Advances in multiple wavelength anomalous diffraction crystallography

In only a few years, multiple wavelength anomalous diffraction (MAD) phasing has advanced from an esoteric technique used in only a few favorable cases to the method of choice for solving new macromolecular structures. Before 1994, MAD phasing had been used for fewer than a dozen new structure determinations. In 1999 alone, well over 100 new structures were determined by MAD phasing. The meteoric rise in MAD applications resulted from the availability of new synchrotron beamlines, equipped with low bandpass optics, fast readout detectors, cryogenic cooling and user-friendly interfaces. The power of MAD phasing has been amplified by the availability of new computer programs for locating the positions of the anomalous scattering atoms and for calculating phases from the experimental data. Phasing by anomalous scattering techniques has been applied to structures as large as 640 kDa and 120 selenium atoms in the asymmetric unit. The practical size limitation for application of MAD phasing techniques has not yet been encountered.     

Selenomethionine incorporation into a protein by cell-free synthesis

Multi-wavelength anomalous diffraction phasing is especially useful for high-throughput structure determinations. Selenomethionine substituted proteins are commonly used for this purpose. However, the cytotoxicity of selenomethionine drastically reduces the efficiency of its incorporation in in vivo expression systems. In the present study, an improved E. coli cell-free protein synthesis system was used to incorporate selenomethionine into a protein, so that highly efficient incorporation could be achieved. A milligram quantity of selenomethionine-containing Ras was obtained using the cell-free system with dialysis. The mass spectrometry analysis showed that more than 95% of the methionine residues were substituted with selenomethionine. The crystal of this protein grew under the same conditions and had the same unit cell constants as those of the native Ras protein. The three-dimensional structure of this protein, determined by multi-wavelength anomalous diffraction phasing, was almost the same as that of the Ras protein prepared by in vivo expression. Therefore, the cell-free synthesis system could become a powerful protein expression method for high-throughput structure determinations by X-ray crystallography.     

Anomalous X-ray diffraction with soft X-ray synchrotron radiation.

Anomalous diffraction with soft X-ray synchrotron radiation opens new possibilities in protein crystallography and materials science. Low-Z elements like silicon, phosphorus, sulfur and chlorine become accessible as new labels in structural studies. Some of the heavy elements like uranium exhibit an unusually strong dispersion at their M(V) absorption edge (lambdaMV = 3.497 A, E(MV) = 3545 eV) and so does thorium. Two different test experiments are reported here showing the feasibility of anomalous X-ray diffraction at long wavelengths with a protein containing uranium and with a salt containing chlorine atoms. With 110 electrons the anomalous scattering amplitude of uranium exceeds by a factor of 4 the resonance scattering of other strong anomalous scatterers like that of the lanthanides at their L(III) edge. The resulting exceptional phasing power of uranium is most attractive in protein crystallography using the multi-wavelength anomalous diffraction (MAD) method. The anomalous dispersion of an uranium derivative of asparaginyl-tRNA synthetase (hexagonal unit cell; a = 123.4 A, c = 124.4 A) has been measured for the first time at 4 wavelengths near the M(V) edge using the beamline ID1 of ESRF (Grenoble, France). The present set up allowed to measure only 30% of the possible reflections at a resolution of 4 A, mainly because of the low sensitivity of the CCD detector. In the second experiment, the dispersion of the intensity of 5 X-ray diffraction peaks from pentakismethylammonium undecachlorodibismuthate (PMACB, orthorhombic unit cell; a = 13.003 A, b = 14.038 A, c = 15.450 A) has been measured at 30 wavelengths near the K absorption edge of chlorine (lambdaK = 4.397 A, EK= 2819.6 eV). All reflections within the resolution range from 6.4 A to 3.4 A expected in the 20 degree scan were observed. The chemical state varies between different chlorine atoms of PMACB, and so does the dispersion of different Bragg peaks near the K-edge of chlorine. The results reflect the performance of the beamline ID1 of ESRF at wavelengths beyond 3 A at the end of 1998. A gain by a factor 100 for diffraction experiments with 4.4 A photons was achieved in Autumn 1999 when two focusing mirrors had been added to the X-ray optics. Further progress is expected from area detectors more sensitive to soft X-rays. Both CCD detectors and image plates would provide a gain of two orders of measured intensity. Image plates would have the additional advantage that they can be bent cylindrically and thus cover a larger solid angle in reciprocal space. In many cases, samples need to be cooled: closed and open systems are presented. A comparison with the state of art of soft X-ray diffraction, as it had been reached at HASYLAB (Hamburg, Germany), and as it is developing at the Brookhaven National Laboratory (USA), is given.     

