Kasim Sader

3D morphology of the human hepatic ferritin mineral core: New evidence for a subunit structure revealed by single particle analysis of HAADF-STEM images

Ferritin, the major iron storage protein, has dual functions; it sequesters redox activity of intracellular iron and facilitates iron turn-over. Here we present high angle annular dark field (HAADF) images from individual hepatic ferritin cores within tissue sections, these images were obtained using spherical aberration corrected scanning transmission electron microscopy (STEM) under controlled electron fluence. HAADF images of the cores suggest a cubic morphology and a polycrystalline (ferrihydrite) subunit structure that is not evident in equivalent bright field images. By calibrating contrast levels in the HAADF images using quantitative electron energy loss spectroscopy, we have estimated the absolute iron content in any one core, and produced a three dimensional reconstruction of the average core morphology. The core is composed of up to eight subunits, consistent with the eight channels in the protein shell that deliver iron to the central cavity. We find no evidence of a crystallographic orientation relationship between core subunits. Our results confirm that the ferritin protein shell acts as a template for core morphology and within the core, small (∼2 nm), surface-disordered ferrihydrite subunits connect to leave a low density centre and a high surface area that would allow rapid turn-over of iron in biological systems.

Preservation of high resolution protein structure by cryo-electron microscopy of vitreous sections.

We have quantitated the degree of structural preservation in cryo-sections of a vitrified biological specimen. Previous studies have used sections of periodic specimens to assess the resolution present, but preservation before sectioning was not assessed and so the damage due particularly to cutting was not clear. In this study large single crystals of lysozyme were vitrified and from these X-ray diffraction patterns extending to better than 2.1 Å were obtained. The crystals were high pressure frozen in 30% dextran, and cryo-sectioned using a diamond knife. In the best case, preservation to a resolution of 7.9 Å was shown by electron diffraction, the first observation of sub-nanometre structural preservation in a vitreous section.

Nucleotide-Dependent Shape Changes in the Reverse Direction Motor, Myosin VI

We have studied the shape of myosin VI, the actin minus-end directed motor, by negative stain and metal shadow electron microscopy. Single particle processing was used to make two-dimensional averages of the stain images, which greatly increases the clarity and allows detailed comparisons with crystal structures. A total of 169,964 particle images were obtained from two different constructs in six different states (four nucleotide states and with and without Ca(2+)). The shape of truncated apo myosin VI was very similar to the apo crystal structure, with the lever arm bent strongly backward and around the motor domain. In the full-length molecule, the C-terminal part of the tail has an additional bend taking it back across the motor domain, which may reflect a regulated state. Addition of ATP, ADP, or ATP-γS resulted in a large change, straightening the molecule from the bent shape and swinging the lever by ∼140°. Although these nucleotides would not be expected to produce the pre-powerstroke state, myosin VI in their presence was most similar to the truncated crystal structure with bound ADP-VO(4), which is thought to show the pre-powerstroke shape. The nucleotide data were therefore substantially different from expectation based on crystal structures. The full-length molecule was almost completely monomeric; only ∼1% were dimers, joined through the ends of the tail. Addition of calcium ions appeared to result in release of the second calmodulin light chain. In negatively stained molecules there was little indication of extended α-helical structure in the tail, but molecules viewed by metal shadowing had a tail ∼3× longer, 29 vs. 9 nm, part of which is likely to be a single α-helix.

Quantification of absolute iron content in mineral cores of cytosolic ferritin molecules in human liver

Abstract The authors present aberration corrected scanning transmission electron microscopy (SuperSTEM) and electron energy loss spectroscopy (EELS) spectrum imaging data for the iron hydroxide mineral cores of cytosolic ferritin molecules in situ within human liver sections measured at a controlled electron dose. The authors have quantified the absolute iron content in individual cores using EELS and have checked the results against similar data obtained from synthetic maghemite nanoparticles of known composition and dimensions. Using these results the authors have then determined the quantitative relationship between the STEM high angle annular dark field (HAADF) image intensity of a ferritin core normalised to the background matrix intensity and the absolute iron content of the core as determined by EELS. This procedure then allows us to perform a rapid assessment of the iron content of any core within a tissue section based on the STEM HAADF image intensity.

Electron Microscopy of Cocatalyst Nanostructures on Semiconductor Photocatalysts

A thorough analytical electron microscopy investigation into the oxidized nickel cocatalyst structure on known photocatalytic materials NiTa2O6 and InTaO4 has been conducted. These photocatalyst materials have previously been reported in the literature to have activity in the splitting of water to form H2, and with the addition of a cocatalyst, such as NiO/Ni core shell nanoparticles, activity has been suggested to improve. Although important implications are placed on the effect of this cocatalyst, its structure has not previously been examined in detail. By using high resolution analytical transmission electron microscopy it has been determined that the structure is not as simple as suggested and, although a core shell metal/metal oxide structure is produced, in neither case is a perfect Ni/NiO composition obtained. In particular, in the case of the InTaO4 based material, the nominal NiO/Ni cocatalyst contains indium diffused into the nanoparticles from the photocatalyst support. The general implications of this result for the design of potential photocatalyst systems are discussed.

Structural characterisation of protein-caged iron minerals in biological systems

In this paper we have collected multiple high angle annular dark field (HAADF) images of purified horse spleen ferritin mineral cores with an aberration corrected STEM (SuperSTEM). The images of individual cores have been separated into groups to determine characteristic views, and averaged to reduce the noise. We have found that these isolated cores do not have the same clear substructure as ferritin cores in plastic-embedded human liver sections (from a patient with haemochromatosis). There is a clear discrepancy between the in situ human liver and purified horse spleen ferritin, but also between Massovers observation of regular substructure in purified horse spleen ferritin by cryo-electron microscopy [1] and these results. A possible basis for this discrepancy is the difference in purification methods used between the two horse spleen ferritin preparations, with the one here purified using CdSO4 precipitation, and the one used by Massover purified without the use of CdSO4.

Aberration corrected STEM and EELS: Atomic scale chemical mapping

Scanning transmission electron microscopy (STEM) has enjoyed a recent surge in activity for two reasons. A long awaited improvement in the STEM performance of conventional TEM/STEM instruments coincided with the advent of aberration correction. This improvement was not for any fundamental reason — from the principle of reciprocity, the STEM resolution should be at least as good as the TEM resolution for the same objective lens. Lattice resolution high angle annular dark field (HAADF) STEM images are now a much more routine part of the analytical arsenal applied to materials characterisation.