Hervé Delacroix

The cubic phases of lipids

Publisher Summary This chapter discusses the cubic phases of lipids. Phases with cubic symmetry is observed in lipid-water systems because the early days of lipid polymorphism, at a time when it seemed all too natural to presume without closer inspection that phases with such high symmetry should consist of spherical micelles of type I or II, orderly packed in a cubic lattice. Crystallographic analyses revealed that cubic symmetry is the attribute of not one lipid phase but of a large family of phases. Moreover, the structure of these phases that took quite a few years to determine turned out to be unexpectedly complex and multifarious. The chapter discusses the structural and physical properties of the cubic phases and of other lipid phases of lower symmetry in a search for general criteria applicable to all lipids.

Spherical plant viruses: interactions in solution, phase diagrams and crystallization of brome mosaic virus

Brome mosaic virus (BMV) is a small icosahedral plant virus of mean diameter 268 A. Interactions between BMV particles in solution were studied by means of small-angle X-ray scattering in order to find crystallization conditions. The interactions between biomacromolecules as large as these viruses have not yet been systematically studied by this method. As it is known that usually proteins crystallize in, or close to, attractive regimes, the interactions between BMV particles in solution were studied as a function of pH, type of salt and size and concentration of polyethylene glycol. An unexpected result of these studies is that the precipitates obtained upon addition of PEG alone or PEG combined with salt were in fact made of microcrystals, which were all characterized by the same series of diffraction peaks, with positions close to those of a centered cubic space group. A phase diagram of the virus as a function of PEG concentration was established by means of microbatch experiments. From the precipitation zones, conditions for crystallization were tested from 5 to 40 mg ml(-1) virus with 3-10%(w/v) PEG 8000 or PEG 20,000. Small crystals were obtained in several conditions after a few days and continued growing for several weeks.

Chapter 1 The Cubic Phases of Lipids

Publisher Summary This chapter describes the cubic phases of lipids. Phases with cubic symmetry have been observed in lipid-water systems since the early days of lipid polymorphism, at a time when it seemed all too natural to presume without closer inspection that phases with such high symmetry should consist of spherical micelles of type I or 11, orderly packed in a cubic lattice. Another important aspect of the cubic phases of lipid-containing systems is their possible biological relevance. In a more general way, the biological significance of lipid polymorphism is a problem often evoked in the past. On account of their remarkable topological properties, the bicontinuous cubic phases are far better candidates for biological speculations than the lamellar and the hexagonal phases.

Combined use of freeze-fracture electron microscopy and X-ray diffraction for the structure determination of three-dimensionally ordered specimens

The cubic phases of lipid‐water systems have been studied by freeze‐fracture electron microscopy. The preservation of the sample structure following cryofixation was verified by low temperature X‐ray diffraction. Different types of fracture planes were identified; all display highly ordered two‐dimensional domains, each subdivided into sub‐domains related to each other by displacements and rotations related to the symmetry of the space group. The images were filtered using cross‐correlation averaging techniques and the filtered images were compared to the corresponding planar sections of the electron density maps. Several conclusions were drawn: 1) when properly cryofixed, as assessed by low temperature X‐ray diffraction, the structure of the sample was well preserved in the replicas; 2) the symmetry of the space group was faithfully reflected in the electron microscope images; 3) the crystallographic orientations of the most frequently identifiedfracture planes coincided with those of the most intense X‐ray reflections indicating that the fracture propagates, preferentially, in regions where the electron density variations are the largest; 4) when different structural models are compatible with X‐ray diffraction data, it is possible to determine the correct model by comparing the filtered images with sections of the corresponding electron density maps; and 5) this approach constitutes a new and powerful tool of general interest for the low resolution study of three‐dimensionally ordered specimens.

Use of cryo-negative staining in tomographic reconstruction of biological objects: application to T4 bacteriophage.

Recent advances in electron microscopy and image analysis techniques have resulted in the development of tomography, which makes possible the study of structures neither accessible to X—ray crystallography nor nuclear magnetic resonance. However, the use of tomography to study biological structures, ranging from 100 to 500 nm, requires developments in sample preparation and image analysis. Indeed, cryo‐electron tomography present two major drawbacks: the low contrast of recorded images and the sample radiation damage. In the present work we have tested, on T4 bacteriophage samples, the use of a new preparation technique, cryo‐negative staining (Adrian et al., 1998), which reduces the radiation damage while preserving a high signal‐to‐noise ratio (De Carlo et al., 2002). Our results demonstrate that the combination of cryo‐negative staining in tomography with standard cryo‐microscopy and single particle analysis results in a methodological approach that could be useful in the study of biological structures ranging in the T4 bacteriophage size.

