Cross-beta order and diversity in nanocrystals of an amyloid-forming peptide

The seven-residue peptide GNNQQNY from the N-terminal region of the yeast prion protein Sup35, which forms amyloid fibers, colloidal aggregates and highly ordered nanocrystals, provides a model system for characterizing the elusively protean cross-beta conformation. Depending on preparative conditions, orthorhombic and monoclinic crystals with similar lath-shaped morphology have been obtained. Ultra high-resolution (<0.5A spacing) electron diffraction patterns from single nanocrystals show that the peptide chains pack in parallel cross-beta columns with approximately 4.86A axial spacing. Mosaic striations 20-50 nm wide observed by electron microscopy indicate lateral size-limiting crystal growth related to amyloid fiber formation. Frequently obtained orthorhombic forms, with apparent space group symmetry P2(1)2(1)2(1), have cell dimensions ranging from /a/=22.7-21.2A, /b/=39.9-39.3A, /c/=4.89-4.86A for wet to dried states. Electron diffraction data from single nanocrystals, recorded in tilt series of still frames, have been mapped in reciprocal space. However, reliable integrated intensities cannot be obtained from these series, and dynamical electron diffraction effects present problems in data analysis. The diversity of ordered structures formed under similar conditions has made it difficult to obtain reproducible X-ray diffraction data from powder specimens; and overlapping Bragg reflections in the powder patterns preclude separated structure factor measurements for these data. Model protofilaments, consisting of tightly paired, half-staggered beta strands related by a screw axis, can be fit in the crystal lattices, but model refinement will require accurate structure factor measurements. Nearly anhydrous packing of this hydrophilic peptide can account for the insolubility of the crystals, since the activation energy for rehydration may be extremely high. Water-excluding packing of paired cross-beta peptide segments in thin protofilaments may be characteristic of the wide variety of anomalously stable amyloid aggregates.     

Spontaneous Exciton Dissociation at Organic Semiconductor Interfaces Facilitated by the Orientation of the Delocalized Electron–Hole Wavefunction

In organic semiconductors, optical excitation does not necessarily produce free carriers. Very often, electron and hole are bound together to form an exciton. Releasing free carriers from the exciton is essential for the functioning of photovoltaics and optoelectronic devices, but it is a bottleneck process because of the high exciton binding energy. Inefficient exciton dissociation can limit the efficiency of organic photovoltaics. Here, nanoscale features that can allow the free carrier generation to occur spontaneously despite being an energy uphill process are determined. Specifically, by comparing the dissociation dynamics of the charge transfer (CT) exciton at two donor–acceptor interfaces, it is found that the relative orientation of the electron and hole wavefunction within a CT exciton plays an important role in determining whether the CT exciton will decompose into the higher energy free electron–hole pair or relax to the lower energy tightly‐bound CT exciton. The concept of the entropic driving force is combined with the structural anisotropy of typical organic crystals to devise a framework that can describe how the orientation of the delocalized electronic wavefunction can be manipulated to favor the energy‐uphill spontaneous dissociation of CT excitons over the energy‐downhill CT exciton cooling.     

Fluid dynamics in bubble column bioreactors: experiments and numerical simulations

Detailed measurements of multiphase flows that prevail in bioreactors tell us that different transport mechanisms are dominating on different observation scales. The consequence in terms of reactor modeling is that the processes on different scales can be treated independently. A three-dimensional, dynamical model is presented that can be used to describe bubble column bioreactors on the reactor scale. It is based on the Navier-Stokes equation system. On the next smaller scale, the dynamics of the gas phase is described in a Lagrangian way, by tracking many bubble clusters or bubbles simultaneously on their way through the reactor. The model is capable of describing bubble columns of different size and can thus be used for scale-up. Its performance is demonstrated with a production-scale beer fermentor. (c) 1996 John Wiley & Sons, Inc.     

Distance metrics for heme protein electron tunneling

There is no doubt that distance is the principal parameter that sets the order of magnitude for electron-tunneling rates in proteins. However, there continue to be varying ways to measure electron-tunneling distances in proteins. This distance uncertainty blurs the issue of whether the intervening protein medium has been naturally selected to speed or slow any particular electron-tunneling reaction. For redox cofactors lacking metals, an edge of the cofactor can be defined that approximates the extent in space that includes most of the wavefunction associated with its tunneling electron. Beyond this edge, the wavefunction tails off much more dramatically in space. The conjugated porphyrin ring seems a reasonable edge for the metal-free pheophytins and bacteriopheophytins of photosynthesis. For a metal containing redox cofactor such as heme, an appropriate cofactor edge is more ambiguous. Electron-tunneling distance may be measured from the conjugated heme macrocycle edge or from the metal, which can be up to 4.8 A longer. In a typical protein medium, such a distance difference normally corresponds to a approximately 1000 fold decrease in tunneling rate. To address this ambiguity, we consider both natural heme protein electron transfer and light-activated electron transfer in ruthenated heme proteins. We find that the edge of the conjugated heme macrocycle provides a reliable and useful tunneling distance definition consistent with other biological electron-tunneling reactions. Furthermore, with this distance metric, heme axially- and edge-oriented electron transfers appear similar and equally well described by a simple square barrier tunneling model. This is in contrast to recent reports for metal-to-metal metrics that require exceptionally poor donor/acceptor couplings to explain heme axially-oriented electron transfers.     

