Crystallite Features of Valonia cellulose by Electron Diffraction and Dark-Field Electron Microscopy

Individual cellulose crystallites from the cell wall of Valonia ventricosa have been studied by electron diffraction and observed by dark-field electron microscopy. These two techniques reveal that the crystalline zones which run along the fibrils are above 1000 Å in length without any longitudinal periodicity. The width of the crystallites covers the width of the microfibrils and ranges from 140 to 180Å without persistent 35 Å subunits. In several instances, the crystalline zones terminate in the manner of a fork with two arms of 30 to 40 Å in width.     

On the polarity of cellulose in the cell wall of Valonia

The orientation of the triclinic phase of cellulose in the cell wall of Valonia ventricosa J. Agardh was investigated by X-ray- and electron-diffraction analysis. In addition to the well-documented uniplanar-axial organization of the cell wall which requires that the a ^* axis should be always perpendicular to the wall surface, the direction of this axis was also found to be pointing outward from the plasma membrane side of the wall. This unidirectionality was persistent throughout the various layers that constitute the cell wall and also for the three microfibrillar orientations that occur in Valonia cell walls. The unidirectionality of the a ^* axis indicates, in particular, that the Valonia cellulose microfibrils are not twisted along their axis. These observations are consistent with a cellulose biosynthetic scheme where a close association exists between terminal-complex orientations and those of the cellulose microfibrils. In this context, the unidirectionality of the a ^* axis of cellulose seems to be related to the restricted mobility of the terminal complexes which are able to slide in the plasma membrane but not to rotate along their long axis.Abbreviations TC terminal complex This work was initiated during a visit of J.F.R at Grenoble in the framework of a France-Québec exchange program. J.S. was recipient of a CNRS fellowship. The diagram in Fig. 8 was kindly drawn for us by Miss Yukie Saito from the Department of Forest Products, the University of Tokyo.     

Action of exo- and endo-type cellulases from Irpex lacteus on Valonia cellulose

Cellulolytic mode of action of the two highly purified exo- and endo-type cellulases from Irpex lacteus on pure Valonia cellulose was investigated. Electron microscopy substantiated that both cellulases are adsorbed preferentially into the internal parts of microfibrils in the network structure of the cellulose at initial stages before enzymatic hydrolysis, and that the adsorption ratio of both cellulases onto the external surfaces of microfibrils increased with incubation time although this tendency was less remarkable with the exo-type cellulase than with the endo-type one. The exo-type cellulase exhibited relatively high activity producing cellobiose throughout 12-h incubation, while the endo-type cellulase produced small amounts of cellooligosaccharides. The degree of polymerization was far more suppressed by the endo-type cellulase than by the exo-type one. Degradation by the cellulases in typical exo- and endo-fashions yielded quite different morphological patterns in the microfibrils. Exo-type cellulase loosened the network structure of microfibrils and made them slightly thinner, while endo-type cellulase caused conspicuous swelling and dissolution of individual microfibrils.     

Electron diffraction of membranes

Summary Electron diffraction conducted on myelin membranes, photosynthetic and photoreceptor membranes yielded spot diffraction patterns indicating an ordered state of membranes; the interplanar spacings being of the order of Å units. It was observed, too, that a membrane specimen accommodates different space structures. Based on these findings it is suggested that membrane functions-like active transport-are exercised through phase transitions; the lattice acts as a coupling agent.     

Electron diffraction from single crystals of DNA.

Crystals of DNA have been grown in a form suitable for study by electron diffraction and electron microscopy. Preliminary electron diffraction patterns of any type that have been obtained from single crystals of highly polymerized DNA. The patterns, obtained from frozen, hydrated crystals with the beam approximately parallel to the DNA strand axis, show a hexagonal geometrical arrangement with a (1,0) Bragg spacing of 23.1 A.     

Electron diffraction analysis of the M412 intermediate of bacteriorhodopsin.

High resolution electron diffraction data have been recorded for glucose-embedded purple membrane specimens in which bacteriorhodopsin (bR) has been trapped by cooling slowly to below--100 degrees C under continuous illumination. Thin films (OD approximately 0.7) of glucose-embedded membranes, prepared as a control, showed virtually 100% conversion to the M state, and stacks of such thin film specimens gave very similar x-ray diffraction patterns in the bR568 and the M412 state in most experiments. To be certain that any measured differences in diffraction intensity would be real, two independent sets of electron diffraction intensities were recorded for near-equatorial, i.e. (hkO), reflections. Little correlation was indeed observed between these two sets for delta F values at low resolution (15-5.0 A, 49 reflections), but the correlation coefficient is approximately 0.3 at high resolution (5.0-3.3 A, 218 reflections). Thus, while most of the measured difference is error, the mean delta F and the correlation coefficient can be used to estimate the smaller, true delta F due to structural changes occurring in the M state. The magnitude of this estimated true mean delta F is equal to what would be produced if approximately five to seven nonhydrogen atoms were moved to structurally uncorrelated (i.e., new) positions in the M state. Movements of a few amino acid side chains, and repositioning of atoms of the retinal group and the associated lysine side chain after trans-cis isomerization, are the most probable causes of the observed intensity changes in the M state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of cellulose crystallinity from powder diffraction diagrams

One‐dimensional (1D) (spherically averaged) powder diffraction diagrams are commonly used to determine the degree of cellulose crystallinity in biomass samples. Here, it is shown using molecular modeling how disorder in cellulose fibrils can lead to considerable uncertainty in conclusions drawn concerning crystallinity based on 1D powder diffraction data alone. For example, cellulose microfibrils that contain both crystalline and noncrystalline segments can lead to powder diffraction diagrams lacking identifiable peaks, while microfibrils without any crystalline segments can lead to such peaks. This leads to false positives, that is, assigning disordered cellulose as crystalline, and false negatives, that is, categorizing fibrils with crystalline segments as amorphous. The reliable determination of the fraction of crystallinity in any given biomass sample will require a more sophisticated approach combining detailed experiment and simulation. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 67–73, 2015.     

Electron diffraction analysis of structural changes in the photocycle of bacteriorhodopsin.

Structural changes are central to the mechanism of light-driven proton transport by bacteriorhodopsin, a seven-helix membrane protein. The main intermediate formed upon light absorption is M, which occurs between the proton release and uptake steps of the photocycle. To investigate the structure of the M intermediate, we have carried out electron diffraction studies with two-dimensional crystals of wild-type bacteriorhodopsin and the Asp96-->Gly mutant. The M intermediate was trapped by rapidly freezing the crystals in liquid ethane following illumination with a xenon flash lamp at 5 and 25 degrees C. Here, we present 3.5 A resolution Fourier projection maps of the differences between the M intermediate and the ground state of bacteriorhodopsin. The most prominent structural changes are observed in the vicinity of helices F and G and are localized to the cytoplasmic half of the membrane.