Charles M. Weeks

The design and implementation of SnB version 2.0

SnB is a direct-methods program based on the Shake-and-Bake methodology. It has been used to solve difficult or large structures that could not be solved by traditional reciprocal-space routines based on the tangent formula. Recently, it has also been used to determine the Se sites in large selenomethionyl-substituted proteins. SnB version 1.5 has been available for several years and is being used regularly in many laboratories. In this paper, we introduce SnB version 2.0, which incorporates a graphical user interface written in Java, a dynamic histogram display, and an interactive Java/VRML-based visualization facility. In addition, it provides the user with several utility routines and a variety of new algorithmic options.

SnB : crystal structure determination via shake-and-bake

Shake-and-bake is a direct-methods phasing algorithm for structure determination based on the minimal principle. SnB is a program based on shake-and-bake that has been used successfully to solve more than a dozen structures in a variety of space groups. The focus of this paper is on the details of this program, including its structure, system requirements, running times and the rationale for coding in a combination of C and Fortran. A summary of successful SnB applications is also provided. These include solving two previously unknown 100-atom structures and re-solving crambin (a structure containing the equivalent of approximately 400 fully occupied atomic positions) for the first time with a direct-methods technique.

The refined three-dimensional structure of 3α,20β-hydroxysteroid dehydrogenase and possible roles of the residues conserved in short-chain dehydrogenases

Abstract Background Bacterial 3 α ,20 β -hydroxysteroid dehydrogenase reversibly oxidizes the 3 α and 20 β hydroxyl groups of steroids derived from androstanes and pregnanes. It was the first short-chain dehydrogenase to be studied by X-ray crystallography. The previous description of the structure of this enzyme, at 2.6 a resolution, did not permit unambiguous assignment of several important groups. We have further refined the structure of the complex of the enzyme with its cofactor, nicotinamide adenine dinucleotide (NAD), and solvent molecules, at the same resolution. Results The asymmetric unit of the crystal contains four monomers each with 253 amino acid residues, 38 water molecules, and 176 cofactor atoms belonging to four NAD molecules — one for each subunit. The positioning of the cofactor molecule has been modified from our previous model and is deeper in the catalytic cavity as observed for other members of both the long-chain and short-chain dehydrogenase families. The nicotinamide-ribose end of the cofactor has several possible conformations or is dynamically disordered. Conclusions The catalytic site contains residues Tyr152 and Lys156. These two amino acids are strictly conserved in the short-chain dehydrogenase superfamily. Modeling studies with a cortisone molecule in the catalytic site suggest that the Tyr152, Lys156 and Ser139 side chains promote electrophilic attack on the (C 20 – O) carbonyl oxygen atom, thus enabling the carbon atom to accept a hydride from the reduced cofactor.

Structure solution by minimal-function phase refinement and Fourier filtering. II. Implementation and applications

The minimal function, R(psi), has been used to provide the basis for a new computer-intensive direct-methods procedure that shows potential for providing fully automatic routine solutions for structures in the 200-400 atom range. This procedure, which has been called shake-and-bake, is an iterative process in which real-space filtering is alternated with phase refinement using a technique that reduces the value of R(psi). It has been successfully tested using experimental data for a dozen known structures ranging in size from 25 to 317 atoms and crystallizing in a variety of space groups. The details of this procedure, the parameters used and the results of these applications are described.

Optimizing Shake-and-Bake for proteins.

Shake-and-Bake is a direct-methods procedure which has provided ab initio solutions for protein structures containing as many as 1000 independent non-H atoms. This algorithm extends the range of conventional direct methods by repetitively, unconditionally and automatically alternating reciprocal-space phase refinement with filtering in real space to impose constraints. The application of SnB to protein-sized molecules is significantly affected by the choice made for certain critical parameters, including the number of peaks used for density modification, the choice of phase-refinement method and the number of refinement cycles. The effects of parameter variation have been studied for six protein structures, all of which are solvable by Shake-and-Bake using data at 1.1 A or higher resolution. Solvability in the resolution range 1.2-1.4 A appears to be enhanced by the presence of heavier atoms (S, Cl). Furthermore, it appears that in this range the ratio of refinement cycles and triplet phase invariants to atoms in the structure must be increased. Large structures lacking atoms of any element heavier than oxygen also require non-traditional parameter values.

X-ray crystallographic analysis of the hydration of A- and B-form DNA at atomic resolution.

