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A V Crewe

Publications and source records attributed to A V Crewe.

17 recordsLinked to original sources
PubMed1987

Scanning transmission electron microscopic examination of the hexagonal bilayer structures formed by the reassociation of three of the four subunits of the extracellular hemoglobin of Lumbricus terrestris.

A fraction obtained by gel filtration at neutral pH of the extracellular Hb of Lumbricus terrestris dissociated either at pH 9.8 or at pH 4.0, consisting of the three subunits D1 (31 kDa), D2 (37 kDa), and T (50 kDa), was found to produce two peaks when subjected to gel filtration on Superose 6 at pH 7. The first peak, which was eluted at a slightly greater volume than the native Hb, consisted of reassociated hexagonal bilayer structures when examined by scanning transmission electron microscopy. The dimensions of the two reassociated hexagonal bilayer structures were a vertex-to-vertex diameter of 25 nm and a height of 16 nm. The difference in size between the hexagonal bilayer structures and the native Hb is the contribution of subunit M, which consists of a single heme-containing chain I (16.75 kDa). Although the reassociated hexagonal bilayer structures have overall dimensions smaller than the 30 nm x 20 nm dimensions of the native Hb, the diameters of the central cavities are not substantially altered. Subtraction of the three-dimensional reconstructions of the reassociated hexagonal bilayer structures from those of the native Hb showed that subunit M was primarily localized at the periphery of Lumbricus Hb. The formation of hexagonal bilayer structures in the complete absence of subunit M provides additional support for the "bracelet" model of the quaternary structure of Lumbricus Hb proposed recently by us in which subunits D1 and D2 were assumed to act as linkers for complexes of subunits M and T or to form a "bracelet" decorated with 12 complexes of subunits M and T.

Animals↗
PubMed1986

Bracelet protein: a quaternary structure proposed for the giant extracellular hemoglobin of Lumbricus terrestris.

The complete dissociation of the hexagonal bilayer structure of Lumbricus terrestris hemoglobin (3900 kDa) at neutral pH, in the presence of urea, guanidine hydrochloride, sodium perchlorate, potassium thiocyanate, sodium phosphotungstate, and sodium phosphomolybdate, followed by gel filtration at neutral pH on Sephacryl S-200 or Superose 6, produced two fragments, II (65 kDa) and III (17 kDa); NaDodSO4/polyacrylamide gel electrophoresis showed that peak II consisted of subunits D1 (31 kDa, chain V), D2 (37 kDa, chain VI), and T (50 kDa, disulfide-bonded trimer of chains II, III, and IV) and that peak II consisted of subunit M (16 kDa, chain I). When dissociation was incomplete, two additional peaks were present, peak Ia eluting at the same volume as the whole hemoglobin and peak Ib (200 kDa). Scanning transmission electron micrographs of peak Ia showed it to consist of whole molecules and of incomplete hexagonal bilayer structures, missing an apparent 1/12th. Peak Ib contained all four subunits but was usually deficient in subunits D1 and D2, was not always in equilibrium with the whole molecule, and could be dissociated further into II and III. The patterns of dissociation observed at neutral pH were very similar to those observed previously at alkaline pH and at acid pH and appear to be incompatible with the generally accepted multimeric model of Lumbricus hemoglobin subunit structure. A model is proposed in which it is postulated that the stoichiometries of some of the subunits need not be constant and that subunits D1 and D2 either form a "bracelet" decorated with complexes of T and M subunits or serve as "linkers" between the latter, to provide the appearance of a two-tiered hexagonal structure. Additional support for the proposed model comes from observations that the fragment II obtained subsequent to dissociation at pH 4, in sodium phosphotungstate, in sodium perchlorate, and in potassium thiocyanate was found to be in equilibrium with a hexagonal bilayer structure IaR(II), whose dimensions were approximately equal to 20% smaller than those of the native hemoglobin.

Animals↗
PubMed1985

The molecular size of Myxicola infundibulum chlorocruorin and its subunits.

The molecular shape and size of the extracellular chlorocruorin of Myxicola infundibulum was determined using scanning transmission electron microscopy and its dissociation in the presence of sodium dodecyl sulfate (SDS) was investigated using polyacrylamide gel electrophoresis. The shape of the chlorocruorin is that of a two-tiered hexagon with a vertex-to-vertex diameter of 29.0-29.5 nm and a height of 19.0-19.7 nm: it appears to be smaller by 5-10% relative to several annelid extracellular hemoglobins examined by scanning transmission electron microscopy. The quaternary structure of the chlorocruorin appears to be sensitive to Ca(II) concentration; dissociation fragments of the whole molecule were observed, consisting of octamers an dimers of one-twelfth subunits. The unreduced chlorocruorin dissociated into two subunits with estimated molecular masses of 23 000 (1) and 60 000 (2); the reduced chlorocruorin dissociated into subunits with estimated molecular masses of 13 000 (I), 14 000 (II) and 30 000 (III). SDS-polyacrylamide gel electrophoresis of reduced subunits 1 and 2 showed that subunit 1 corresponded to subunit III and that subunit 2 dissociated to subunits I and II. Densitometry of the polyacrylamide gels indicates that 85-90% of the Myxicola chlorocruorin consists of disulfide-bonded tetramers of polypeptide chains of about 15 000. Such a pattern of subunit aggregation has not been observed previously in annelid extracellular hemoglobins and chlorocruorins.