Structural analysis of the lyotropic polymorphism of four-stranded aggregates of 2′-deoxyguanosine 3′-monophosphate derivatives

Abstract Guanosine derivatives, dissolved in water, can form chromonic cholesteric and hexagonal phases. The common structural unit is a chiral stack of Hoogsteen-bonded guanosine tetramers. Using optical microscopy, circular dichroism and X-ray diffraction techniques, we have analysed the complex columnar polymorphism of two derivatives of 2′-deoxyguanosine—the 3′-monophosphate d(Gp) and the 3′-monophosphate monoisobutyl ester d(Gp)iBu. As a function of water concentration, both compounds exhibit four different columnar phases: in addition to the well-known cholesteric (Ch) and hexagonal (H) phaseg, a 2-dimensional square (Sq) lattice and a new hexagonal packing have been observed. In such a structure (Hb), two columns are packed in the 3m special positions of co-ordinates 1/3,2/3 and 2/3, 1/3 of the two-dimensional hexagonal unit cell. The unusual phase sequence appears to be related to the presence of strong short range intercolumnar interactions. Moreover, the easing of stacking constraints on increas...

Freeze–fracture electron microscopy of lyotropic lipid systems Quantitative analysis of cubic phases of space group Ia3d (Q230)

Abstract The cubic phases Q230 (space group Ia3d) of type I (oil in water) and type II (water in oil) have been studied by freeze-fracture electron microscopy. The preservation of the sample structure during cryofixation was verified by low temperature X-ray diffraction. Three types of fracture planes were clearly identified in the type I system and two in the type II system. These fracture planes showed two-dimensionally ordered domains, each subdivided into subdomains related to each other by displacements and rotations compatible with the symmetry of the space group. The images were filtered using correlation averaging techniques and the filtered images were compared with the corresponding sections of the electron density map. Several conclusions were drawn: (i) The structures of the samples had been preserved after quenching, fracturing and replicating processes, (ii) The electron microscope images were fully compatible with the postulated space group symmetries. (iii) The fracture planes coincide wit...

Time-resolved scattering investigations of brome mosaic virus microcrystals appearance.

The behavior of brome mosaic virus in solution as a function of physico-chemical conditions has already been characterized by Small Angle X-ray Scattering studies. The most striking result was that the precipitates induced by the addition of polyethylene glycol were in fact made of microcrystals. This result was reinvestigated on the ID2 beamline at ESRF (Grenoble, France) to determine whether there was an amorphous state before the organized one, and measure the necessary period of latency for the microcrystals to form. The stopped-flow device associated to the high brilliance of the beamline enabled us to characterize the growth of the diffraction peaks as a function of time.

A unified approach for high throughput analysis of real-time biomolecular interactions in surface plasmon resonance and fluorescence imaging

The analysis of real-time biomolecular interactions (observation is performed as the biological interaction occurs) provides information on the formation of target/probe complexes, particularly on their dynamic behaviours. Namely, it allows the determination of the affinity constant, a static value that characterizes the interaction properties, using two dynamic values, the association and dissociation constants. Such dynamic behaviour can be assessed either with surface plasmon resonance (SPR) or uorescence-based biosensors. The challenging issue is the automatic extraction and analysis of the interaction signal for each spotted probe on the biosensor in a highthroughput framework (hundreds of probes). This paper addresses such issue and develops a uniffied approach for analyzing the image data provided by the above-mentioned technologies. A mathematical modelling of the image data allowed building-up a virtual biosensor able to simulate biologic experiences related to various possible parameters (level of signal and noise, presence of artefacts, surface functionalization, spotting heterogeneity). Based on such simulation, a generic and automated approach combining 3D mathematical morphology and spatio-temporal classiffication is proposed for detecting the interacting probes, segmenting the regions of effective signal, and characterizing the associated affinity constants. The developed method has been assessed both qualitatively and quantitatively on simulated and experimental datasets and showed accurate results (maximum error of 7% for the most difficult cases in terms of noise and surface functionalization).

DEFPARAM: a program package for aligning elliptical sections of biological objects containing an n-fold symmetry

A program package has been developed to align automatically images of biological objects containing an n-fold symmetry, and to remove the distortions induced on their circular shape by the microtomy. It uses an original procedure based on correlation techniques and replaces usual manual processing. Examples of direct averaging of transverse sections of biological objects are given to illustrate the programs capabilities.