Abstract
The anaphase-promoting complex (APC/C) is a large E3 ubiquitin ligase that regulates progression through specific stages of the cell cycle by coordinating the ubiquitin-dependent degradation of cell-cycle regulatory proteins. Depending on the species, the active form of the APC/C consists of 14-15 different proteins that assemble into a 20-subunit complex with a mass of approximately 1.3 MDa. A hybrid approach of single-particle electron microscopy and protein crystallography of individual APC/C subunits has been applied to generate pseudo-atomic models of various functional states of the complex. Three approaches for assigning regions of the EM-derived APC/C density map to specific APC/C subunits are described. This information was used to dock atomic models of APC/C subunits, determined either by protein crystallography or homology modelling, to specific regions of the APC/C EM map, allowing the generation of a pseudo-atomic model corresponding to 80% of the entire complex.
Highlights
The active form of the APC/C consists of 14–15 different proteins that assemble into a 20-subunit complex with a mass of approximately 1.3 MDa
Three approaches for assigning regions of the EM-derived APC/C density map to specific APC/C subunits are described. This information was used to dock atomic models of APC/C subunits, determined either by protein crystallography or homology modelling, to specific regions of the APC/C EM map, allowing the generation of a pseudo-atomic model corresponding to 80% of the entire complex
We describe an example of molecular replacement in which we use atomic models to interpret electron-density maps determined using single-particle electron-microscopy data
Summary
The active form of the APC/C consists of 14–15 different proteins that assemble into a 20-subunit complex with a mass of approximately 1.3 MDa. A hybrid approach of single-particle electron microscopy and protein crystallography of individual APC/C subunits has been applied to generate pseudo-atomic models of various functional states of the complex. Our research has been aimed at obtaining atomic structures of individual APC/C subunits and to define how these subunits are organized within the whole complex as a means to understand how the APC/C recognizes its substrates and catalyses the assembly of polyubiquitin chains. To apply the hybrid approach to generate pseudoatomic structures of the APC/C we have determined atomic models of most of the large APC/C subunits through a combination of protein crystallography and homology modelling. We lack structural information on some of the smaller APC/C subunits (Table 1)
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