The largest and one of the most complex ATP-dependent allosteric phosphofructokinase (Pfk) has been found in the methylotrophic yeast, Pichia pastoris. The enzyme is a hetero-oligomer (similar to 1 MDa) composed of three distinct subunits (alpha, beta and gamma) with molecular masses of 109, 104 and 41 kDa, respectively. While the alpha-and beta-subunits show sequence similarities to other phosphofructokinase subunits, the gamma-subunit does not show high homology to any known protein in the databases. We have determined the first quaternary structure of P. pastoris phosphofructokinase by 3D electron microscopy. Random conical techniques and tomography have been instrumental to ascertain the quality of the sample preparations for structural studies and to obtain a reliable 3D structure. The final reconstruction of P. pastoris Pfk resembles its yeast counterparts with four additional densities, assigned to four gamma-subunits, bridging the N-terminal domains of the four pairs of alpha- and beta-subunits. Our data has evidenced novel interactions between the gamma- and the alpha-subunits comparable in intensity to the interactions, shown by cross-linking and limited proteolytic degradation experiments, between the gamma- and beta-subunits. The structural data provides clear insights into the allosteric fine-tuned regulation of the enzyme by ATP and AMP observed in this yeast species. (C) 2009 Elsevier Inc. All rights reserved.