Imaging vesicular dispersions with cold-stage electron microscopy

A fast-freeze, cold-stage transmission electron microscopy technique which can incorporate in situ freeze-drying of the sample is described. Its use in elucidating structure in unstained and stained, hydrated and freeze-dried, aqueous vesicular dispersions of biological and chemical interest is demonstrated with vesicles of l-α-phosphatidylcholine (bovine phosphatidylcholine) and of the synthetic surfactant sodium 4-(1′-heptylnonyl)benzenesulfonate (SHBS). The contrast features observed in transmission electron microscope images of frozen, hydrated samples are identified and explained with the dynamical theory of electron diffraction. Radiolysis by the electron beam is shown to increase contrast in vesicle images and to change their structure and size. Micrographs illustrate the freeze-drying of a dispersion in the microscope; the process causes vesicles to shrink and collapse.     

Computer controlled cryo-electron microscopy--TOM² a software package for high-throughput applications

Automated data acquisition expedites structural studies by electron microscopy and it allows to collect data sets of unprecedented size and consistent quality. In electron tomography it greatly facilitates the systematic exploration of large cellular landscapes and in single particle analysis it allows to generate data sets for an exhaustive classification of coexisting molecular states. Here we describe a novel software philosophy and architecture that can be used for a great variety of automated data acquisition scenarios. Based on our original software package TOM, the new TOM(2) package has been designed in an object-oriented way. The whole program can be seen as a collection of self-sufficient modules with defined relationships acting in a concerted manner. It subdivides data acquisition into a set of hierarchical tasks, bonding data structure and the operations to be performed tightly together. To demonstrate its capacity for high-throughput data acquisition it has been used in conjunction with instrumentation combining the latest technological achievements in electron optics, cryogenics and robotics. Its performance is demonstrated with a single particle analysis case study and with a batch tomography application.     

The glucan–chitin complex in Saccharomyces cerevisiae. IV. The electron diffraction of crustacean and yeast cell wall chitin

Crustacean and yeast cell wall chitin were analyzed by means of transmission electron microscopy and selected-area diffraction. Single fibrils 8–25 nm wide have been observed in the micrographs of crustacean chitin. Analysis of a series of diffraction patterns obtained from thin crustacean chitin platelets yielded results which were in a better agreement with the theoretical structural model than those measured earlier. In this respect electron diffraction is shown to be superior to the more commonly used x-ray diffraction. Yeast cell wall chitin had a less perfect structure than the crustacean chitin. Single fibrils were not observed on the micrographs and electron diffraction patterns did not show any preferred fiber orientation. The evaluation of electron-diffraction patterns of both the primary septum and the adjacent circular zone of scar ring led to the conclusion that α-chitin is present in both these parts of the mother bud scar.     

Electron crystallography of macromolecular periodic arrays on phospholipid monolayers

Electron crystallography has the potential of yielding structural information equivalent to x-ray diffraction. The major difficulty has been preparing specimens with the required structural order and size for diffraction and imaging in the electron microscope. 2D crystallization on phospholipid monolayers is capable of fulfilling both of these requirements. Crystals can form as a result of specific interactions with a protein's ligand or an analog, suitably linked to a lipid tail; or on a surface of complementary head-group charge. With such choices, the availability of a suitable lipid is limited only by synthetic chemistry. Ultimately, it is the quality and regularity of the protein-protein interactions that determine the crystalline order, as it is with any protein crystal. In the case of streptavidin, the monolayer crystal diffracts beyond 2.5 Å. A 3 Å projection map reconstructed from electron diffraction amplitudes and phases from images shows density which can be interpreted as β-sheets and clusters of side chains. It remains to be shown that the monolayer crystals are flat and diffract as well at high tilt angle as untilted. Technological issues such as charging must be resolved. With parallel advances in data collection and processing, electron crystallography of monolayer macromolecular crystals will eventually take its place beside x-ray crystallography and NMR as a routine and efficient structural technique.     

