Crystallization of 70 S ribosomes and 30 S ribosomal subunits from Thermus thermophilus

Well-ordered three-dimensional crystals of 70 S ribosomes and 30 S ribosomal subunits from extremely thermophilic bacteria Thermus thermophilus have been obtained. Positively stained thin sections of the crystals have been analyzed by electron microscopy. Redissolved crystalline ribosomes and small ribosomal subunits reveal sedimentation constants of 70 S and 30 S, respectively, and are functionally active in the poly(U)-system.     

The innermost chorionic layer of Drosophila. I. The role of chorin octamers in the formation of a family of interdigitating crystalline plates

The innermost chorionic layer (ICL) within egg shells of Drosophila melanogaster is composed of thin, abutting three-dimensional crystalline plates which form a closed, membrane-like sheath. Collectively, the crystals within the sheath appear to form a family of related three-dimensional crystals in space group C222; however, specimens prepared for electron microscopy are actually two-dimensional crystals in c222. The projected structures of the negatively stained crystals have been studied by minimal dose electron microscopy employing image reconstruction methods. Thin sections indicate that unit cells within the ICL are composed of paired layers; top and bottom layers are related by centrally located 2-fold axes, aligned parallel to the surface of the ICL. The most probable structural unit of the crystals is a tetramer of chorin dimers with a point group symmetry of 222, which is denoted a chorin octamer. Projection maps were computed from average transforms of two-dimensional crystals for delta (the primitive unit cell angle) equal to 84 degrees, 90 degrees and 97 degrees (+/- 1.5 degrees). The maps indicate that the molecular transitions responsible for the observed family of crystals involve concerted intramolecular rearrangements about molecular 2-fold axes. The significance in vivo of the family of crystals within the ICL is not known; however, structural considerations suggest that the observed polymorphism may reflect one facet of an intrinsic bonding flexibility of the ICL octamer that may play a role in the formation of interplate junctions and the assembly of a continuous closed sheath. The ICL may therefore serve as a structural bridge between the vitelline membrane-wax layer and the endochondrial floor, allowing the larva to shed the inner egg shell layers during hatching.     

Projection structure of the bacterial oxalate transporter OxlT at 3.4A resolution

OxlT is a bacterial transporter protein with 12 transmembrane segments that belongs to the Major Facilitator Superfamily of transporters. It facilitates the exchange of oxalate and formate across the membrane of the Gram-negative bacterium Oxalobacter formigenes. From an electron crystallographic analysis of two-dimensional, tube-like crystals of OxlT, we have previously determined the three-dimensional structure of this transporter at 6.5 A resolution. Here, we report conditions to obtain crystalline, two-dimensional sheets of OxlT with diameters exceeding 2 microm. Images of the crystalline sheets were recorded at liquid nitrogen temperatures on a transmission electron microscope equipped with a field-emission gun, operated at 300 kV. Computed optical diffraction patterns from the best images display measurable reflections to about 3.4A, and electron diffraction patterns show spots to about 3.2 A resolution in the best cases. As in the case of the tube-like crystals, the new crystalline sheets also belong to the p22(1)2(1) symmetry group. However, the unit cell dimensions of 102.7A x 67.3 A are significantly smaller in one direction than those previously observed with the tube-like crystals that display unit cell dimensions of 100.3A x 79.0 A. Different regions of OxlT are involved in intermolecular contacts in the two types of crystals, and the improved resolution of the sheet crystals appears to be mainly attributable to this tighter packing of the monomers within the unit cell.     

Crystallization and preliminary x-ray diffraction study of the flavoprotein NADH peroxidase from Streptococcus faecalis 10C1

NADH peroxidase from Streptococcus faecalis 10C1 has been crystallized from ammonium sulfate solutions using the hanging drop vapor diffusion method. Depending on pH, the crystals grew in the orthorhombic space group I222 or one of its subgroups P222 or P2(1)2(1)2 (or one of its two permutations). In both cases the unit cell axes are a = 76.6 A, b = 132.9 A, and c = 145.7 A. There are two monomers/asymmetric unit in the body-centered crystal form and four in the primitive one. The enzyme is catalytically active in the crystalline state. The crystals diffract to at least 2.5 A resolution; they are stable in the x-ray beam and hence suitable for detailed three-dimensional structure determination.     

