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Biomedical subjects

W Chiu

Publications and source records attributed to W Chiu.

At least 127 records · Page 7Linked to original sources
PubMed1984

Electron imaging of crotoxin complex thin crystal at 3.5 A.

Crotoxin complex forms thin crystals which are suitable for electron crystallographic analysis. We have used a 100 kV electron microscope equipped with a superconducting lens to image this crystal embedded in glucose. Optical diffraction analysis of the micrographs show unambiguously a structural resolution of 3.9 A which has not been obtained with the conventional microscope at room temperature. A density map with a nominal resolution of 3.5 A has been synthesized from these images by computer processing techniques.

Animals↗
PubMed1984

Specimen preparative methods for electron crystallography of soluble proteins.

Technical factors which influence the choice of specimen preparation method for electron crystallographic study of thin crystals of soluble proteins are discussed. Ice embedding appears to be the most desirable choice of preparation method. However, in terms of the yield of major structural information, we conclude that negative stain remains a useful method for low resolution and glucose embedding for high resolution.

Animals↗
PubMed1984

Experimental strategy in three-dimensional structure determination of crotoxin complex thin crystal.

Electron images and diffraction patterns of crotoxin complex thin crystals contain high-resolution structural information in projection at both room and low temperatures. This paper outlines our strategy for collecting three-dimensional amplitude and phase data from these uniquely well-ordered toxin thin crystals. Preliminary experiments in support of the feasibility of this approach are presented.

Animals↗
PubMed1984

Estimates of validity of projection approximation for three-dimensional reconstructions at high resolution.

As spatial frequency increases, the electron microscope "image" deviates increasingly from a true projection of the specimen's structure. This is due to the finite radius of the Ewald sphere. Quantitative estimates of these deviations of the reconstruction from the true projection are presented for a range of accelerating voltages, spatial frequencies, specimen thicknesses, and specimen tilts.

Data Display↗
PubMed1984

Quantitative assessment of radiation damage in a thin protein crystal.

Radiation damage is a limiting factor for high resolution structural determination of protein crystals. We have used the median and quartile values of Ne exposures and the structural disordering factor (delta B) to describe the crystalline disordering due to radiation damage in thin crystals of crotoxin complex embedded in glucose at room and low temperatures and in ice embedded crystals. By approximating the radiation damage to follow first order kinetics, we applied a correction to the diffraction intensities from patterns with high accumulated exposures. We used the structural similarity factor (R) to show that, for some data, the effectiveness of the correction can be as good as those in protein X-ray crystallography.

Crotalid Venoms↗
PubMed1983

Structural analysis of T4 DNA helix destabilizing protein (gp32 I) crystal by electron microscopy.

Low dose electron diffraction and imaging techniques have been applied to the study of the crystalline structure of gp32*I, a DNA helix destabilizing protein derived from bacteriophage T4 gene 32 protein. A quantitative analysis of intensities from electron diffraction patterns from tilted, multilayered gp32*I crystal has provided the unit cell thickness of the crystal. The three-dimensional phases indicate that the space group P2(1)2(1)2. By taking into account the unit cell volume and the solvent content in the crystal, it was deduced that there is one gp32*I molecule in each asymmetric unit. A projected density map of unstained, glucose-embedded gp32*I crystal was synthesized with amplitudes from electron diffraction intensities and phases from electron images with reflections out to 7.6 A. Because of the similarity in the scattering density between glucose and protein, this projected map cannot be interpreted with certainty. A low resolution three-dimensional reconstruction shows that the protein molecule is about 90 A long and about 20 A in diameter. Because the dimer is formed around a dyad axis, the protein molecules comprising it must be arranged head-to-head. This dimeric arrangement of the proteins in the unit cell may be implicated as one of the conformational states of this protein in solution.

Crystallography↗
PubMed1983

Low dose electron microscopy of the crotoxin complex thin crystal.

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.

Animals↗
PubMed1982

Perspectives and outlook for electron microscopy in biology in general.

The possibilities for improving methods of electron microscopy with the aim of visualizing still finer details of biological relevance are discussed with respect to different goals: discovering and defining new structures in situ, in the cell, in relation to functions, and investigating the fine structure of already morphologically and biochemically defined structures which are available in isolated and purified form. For each of these goals the importance of the inherent limitations are considered: lack of definition due to the unknown nature of heavy metal staining, deformations caused by the physical events of specimen preparation, and radiochemical alterations induced by the electron beam. Methods of investigating and overcoming these limitations by new cryotechniques, new imaging modes and improved crystallographic techniques are outlined. Emphasis is given to relevant biological phenomena, where electron microscopy is potentially the method of choice, e.g., those related to functional proteins situated in and on biological membranes and where relatively large conformational changes are supposed to have occurred to explain the functions.

Animals↗
PubMed1977

Factors affecting high resolution fixed-beam transmission electron microscopy.

