The structure of nucleosomes that contain the cenH3 histone variant has been 
controversial. In budding yeast, a single right-handed cenH3/H4/H2A/H2B tetramer 
wraps the ∼80-bp Centromere DNA Element II (CDE II) sequence of each centromere 
into a 'hemisome'. However, attempts to reconstitute cenH3 particles in vitro 
have yielded exclusively 'octasomes', which are observed in vivo on chromosome 
arms only when Cse4 (yeast cenH3) is overproduced. Here, we show that Cse4 
octamers remain intact under conditions of low salt and urea that dissociate H3 
octamers. However, particles consisting of two DNA duplexes wrapped around a 
Cse4 octamer and separated by a gap efficiently split into hemisomes. Hemisome 
dimensions were confirmed using a calibrated gel-shift assay and atomic force 
microscopy, and their identity as tightly wrapped particles was demonstrated by 
gelFRET. Surprisingly, Cse4 hemisomes were stable in 4 M urea. Stable Cse4 
hemisomes could be reconstituted using either full-length or tailless histones 
and with a 78-bp CDEII segment, which is predicted to be exceptionally stiff. We 
propose that CDEII DNA stiffness evolved to favor Cse4 hemisome over octasome 
formation. The precise correspondence between Cse4 hemisomes resident on CDEII 
in vivo and reconstituted on CDEII in vitro without any other factors implies 
that CDEII is sufficient for hemisome assembly.