Single Isomorphous Replacement Phases Research Articles

Single isomorphous replacement phasing of selenomethionine-containing proteins using UV-induced radiation damage

The most commonly used heavy-atom derivative, selenium, requires the use of a tunable beamline to access the Se K edge for experimental phasing using anomalous diffraction methods, whereas X-ray diffraction experiments for selenium-specific ultraviolet radiation-damage-induced phasing can be performed on fixed-wavelength beamlines or even using in-house X-ray sources. Several nonredundant X-ray diffraction data sets were collected from three different selenomethionine (Mse) derivatized protein crystals at energies far below the absorption edge before and after exposing the crystal to ultraviolet (UV) radiation using 266 nm lasers of flux density 1.7 × 10¹⁵ photons s⁻¹ mm⁻² for 10-50 min. A detailed analysis revealed that significant changes in diffracted intensities were induced by ultraviolet irradiation whilst retaining crystal isomorphism. These intensity changes allowed the crystal structures to be solved by the radiation-damage-induced phasing (RIP) technique. Inspection of the crystal structures and electron-density maps demonstrated that covalent bonds between selenium and carbon at all sites located in the core of the proteins or in a hydrophobic environment were much more susceptible to UV radiation-induced cleavage than other bonds typically present in Mse proteins. The rapid UV radiation-induced bond cleavage opens a reliable new paradigm for phasing when no tunable X-ray source is available. The behaviour of Mse derivatives of various proteins provides novel insights and an initial basis for understanding the mechanism of selenium-specific UV radiation damage.

Pseudomonas aeruginosa LD-Carboxypeptidase, a Serine Peptidase with a Ser-His-Glu Triad and a Nucleophilic Elbow*

LD-Carboxypeptidases (EC 3.4.17.13) are named for their ability to cleave amide bonds between l- and d-amino acids, which occur naturally in bacterial peptidoglycan. They are specific for the link between meso-diaminopimelic acid and d-alanine and therefore degrade GlcNAc-MurNAc tetrapeptides to the corresponding tripeptides. As only the tripeptides can be reused as peptidoglycan building blocks, ld-carboxypeptidases are thought to play a role in peptidoglycan recycling. Despite the pharmaceutical interest in peptidoglycan biosynthesis, the fold and catalytic type of ld-carboxypeptidases are unknown. Here, we show that a previously uncharacterized open reading frame in Pseudomonas aeruginosa has ld-carboxypeptidase activity and present the crystal structure of this enzyme. The structure shows that the enzyme consists of an N-terminal beta-sheet and a C-terminal beta-barrel domain. At the interface of the two domains, Ser(115) adopts a highly strained conformation in the context of a strand-turn-helix motif that is similar to the "nucleophilic elbow" in alphabeta-hydrolases. Ser(115) is hydrogen-bonded to a histidine residue, which is oriented by a glutamate residue. All three residues, which occur in the order Ser-Glu-His in the amino acid sequence, are strictly conserved in naturally occurring ld-carboxypeptidases and cannot be mutated to alanines without loss of activity. We conclude that ld-carboxypeptidases are serine peptidases with Ser-His-Glu catalytic triads.

Structure and Properties of the C-terminal Domain of Insulin-like Growth Factor-binding Protein-1 Isolated from Human Amniotic Fluid

Insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) regulates the activity of the insulin-like growth factors in early pregnancy and is, thus, thought to play a key role at the fetal-maternal interface. The C-terminal domain of IGFBP-1 and three isoforms of the intact protein were isolated from human amniotic fluid, and sequencing of the four N-terminal polypeptide chains showed them to be highly pure. The addition of both intact IGFBP-1 and its C-terminal fragment to cultured fibroblasts has a similar stimulating effect on cell migration, and therefore, the domain has a biological activity on its own. The three-dimensional structure of the C-terminal domain was determined by x-ray crystallography to 1.8 Angstroms resolution. The fragment folds as a thyroglobulin type I domain and was found to bind the Fe(2+) ion in the crystals through the only histidine residue present in the polypeptide chain. Iron (II) decreases the binding of intact IGFBP-1 and the C-terminal domain to IGF-II, suggesting that the metal binding site is close to or part of the surface of interaction of the two molecules.

