The COP9 signalosome (CSN) is a regulator of the ubiquitin (Ub) proteasome 
system (UPS). In the UPS, proteins are Ub-labeled for degradation by Ub ligases 
conferring substrate specificity. The CSN controls a large family of Ub ligases 
called cullin-RING ligases (CRLs), which ubiquitinate cell cycle regulators, 
transcription factors and DNA damage response proteins. The CSN possesses 
structural similarities with the 26S proteasome Lid complex and the translation 
initiation complex 3 (eIF3) indicating similar ancestry and function. Initial 
structures were obtained 14years ago by 2D electron microscopy (EM). Recently, 
first 3D molecular models of the CSN were created on the basis of negative-stain 
EM and single-particle analysis, mostly with recombinant complexes. Here, we 
compare deneddylating activity and structural features of CSN complexes purified 
in an elaborate procedure from human erythrocytes and efficiently pulled down 
from mouse Flag-CSN2 B8 fibroblasts. In an in vitro deneddylation assay both the 
human and the mouse CSN complexes deneddylated Nedd8-Cul1 with comparable rates. 
3D structural models of the erythrocyte CSN as well as of the mouse Flag-CSN 
were generated by negative stain EM and by cryo-EM. Both complexes show a 
central U-shaped segment from which several arms emanate. This structure, called 
the horseshoe, is formed by the PCI domain subunits. CSN5 and CSN6 point away 
from the horseshoe. Compared to 3D models of negatively stained CSN complexes, 
densities assigned to CSN2 and CSN4 are better defined in the cryo-map. Because 
biochemical and structural results obtained with CSN complexes isolated from 
human erythrocytes and purified by Flag-CSN pulldown from mouse B8 fibroblasts 
are very similar, Flag-CSN pulldowns are a proper alternative to CSN preparation 
from erythrocytes.