The structure of a phytocyanin, the basic blue protein from cucumber, refined at 1.8 A resolution.

The crystal structure of the cucumber basic protein (CBP), a type 1 or blue copper protein, has been refined at 1.8 A resolution. The molecule resembles other blue copper proteins in having a Greek key beta-barrel structure, except that the barrel is open on one side and is better described as a "beta-sandwich" or "beta-taco". The Cu atom has the normal blue copper NNSS' co-ordination with bond lengths Cu-N(His39) = 1.93 A, Cu-S(Cys79) = 2.16 A, Cu-N(His84) = 1.95 A, Cu-S(Met89) = 2.61 A. The Cu-S(Met) bond is the shortest so far observed in a blue copper protein. A disulphide link, (Cys52)-S-S-(Cys85), appears to play an important role in stabilising the molecular structure. It is suggested that the polypeptide fold is typical of a sub-family of blue copper proteins (phytocyanins) as well as a non-metalloprotein, ragweed allergen Ra3, with which CBP has a high degree of sequence identify. The proteins currently identifiable as phytocyanins are CBP, stellacyanin, mavicyanin, umecyanin, a cucumber peeling cupredoxin, a putative blue copper protein in pea pods, and a blue copper protein from Arabidopsis thaliana. In all except CBP and the pea-pod protein, the axial methionine ligand normally found at blue copper sites is replaced by glutamine. The structure of CBP was originally solved by the multiple wavelength anomalous scattering method, using data recorded at four wavelengths. All these data were included in the restrained least squares refinement. The final model comprises 96 amino acid residues, 122 solvent molecules and a copper atom. Several residues are modelled as having more than one conformation. The residual R is 0.141 for 41,910 observations (including Bijvoet-related observations) of 8.142 unique reflections in the resolution range 7 to 1.8 A.

Laue diffraction study on the structure of cytochrome c peroxidase compound I.

BACKGROUND: Cytochrome c peroxidase from yeast is a soluble haem-containing protein found in the mitochondrial electron transport chain where it probably protects against toxic peroxides. The aim of this study was to obtain a reliable structure for the doubly oxidized transient intermediate (termed compound I) in the reaction of cytochrome c peroxidase with hydrogen peroxide. This intermediate contains a semistable free radical on Trp191, and an oxyferryl haem group. RESULTS: Compound I was produced in crystals of yeast cytochrome c peroxidase by reacting the crystalline enzyme with hydrogen peroxide in a flow cell. The reaction was monitored by microspectrophotometry and Laue crystallography in separate experiments. A nearly complete conversion to compound I was achieved within two minutes of the addition of hydrogen peroxide, and the concentration of the intermediate remained at similar levels for an additional half an hour. The structure of the intermediate was determined by Laue diffraction. The refined Laue structure for compound I shows clear structural changes at the peroxide-binding site but no significant changes at the radical site. The photographs were processed with a new software package (LEAP), overcoming many of the former problems encountered in extracting structural information from Laue exposures. CONCLUSIONS: The geometry of the haem environment in this protein allows structural changes to be extremely small, similar in magnitude to those observed for the Fe2+/Fe3+ transition in cytochrome c. The results suggest that these molecules have evolved to transfer electrons with a minimal need for structural adjustment.

Structure determination and refinement of homotetrameric hemoglobin from Urechis caupo at 2.5 A resolution.

A 5 A resolution multiple isomorphous replacement solution for hemoglobin isolated from Urechis caupo revealed a previously unobserved quaternary structure for tetrameric hemoglobin [Kolatkar, Meador, Stanfield & Hackert (1988). J. Biol. Chem. 263(7), 3462-3465]. We report here the structure of Urechis hemoglobin in the cyanomet state refined to 2.5 A resolution by simulated annealing yielding R = 0.148 for reflections F greater than 3 sigma between 5.0 and 2.5 A resolution. The starting model was fitted to a map originally derived from multiple-wavelength anomalous-dispersion phases to 3 A resolution that was then subjected to cyclic twofold molecular averaging and solvent flattening. Structural analysis of the resultant model shows that the unique quaternary assemblage is possible due to several favorable interactions between subunits, including salt links, hydrophobic pockets and interactions mediated by bound water. The tetramer is stabilized by subunit-subunit interactions between the G/H turns and D helices within the crystallographic dimer, and the A/B turn regions and E helices between subunits related by a molecular twofold axis. Interestingly, each subunit has one cysteine residue (Cys21) located in the A/B turn. These twofold-related cysteinyl residues are near enough to one another to form a disulfide bridge but do not.

Crystal structure of core streptavidin determined from multiwavelength anomalous diffraction of synchrotron radiation.

A three-dimensional crystal structure of the biotin-binding core of streptavidin has been determined at 3.1-A resolution. The structure was analyzed from diffraction data measured at three wavelengths from a single crystal of the selenobiotinyl complex with streptavidin. Streptavidin is a tetramer with subunits arrayed in D2 symmetry. Each protomer is an 8-stranded beta-barrel with simple up-down topology. Biotin molecules are bound at one end of each barrel. This study demonstrates the effectiveness of multiwavelength anomalous diffraction (MAD) procedures for macromolecular crystallography and provides a basis for detailed study of biotin-avidin interactions.

Crystal structure of Clostridium acidi-urici ferredoxin at 5-A resolution based on measurements of anomalous X-ray scattering at multiple wavelengths.

