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1.
Thin, three-dimensional crystals of CaATPase have been studied at high resolution by electron crystallography. These crystals were grown by adding purified CaATPase to appropriate concentrations of lipid, detergent and calcium. A thin film of crystals was then rapidly frozen and maintained in the frozen-hydrated state during electron microscopy. The resulting electron diffraction patterns extend to 4.1 A resolution and images contain phase data to 6 A resolution. By combining Fourier amplitudes from electron diffraction patterns with phases from images, a density map has been calculated in projection. Comparison of this map from unstained crystals with a previously determined map from negatively stained crystals reveals distinct contributions from intramembranous and extramembranous protein domains. On the basis of this distinction and of the packing of molecules in the crystal, we have proposed a specific arrangement for the ten alpha-helices that have been suggested as spanning the bilayer. 相似文献
2.
Alignment and merging of electron microscope images of frozen hydrated crystals of the T4 DNA helix destabilizing protein gp32*I. 总被引:1,自引:0,他引:1
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Low dose cryoelectron microscopy has been used to record images and electron diffraction patterns of frozen hydrated crystals of the single-stranded DNA binding protein gp32*I. Fourier transforms from 13 image areas, corresponding to approximately 40,000 unit cells, were aligned by a minimal phase residual search and merged by vector addition in reciprocal space. Phases from the resulting composite transform were combined with amplitudes from electron diffraction patterns to reconstruct the projected mass density of the gp32*I crystal at 8.4 A resolution. 相似文献
3.
High-resolution spot-scan electron microscopy of microcrystals of an alpha-helical coiled-coil protein 总被引:1,自引:0,他引:1
We describe the electron microscopy of a crystalline assembly of an alpha-helical coiled-coil protein extracted from the ootheca of the praying mantis. Electron diffraction patterns of unstained crystals show crystal lattice sampling of the coiled-coil molecular transform to a resolution beyond 1.5 A. Using a "spot-scan" method of electron imaging, micrographs of unstained crystals have been obtained that visibly diffract laser light from crystal spacings as small as 4.3 A. A projection map was calculated to 4 A using electron diffraction amplitudes and phases from computer-processed images. The projection map clearly shows modulations in density arising from the 5.1 A alpha-helical repeat, the first time this type of modulation has been revealed by electron microscopy. The crystals have p2 plane group symmetry with a = 92.4 A, b = 150.7 A, y = 92.4 degrees. Examination of tilted specimens shows that c is approximately 18 A, indicating that the unit cell is only one molecule thick. A preliminary interpretation shows tightly packed molecules some 400 A long lying with their long axes in the plane of the projection. The molecules have a coiled-coil configuration for most of their length. The possible modes of packing of the molecules in three dimensions are discussed. 相似文献
4.
Summary Single crystals from adult human peritubular dentine were studied by high-resolution transmission electron microscopy. Periodic fringe patterns were obtained from which the exact shape of the inorganic crystals were deduced. The crystals were found to have a mean length of 36.00±1.87 nm, a mean width of 25.57±1.37 nm, and a mean thickness of 9.76±0.69 nm. They consisted of platelets with a mean width-to-thickness ratio of 2.61, each being a flattened hexagonal prism of hydroxyapatite. Such conclusions are based upon a) the electron diffraction patterns that we obtained, and b) our comparison of the values of the periodic, equidistant fringes seen along different planes of sectioning with the corresponding theoretical values for hydroxyapatite. 相似文献
5.
The crotoxin complex from Crotalus d. terrificus rattlesnake venom was crystallized in the form of thin platelets. These crystals were prepared by the glucose embedding technique and examined by low dose electron microscopy. Electron diffraction patterns and images have been recorded to 2.2 and 4.5 A, respectively. By a combination of electron and X-ray diffraction techniques, the space group of this crystal was determined to be P4(2)22 with eight crotoxin complex molecules in one unit cell with dimensions of 38.8 A x 38.8 A x 256.8 A. The Patterson maps and the symmetry reliability factors calculated from the electron diffraction intensities clearly showed the existence of three types of electron diffraction patterns in different crystals. The phases in the computer-calculated transform of the low dose images also show the variation in symmetry among crystals. These phenomena are explained by the presence of crystals consisting of one-half, three-quarter and one unit cell in thickness. The interpretation of the computer reconstructed two-dimensional density map was limited, partly because of the similarity in density between the protein and the embedding glucose and partly because of the non-uniqueness in relating projected structure to the three-dimensional structure. 相似文献
6.
