Dimensions of active cytochrome c oxidase in reconstituted liposomes using a gold ball shadow width standard: a freeze‐etch electron microscopy study

Abstract

SUMMARY The preparation and characterization of a distribution of gold balls on a thin, flat carbon film is described. The relation of the platinum carbon shadow width distribution means to a gold ball size is reported. Freeze‐etched cytochrome oxidase vesicles and gold ball calibration grids were simultaneously shadowed with platinum/carbon. The shadow width distribution of the cytochrome oxidase located in and spanning the membrane was measured. The membrane fracture face edge and cross‐fractured bilayer membrane edge were also measured. Dimensions of the cytochrome oxidase were found to be 5·8 ± 0·3 nm in diameter parallel to the membrane and 8·2 ± 0·3 nm long across the membrane. The bilayer membrane dimensions were 3·0 ± 0·3 nm for the half bilayer and 5·8 ± 0·3 nm for the cross‐fractured bilayer membrane edge thickness. The length of the cytochrome oxidase was observed to span the bilayer membrane. Previous X‐ray diffraction measurements on similar hydrated liquid crystalline artificial membranes were found to be in good agreement with the freeze‐etched results. Membrane widths from thin‐sectioned cytochrome oxidase vesicles were measured and found to be 5·8–5·9 nm in non‐post‐stained sections. Post‐staining with uranyl acetate and/or lead citrate was shown to increase this average thickness. The technique of freeze‐etching electron microscopy in conjunction with the gold ball shadow width calibration experiment has been shown to provide accurate and precise measurements of membranes and a functional intramembrane protein in a hydrated non‐crystalline sample.

Quantification of particle sizes with metal replication under standard freeze‐etching conditions: A gold ball standard for calibrating shadow widths was used to measure freeze‐etched globular proteins

“…The shadow width and metal-coated particle width distributions tested by a chi square test differ significantly from a normal distribution but not from a log normal distribution. This work also confirms the earlier work of Ruben and Telford (1980) that freeze-etched hydrated particles retain an -0.7 nm layer of hydration absent in negatively stained preparations. The "geometric assumption" that each replicated particle creates a shadow width the same size as the metal-coated particle diameter in Figure 1 was also tested.…”

“…Figure 17a shows gold balls replicated at a 45" angle; their shadow widths were in general larger that the Pt-C-coated gold ball diameters. That shadow widths are greater than the Pt-C-coated gold balls has been shown for substrate temperatures of -20°C (Ruben and Telford, 1980), --lOO"C, and now -185°C. Figure 17b shows gold balls replicated at an 80" angle; Particle shadows disappeared when viewing the gold ball replica in an untilted condition.…”

Ultrathin (1 nm) vertically shadowed platinum‐carbon replicas for imaging individual molecules in freeze‐etched biological DNA and material science metal and plastic specimens

“…5c,d). The Pt-C films deposited on mica at -185°C were not composed of the particulate metal nuclei that have been seen in Pt-C films made at warmer temperatures (Ruben et al, 1976;Ruben and Telford, 1980;Ruben, 1981a-c), but their granularity is different. The granularity of all of the Pt-C films in Figure 5 is composed of Pt-C filament structures, which are readily seen in Figure 5c,d and can also be found in Figure 5a,b, the low-angle deposition films.…”

Freeze-fracture studies on barley plastid membranes IV. Analysis of freeze-fracture particle size and shape

“…Their size tends to be overestimated due to shadowing and decoration effects (18) as well as by the possibility of adsorbed layers of water (16). Particles tend to be reduced in size, particularly in their larger dimension, due to plastic deformation.…”