The crystal structure of Clostridium acidi-urici ferredoxin has been determined using multiple wavelength anomalous diffraction (MAD) techniques at 5.0-A resolution. The electron density map shows striking similarity to a map of Peptococcus aerogenes ferredoxin computed at the same resolution from the atomic coordinates reported by Adman et al. (Adman, E. T., Sieker, L. C., and Jensen, L. H. (1973) J. Biol. Chem. 248, 3987-3996). Such similarity is expected from the high degree of identity between amino acid sequences of the two proteins. The use of MAD methods has in the relatively recent past become a practical possibility due to instrumental advances enabling the collection of accurate data at several wavelengths at synchrotrons and due to theoretical and computational advances that facilitate the analysis of these data for the determination of phases. These methods hold great promise as an alternative to the multiple isomorphous replacement method in macromolecular structure determination. The present report represents one of the first applications of the MAD techniques to the determination of the structure of a protein which was previously unknown in detail.

Phase determination by multiple-wavelength x-ray diffraction: crystal structure of a basic "blue" copper protein from cucumbers.

A novel x-ray diffraction technique, multiple-wavelength anomalous dispersion (MAD) phasing, has been applied to the de novo determination of an unknown protein structure, that of the "blue" copper protein isolated from cucumber seedlings. This method makes use of crystallographic phases determined from measurements made at several wavelengths and has recently been made technically feasible through the use of intense, polychromatic synchrotron radiation together with accurate data collection from multiwire electronic area detectors. In contrast with all of the conventional methods of solving protein structures, which require either multiple isomorphous derivatives or coordinates of a similar structure for molecular replacement, this technique allows direct solution of the classical "phase problem" in x-ray crystallography. MAD phase assignment should be particularly useful for determining structures of small to medium-sized metalloproteins for which isomorphous derivatives are difficult or impossible to make. The structure of this particular protein provides new insights into the spectroscopic and redox properties of blue copper proteins, an important class of metalloproteins widely distributed in nature.

Crystallographic structure analysis of lamprey hemoglobin from anomalous dispersion of synchrotron radiation.

The molecular structure of lamprey hemoglobin was previously determined and refined by conventional crystallographic analysis. In this study, the structural analysis has been repeated in the course of developing the method of multiwavelength anomalous diffraction (MAD) for phase determination. New experimental and analytical procedures that were devised to perform this determination should have general applicability. These include an experimental design to optimize signal strength and reduce systematic errors, experimental evaluation of anomalous scattering factors, and a least-squares procedure for analyzing the MAD data. MAD phases for the structure at 3 A resolution are as accurate overall as the multiple isomorphous replacement (MIR) phases determined previously.

Structure and specificity of antibody molecules.

The structure of the Fab' fragment of a human myeloma protein (IgG1 (lambda) New) has been determined by X-ray crystallographic analysis to a nominal resolution of 0.2 nm. Each of the structure subunits corresponding to the variable and to the constant homology regions of the light and heavy polypeptide chains contains two irregular beta-sheets which are roughly parallel to each other and surround a tighly packed interior of hydrophobic side chains. The regions of the hypervariable sequences in the light and heavy chains occur in close spatial proximity at one end of the molecule, defining the active site of IgG New. The role of these hypervariable regions in defining the size and shape of the active site of different immunoglobulins is discussed on the basis of the three-dimensional model of Fab' New. Several ligands that bind to the active centre of IgG New have been used to obtain crystalline ligand-Fab' New complexes which were investigated by difference Fourier maps. These studies are analysed in terms of the biological function and specificity of antibodies.

The three-dimensional structure of the fab' fragment of a human myeloma immunoglobulin at 2.0-angstrom resolution.

The structural analysis of the Fab' fragment of human myeloma immunoglobulin IgGl(lambda) New has been extended to a nominal resolution of 2.0 A. Each of the structural subunits corresponding to the variable and to the constant homology regions of the light and heavy chains contains two irregular beta-sheets which are roughly parallel to each other and surround a tightly packed interior of hydrophobic side chains. About 50-60% of the amino-acid residues are included in beta-pleated sheets. Sequence alignments between the homology regions of Fab' New obtained by comparison of their three-dimensional structures are given. Some of the sequence variations observed in light and heavy chains and the role of the regions of hypervariable sequence in defining the size and shape of the active site of different immunoglobulin molecules are discussed on the basis of the three-dimensional model of Fab' New.

Three-dimensional structure of the Fab' fragment of a human immunoglobulin at 2,8-A resolution.

The structure of the Fab' fragment of a human myeloma immunoglobulin was determined by x-ray crystallographic analysis at 2.8-A resolution. The Fourier map of the electron density was correlated with the aminoacid sequence to obtain a three-dimensional model. Four globular subunits, which correspond to the homology regions of the light and heavy chains, are arranged in a tetrahedral configuration. These subunits closely resemble each other, sharing a basic pattern of polypeptide chain folding. In each subunit, long sequences of tightly packed, hydrogen bonded polypeptide chain run parallel to the major axis of the subunit. No helical conformation can be seen. Different patterns of interchain disulfide linkage and unusual intrachain disulfide bonds that have been observed in other immunoglobulins can be explained with this model. The regions of hypervariable sequences in the light and heavy chains occur at one end of the molecule, in close spatial proximity.