Diaz-Avalos R Long C Fontano E Balbirnie M Grothe R Eisenberg D Caspar DL 《Journal of molecular biology》2003,330(5):1165-1175
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. 相似文献
7.
Three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex from pea chloroplasts 总被引:2,自引:0,他引:2
W Kühlbrandt 《Journal of molecular biology》1987,194(4):757-762
Two forms of three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex from pea have been obtained. Crystals of one form grew as hexagonal plates measuring up to 150 micron across and 2 to 3 micron in thickness. Electron diffraction patterns of thin hexagonal plates showed sharp reflections to a resolution of 3.7 A on a hexagonal reciprocal lattice. The unit cell in projection (a = 127.0 A) and the symmetry of the diffraction pattern (6 mm) suggested that the hexagonal plates were highly ordered stacks of two-dimensional crystals suitable for structure analysis by electron microscopy and image processing. Crystals of a second form grew as dark green octahedra measuring roughly 0.5 mm across. Low-resolution X-ray diffraction patterns suggested a large cubic unit cell (a = 390 A). SDS/polyacrylamide gel electrophoresis of single octahedral crystals showed the same polypeptide composition as the starting solution, one major band at 24,000 apparent molecular weight and two satellite bands of 23,000 and 23,500 apparent molecular weight. 相似文献
8.
Nobuo Kato Yoshichika Arakawa Tsuyoshi Sugiyama Hideo Ito Setsuko Naito Nobuo Kido Michio Ohta Kyoyu Sasaki 《Microbiology and immunology》1994,38(8):629-637
Various chemotypes (Re, Rd2, Rd1P–, Rd1, RcP–, Rc, Rb3, Rb2, Rb1, and Ra) of R-form lipopolysaccharides (LPSs) of Salmonella spp. were crystallized by treatment with 70% ethanol containing 250 mM MgCl2, and crystals of the LPSs were observed electron microscopically and analyzed by electron diffraction and synchrotron X-ray diffraction. All the LPSs tested formed three-dimensional crystals showing very similar shapes; hexagonal plate, solid column, discoid, square or rectangular plate, lozenge plate and truncated hexangular or rectangular pyramid forms. Electron diffraction patterns from the hexagonal plate crystals of all these LPSs obtained by electron irradiation from the direction perpendicular to the basal plane showed that they consist of hexagonal lattices with the lattice constant of 4.62 Å. The crystals of all the LPSs thus formed gave ring-like X-ray diffraction patterns because of their small sizes. The long-axis values were calculated from the X-ray diffraction patterns from crystals of all the LPSs in the low-angle region and they corresponded roughly to the length of the proposed primary chemical structures of the R cores of the LPSs. The volume occupied by a single molecule of all the LPSs were calculated from the molecular weights based on the proposed structures and the crystallographic data obtained by electron diffraction, X-ray diffraction, and density determination. 相似文献
9.
The goal of time-resolved crystallographic experiments is to capture dynamic "snapshots" of molecules at different stages of a reaction pathway. In recent work, we have developed approaches to determine determined light-induced conformational changes in the proton pump bacteriorhodopsin by electron crystallographic analysis of two-dimensional protein crystals. For this purpose, crystals of bacteriorhodopsin were deposited on an electron microscopic grid and were plunge-frozen in liquid ethane at a variety of times after illumination. Electron diffraction patterns were recorded either from unilluminated crystals or from crystals frozen as early as 1 ms after illumination and used to construct projection difference Fourier maps at 3.5-A resolution to define light-driven changes in protein conformation. As demonstrated here, the data are of a sufficiently high quality that structure factors obtained from a single electron diffraction pattern of a plunge-frozen bacteriorhodopsin crystal are adequate to obtain an interpretable difference Fourier map. These difference maps report on the nature and extent of light-induced conformational changes in the photocycle and have provided incisive tools for understanding the molecular mechanism of proton transport by bacteriorhodopsin. 相似文献
10.