Structure and single crystal spectroscopy of Green Fluorescent Proteins

Usually, spectroscopic data on proteins in solution are interpreted at molecular level on the basis of the three-dimensional structures determined in the crystalline state. While it is widely recognized that the protein crystal structures are reliable models for the solution 3D structures, nevertheless it is also clear that sometimes the crystallization process can introduce some "artifacts" that can make difficult or even flaw the attempt to correlate the properties in solution with those in the crystalline state. In general, therefore, it would be desirable to identify some sort of control. In the case of the spectroscopic properties of proteins, the most straightforward check is to acquire data not only in solution but also on the crystals. In this regard, the Green Fluorescent Protein (GFP) is an interesting case in that a massive quantity of data correlating the spectroscopic properties in solution with the structural information in the crystalline state is available in literature. Despite that, a relatively limited amount of spectroscopic studies on single crystals of GFP or related FPs have been described. Here we review and discuss the main spectroscopic (in solution) and structural (in crystals) studies performed on the GFP and related fluorescent proteins, together with the spectroscopic analyses on various FPs members in the crystalline state. One main conclusion is that "in cristallo" spectroscopic studies are useful in providing new opportunities for gathering information not available in solution and are highly recommended to reliably correlate solution properties with structural features. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.     

Mass Determination of the Unit Cell of the Innermost Chorionic Layer in Drosophilidae by Scanning Transmission Electron Microscopy

The innermost chorionic layer (ICL) in eggshells of Drosophila melanogaster is a naturally occurring patchwork of thin three-dimensional crystalline plates located between the inner endochorion and the vitelline envelope. The mass-per-unit area of the ICL has been measured from scanning transmission electron microscope images of isolated unstained material and it was possible to distinguish up to four layers with the majority of the crystalline sheets being one to three layers thick. Taking into account the unit cell areas for the different crystals, we have estimated the mean ICL subunit sizes to be 36 kDa for Drosophila melanogaster, 35 kDa for Drosophila auraria, and 33 kDa for Drosophila teissieri. The results suggest that the three different Drosophilidae species have very similar average subunit masses.     

Spontaneous formation of two-dimensional and three-dimensional cholesterol crystals in single hydrated lipid bilayers

Grazing incidence x-ray diffraction measurements were performed on single hydrated bilayers and monolayers of Ceramide/Cholesterol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocyholine at varying concentrations. There are substantial differences in the phase and structure behavior of the crystalline domains formed within the bilayers relative to the corresponding monolayers, due to interactions between the opposing lipid leaflets. Depending on the lipid composition, these interactions lead to phase separation and formation of cholesterol crystals. The cholesterol and ceramide/cholesterol mixed phases were further characterized at 37°C by immunolabeling with specific antibodies recognizing ordered molecular arrays of cholesterol. Previous studies have shown that cholesterol may nucleate in artificial membranes to form thick two-dimensional bilayer crystals. The study herein demonstrates further growth of cholesterol into three-dimensional crystals. We believe that these results may provide further insight into the formation of cholesterol crystals in early stages of atherosclerosis inflammation.     

A helical processing pipeline for EM structure determination of membrane proteins

Electron crystallography plays a key role in the structural biology of integral membrane proteins (IMPs) by offering one of the most direct means of providing insight into the functional state of these molecular machines in their lipid-associated forms, and also has the potential to facilitate examination of physiologically relevant transitional states and complexes. Helical or tubular crystals, which are the natural product of proteins crystallizing on the surface of a cylindrical vesicle, offer some unique advantages, such as three-dimensional (3D) information from a single view, compared to other crystalline forms. While a number of software packages are available for processing images of helical crystals to produce 3D electron density maps, widespread exploitation of helical image reconstruction is limited by a lack of standardized approaches and the initial effort and specialized expertise required. Our goal is to develop an integrated pipeline to enable structure determination by transmission electron microscopy (TEM) of IMPs in the form of tubular crystals. We describe here the integration of standard Fourier-Bessel helical analysis techniques into Appion, an integrated, database-driven pipeline.     

