Phosphofructokinase plays a key role in the regulation of the glycolytic pathway and is responsible for the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate. Although the structure of the bacterial enzyme is well understood, the knowledge is still quite limited for higher organisms given the larger size and complexity of the eukaryotic enzymes. We have studied phosphofructokinase from Saccharomyces cerevisiae in the presence of fructose 6-phosphate by cryoelectron microscopy and image analysis of single particles and obtained the structure at 10.8Angstrom resolution. This was achieved by optimizing the illumination conditions to obtain routinely 8-Angstrom data from hydrated samples in an electron microscope equipped with an LaB6 and by improving the image alignment techniques. The analysis of the structure has evidenced that the homology of the subunits at the sequence level has transcended to the structural level. By fitting the X-ray structure of the bacterial tetramer into each dimer of the yeast octamer the putative binding sites for fructose 6-phosphate were revealed. The data presented here in combination with molecular replacement techniques have served to provide the initial phases to solve the X-ray structure of the yeast phosphofructokinase. (C) 2003 Elsevier Inc. All rights reserved.