Position and Orientation of Phalloidin in F-Actin Determined by X-Ray Fiber Diffraction Analysis

Knowledge of the phalloidin binding position in F-actin and the relevant understanding of the mechanism of F-actin stabilization would help to define the structural characteristics of the F-actin filament. To determine the position of bound phalloidin experimentally, x-ray fiber diffraction data were obtained from well-oriented sols of F-actin and the phalloidin-F-actin complex. The differences in the layer-line intensity distributions, which were clearly observed even at low resolution (8 Å), produced well-resolved peaks corresponding to interphalloidin vectors in the cylindrically averaged difference-Patterson map, from which the radial binding position was determined to be ∼10 Å from the filament axis. Then, the azimuthal and axial positions were determined by single isomorphous replacement phasing and a cross-Patterson map in radial projection to be ∼84° and 0.5 Å relative to the actin mass center. The refined position was close to the position found by prior researchers. The position of rhodamine attached to phalloidin in the rhodamine-phalloidin-F-actin complex was also determined, in which the conjugated Leu(OH) 7 residue was found to face the outside of the filament. The position and orientation of the bound phalloidin so determined explain the increase in the interactions between long-pitch strands of F-actin and would also account for the inhibition of phosphate release, which might also contribute to the F-actin stabilization. The method of analysis developed in this study is applicable for the determination of binding positions of other drugs, such as jasplakinolide and dolastatin 11.

Structure determination of the head-tail connector of bacteriophage phi29.

The head-tail connector of bacteriophage phi29 is composed of 12 36 kDa subunits with 12-fold symmetry. It is the central component of a rotary motor that packages the genomic dsDNA into preformed proheads. This motor consists of the head-tail connector, surrounded by a phi29-encoded, 174-base, RNA and a viral ATPase protein, both of which have fivefold symmetry in three-dimensional cryo-electron microscopy reconstructions. DNA is translocated into the prohead through a 36 A diameter pore in the center of the connector, where the DNA takes the role of a motor spindle. The helical nature of the DNA allows the rotational action of the connector to be transformed into a linear translation of the DNA. The crystal structure determination of connector crystals in space group C2 was initiated by molecular replacement, using an approximately 20 A resolution model derived from cryo-electron microscopy. The model phases were extended to 3.5 A resolution using 12-fold non-crystallographic symmetry averaging and solvent flattening. Although this electron density was not interpretable, the phases were adequate to locate the position of 24 mercury sites of a thimerosal heavy-atom derivative. The resultant 3.2 A single isomorphous replacement phases were improved using density modification, producing an interpretable electron-density map. The crystallographically refined structure was used as a molecular-replacement model to solve the structures of two other crystal forms of the connector molecule. One of these was in the same space group and almost isomorphous, whereas the other was in space group P2(1)2(1)2. The structural differences between the oligomeric connector molecules in the three crystal forms and between different monomers within each crystal show that the structure is relatively flexible, particularly in the protruding domain at the wide end of the connector. This domain probably acts as a bearing, allowing the connector to rotate within the pentagonal portal of the prohead during DNA packaging.

Human class II MHC molecule HLA-DR1: X-ray structure determined from three crystal forms.

The three-dimensional structure of the extracellular region of a 60 kDa class II major histocompatibility glycoprotein, HLA-DR1, was determined to 3.3 A by X-ray crystallography using three crystal forms, each containing two molecules per asymmetric unit. Phases were initially determined to 4.2 A using two crystal forms both containing DR1 from human lymphocytes complexed with a mixture of endogenous peptides. One of these crystal forms also contained a 28 kDa superantigen, Staphylococcus aureus enterotoxin B (SEB), bound to each DR1 molecule. Single-isomorphous replacement phasing followed by iterative two- and fourfold non-crystallographic real-space averaging between the two crystal forms resulted in 4.2 A resolution electron-density maps from which the paths of the polypeptides could be traced. Cryocrystallography and synchrotron radiation were then used to extend the resolution to 3.3 A for the two lymphocyte-derived crystal forms and for a third crystal form grown from DR1 produced in insect cells and complexed in vitro with a specific antigenic peptide. Iterative sixfold non-crystallographic real-space averaging resulted in an electron- density map into which 340 of 371 residues could be fit unambiguously. Crystal contacts and the existence of a parallel dimer of the DR1 alphabeta heterodimer in the three crystal forms are discussed.

