Improved structural detail in freeze‐fracture replicas: High‐angle shadowing of gap junctions cooled below −170°C and protected by liquid nitrogen—cooled shrouds

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Abstract
In conventional freeze-fracture replicas, precise complementarity of membrane faces is seldom achieved. In a model system frequently used to evaluate replica quality, vertebrate gap junctions are usually visualized as patches of 8–10 nm P-face intramembrane particles separated by 1–2 nm spaces, while E-face images are represented by 4–6 nm conical pits separated by 5–7 nm wide membrane ridges. However, that disparity in sizes of particles versus pits, as well as the disparity in the widths of the spaces separating particles versus pits, suggests that a significant reduction in complementarity of membrane faces has occurred. In this investigation, a JEOL JFD-9000 freeze-etch machine was modified so that fracturing and replication could be performed at temperatures much colder than commonly employed. With the addition of cryopumps to improve overall vacuum and the installation of optically tight LN2-cooled shrouds surrounding the specimen and the knife, water vapor contamination arising from all sources within the vacuum chamber was reduced substantially, allowing replicas to be made at temperatures down to −185°C. With the specimen at these much colder temperatures, water vapor released by the heat of cleaving was also reduced significantly, providing additional improvement in replica quality. In addition, with higher shadowing angles (< 60°) and with the specimen at a much lower temperature, the grain size of the platinum film was noticeably reduced, thereby improving resolution at the molecular level. Under these improved conditions, replicas of rat liver gap junctions revealed that many of the P-face IMPs were tubes 6–7 nm in diameter, but that other IMPs had been stretched and distorted by the fracturing process. More important, however, these high resolution replicas revealed that the replicas of the E-face pits represented three-dimensional molecular casts of the transmembrane proteins comprising the connexon hexamer. This means that before they were replicated, the E-face pits faithfully maintained the shape that the IMPs had before fracturing. These more detailed images revealed a new structure in the center of each E-face pit: a 2–3 nm “peg” that may represent the frozen aqueous matrix of the connexon ion channel that remained after elastic extraction of the surrounding six connexin molecules. Thus, high-angle shadowing at very low specimen temperature under virtually non-contaminating conditions has revealed a new level of detail for membrane structure in freeze-fracture replicas.