The CCR4-NOT complex was purified according to a modified TAP-protocol using both affinity tags of the TAP-tag on the N-terminus of CAF1. This yielded a complex that consisted of three components, which we identified by mass spectrometry as NOT1, CAF1 and CCR4 (A, right panel). However, several other subunits, which were previously identified as being part of the CCR4-NOT-complex (NOT2–5, CAF130, CAF40) were only present after the TEV cleavage (A, left panel), but were absent after the calmoduline purification (A, right panel). Especially, most of the subunits of the NOT-subcomplex were lost and the band of the NOT1 protein was relatively weak, indicating a recovery of NOT1 in sub-stoichiometric amounts. Therefore, we changed the bait to the NOT1-protein, which is the scaffolding protein for the NOT-subcomplex. Similar to the tag on CAF1, the purification yielded a complex where most of the proteins were present after the TEV cleavage (B, left panel) but with an increased amount of recovered complex (not shown). Next, we considered that binding of this complex to the Calmodulin beads followed by chelating of Calcium ions with EGTA might interfere with the complex integrity. Therefore, we replaced the calmodulin purification with a glycerol gradient that is much milder. This yielded a larger complex, which contained all the expected nine components of the CCR4-NOT-complex, namely NOT1–5, CCR4, CAF1, CAF40 and CAF130 (B, right panel).
Fig. 1 Biochemical characterization of the purification of the CCR4-NOT complex. (A) Coomassie stained SDS-gels of tandem affinity purification with CAF1 as bait after TEV cleavage (left) and after calmodulin purification (right). (B) Coomassie stained SDS-gels (more ...)
Electron microscopy of negatively stained samples showed a large complex with some heterogeneity in size (A, left panel). This heterogeneity was confirmed by native gels, which showed two major bands of 1.0 MDa and 0.7 MDa (C, arrows). The fact that the two species detected in the native gel, originated from the same fraction of the glycerol gradient, suggested an inherent instability of the complex. In order to stabilize the complex for further structural studies, the sample was cross-linked with glutaraldehyde (GraFix protocol; 
). The fractions containing NOT1 were identified with a dot-blot-assay against the TAP-tag at NOT1 (D). The blot showed that the NOT1-containing cross-linked complex had shifted towards higher glycerol concentrations, indicating the recovery of a somewhat larger complex. Electron microscopy of negatively stained samples confirmed that the cross-linked CCR4-NOT-complex was more homogeneous than the non-cross-linked complex. Furthermore, it was virtually free of random aggregates implying that the shift was not due to random-aggregation.
Fig. 2 Electron microscopic analysis of the CCR4-NOT complex. (A) Micrographs of negatively stained CCR4-NOT-complex without cross-linking (left) and with cross-linking (right). (B) Two-dimensional averages of the CCR4-NOT complex (top row) with matching projections (more ...)
After cross-linking, the homogeneity was sufficient for further structural analysis by electron microscopy and image processing. By using RCT [21,22]
maximum likelihood sorting 
and projection matching 
, we could determine a three-dimensional map of a typical conformation of the 9-subunit complex. Projections of this map matched with 2D-averages calculated from particles that were assigned with the same Euler angles (B) highlighting the validity of the map. The resolution was 33 Å at FSC = 0.5 or 31 Å at FSC = 0.14 (C). The map revealed a flat, L-shaped particle () with two arms of similar length (180 Å and 190 Å). The shorter arm accounted for ca. 600 kDa, whereas the longer, thinner arm accounted for ca. 300 kDa and the hinge domain for approximately 100 KDa. Consequently, the shorter arm together with the hinge would be large enough to account for the 700 kDa complex that we observed in native gels. Considering that NOT1 was used as bait, it is likely, that the 700 kDa sub-complex contained the NOT1-protein.
Surface representation of the three-dimensional map. The tentative positions of the subunits are indicated. The positions are mainly based on size considerations and require further experimental validation.
The relative arrangement of both arms varied between different reconstructions indicating flexibility of the whole assembly and explaining the limited resolution of the map. Both arms form an accessible cavity in their centre, which could provide an extensive platform for controlled interaction with RNA and accessory regulating factors.
The current knowledge on binary protein-protein interactions and the lack of high-resolution structures of individual subunits and sub-complexes leave ambiguities in the interpretation of the map of the CCR4-NOT-complex. Considering that NOT1 is the major scaffolding protein to which the other NOT proteins bind 
suggests that the shorter arm is mainly formed by the NOT-proteins, which account for ca. 500 kDa. Furthermore, NOT1 is in contact with CAF1, which binds to CCR4 
. This could place CCR4 at the strategic position in the hinge-domain, which has a similar shape and dimension as the catalytic domain of the human homologue of CCR4 
. It is likely that the remaining CAF40 and CAF130 account for most of the longer arm, which agrees with the lack of direct interactions between CAF40 and CAF130 with the NOT2–5 proteins.