Three-dimensional diffractive imaging for crystalline monolayers with one-dimensional compact support

The use of a compact support constraint along the beam direction is considered as a solution to the phase problem for diffraction by two-dimensional protein crystals. Specifically we apply the iterative Gerchberg-Saxton-Fienup algorithm to simulated three-dimensional transmission electron diffraction data from monolayer organic crystals. We find that oversampling along the reciprocal-lattice rods (relrods) normal to the monolayer alone does not solve the phase problem in this geometry in general. However, based on simulations for a crystalline protein monolayer (lysozyme), we find that convergence is obtained in three dimensions if phases are supplied from a few high resolution electron microscope images recorded at small tilts to the beam direction. In the absence of noise, amplitude-weighted phase residuals of around 5 degrees, and a cross-correlation coefficient of 0.96 between the true and estimated potential are obtained if phases are included from images at tilts of up to 15 degrees. The performance is almost as good in the presence of noise at a level that is comparable to that commonly observed in electron crystallography of proteins. The method should greatly reduce the time and labor needed for data acquisition and analysis in cryo-electron microscopy of organic thin crystals by avoiding the need to record images at high tilt angles.     

Catalytic activity of rabbit skeletal muscle aldolase in the crystalline state

The monoclinic crystalline form of aldolase from rabbit skeletal muscle grown at 29 degrees C is catalytically active in the direction of aldol cleavage. Activity was assayed for in a crystallization buffer containing 45% saturated ammonium sulfate using chemically unmodified single crystals cut to precise dimensions. Diffusion effects on velocities from assays employing aldolase crystals do not appear to be limiting when cut single crystals are crushed. Assays of crushed crystals are linear with respect to both time and enzyme concentration. Kinetic constants are reported for both substrates fructose 1-phosphate and fructose 1,6-phosphate. Maximal velocities and binding constants determined differ by no more than a factor of 2 between the crystalline and the soluble state of the enzyme. Analysis of the kinetic constants for fructose 1-phosphate as substrate shows that binding of substrate does not change in going to the crystalline state. Release of product is reduced roughly 2-fold in the crystalline state. A similar conclusion can be reached in the case of fructose 1,6-phosphate as substrate provided the "on" steps of substrate and product are only diffusion limited but independent of the physical state of the enzyme. It is not possible to distinguish between a more sluggish conformational change during catalysis or simply tighter product binding in the crystalline state as compared to the soluble enzyme state.     

Structural differences in solution and crystalline forms of met-myoglobin

For several decades X-ray diffraction studies have been the paragon of biological structure studies at atomic resolution. Diffraction provides three-dimensional structure information, which is essential to our fundamental understanding of protein function. However, since X-ray diffraction cannot be done to atomic resolution on proteins in their native solution or membrane-bound state, the possibility exists that the conformations of the protein in crystals are slightly different from the conformations in solution, and attempts to interpret details of the structure may be misleading and without physiological relevance. In this paper, we show that this concern is justified for a familiar protein, myoglobin. Performing X-ray absorption fine structure experiments on both solution and crystalline met-myoglobin (met-Mb), we find significant differences in the local environment of the iron between the two states. Specifically, the average iron-nearest neighbor atom distance in the crystalline form is 0.05 A shorter than that in the solution form, and the iron-nearest neighbor bond is more rigid in the crystalline met-Mb. Possible artifactual explanations for the differences have been ruled out.     

High pressure macromolecular crystallography: the 140-MPa crystal structure at 2.3 A resolution of urate oxidase, a 135-kDa tetrameric assembly

We report the three-dimensional structure determined by high-pressure macromolecular crystallography (HPMX) of a 135-kDa homo-tetrameric enzyme, urate oxidase from Aspergillus flavus complexed with its potent inhibitor 8-azaxanthin. Urate oxidase crystals are quite sensitive to pressure, as three-dimensional order is lost at about 180 MPa. A highly complete 2.3 A resolution data set was collected at 140 MPa, close to the critical pressure. Crystal structures at atmospheric pressure and at high pressure were refined in the orthorhombic space group I222 with final crystallographic R factors 14.1% and 16.1%, respectively. The effect of pressure on temperature factors, ordered water molecules, hydrogen bond lengths, contacts, buried surface areas as well as cavity volume was investigated. Results suggest that the onset of disruption of the tetrameric assembly by pressure has been captured in the crystalline state.     