An experimental and theoretical characterization of a fixed-beam transmission electron microscope with a field emission gun has been made with regard to the factors of electron beam brightness, spatial and temporal coherence of the incident electrons, objective lens current fluctuation, mechanical stability, and specimen contamination. It has been found that mechanical stability and temporal coherence are the primary factors that prevent the contrast transfer function from extending to 2.0 A in our microscope. Different amorphous thin films have also been used in order to compare their suitability for testing the imaging capability of the microscope at atomic resolution.

Microscopy, Electron↗
PubMed1976

Photoaffinity site-specific covalent labeling of human corticosteroid-binding globulin.

A method was developed for the synthesis of high-specific-activity 21-diazo-21-[6,7-(3)H]deoxycorticosterone, an analog of corticosterone. This analog was used as a photoaffinity label of a high affinity steroid-binding protein, human corticosteroid-binding globulin. Based on direct binding studies and crosscompetition experiments, this diazo derivative exhibited the requisite affinity (within a factor of 1.5 times that of corticosterone) and site specificity to qualify as an affinity labeling legand. Irradiation of corticosteroid-binding globulin with the 21-diazo derivative resulted in irreversible binding to corticosteroid-binding globulin, identified by polyacrylamide gel electrophoresis. Specificity of covalent binding to corticosteroid-binding globulin was established by competition analysis with various steroids. Irreversibility of photodependent binding was shown by persistence of the complex on electrophoresis (in contrast to the noncovalently linked complex), and resistance to exchange with corticosterone or pregnanediol and to solvent extraction. Site specificity of covalent binding was inferred from the effects of a scavenger, Tris-HC1, and fluorescence quenching of a neighboring tryptophan.

Affinity Labels↗
PubMed1975

Single atom image contrast: conventional dark-field and bright-field electron microscopy.

Single atom inage intensities are calculated for bright-field and various dark-field modes, including the ideal beam stop, a wire beam stop, tilted illumination and a displaced aperture. Comparisons of scattering amplitudes and elastic scattering cross-sections are made with different object potentials and scattering formulations. The image contrast for one mercury atom (Z equals 80) on a column of carbon atoms (Z equals 6) as the substrate is also discussed for both the bright-field and the various dark-field situations.

Carbon↗
PubMedDate not supplied

Preliminary electron crystallographic analysis of ice-embedded tropomyosin crystals.

Electron images and diffraction patterns of ice-embedded tropomyosin crystalline sheets have been recorded at 100 and 400 kV. Optical diffractograms from the images indicated an elongated, centered unit cell with a = 799.2 +/- 10.6 A, b = 55.1 +/- 3.5 A. Evaluation of the phases in the computed Fourier transforms up to 7 A resolution revealed the presence of symmetry axes consistent with two-dimensional space group cmm. Electron diffraction patterns show diffuse arcs and discrete sampling at a resolution of 5.1 A, arising from the alpha-helical coiled-coil features of the molecule. These results demonstrate that tropomyosin thin sheets are highly ordered and suggest that retrieval of its high-resolution three-dimensional structure may be feasible by electron crystallography.

Animals↗
PubMedDate not supplied

4-A projection map of bacteriophage T4 DNA helix-destabilizing protein (gp32*I) crystal by 400-kV electron cryomicroscopy.

Ice-embedded crystals of bacteriophage T4 DNA helix-destabilizing protein gp32*I were imaged by computer-controlled spot-scanning on a 400-kV electron cryomicroscope. gp32*I crystals generally have different steps of thickness within a crystal; each step can have different symmetry. Multivariate statistical analysis enabled us to unambiguously select spot-scan images that consist entirely of one motif which were processed subsequently by crystallographic Fourier-averaging techniques. The computed phases of the resulting reflections were evaluated for symmetry in projection, and some of those images were correlated with independent thickness measurements of freeze-dried samples of the same crystals. The structure factors with pgg symmetry from nine spot-scan images were merged, and the mean figure of merit of merged phases was better than 0.9 for data at resolution up to 4 A. A projection map was generated and showed multiple density peaks that corresponded to the high-resolution features of gp32*I.

Bacteriophage T4↗
PubMedDate not supplied

Applications of a slow-scan CCD camera in protein electron crystallography.

A Gatan 1024 x 1024 slow-scan charge-coupled device (CCD) camera has been interfaced to a JEOL4000EX electron cryomicroscope and explored for its usefulness in the electron crystallographic analysis of thin, glucose-embedded crystals of crotoxin complex kept at -125 degrees C. We show that the camera allows for an on-line assessment of the crystals' crystallinity, flatness, and thickness. Intensities obtained from electron diffraction patterns acquired with the camera have been statistically analyzed and were found to be consistent with theoretically expected values. A quantitative analysis of the diffraction intensity as function of the accumulated electron dose suggests the possibility of recording up to 250 diffraction patterns with 3.5 A resolution from a single crotoxin complex crystal 128 A thick. Tilt series of 125 electron diffraction patterns with 3.5 A data acquired from a single crystal are shown to be practically feasible. The current study demonstrates for the first time the effectiveness of using a slow-scan CCD camera for electron diffraction data collection from thin protein crystals at near atomic resolution.

Crotoxin↗