Observation of inverted cubic phase in hydrated dioleoylphosphatidylethanolamine membranes

We report the observation of an inverted cubic phase in aqueous dispersions of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) by small-angle X-ray diffraction. DOPE is a paradigm in the study of nonlamellar phases in biological systems: it exhibits a well-known phase transition from the lamellar (L alpha) to the inverted hexagonal phase (HII) as the temperature is raised. The transition is observed to occur rapidly when a DOPE dispersion is heated from 2 degrees C, where the L alpha phase is stable, to 15 degrees C, where the HII phase is stable. We report on the induction of a crystallographically well-defined cubic lattice that is slowly formed when the lipid dispersion is rapidly cycled between -5 and 15 degrees C hundreds of times. Once formed, the cubic lattice is stable at 4 degrees C for several weeks and exhibits the same remarkable metastability that characterizes other cubic phases in lipid-water systems. X-ray diffraction indicates that the cubic lattice is most consistent with either the Pn3m or Pn3 space group. Tests of lipid purity after induction of the cubic indicate the lipid is at least 98% pure. The cubic lattice can be destroyed and the system reset by cycling the specimen several times between -30 and 2 degrees C. The kinetics of the formation of the cubic are dependent on the thermal history of the sample, overall water concentration, and the extreme temperatures of the cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physical properties of glycosyl diacylglycerols. 2. X-ray diffraction studies of a homologous series of 1,2-Di-O-acyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerols

X-ray diffraction methods were used to characterize the thermotropic polymorphism exhibited by aqueous dispersions of a homologous series of 1,2-O-acyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerols. Upon cooling from temperatures at which the acyl chains of these lipids are melted, all of these compounds form structures that exhibit both low-angle and wide-angle diffraction patterns consistent with the formation of lamellar L beta gel phases. After a suitable protocol of low-temperature annealing, complex diffraction patterns consistent with the formation of highly ordered, lamellar, crystal-like phases are obtained. These patterns are similar for all of the compounds studied, suggesting that the unit cell structure is invariant. The assumption that the unit cell structure is invariant permits the assignment of phases to the diffraction orders, thereby making possible the construction of electron density profiles. These electron density profiles indicate that the crystal-like phases of these lipids are poorly hydrated structures with the hydrocarbon chains inclined at 35 degrees to the bilayer normal. The diffraction patterns of the crystal-like phases of these lipids changed abruptly at the calorimetrically determined phase transition temperatures to those characteristic of either lamellar liquid crystalline phases (N less than or equal to 17) or inverted nonbilayer phases. With these X-ray diffraction data we demonstrate that, at elevated temperatures, the shorter chain homologues (N less than or equal to 16) form cubic phases of the Pn3m space group, whereas the longer chain compounds form inverted hexagonal phases.     

Evidence of cholesterol accumulated in high curvature regions: implication to the curvature elastic energy for lipid mixtures