Structure determination of aldose reductase: joys and traps of local symmetry averaging

The structure of aldose reductase, a monomeric enzyme of 314 amino acids which crystallizes in space group P1 with four monomers per asymmetric unit, has been solved using a combination of single isomorphous replacement (SIR), solvent flattening and local symmetry averaging. The self rotation showed evidence of 222 local symmetry. The map calculated from the original single isomorphous replacement phases showed a clear solvent envelope but was uninterpretable. A first averaging attempt failed because the molecular envelope obtained from the SIR map weighted with monomer correlation was too small and the averaging was biased by low-resolution truncation. A second attempt with an enlarged envelope and including low-resolution reflections succeeded in refining phases at 3.5 A resolution but failed to extend them correctly. Rigid-body refinement of a partial model based on the 3.5 A map calculated from refined phases showed significant departures from the 222 symmetry. A third averaging attempt using the improved symmetry succeeded in producing a clear map with phases extended to 3.07 A resolution. This map revealed a (beta/alpha)(8) fold, not previously found in NADPH-dependent enzymes. This work shows the importance of mask definition and local symmetry elements accuracy for averaging, and describes a method for improving these parameters.

Structure determination of monoclinic canine parvovirus.

The three-dimensional structure of the single-stranded DNA canine parvovirus has been determined to 3.25 A resolution. Monoclinic crystals belonging to space group P2(1) (a = 263.1, b = 348.9, c = 267.2 A, beta = 90.82 degrees) were selected for data collection using primarily the Cornell High Energy Synchrotron Source and oscillation photography. There was one icosahedral particle per crystallographic asymmetric unit, giving 60-fold redundancy. The particle orientations in the unit cell were determined with a rotation function. The rough positions of the particles in the unit cell were estimated by considering the packing of spheres into the P2(1) crystal cell. More accurate particle centers were determined from Harker peaks in a Patterson function. Hollow-shell models were used to compute phases to 20 A resolution. The radii of the models were based on packing considerations, the fit of spherical shells to the low-resolution X-ray data and low-angle solution scattering data. The phases were extended to 9 A resolution using molecular replacement real-space averaging. These were then used to determine the heavy-atom position of a K2PtBr6 derivative, for which only 5% of the theoretically observable reflections had been recorded. The center of gravity of the 60 independent heavy-atom sites gave an improved particle center position. Single isomorphous replacement phases to 8 A resolution were then calculated with the platinum derivative. These were used to initiate phase improvement and extension to 3.25 A resolution using density averaging and Fourier back-transformation in steps of one reciprocal lattice point at a time. The resulting electron density map was readily interpretable and an atomic model was built into the electron density map on a PS390 graphics system using the FRODO program. The R factor prior to structure refinement for data between 5.0 and 3.25 A was 36%.

Refined crystal structure of Cd, Zn metallothionein at 2·0 Å resolution

The crystal structure of Cd5,Zn2-metallothionein from rat liver has been refined at 2.0 A resolution of a R-value of 0.176 for all observed data. The five Cd positions in the asymmetric unit of the crystal create a pseudo-centrosymmetric constellation about a crystallographic 2-fold axis. Consequently, the distribution of anomalous differences is almost ideally centrosymmetric. Therefore, the previously reported metal positions and the protein model derived therefrom are incorrect. Direct methods were applied to the protein amplitudes to locate the Cd positions. The new positions were used to calculate a new electron density map based on the Cd anomalous scattering and partial structure to model the metal clusters and the protein. Phases calculated from this model predict the positions of three sites in a (NH4)2WS4 derivative. Single isomorphous replacement phases calculated with these tungsten sites confirm the positions of the Cd sites from the new direct methods calculations. The refined metallothionein structure has a root-mean-square deviation of 0.016 A from ideality of bonds and normal stereochemistry of phi, phi and chi torsion angles. The metallothionein crystal structure is in agreement with the structures for the alpha and beta domains in solution derived by nuclear magnetic resonance methods. The overall chain folds and all metal to cysteine bonds are the same in the two structure determinations. The handedness of a short helix in the alpha-domain (residues 41 to 45) is the same in both structures. The crystal structure provides information concerning the metal cluster geometry and cysteine solvent accessibility and side-chain stereochemistry. Short cysteine peptide sequences repeated in the structure adopt restricted conformations which favor the formation of amide to sulfur hydrogen bonds. The crystal packing reveals intimate association of molecules about the diagonal 2-fold axes and trapped ions of crystallization (modeled as phosphate and sodium). Variation in the chemical and structural environments of the metal sites is in accord with data for metal exchange reactions in metallothioneins.

