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GraFix: Stabilization of Fragile Macromolecular Complexes for Single Particle Cryo-EM

2010; Academic Press; Linguagem: Inglês

10.1016/s0076-6879(10)81005-5

1557-7988

Holger Stark,

Photosynthetic Processes and Mechanisms

Here, we review the GraFix (Gradient Fixation) method to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy (cryo-EM). During GraFix, macromolecules undergo a weak, intramolecular chemical cross-linking while being purified by density gradient ultracentrifugation. GraFix-stabilized particles can be used directly for negative-stain cryo-EM or, after a brief buffer-exchange step, for unstained cryo-EM. This highly reproducible method has proved to dramatically reduce problems in heterogeneity due to particle dissociation during EM grid preparation. Additionally, there is often an appreciable increase in particles binding to the carbon support film. This and the fact that binding times can be drastically increased, with no apparent disruption of the native structures of the macromolecules, makes GraFix a method of choice when preparing low-abundance complexes for cryo-EM. The higher sample quality following GraFix purification is evident when examining raw images, which usually present a low background of fragmented particles, good particle dispersion, and high-contrast, well-defined particles. Setting up the GraFix method is straightforward, and the resulting improvement in sample homogeneity has been beneficial in successfully obtaining the 3D structures of numerous macromolecular complexes by cryo-EM in the past few years.