An Isomorphous Replacement Method for Phasing Twinned Structures

A linear least-squares formulation of the method of isomorphous replacement is presented. With data from untwinned crystals, this approach is shown to be equivalent to the phasing representation developed by Hendrickson & Lattman [Acta Cryst. (1970). B26, 136-143]. A general method for calculating the most probable phase is described and applied to the higher- dimensional problem of phase determination for twinned structures. A method for calculating the best phase with intensity data from twinned crystals is also presented. The dependences of these phasing procedures on the number of derivatives and accuracy of the data sets are evaluated in test calculations

The Bacterial Photosynthetic Reaction Center as a Model for Membrane Proteins

Membrane proteins participate in many fundamental cellular processes. Until recently, an understanding of the function and properties of membrane proteins was hampered by an absence of structural information at the atomic level. A landmark achievement toward understanding the structure of membrane proteins was the crystallization (1) and structure determination (2-5) the photosynthetic reaction center (RC) from the purple bacteria Rhodopseudomonas viridis, followed by that of the RC from Rhodobacter sphaeroides (6-17). The RC is an integral membrane protein-pigment complex, which carries out the initial steps of photosynthesis (reviewed in 18). RCs from the purple bacteria Rps. viridis and Rb. sphaeroides are composed of three membrane-associated protein subunits (designated L, M, and H), and the following cofactors: four bacteriochlorophylls (Bchl or B), two bacteriopheophytins (Bphe or [phi]), two quinones, and a nonheme iron. The cofactors are organized into two symmetrical branches that are approximately related by a twofold rotation axis (2, 8). A central feature of the structural organization of the RC is the presence of 11 hydrophobic [alpha]-helixes, approximately 20-30 residues long, which are believed to represent the membrane-spanning portion of the RC (3, 9). Five membrane-spanning helixes are present in both the L and M subunits, while a single helix is in the H subunit. The folding of the L and M subunits is similar, consistent with significant sequence similarity between the two chains (19-25). The L and M subunits are approximately related by the same twofold rotation axis that relates the two cofactor branches. RCs are the first membrane proteins to be described at atomic resolution; consequently they provide an important model for discussing the folding of membrane proteins. The structure demonstrates that [alpha]-helical structures may be adopted by integral membrane proteins, and provides confirmation of the utility of hydropathy plots in identifying nonpolar membrane-spanning regions from sequence data. An important distinction between the folding environments of water-soluble proteins and membrane proteins is the large difference in water concentration surrounding the proteins. As a result, hydrophobic interactions (26) play very different roles in stabilizing the tertiary structures of these two classes of proteins; this has important structural consequences. There is a striking difference in surface polarity of membrane and water-soluble proteins. However, the characteristic atomic packing and surface area appear quite similar. A computational method is described for defining the position of the RC in the membrane (10). After localization of the RC structure in the membrane, surface residues in contact with the lipid bilayer were identified. As has been found for soluble globular proteins, surface residues are less well conserved in homologous membrane proteins than the buried, interior residues. Methods based on the variability of residues between homologous proteins are described (13); they are useful (a) in defining surface helical regions of membrane and water-soluble proteins and (b) in assigning the side of these helixes that are exposed to the solvent. A unifying view of protein structure suggests that water-soluble proteins may be considered as modified membrane proteins with covalently attached polar groups that solubilize the proteins in aqueous solution

Structure of the Reaction Center from Rhodopseudomonas sphaeroides R-26: The Cofactors

The three-dimensional structure of the cofactors of the reaction center of Rhodobacter sphaeroides R-26 has been determined by x-ray diffraction and refined at a resolution of 2.8 Å with an R value of 26%. The main features of the structure are similar to the ones determined for Rhodopseudomonas viridis [Michel, H., Epp, O. & Deisenhofer, J. (1986) EMBO J. 5, 2445-2451]. The cofactors are arranged along two branches, which are approximately related to each other by a 2-fold symmetry axis. The structure is well suited to produce light-induced charge separation across the membrane. Most of the structural features predicted from physical and biochemical measurements are confirmed by the x-ray structure

Structure of the Reaction Center from Rhodopseudomonas sphaeroides R-26 and 2.4.1: Symmetry Relations and Sequence Comparisons between Different Species

