PMC

Coomassie-stained gels and gelfiltration profiles of Mtw1 complex and Ndc80 complex individually, or after combination (5 μM each). Asterisk indicates Hsp70 contamination in the COMA complex sample.

A. Expression and purification of the Ndc80 kinetochore complex. The Ndc80-Nuf2 and Spc24-Spc25 heterodimers are expressed individually and after combination the reconstituted full complex is purified by SEC. B. Coomassie-stained gels of gelfiltration runs of Mtw1 complex and Ndc80 complex alone, or after combination (9 μM each). C. Gelfiltration profile corresponding to B. D. Coomassie-stained gels of Mtw1 complex and purified Spc24/25 head domain alone, or after combination (6 μM each). E. Gelfiltration profile corresponding to D.

A. COMA-flag complex (lane1) was immobilized on M2 beads. Mtw1 (lane2) and Ndc80 complexes (lane 3) were pre-mixed and incubated with control-beads (lane 4) or COMA-flag beads. After washing, bound complexes were eluted with Flag peptide (lanes 5–7). B. Pull-down experiment using 6xHis tagged MN-subcomplex on Ni-NTA agarose and and FLAG-tagged COMA complex. Note co-elution of the complexes from the Ni-NTA beads. Asterisk indicates Nnf1 truncation product C. Pull-down experiment using 6xHis tagged DN-subcomplex on Ni-NTA agarose and FLAG-tagged COMA complex. The COMA complex fails to interact with the DN subcomplex. Asterisk denotes Nsl1 truncation product.

A. Negative stain EM image of the Mtw1 complex. Note the heterogeneous composition of the sample. White circles correspond to particles chosen for further analysis. B. Selected class averages showing the architecture and dimensions of the complex. The complex is a bi-lobed rod, with one large and one small globular domain. C. Coiled-coil predictions for the protein subunits of the complex. Grey regions correspond to greater than 50% coiled-coil probability. D. Entire complement of class averages obtained for the complex. They all display the bi-lobed architecture, with most heterogeneity occurring in the size of the larger globular domain.

A. Reconstitution of the Ctf19-Okp1-Mcm21-Ame1 complex (COMA). Coomassie stained gel of different stages of the purification. Right panel: The four polypeptides of the complex are resolved on large SDS-PAGE gels. B. Pull-down experiment with immobilized COMA complex. Different amounts of recombinant COMA complex (lane 1) were immobilized on Anti-FLAG M2 affinity gel and incubated with Mtw1, Ndc80 or Dam1 complex (lane 2). Control pull-downs contained only M2 beads (lane 3). After washing, bound complexes were eluted with FLAG-peptide (lane 4–6). Note that the Mtw1 complex co-elutes with COMA from the beads. C. COMA-Flag complex (lane 1) was incubated with His-tagged Mtw1-, Ndc80 or Dam1 complex which were immobilized on Ni-NTA beads (lane 2, lane 3 denotes Ni-NTA beads as a negative control). After washing, bound complexes were eluted with imidazole (lanes 4–6). Note that the COMA complex is co-eluted with the Mtw1 complex from Ni-NTA beads.

A. Expression and purification of the Mtw1 complex. The coomassie-stained gel shows different steps of the purification scheme: control extract and extract after induction with IPTG, eluate from Ni-NTA beads and purified fraction after SEC. Note the presence of degradation products of the Dsn1p subunit. B. Expression and purification of a complex with an N-terminally truncated Dsn1p subunit. C. Expression and purification of a stable Dsn1-Nsl1 heterodimer. IEX denotes anion exchange chromatorgraphy. Asterisks in A, B and C indicate a contamination with the E. coli Hsp70 chaperone D. Expression and purification of Mtw1-Nnf1 heterodimer. E. SEC runs of Dsn1p-Nsl1p and Mtw1p-Nnf1p subcomplexes (8 μM each) and of the full complex after reconstitution. F. Coomassie-stained SDS-PAGE of fractions from E. Asterisks indicate an Nnf1 truncation product.