Seeded Microtubule Growth for Cryoelectron Microscopy of End-Binding Proteins
End-binding proteins (EBs) have the ability to autonomously track the ends of growing microtubules, where they recruit several proteins that control various aspects of microtubule cytoskeleton organization and function. The structural nature of the binding site recognized by EBs at growing microtubule ends has been a subject of debate. Recently, a fluorescence microscopy assay used for the study of dynamic end tracking in vitro was adapted for cryoelectron microscopy (cryo-EM). In combination with single-particle reconstruction methods, this modified assay was used to produce the first subnanometer-resolution model of how the microtubule-binding domain of EBs binds to microtubules grown in the presence of GTPγS. A GTPγS microtubule can be considered a static mimic of the transiently existing binding region of EBs at a microtubule end growing in the presence of GTP. Here we describe in detail the procedure used to generate these samples. It relies on the polymerization of microtubules from preformed stabilized and quantum dot-labeled microtubule seeds. This allows the cryo-EM analysis of proteins bound to paclitaxel-free microtubules. It provides freedom for using different GTP analogues during microtubule elongation independent of their nucleation properties. This assay could also be useful for the cryo-EM analysis of other microtubule-associated proteins.
Key wordscryo-EM Microtubule structure GMP-CPP seeds GTPγS EB1 Mal3 Kinesin
We thank Julia Cope, Rachel A. Santarella, and Cindi L. Schwartz for training in electron microscope operation and cryoelectron microscope sample preparation. We thank the electron microscopy facility at EMBL Heidelberg for support. S.P.M. was supported by Marie Curie (PIEF-GA-2009-253043) and EMBO (ALTF 1032-2009) fellowships. F.J.F. was supported by an EMBO Long-Term Fellowship (ALTF 219-2011). A.H. and the Boulder 3-D lab are supported by grant P41-GM103431 (NIH-NIGMS).