On the relationship between the protein structure and protein dynamics

Recently, we have developed a method (Shih et al., Proteins: Structure, Function, and Bioinformatics 2007;68: 34-38) to compute correlation of fluctuations of proteins. This method, referred to as the protein fixed-point (PFP) model, is based on the positional vectors of atoms issuing from the fixed point, which is the point of the least fluctuations in proteins. One corollary from this model is that atoms lying on the same shell centered at the fixed point will have the same thermal fluctuations. In practice, this model provides a convenient way to compute the average dynamical properties of proteins directly from the geometrical shapes of proteins without the need of any mechanical models, and hence no trajectory integration or sophisticated matrix operations are needed. As a result, it is more efficient than molecular dynamics simulation or normal mode analysis. Though in the previous study the PFP model has been successfully applied to a number of proteins of various folds, it is not clear to what extent this model will be applied. In this article, we have carried out the comprehensive analysis of the PFP model for a dataset comprising 972 high-resolution X-ray structures with pairwise sequence identity or=0.5. Our result shows that the fixed-point model is indeed quite general and will be a useful tool for high throughput analysis of dynamical properties of proteins.     

Liquid-like water confined in stacks of biological membranes at 200 k and its relation to protein dynamics

Confined water is of considerable current interest owing to its biophysical importance and relevance to cryopreservation. It can be studied in its amorphous or supercooled state in the "no-man's land", i.e., in the temperature range between 150 and 235 K, in which bulk water is always crystalline. Amorphous deuterium oxide (D(2)O) was obtained in the intermembrane spaces of a stack of purple membranes from Halobacterium salinarum by flash cooling to 77 K. Neutron diffraction showed that upon heating to 200 K the intermembrane water space decreased sharply with an associated strengthening of ice diffraction, indicating that water beyond the first membrane hydration layer flowed out of the intermembrane space to form crystalline ice. It was concluded that the confined water undergoes a glass transition at or below 200 K to adopt an ultraviscous liquid state from which it crystallizes to form ice as soon as it finds itself in an unconfined, bulk-water environment. Our results provide model-free evidence for translational diffusion of confined water in the no-man's land. Potential effects of the confined-water glass transition on nanosecond membrane dynamics were investigated by incoherent elastic neutron scattering experiments. These revealed no differences between flash-cooled and slow-cooled samples (in the latter, the intermembrane space at temperatures <250 K is occupied only by the first membrane hydration layers), with dynamical transitions at 150 and 260 K, but not at 200 K, suggesting that nanosecond membrane dynamics are not sensitive to the state of the water beyond the first hydration shell at cryotemperatures.     

5''-Nucleotidases in rat heart. Evidence for the occurrence of two soluble enzymes with different substrate specificities.

Chromatography of soluble proteins from rat heart on phosphocellulose columns separates two 5'-nucleotidases. The first to emerge from the column shows a preference for AMP over IMP as substrate, whereas the second shows a preference for IMP over AMP. The properties of the IMP-preferring enzyme, including the conditions under which it is eluted from phosphocellulose columns, show it to be the enzyme studied by Itoh, Oka & Ozasa [Biochem. J. (1986) 235, 847-851]. The kinetic properties of the AMP-preferring enzyme indicate that it is likely to be the enzyme responsible for the production of adenosine under conditions of hypoxia and increased work load, and with metabolic stresses such as a high load of acetate.     

Enamel rod relations in the developing rat incisor

Scanning electron microscopic and X-ray diffraction studies have shown that mandibular rat incisor teeth have two sets of rods which decussate at angles between 60 and 80% in both the most immature zone and the zone just beyond the opaque margin. A less well oriented interrod enamel component was found at right angles to both sets of rods. The information provides additional views of this complex tissue. Furthermore, it has been shown that the Wistar rat incisal enamel ultrastructure facilitates the use of X-ray diffraction to determine molecular relations between the crystals and matrix constituents as the rods are not all at 90 degrees to one another.     

Chemical and physical properties of radionuclides

There are many radionuclides with a wide range of energies and half-lives available for use as non-sealed radiotherapeutic agents. To date, no single radionuclide has emerged as being clearly superior to all others, in the way that ^99mTc predominates in diagnostic imaging. It is unlikely that one will emerge. Instead, if a particular application demands certain decay properties, the radionuclide which will be used will be the one for which appropriate chemistry can be developed and which can be produced and distributed most economically.     

Direct determination of crystallographic phases for diffraction data from phospholipid multilamellar arrays.