We have determined single crystal structures of an A-DNA decamer and a B-DNA dodecamer at 0.83 and 0.95 A, respectively. The resolution of the former is the highest reported thus far for any right-handed nucleic acid duplex and the quality of the diffraction data allowed determination of the structure with direct methods. The structures reveal unprecedented details of DNA fine structure and hydration; in particular, we have reexamined the overall hydration of A- and B-form DNA, the distribution of water around phosphate groups, and features of the water structure that may underlie the B to A transition.

Valinomycin Crystal Structure Determination by Direct Methods

The conformation of an uncomplexed form of the antibiotic valinomycin (C54N6O18H90) has been determined by direct methods including a novel technique for strong enantiomorph discrimination via the calculation and systematic analysis of cosine invariants of a special type. The intramolecular hydrogen bonding scheme and the isopropyl group stereochemistry of uncomplexed valinomycin are compatible with interpretations of spectral measurements for the complexed and uncomplexed molecule in solution but are different from any previously proposed structure. The simple conformational change of a hydrogen bond shift, which could be induced by the process of potassium ion complexing, transforms the uncomplexed into the complexed structure.

Structure solution by minimal-function phase refinement and Fourier filtering. I. Theoretical basis

Eliminating the N atomic position vectors rj, j = 1, 2, ..., N, from the system of equations defining the normalized structure factors EH yields a system of identities that the EHs must satisfy, provided that the set of EHs is sufficiently large. Clearly, for fixed N and specified space group, this system of identities depends only on the set [H], consisting of n reciprocal-lattice vectors H, and is independent of the crystal structure, which is assumed for simplicity to consist of N identical atoms per unit cell. However, for a fixed crystal structure, the magnitudes magnitude of /EH/ are uniquely determined so that a system of identities is obtained among the corresponding phases psi H alone, which depends on the presumed known magnitudes magnitude of /EH/ and which must of necessity be satisfied. The known conditional probability distributions of triplets and quartets, given the values of certain magnitudes magnitude of /E/, lead to a function R(psi) of phases, uniquely determined by magnitudes magnitude of /E/ and having the property that RT < 1/2 < RR, where RT is the value of R(psi) when the phases are equal to their true values, no matter what the choice of origin and enantiomorph, and RR is the value of R(psi) when the phases are chosen at random. The following conjecture is therefore plausible: the global minimum of R(psi), where the phases are constrained to satisfy all identities among them that are known to exist, is attained when the phases are equal to their true values and is thus equal to RT.(ABSTRACT TRUNCATED AT 250 WORDS)

Towards automated protein structure determination: BnP, the SnB-PHASES interface

Abstract The direct-methods program SnB provides an efficient means for solving protein substructures containing many heavy-atom sites (current record: 160). In order to meet the high-throughput requirements of structural genomics projects, substructure determination needs to be tightly integrated with other aspects of the protein-phasing process. This has been accomplished through the design of a common Java interface, BnP, for SnB and components of PHASES, a popular and proven program suite that provides all the tools necessary to proceed from substructure refinement to the computation of an unambiguous protein electron-density map. Therefore, BnP will facilitate a high degree of automation and enable rapid structure determination by both experienced and novice crystallographers.

Molecular conformation and protein binding affinity of progestins.

Analysis of X-ray data concerning 277 estranes, androstanes, and pregnanes and comparison with progesterone receptor binding data have prompted the following observations. In general: 1. The flexibility of natural steroid hormones permits them to take up conformations optimal for binding to sites on proteins that vary in individual structural requirements. 2. When substituents strain the fused ring system, the strain will be delocalized and often transmitted to the most flexible point of the molecule, thus giving rise to conformational transmission effects. Consequently, substituents will generally stabilize a specific conformation, limiting protein interaction and enhancing a specific hormone response. 3. Hydrogen bond patterns in crystals can be used to predict points of active site attachment. 4. Distortions resulting from crystal packing forms are insignificant. Progestin receptor binding affinity: 5. Complementarity of fit is not specific on the alpha and beta faces of the B, C, and D rings. 6. The delta4-3-one composition is the only consistently required element. 7. Five of the eight highest-affinity binders have inverted A rings. Others may be easily converted to it. 8. The inverted A ring is proposed as the optimal conformation and primary factor controlling binding. 9. An A ring binding pattern is apparent in other steroidal hormones. 10. The D-ring region is open to contribute to conformational change in the receptor or genome interaction.