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

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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.