Inwardly-rectifying potassium (Kir) channels regulate membrane electrical excitability and K+ transport in many cell types where they control such diverse processes as heart rate, vascular tone, insulin secretion and salt/fluid balance. Their physiological importance is highlighted by the fact that genetically inherited defects in Kir channels are responsible for a wide-range of channelopathies. To elucidate how channel function becomes defective in the disease state requires a detailed understanding of channel structure in both the open and closed states, but to date detailed information about the open state structure of the Kir channel is lacking. In this work, we have used EM analysis of 2D crystals of a prokaryotic Kir channel trapped in an open state and compared these results with an open state structure of the same channel that our lab recently determined by X-ray crystallography at 3Å resolution. Intriguingly, the projection maps from the EM experiments suggest a larger opening of the pore in the 2D crystal form compared to that observed in the 3D crystal structure. The organization of these two crystal forms is different and suggests that the 2D crystals may permit stabilisation of an open state structure that is not compatible with 3D crystallisation. These results not only have major implications for our understanding of the open state structure of the Kir channel, but more importantly they demonstrate the general utility and importance of methods such as electron microscopy and 2D crystallography for the study of membrane protein structure.