Photosynthetic reaction centers from purple bacteria exhibit an approximate twofold symmetry axis, which relates both the cofactors and the L and M subunits. For the reaction center from Rhodobacter sphaeroides, deviations from this twofold symmetry axis have been quantitated by superposing, by a 180 degrees rotation, the cofactors of the B branch onto the A branch and the M subunit onto the L subunit. An alignment of the sequences of the L and M subunits from four purple bacteria, one green bacterium, and the D_1 and D_2 subunits of a photosystem II-containing green alga is presented. The residues that are conserved in all six species are shown in relation to the structure of Rb. sphaeroides and their possible role in the function of the reaction center is discussed. A method is presented for characterizing the exposure of α-helices to the membrane based on the periodicity of conserved residues. This method may prove useful for modeling the three-dimensional structures of membrane proteins

Quantitative Trait Locus Analysis of Morphogenetic and Developmental Traits in an SSR and AFLP-Based Genetic Map of White Clover (\u3cem\u3eTrifolium Repens\u3c/em\u3e L.)

Molecular marker-assisted plant breeding is a key target for the temperate legume pasture crop white clover (Trifolium repens L.). The first genetic linkage map of white clover has been constructed using self-fertile mutants to derive an intercross based fourth and fifth generation inbred parental genotypes (F2[I.4R x I.5J]). The framework map was constructed using simple sequence repeat (TRSSR) and amplified fragment length polymorphism (AFLP) markers. Eighteen linkage groups (LG) corresponding to the anticipated 16 chromosomes of white clover (2n = 4x = 32), with a total map length of 825 cM were derived from a total of 135 markers (78 TRSSR loci and 57 AFLP loci). The F2(I.4R x I.5J) family has been subjected to intensive phenotypic analysis for a range of morphogenetic and developmental traits over several years at IGER, Aberystwyth, Wales and East Craigs, near Edinburgh, Scotland. The resulting phenotypic data were analysed independently to identify QTL (quantitative trait loci) for the various traits, using single marker regression (SMR), interval mapping (IM) and composite interval mapping (CIM) techniques. Multiple coincident QTL regions were identified from the different years and different sites for the same or related traits. The data were reanalysed using a meta-analysis across years and sites and Best Linear Unbiased Estimates (BLUEs) were derived for the plant spread, petiole length, leaf width, leaf length, leaf area, internode length, plant height and flowering date traits. A total of 24 QTLs were identified on 10 of the linkage groups. Three regions on LGs 2, 7 and 12 all demonstrated overlapping QTLs for multiple traits (Figure 1). A meta-analysis approach can quickly identify regions of the genome that control the trait in a robust predictable manner across multiple spatial and temporal replication for rapid targeted genetic enhancement via marker-assisted breeding. This first genetic dissection of agronomic traits in white clover provides the basis for comparative trait-mapping studies and the enhanced development and implementation of marker-assisted breeding strategies

Structure of the Reaction Center from Rhodopseudomonas sphaeroides R-26 and 2.4.1: Protein-Cofactor (Bacteriochlorophyll, Bacteriopheophytin and Carotenoid) Interactions

The three-dimensional structures of the cofactors and protein subunits of the reaction center (RC) from the carotenoidless mutant strain of Rhodobacter sphaeroides R-26 and the wild-type strain 2.4.1 have been determined by x-ray diffraction to resolutions of 2.8 Å and 3.0 Å with R values of 24% and 26%, respectively. The bacteriochlorophyll dimer (D), bacteriochlorophyll monomers (B), and bacteriopheophytin monomers (φ) form two branches, A and B, that are approximately related by a twofold symmetry axis. The cofactors are located in hydrophobic environments formed by the L and M subunits. Differences in the cofactor-protein interactions between the A and B cofactors, as well as between the corresponding cofactors of Rb, sphaeroides and Rhodopseudomonas viridis [Michel, H., Epp, O. & Deisenhofer, J. (1986) EMBO J. 3, 2445-2451], are delineated. The roles of several structural features in the preferential electron transfer along the A branch are discussed. Two bound detergent molecules of beta-octyl glucoside have been located near B_A and B_B. The environment of the carotenoid, C, that is present in RCs from Rb. sphaeroides 2.4.1 consists largely of aromatic residues of the M subunit. A role of B_B in the triplet energy transfer from D to C and the reason for the preferential ease of removal of B_B from the RC is proposed