Ruggiero A Chambery A Di Maro A Pisante M Parente A Berisio R 《Protein and peptide letters》2007,14(1):97-100
PD-L4, a type 1 ribosome inactivating protein from Phytolacca dioica leaves, has been successfully crystallized using vapour diffusion methods and PEG 4000 as a precipitant agent. In addition, crystals of a PD-L4 mutant, which has been recently observed to have a lower polynucleotide-adenosine glycosidase activity on DNA, rRNA and poly (A) substrates, have been obtained. To gather information on PD-L4 reaction mechanism both forms have been co-crystallized with adenine, the major product of their catalytic reaction. Diffraction patterns extend to atomic resolution and crystals belong to the orthorhombic P2(1)2(1)2(1) space group, with one molecule in the asymmetric unit. Structure determination has been achieved using molecular replacement; preliminary electron density maps have clearly given evidence of adenine binding. 相似文献
11.
A two-dimensionally large and thin crystal has been obtained from gp321I, a proteolytically digested product of a DNA helix-destabilizing protein coded by gene 32 in bacteriophage T4. High-resolution electron diffraction patterns (~3.7Å) are recorded from both unstained and stained protein crystals embedded in glucose. The crystal is of orthorhombic space group with . 相似文献
12.
Single crystals of Bombyx mori silk fibroin in the metastable silk I polymorph have been produced using a new foaming technique. Foams of silk protein are generated by bubbling pure nitrogen gas through an aqueous solution of regenerated silk fibroin. The foamed material is collected, dried, and then sonicated to yield individual crystals which were examined using transmission electron microscopy and electron diffraction. It is found that slightly acidic conditions in the solution from which the foam was generated favor the formation of silk II while neutral to slightly basic solutions favor silk I formation. More dilute solutions favor the formation of silk II while more concentrated solutions (about 7 wt.% or greater) favor the formation of silk I. X-ray powder diffraction patterns from the dried silk I foams displayed features highly indicative of silk I. We also report the first single crystal electron diffraction patterns of silk I. These patterns indicate a large unit cell, possibly 22.66 x 5.70 x 20.82 A. with six chains of six residues, Gly-Ala-Gly-Ala-Gly-Ser. Although we have not fully characterized this complex structure it appears that the chain is nearly fully extended and thus our data is consistent with models possessing general features similar to those proposed by Fossey SA, Nemethy G, Gibson KD, Scheraga HA. (Biopolymers 1991;31:1529-1541). 相似文献
13.
Electron diffraction patterns obtained from single crystals of amylose complexed with n-butanol are reported. The crystals were examined in the frozen state, after quench-freezing in liquid nitrogen in order to maintain the complexed state in the electron microscope. The patterns may be indexed in the base plane with an orthorhombic unit cell of dimensions a = 27.0 ± 0.2 Å and b = 26.4 ± 0.2 Å, and the symmetry of the patterns is consistent with the P212121 space group. The relationship between the orthorhombic patterns and the pseudohexagonal patterns obtained by previous workers from dried crystals is discussed. 相似文献
14.
Preliminary morphological and X-ray diffraction studies of the crystals of the DNA cetyltrimethylammonium salt.
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V D Osica T L Pyatigorskaya O F Polyvtsev A T Dembo M O Kliya V N Vasilchenko B I Verkin B Y Sukharevskya 《Nucleic acids research》1977,4(4):1083-1096
Double-stranded DNA molecules (molecular weight 2.5 X 10(5) - 5 X 10(5) daltons) have been crystallized from water-salt solutions as cetyltrimethylammonium salts (CTA-DNA). Variation of crystallization conditions results in a production of different types of CTA-DNA crystals: spherulits, dendrites, needle-shaped and faceted rhombic crystals, the latter beeing up to 0.3 mm on a side. X-ray diffraction data indicate that DNA molecules in the crystals form a hexagonal lattice which parameters vary slightly with the morphological type of the crystal. Comparison of the melting curves of the DNA preparation before and after crystallization suggests that DNA molecules are partially fractionated in the course of crystallization. Crystals of the CTA-DNA-proflavine complex have also been obtained. 相似文献
15.
Ferrochelatase activity in wild-type and mutant strains of Spirillum itersonii. Solubilization with chaotropic reagents 总被引:5,自引:0,他引:5
New low-angle X-ray diffraction data have been obtained from nerve myelin after rehydration. The X-ray patterns show the first six orders of diffraction of a lamellar repeat unit of about 100 Å. Direct methods of structure analysis have been used to determine uniquely the phases of the first three orders of diffraction. The electron density profile of rehydrated nerve myelin has been obtained on an absolute electron density scale and is compared with the electron density profile of normal nerve myelin at the same resolution of 16–17 Å. Possible electron-density profiles of rehydrated nerve myelin at a resolution of 8 Å are shown. 相似文献
16.
