Developing a Modular Scaffold to Drive the Symmetric Assembly of Cellular Proteins for Single Particle Cryo-EM Imaging
EMSL Project ID
49768
Abstract
High resolution structural studies can provide exceptionally valuable information on the workings of cellular components and molecular machines, many of which play critical roles in biological energy conversions and other environmentally important processes.Recently, a number of technological advancements in the area of single particle cryo-electron microscopy (cryo-EM) have brought that technique to the forefront of macromolecular structural studies. In particular, several recent studies have demonstrated atomic or near-atomic resolutions (in the 3-angstrom or better range). It is particularly noteworthy that the recent successes come from studies on macromolecular assemblies that are very large (e.g. ribosomes) and often highly symmetric (e.g. viral capsids). Indeed, the major challenge at present for fully exploiting cryo-EM for routine structural studies is how to attack problems where the protein or protein complex is *not* particularly large or symmetric; macromolecular assemblies smaller than about 200 kDa offer poor tradeoffs in terms of signal-to-noise ratio vs. radiation damage by high energy electrons.
Concurrent with the new revolution in cryo-EM, methods have been developed in the last several years, particularly in the Yeates laboratory, for creating novel, highly symmetric protein architectures (such as cubic cages) through protein design. This proposal seeks to use our emerging methods for designing highly symmetric protein assemblies to overcome the size and symmetry requirements that currently limit cryo-EM. This major technological advance will ultimately enable high resolution structural studies on diverse biological systems related to energy and the environment, while simultaneously advancing the broad scientific area of biological imaging and EMSL's capacity there.
The target requirements for our design work are: a designed self-assembling protein system that is symmetric, rigid, and modular in such a way that it can be used to drive the symmetric co-assembly of an otherwise arbitrary protein or protein complex of unknown structure and typical size (i.e. 20 - 200 kDa). This will be achieved by modifying recently designed protein cages by genetic fusion to DARPins; DARPins are a system of mainly alpha-helical proteins that have been developed by others as selective binders (by amino acid changes in the loops of the DARPin) to other proteins of interest using powerful selection techniques such as phage display. The alpha helical nature of the DARPins makes them suitable for rigid attachment to designed protein cages that have alpha helical termini, while the selectable binding properties of DARPins provides a facile route for obtaining DARPin sequences that bind rigidly to a given target protein. The combined features of this system will therefore provide a way to symmetrically co-assemble target proteins of interest in a rigid and highly symmetric fashion onto designed protein cages through interaction with DARPins. This would represent a new and high impact achievement in protein design and cryo-EM. The protein design work will rely on expertise in the Yeates laboratory at UCLA in the BER-funded DOE Institute for Genomics and Proteomics, while the important work in testing and defining useful scaffolds for cryo-EM will rely on expertise and instrumentation at EMSL
Project Details
Project type
Large-Scale EMSL Research
Start Date
2017-10-01
End Date
2018-09-30
Status
Closed
Released Data Link
Team
Principal Investigator