Recent experiments suggested that cholesterol and other lipid components of high negative spontaneous curvature facilitate membrane fusion. This is taken as evidence supporting the stalk-pore model of membrane fusion in which the lipid bilayers go through intermediate structures of high curvature. How do the high-curvature lipid components lower the free energy of the curved structure? Do the high-curvature lipid components modify the average spontaneous curvature of the relevant monolayer, thereby facilitate its bending, or do the lipid components redistribute in the curved structure so as to lower the free energy? This question is fundamental to the curvature elastic energy for lipid mixtures. Here we investigate the lipid distribution in a monolayer of a binary lipid mixture before and after bending, or more precisely in the lamellar, hexagonal, and distorted hexagonal phases. The lipid mixture is composed of 2:1 ratio of brominated di18:0PC and cholesterol. Using a newly developed procedure for the multiwavelength anomalous diffraction method, we are able to isolate the bromine distribution and reconstruct the electron density distribution of the lipid mixture in the three phases. We found that the lipid distribution is homogenous and uniform in the lamellar and hexagonal phases. But in the distorted hexagonal phase, the lipid monolayer has nonuniform curvature, and cholesterol almost entirely concentrates in the high curvature region. This finding demonstrates that the association energies between lipid molecules vary with the curvature of membrane. Thus, lipid components in a mixture may redistribute under conditions of nonuniform curvature, such as in the stalk structure. In such cases, the spontaneous curvature depends on the local lipid composition and the free energy minimum is determined by lipid distribution as well as curvature.     

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.     

Phytosphingosine ceramide mainly localizes in the central layer of the unique lamellar phase of skin lipid model systems

Understanding the lipid arrangement within the skin’s outermost layer, the stratum corneum (SC), is important for advancing knowledge on the skin barrier function. The SC lipid matrix consists of ceramides (CERs), cholesterol, and free fatty acids, which form unique crystalline lamellar phases, referred to as the long periodicity phase (LPP) and short periodicity phases. As the SC lipid composition is complex, lipid model systems that mimic the properties of native SC are used to study the SC lipid organization and molecular arrangement. In previous studies, such lipid models were used to determine the molecular organization in the trilayer structure of the LPP unit cell. The aim of this study was to examine the location of CER N-(tetracosanoyl)-phytosphingosine (CER NP) in the unit cell of this lamellar phase and compare its position with CER N-(tetracosanoyl)-sphingosine (CER NS). We selected CER NP as it is the most prevalent CER subclass in the human SC, and its location in the LPP is not known. Our neutron diffraction results demonstrate that the acyl chain of CER NP was positioned in the central part of the trilayer structure, with a fraction also present in the outer layers, the same location as determined for the acyl chain of CER NS. In addition, our Fourier transformed infrared spectroscopy results are in agreement with this molecular arrangement, suggesting a linear arrangement for the CER NS and CER NP. These findings provide more detailed insight into the lipid organization in the SC lipid matrix.     

Temperature- and pressure-dependent phase behavior of monoacylglycerides monoolein and monoelaidin.

We used x-ray and neutron diffraction to study the temperature- and pressure-dependent structure and phase behavior of the monoacylglycerides 1-monoelaidin (ME) and 1-monoolein (MO) in excess water. The monoacylglycerides were chosen for investigation of their phase behavior because they exhibit mesomorphic phases with one-, two-, and three-dimensional periodicity, such as lamellar, an inverted hexagonal and bicontinuous cubic phases, in a rather easily accessible temperature and pressure range. We studied the structure, stability, and transformations of the different phases over a wide temperature and pressure range, explored the epitaxial relations that exist between different phases, and established a relationship between the chemical structure of the lipid molecules and their phase behavior. For both systems, a temperature-pressure phase diagram has been determined in the temperature range from 0 to 100 degrees C at pressures from ambient up to 1400 bar, and drastic differences in phase behavior are found for the two systems. In MO-water dispersions, the cubic phase Pn3m extends over a large phase field in the T,p-plane. At temperatures above 95 degrees C, the inverted hexagonal phase is found. In the lower temperature region, a crystalline lamellar phase is induced at higher pressures. The phases found in ME-water include the lamellar crystalline Lc phase, the L beta gel phase, the L alpha liquid-crystalline phase, and two cubic phases belonging to the crystallographic space groups Im3m and Pn3m. In addition, the existence of metastable phases has been exploited. Between coexisting metastable cubic structures, a metric relationship has been found that is predicted theoretically on the basis of the curvature elastic energy approximation only.     

