Premium
Complex assembly, crystallization and preliminary X‐ray crystallographic analysis of the human Rod–Zwilch–ZW10 (RZZ) complex
Author(s) -
Altenfeld Anika,
Wohlgemuth Sabine,
Wehenkel Annemarie,
Vetter Ingrid R.,
Musacchio Andrea
Publication year - 2015
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x15004343
Subject(s) - protein subunit , crystallization , biology , crystallography , microtubule , recombinant dna , cytoplasm , mitosis , chlamydomonas reinhardtii , microbiology and biotechnology , chemistry , gene , biochemistry , organic chemistry , mutant
The spindle‐assembly checkpoint (SAC) monitors kinetochore–microtubule attachment during mitosis. In metazoans, the three‐subunit Rod–Zwilch–ZW10 (RZZ) complex is a crucial SAC component that interacts with additional SAC‐activating and SAC‐silencing components, including the Mad1–Mad2 complex and cytoplasmic dynein. The RZZ complex contains two copies of each subunit and has a predicted molecular mass of ∼800 kDa. Given the low abundance of the RZZ complex in natural sources, its recombinant reconstitution was attempted by co‐expression of its subunits in insect cells. The RZZ complex was purified to homogeneity and subjected to systematic crystallization attempts. Initial crystals containing the entire RZZ complex were obtained using the sitting‐drop method and were subjected to optimization to improve the diffraction resolution limit. The crystals belonged to space group P 3 1 (No. 144) or P 3 2 (No. 145), with unit‐cell parameters a = b = 215.45, c = 458.7 Å, α = β = 90.0, γ = 120.0°.