V. D. Osica B. Ya. Sukharevsky V. N. Vasilchenko B. I. Verkin O. F. Polyvtsev 《Molecular biology reports》1976,3(1):73-79
Fragments of calf thymus DNA have been crystallized by precipitation from water-salt solutions, containing 2-methylpentane-2,4-diol (MPD). DNA crystals usually take the form either of spherulites up to 100 in diameter or of needles with the length up to 50 . No irreversible denaturation of DNA occurs during the crystallization process. X-ray diffraction from dense slurries of DNA crystals yields crystalline powder patterns. 相似文献
17.
Brigitte Zimmer H. Hasko Paradies Günther Werz 《Biochemical and biophysical research communications》1977,74(4):1496-1500
Crystals of D-ribulose-1,5-biphosphate carboxylase (E.C. 4.1.1.39), naturally occurring in the extraplasmatic space of the unicellular green algae (Dasycladaceae), were studied by means of electron microscopy and optical diffraction. Optical diffraction patterns were obtained from thin sections. It is shown that the crystals are composed of cubic unit cells with α ~ 31.5 nm. The density of the crystals was estimated as 1.07 ± 0.005 g/ml, a value that gives evidence of the presence of 12 enzyme molecules per unit cell. Optical diffraction studies of the thin sectioned crystals revealed 4mm -symmetry with four 2-fold rotation axes, resulting in at least a 222-symmetry. 相似文献
18.
Obtaining large, flat, well ordered crystals represents the key to structure determination by electron crystallography. Multilamellar crystals of Ca(2+)-ATPase are a good candidate for this methodology, and we have optimized methods of crystallization and of preparation for cryoelectron microscopy. In particular, high concentrations of glycerol were found to prevent nucleation and to reduce stacking; thus, by seeding solutions containing 40% glycerol, we obtained thin crystals that were 5-30 microns in diameter and 2-10 unit cells thick. We found that removing vesicles and minimizing concentrations of divalent cations were critical to preparing flat crystals in the frozen-hydrated state. Finally, we developed two methods for determining the number of lamellae composing individual crystals, information that is required for structure determination of this crystal form. The first method, using low magnification images of freeze-dried crystals, is more practical in our case. Nevertheless, the alternative method, involving analysis of Laue zones from electron diffraction patterns of slightly tilted crystals, may be of general use in structure determination from thin, three-dimensional crystals. 相似文献
19.
Preliminary X-ray crystallographic studies of pig kidney fructose-1,6-bisphosphatase 总被引:2,自引:0,他引:2
B A Seaton R L Campbell G A Petsko D R Rose I Edelstein F Marcus 《The Journal of biological chemistry》1984,259(14):8915-8916
Preliminary x-ray data have been obtained from large single crystals of pig kidney fructose-1,6-bisphosphatase, grown from polyethylene glycol. The crystals have the symmetry of space group P3(1)21 or its enantiomorph P3(2)21, contain two subunits of the 146,000-dalton tetramer/asymmetric unit, and diffract to 2.9-A resolution on still photographs. The unit cell dimensions are a = b = 132.5 A and c = 68.0 A. Small single crystals have been grown in the presence of the inhibitor fructose 2,6-bisphosphate, with and without the allosteric effector AMP added. Crystals grown in the presence of both ligands are isomorphous with native crystals and generate diffraction patterns that show significant intensity changes. 相似文献
20.
The crystalline structure of polyester 6 6 was studied by means of X-ray and electron diffraction and real-space electron microscopy. An orthorhombic unit cell containing eight chain segments with a quasi planar zigzag conformation was derived. The chain axis projection could be defined by a small rectangular cell containing only two molecular segments. Simulation of electron diffraction patterns indicates that molecular segments were arranged with azimuthal angles close to +/-46 degrees . X-ray diffraction patterns suggested that the large dimensions of the unit cell were a consequence of a slight shift between neighboring chains that improved the electrostatic interactions. Chain-folded lamellar crystals were obtained by isothermal crystallization of dilute n-hexanol or n-octanol solutions. The crystalline habit was studied, and the influence of temperature was evaluated. A regular folding surface was observed by using polyethylene decoration techniques. Lamellar crystals were easily degraded with different lipases. A preferential enzymatic attack was, in some cases, observed to occur in the crystal edges, giving rise to highly irregular borders with a fringed texture. 相似文献