Rational design of lipid for membrane protein crystallization

The lipidic cubic phase has been used to grow crystals of membrane proteins for high-resolution structure determination. However, the original, so-called, in meso method does not work reliably at low temperatures, where proteins are generally more stable, because the hosting lipid turns solid. The need existed therefore for a lipid that forms the cubic phase and that supports crystal growth at low temperatures. We created a database of phase diagrams and used it to design such a lipid. X-ray diffraction showed that the new lipid exhibits designed phase behavior. Further, it produces diffraction quality membrane protein crystals by the in meso method at 6 degrees C. This demonstrates that lipidic materials, like their protein counterparts are amenable to rational design. The same approach as used in this study should find application in extending the range of membrane proteins crystallizable by the in meso method and in tailoring transport of cubic phases for controlled delivery and uptake.     

Phase properties of binary mixtures of monogalactosyldiacylglycerols differing in hydrocarbon chain substituents dispersed in aqueous systems

Monogalactosyldiacylglycerols isolated from spinach leaves contain a high proportion of polyunsaturated fatty acyl substituents and form hexagonal-II structures when dispersed in excess water. Catalytic hydrogenation of the lipid in the presence of Adam's catalyst completely saturates the hydrocarbon chains and the lipid forms typical open sheet bilayer structures in water at 20°C. Binary mixtures of the native and hydrogenated lipid tend to phase separate at 20°C. Freeze-fracture electron microscopy reveals lamellar phase lipid indispersed with regions of hexagonal-II structure and the proportions of each reflect the composition of the mixture. X-ray diffraction in both wide- and low-angle regions show that the saturated lipid forms the typical stable gel-phase structure in mixtures that are allowed to equilibrate over three days at 20°C. The phase transition behaviour of binary mixtures of the two galactolipids was investigated by differential scanning calorimetry and fluorescence probe methods. Thermal studies indicate that the phase-separated gel structure undergoes an anomalous transition compared with the saturated pure lipid in that the transition temperature is reduced from about 57°C to 41°C and the enthalpy of the transition is also somewhat reduced. Furthermore, the transition appears to involve the conversion of the completely phase-separated system into bilayer coexisting with phases intermediate between bilayer and hexagonal-II. A homogeneous hexagonal-II phase is presumably formed at higher temperatures. The thermal and structural studies were consistent with fluorescence polarization measurements of 1,6-diphenyl-1,3,5-hexatriene interpolated into the hydrocarbon domain of the structure.     

The phase behavior of cationic lipid-DNA complexes

We present a theoretical analysis of the phase behavior of solutions containing DNA, cationic lipids, and nonionic (helper) lipids. Our model allows for five possible structures, treated as incompressible macroscopic phases: two lipid-DNA composite (lipoplex) phases, namely, the lamellar (L(alpha)(C)) and hexagonal (H(II)(C)) complexes; two binary (cationic/neutral) lipid phases, that is, the bilayer (L(alpha)) and inverse-hexagonal (H(II)) structures, and uncomplexed DNA. The free energy of the four lipid-containing phases is expressed as a sum of composition-dependent electrostatic, elastic, and mixing terms. The electrostatic free energies of all phases are calculated based on Poisson-Boltzmann theory. The phase diagram of the system is evaluated by minimizing the total free energy of the three-component mixture with respect to all the compositional degrees of freedom. We show that the phase behavior, in particular the preferred lipid-DNA complex geometry, is governed by a subtle interplay between the electrostatic, elastic, and mixing terms, which depend, in turn, on the lipid composition and lipid/DNA ratio. Detailed calculations are presented for three prototypical systems, exhibiting markedly different phase behaviors. The simplest mixture corresponds to a rigid planar membrane as the lipid source, in which case, only lamellar complexes appear in solution. When the membranes are "soft" (i.e., low bending modulus) the system exhibits the formation of both lamellar and hexagonal complexes, sometimes coexisting with each other, and with pure lipid or DNA phases. The last system corresponds to a lipid mixture involving helper lipids with strong propensity toward the inverse-hexagonal phase. Here, again, the phase diagram is rather complex, revealing a multitude of phase transitions and coexistences. Lamellar and hexagonal complexes appear, sometimes together, in different regions of the phase diagram.     

