refinement shows a new arrangement of the previously poorly defined residues 31 to 34. Multiple conformations are observed for four cysteine residues and an MPD oxygen atom. The electron density indicates that disulfide bonds between Cys12 and Cys65 and between Cys29 and Cys48 have two distinct side-chain conformations. A molecule of MPD bridges neighboring protein molecules in the crystal lattice, and both MPD enantiomers are present in the crystal. A total of 125 water molecules per molecule of protein are included in the final model with B-values ranging from 11 to 52 A2 and occupancies from unity down to 0.4. Comparisons between the 1.2 A and 1.8 A models, including the bound water structure and crystal packing contacts, are emphasized.