Three-dimensional reconstruction of innermost chorion layer from Drosophila melanogaster

A low-resolution three-dimensional structure of the crystalline innermost chorion layer (ICL) has been calculated from electron microscope images of tilted negatively stained crystals. The isolated ICL is a single layer, about 12 nm thick and appears to be made up of two types of subunits, each approximately 3 nm in diameter, arranged regularly as groups of four heterodimers in space group C222. Linking density between these groups of subunits, maintaining the integrity of the layer, appears to be confined mainly to the outer surfaces of the ICL.     

Three-dimensional reconstruction of innermost chorion layer from Drosophila melanogaster

A low-resolution three-dimensional structure of the crystalline innermost chorion layer (ICL) has been calculated from electron microscope images of tilted negatively stained crystals. The isolated ICL is a single layer, about 12 nm thick and appears to be made up of two types of subunits, each approximately 3 nm in diameter, arranged regularly as groups of four heterodimers in space group C222. Linking density between these groups of subunits, maintaining the integrity of the layer, appears to be confined mainly to the outer surfaces of the ICL.     

Detection of membrane protein two-dimensional crystals in living cells

It is notoriously difficult to grow membrane protein crystals and solve membrane protein structures. Improved detection and screening of membrane protein crystals are needed. We have shown here that second-order nonlinear optical imaging of chiral crystals based on second harmonic generation can provide sensitive and selective detection of two-dimensional protein crystalline arrays with sufficiently low background to enable crystal detection within the membranes of live cells. The method was validated using bacteriorhodopsin crystals generated in live Halobacterium halobium bacteria and confirmed by electron microscopy from the isolated crystals. Additional studies of alphavirus glycoproteins indicated the presence of localized crystalline domains associated with virus budding from mammalian cells. These results suggest that in vivo crystallization may provide a means for expediting membrane protein structure determination for proteins exhibiting propensities for two-dimensional crystal formation.     

Improving Solar Cells’ Light Trapping by the Low Loss Interface Photonic Crystals

Improving the silicon layer’s optical absorption is a key research point for crystalline silicon based thin film solar cells. Light trapping is a method widely adopted to achieve this research purpose. In this paper, we propose low loss interface photonic crystals layer (IPC), which is sandwiched between the crystalline silicon layer and the cover layer. The low loss interface photonic crystals layer could boost the light trapping efficiency significantly. The mechanism is that the smaller refraction index difference between silicon layer and the low loss interface photonic crystals layer could reduce the light’s interface reflection. Taking advantage of the coupling calculation by optical and electrical simulations, solar cell’s absorption efficiency and electrical performance parameters are obtained. Compared with optimized reference group, the maximum output power of the proposed solar cell could be improved by 6.44%. The result indicates that the proposed low loss interface photonic crystals layer is applicable for light’s trapping in crystalline silicon thin film solar cells.

     

Autofluorescent chalcedony in human brains from elderly patients

Abstract

Chalcedony, a microcrystalline form of silica (SiO₂), has been found in the human brains of elderly patients by using a standard optical petrographic microscope. We document here our visualization of chalcedony using a Leica TCS – SP2 confocal laser scanning microscope. Sections of human brain were collected after autopsy from elderly patients. The autofluorescent character of chalcedony allowed us to obtain three-dimensional images of the crystals and mature prismatic quartz (chalcedony) was observed. Chalcedony occurred as rhombohedral (trigonal) crystals approximately 30 μm in size distributed in patches or aggregates. A less mature silica polymorph of about 1–2 μm in size was detected near the crystals. This is the first time that biogenically-produced crystalline mineral as autofluorescent crystal aggregates has been observed in the human central nervous system of elderly patients using confocal laser scanning microscopy.