A rhombohedral phase of lipid containing a membrane fusion intermediate structure

We constructed the electron density distribution from the x-ray diffraction of a phase of phospholipid that exhibited rhombohedral symmetry. To determine the phases of the diffraction amplitudes, we first extended the well-known one-dimensional swelling method for planar bilayers to a three-dimensional method applicable to a layered system containing in-plane structures, such as rhombohedral structures. The complete phase determination was accomplished by a combination of the swelling method and Luzzati's pattern recognition method. The constructed electron density distribution showed that in each unit cell, two apposed monolayers merged across the water layer and developed into an hourglass structure consistent with a postulated membrane fusion intermediate state called a stalk. The observation of the stalk structure lends a strong support to the stalk hypothesis for membrane fusion and opens a way to measure the structural parameters in the fusion pathway.     

Crystal structure of human coactosin-like protein

Human coactosin-like protein is an actin filament binding protein but does not bind to globular actin. It associates with 5-Lipoxygenase both in vivo and in vitro, playing important roles in modulating the activities of actin and 5-Lipoxygenase. Coactosin counteracts the capping activity of capping protein which inhibits the actin polymerization. We determined the crystal structures of human coactosin-like protein by multi-wavelength anomalous dispersion method. The structure showed a high level of similarity to ADF-H domain, although their amino acid sequences share low degree of homology. A few conserved hydrophobic residues that may contribute to the folding were identified. This structure suggests coactosin-like protein bind to F-actin in a different way from ADF/Cofilin family. Combined with the information from previous mutagenesis studies, the binding sites for F-actin and 5-Lipoxygenase were analyzed, respectively. These two sites are quite close, which might prevent F-actin and 5-Lipoxygenase from binding to coactosin simultaneously.     

Phase separation in dipalmitoyl phosphatidylcholine bilayers induced by ionophores and binary electrolytes

Thermotropic behavior of unsonicated aqueous dispersion of dipalmitoyl phosphatidylcholine (DPPC) has been studied by scanning microcalorimetry and fluorescent probe method. Phase separation in the lipid bilayers was observed for systems containing ionophores (valinomycin, dinactin) and 1 : 1 electrolytes (NaCl, KCl, RbCl, CsCl). The ratio of lipid phases coexisting in the systems appeared to be dependent on the concentration of the electrolytes. Changes in the thermotropic properties of the lipid phase induced by valinomycin were observed when K+ and Rb+ ions-forming complexes with the ionophore were present in the systems. The latter phenomenon was not found for the systems containing dinactin possessing a lower ability for complex formation with the cations.     

A new method for determining the phase in the X-ray diffraction structure analysis of phosphatidylcholine/alcohol

A new method based on a sampling theorem is proposed for determining the phase in the X-ray diffraction analysis of the structure of phospholipid systems. The thickness of a lipid layer is changed by changing the length of hydrocarbon chains in order to rebuild the continuous transform from the scattering amplitudes. By employing this method, the phases were accurately determined in a structure analysis of nine phospholipid/alcohol systems at the interdigitated gel phase. The nine systems are dimyristoylphosphatidylcholine(DMPC)/propanol, DPPC/methanol, DPPC/ethanol, DPPC/propanol, DPPC/butanol, distearoylphosphatidylcholine(DSPC)/methanol, DSPC/ethanol, DSPC/propanol and DSPC butanol systems.     

P99-T A Simple Method to Quantitate Selenomethionine Incorporation into Proteins

Selenomethionine substitution is the preferred method for preparing heavy-atom derivatives of proteins for crystal structure determination using the multi-wavelength anomalous diffraction phasing method. This approach allows researchers to take advantage of the anomalous signal from a number of diverse atoms. We recently published a protocol describing a number of variables that play a role in determining incorporation efficiency of selenomethionine into mammalian expression systems.¹ Here we describe, in detail, a simple method for assessing selenomethionine substitution by replacement of methionine in homogeneous protein preparations. Using matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF) technology following trypsin proteolysis of the recombinant protein, we are able to evaluate variables that play roles in affecting selenomethionine incorporation. Examples will illustrate (a) the ease of identification of modified peptides containing the selenomethione and (b) relative quantitation of such peptides when compared with the control, unmodified peptides.