Septins are highly conserved and essential eukaryotic cytoskeletal proteins that 
interact with the inner plasma membrane. They are involved in essential 
functions requiring cell membrane remodeling and compartmentalization, such as 
cell division and dendrite morphogenesis, and have been implicated in numerous 
diseases. Depending on the organisms and on the type of tissue, a specific set 
of septins genes are expressed, ranging from 2 to 13. Septins self-assemble into 
linear, symmetric rods that can further organize into linear filaments several 
microns in length. Only a subset of human septins has been described at high 
resolution by X-ray crystallography (Sirajuddin et al., 2007). Electron 
microscopy (EM) has proven to be a method of choice for analyzing the molecular 
organization of septins. It is possible to localize each septin subunit within 
the rod complex using genetic tags, such as maltose-binding protein or green 
fluorescent protein, to generate a visible label of a specific septin subunit in 
EM images that are processed using single-particle EM methodology. In this 
chapter we present, in detail, the methods that we have used to analyze the 
molecular organization of budding yeast septins (Bertin et al., 2008). These 
methods include purification of septin complexes, sample preparation for EM, and 
image processing procedures. Such methods can be generalized to analyze the 
organization of septins from any organism.