Refined crystal structure of Cd, Zn metallothionein at 2.0Åresolution

The crystal structure of Cd 5,Zn 2-metallothionein from rat liver has been refined at 2.0Åresolution of a R-value of 0.176 for all observed data. The five Cd positions in the asymmetric unit of the crystal create a pseudo-centrosymmetric constellation about a crystallographic 2-fold axis. Consequently, the distribution of anomalous differences is almost ideally centrosymmetric. Therefore, the previously reported metal positions and the protein model derived therefrom are incorrect. Direct methods were applied to the protein amplitudes to locate the Cd positions. The new positions were used to calculate a new electron density map based on the Cd anomalous scattering and partial structure to model the metal clusters and the protein. Phases calculated from this model predict the positions of three sites in a (NH 4) 2WS 4 derivative. Single isomorphous replacement phases calculated with these tungsten sites confirm the positions of the Cd sites from the new direct methods calculations. The refined metallothionein structure has a root-mean-square deviation of 0.016Åfrom ideality of bonds and normal stereochemistry of φ, ϑ and χ torsion angles. The metallothionein crystal structure is in agreement with the structures for the α and β domains in solution derived by nuclear magnetic resonance methods. The overall chain folds and all metal to cysteine bonds are the same in the two structure determinations. The handedness of a short helix in the α-domain (residues 41 to 45) is the same in both structures. The crystal structure provides information concerning the metal cluster geometry and cysteine solvent accessibility and side-chain stereochemistry. Short cysteine peptide sequences repeated in the structure adopt restricted conformations which favor the formation of amide to sulfur hydrogen bonds. The crystal packing reveals intimate association of molecules about the diagonal 2-fold axes and trapped ions of crystallization (modeled as phosphate and sodium). Variation in the chemical and structural environments of the metal sites is in accord with data for metal exchange reactions in metallothioneins.

Crystal structure of muconolactone isomerase at 3.3 Å resolution

The crystal structure of muconolactone isomerase from Pseudomonas putida, a unique molecule with ten 96 amino acid subunits and 5-fold, and 2-fold symmetries, has been solved at 3.3 Å resolution. The non-crystallographic symmetries were used to refine the initial single isomorphous replacement phases and produce an interpretable 10-fold averaged map. The backbone trace is complete and confirmed by the amino acid sequence fit. Each subunit is composed of a body with two α-helices and an antiparallel twisted β-sheet of four strands, and an extended arm. The helices and the sheet fold to form a two-layered structure with an enclosed hydrophobic core and a partially formed putative active site pocket. The C-terminal arm of another subunit related by a local dyad symmetry extends over the core to complete this pocket. The decameric protein is almost spherical, with the helices forming the external coat. There is a large hydrophilic cavity in the center with open ends along the 5-fold axis. Molecular interactions between subunits are extensive. Each subunit contacts four neighbors and loses nearly 40% of its solvent contact area on oligomerization.

Structure determination of crystalline lobster d-glyceraldehyde-3-phosphate dehydrogenase

Single crystal X-ray data were collected on film for the holoenzyme of lobster d-glyceraldehyde-3-phosphate dehydrogenase to 3·0 Å resolution. Films of potassium tetraiodomercurate, K 2HgI 4, comprising a complete low resolution set, with some additional high resolution terms, were given to us by Drs H. C. Watson and L. J. Banaszak. A 3·0 Å high resolution data set was collected of a p-chloromercuri-phenylsulfonate derivative. All these films were processed on a computer controlled Optronics film scanner. The K 2HgI 4 derivative difference Patterson was initially interpreted in terms of four single sites, one for each polypeptide chain, consistent with the previously determined molecular 222 symmetry. Single isomorphous replacement phases were then sufficient to identify other heavy atom sites. Least-squares refined parameters were used to give multiple isomorphous replacement phases at low resolution, and single isomorphous replacement phases at high resolution. The resultant electron density map was oriented along the molecular 2-fold axes and then averaged over all four equivalent subunits. This process produced a much improved electron density map, which could easily be interpreted in terms of a single polypeptide chain per subunit consistent with the known amino acid sequence. The use of non-crystallographic symmetry to improve the electron density map is equivalent to the molecular replacement method. A comparison is also made with other dehydrogenases.