Direct determination of crystallographic phases based on probabilistic of sigma 1 and sigma 2 "triplet" structure invariants has been found to be an effective technique for structure analysis with lamellar x-ray or electron diffraction intensity data from phospholipids. In many cases, nearly all phase values are determined, permitting a structure density (electron density for x-ray diffraction; electrostatic potential for electron diffraction) map to be calculated, which is directly interpretable in terms of known bilayer lipid structure. The major source of error is found to be due to the distortion of observed electron diffraction intensity data by incoherent multiple scattering, which can significantly affect the appearance of the electrostatic potential map, but not the success of the phase determination, as long as the observed Patterson function can be interpreted.     

Low resolution structure of human erythrocyte glutathione reductase

Glutathione reductase from human erythrocytes is a dimeric flavoenzyme with a molecular weight of 100,000. X-ray diffraction analysis using the isomorphous replacement technique with four heavy-atom derivatives yielded an electron density map at 6 Å resolution with a figure of merit of 0.88. Only minor cuts had to be made in the electron density map to isolate one molecule. The dimer interface is on a crystallographic 2-fold axis. Each subunit can be subdivided into three domains: I, II and III, which are aggregated in such a way that deep clefts are formed on opposite sides of the subunit. These clefts accommodate the substrate glutathione, binding to domain III, and the oxidized cofactor NADP, binding to domain I in a similar extended conformation as NAD binds to the dehydrogenases. The shortest connection between the centres of the nicotinamide ring of NADP and the cystine of oxidized glutathione is 18 Å long and goes along the interface between domains II and III right through the centre of the subunit. Presumably, FAD binds to domain II and its isoalloxazine ring bridges the gap between NADP and glutathione.     

Electron-counting MicroED data with the K2 and K3 direct electron detectors

Microcrystal electron diffraction (MicroED) uses electron cryo-microscopy (cryo-EM) to collect diffraction data from small crystals during continuous rotation of the sample. As a result of advances in hardware as well as methods development, the data quality has continuously improved over the past decade, to the point where even macromolecular structures can be determined ab initio. Detectors suitable for electron diffraction should ideally have fast readout to record data in movie mode, and high sensitivity at low exposure rates to accurately report the intensities. Direct electron detectors are commonly used in cryo-EM imaging for their sensitivity and speed, but despite their availability are generally not used in diffraction. Primary concerns with diffraction experiments are the dynamic range and coincidence loss, which will corrupt the measurement if the flux exceeds the count rate of the detector. Here, we describe instrument setup and low-exposure MicroED data collection in electron-counting mode using K2 and K3 direct electron detectors and show that the integrated intensities can be effectively used to solve structures of two macromolecules between 1.2 Å and 2.8 Å resolution. Even though a beam stop was not used with the K3 studies we did not observe damage to the camera. As these cameras are already available in many cryo-EM facilities, this provides opportunities for users who do not have access to dedicated facilities for MicroED.     

The Structure and Mineralization of the Carapace of the Crab (Cancer pagurus L.) 3. The epicuticle

Decalcified and undecalcified preparations of the crab Cancer pagurus in the intermoult condition were studied to examine the mineralization and structure of the epicuticle, using light microscopic, electron microscopic, and microradiographic methods. The epicuticle was found to be composed of two layers, one superficial membrane, and one thicker layer, measuring 1-2 μm. From the base layer spines or microtrichia projected. These were approximately 18 μm long and built like the remainder of the epicuticle. The spines and the base layer of the epicuticle contained vertical canals which in undecalcified sections accomodated columns of crystals. These canals were the only location in which minerals occurred in the epicuticle. In decalcified preparations filamentous strands were observed in the canals. Elsewhere in the epicuticular tissue no fibrillar structure was observed. The canals and their contents seemed to extend across the junctional zone between the epicuticle and the exocuticle.     

ECE measurements via B-X-O mode conversion: A proposal to diagnose the q profile in spherical tokamaks

Generation of electron Bernstein waves by the ordinary-extraordinary-Bernstein (O-X-B) mode conversion process has been successfully demonstrated on W7-AS. According to Kirchoff’s law, the inverse process of plasma EC emission by B-X-O mode conversion at particular angles must take place in tokamak plasmas. The optical depth at electron cyclotron harmonics is generally very high for electron Bernstein waves in tokamak plasmas. Consequently the O-mode ECE spectrum measured below the plasma frequency will show steps in the emitted power when each EC harmonic coincides with the upper hybrid resonance zone, where the mode conversion occurs, giving a local measurement of the relationship between the total magnetic field and plasma density. In a spherical tokamak, there are several EC harmonics below the plasma frequency, so several such steps can be observed via the B-X-O mode conversion mechanism. This is a very promising way to get information about the q profile in ST plasmas.