Animals↗
PubMed1979

Dark field imaging of biological macromolecules with the scanning transmission electron microscope.

A scanning transmission electron microscope (STEM) equipped with a field emission gun has been employed for the examination of biological macromolecules at high resolution. The quality of micrographs obtained with the STEM is dependent upon the quality of the substrate used to support biological objects because the image contrast in dark field is proportional to the mass density of the specimen. In order to reduce deleterious effects of the substrates on the image quality, we have developed a method of fabricating substrates consisting of very thin, very clean carbon films supported on very clean fenestrated plastic films. These films are approximately 15 A thick. Well-known biological macromolecules such as glutamine synthetase and tobacco mosaic virus (both stained) and low-density lipoprotein and ferritin (both unstained were placed on these substrates and examined with the STEM by using various modes of contrast. The micrographs obtained by using the dark field mode of contrast employing an annular detector were free from phase contrast, as expected. Using this contrast mode, we have been able to directly observe (in-focus) 2.5- to 4.4-A lattice spacings in the ferritin core. The effect of electron radiation damage on the helical structure of tobacco mosaic virus was also examined. Micrographs as well as corresponding optical diffraction patterns obtained with moderately low doses showed very clear helical structure from both sides of the virus. In addition, the (11.5 A)(-1) layer lines indicated the effective resolution attained on these particles.

Ferritins↗
PubMed1976

The use of backscattered electrons for imaging purposes in a scanning electron microscope.

It is shown that the use of a very large detector for backscattered electrons can provide a signal comparable to that obtained in the secondary emission mode and that the resolution available is also comparable. A technique is described whereby the difference is taken between these two signals, and the use of this difference signal can significantly enhance surface details. It is believed that the use of such a signal should ultimately prove advantageous in increasing resolution.

Animals↗
PubMed1976

Very low voltage electron microscopy.

We conclude that a 150 V scanning microscope with a resolution of 10 A is quite feasible and could have considerable value. It might consist of a field emission source, an electron gun to decelerate the electrons, a condenser lens to produce a parallel beam, a multipole corrector and a short focal length objective lens. Electrons reflected from the specimen surface would pass through a spectrometer whose principal features would be a large collecting power and low (1/200) energy resolution. Finally, we should add that such a microscope presents a considerable challenge and new opportunities for the electron optician in both lens and spectrometer design. We cannot refrain from pointing out that the Scherzer theorem does not necessarily hold for such a lens since the constraints of the theorem do not apply to this case.

Microscopy, Electron, Scanning↗
PubMed1975

High resolution imaging properties of the STEM.

The effect of the finite size of the atom on the resolution of the STEM is investigated. When the probe size becomes comparable to the size of the atom, the quality of the image depends on the scattering properties of the atom as well as the distribution of electrons in the probe. A technique for calculating the image of a single atom is developed by expanding the scattering amplitude. This allows the image of an atom or its spatial frequency to be expanded into various components. The specific case of dark field contrast formed with elastically scattered electrons is considered. The coefficients of the components are evaluated for carbon and thorium using complex scattering amplitudes derived from relativistic Hartree-Fock-Slater wavefunctions. The coefficients are evaluated for a 100 keV microscope using an immersion type objective lens whose aperture is limited to 12 mrad by primary spherical aberration and a 100 keV microscope using the same objective lens in conjunction with a corrector lens for spherical aberration. Secondary spherical aberration limits the objective aperture of the corrected microscope to 30 mrad.

Carbon↗
PubMed1974

Scanning transmission electron microscopy at high resolution.

We have shown that a scanning transmission electron microscope with a high brightness field emission source is capable of obtaining better than 3 A resolution using 30 to 40 keV electrons. Elastic dark field images of single atoms of uranium and mercury are shown which demonstrate this fact as determined by a modified Rayleigh criterion. Point-to-point micrograph resolution between 2.5 and 3.0 A is found in dark field images of micro-crystallites of uranium and thorium compounds. Furthermore, adequate contrast is available to observe single atoms as light as silver.

Crystallography↗