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is a unique interdisciplinary collaboration between NIH and FEI, an electron microscope manufacturer, aimed at developing general strategies that allow acceleration of structure determination of macromolecular assemblies using cryo-electron microscopy, X-ray diffraction and nuclear magnetic resonance spectroscopy methods.
The “Living Lab” concept is an emerging strategy for driving innovation in a global context where there is opportunity to share resources to solve difficult problems that require expertise contributed by multiple institutions. A central goal of the collaborative research is to derive “workflows”, by which is meant a set of robust, generalizable procedures that allow completion of the specified tasks with a high level of reproducibility and success. The Living Lab is not a service core type of activity. Instead, the central goal of all research conducted is to use a few carefully chosen examples to develop synergy between complementary methods in structural biology as they apply to advancing the resolution and speed with which the structures of dynamic protein assemblies can be studied at the highest possible resolutions.
Successful determination of the structures of macromolecular complexes requires advanced expertise in a range of disciplines. Although X-ray crystallography, nuclear magnetic resonance spectroscopy and cryo-electron microscopic methods are now widely used to obtain structural information on proteins and nucleic acids, they each have specific requirements that limit their application to specialized types of samples. Thus, current application of X-ray crystallographic methods requires that well-ordered 3D crystals are prepared from homogeneous, purified protein preparations. Application of existing nuclear magnetic resonance spectroscopic methods is generally limited to small protein complexes with sizes below 50 kD. The central challenge in structural biology is that the vast majority of macromolecular complexes of biomedical interest are not well-ordered, are often larger than 50 kD, and are far too heterogeneous to form 3D crystals suitable for X-ray crystallography. There is, therefore, a genuine need to find new approaches that overcome the limitations of existing X-ray and nuclear magnetic resonance spectroscopic methods to analyze structures, in the native states, of these large, heterogeneous complexes which are of central interest in modern biomedical research.
It has been recognized for over two decades that cryo-electron microscopy can potentially fill this gap in structural biology as it relates to analysis of these heterogeneous protein complexes. Nevertheless, numerous questions remained whether the technology could achieve resolutions in the 3 Å to 10 Å range that are important in structural biology, or whether sufficiently powerful methods for image analysis could be developed to handle the complexity of analyzing microscopic images from poorly ordered, heterogeneous complexes. The situation has, however, changed dramatically over the last five years. A series of technical advances made at many research institutions world-wide have established that cryo-electron microscopic methods can achieve resolutions as high as about 3 Å in specialized cases of highly ordered, symmetric assemblies, and that it is also possible to tackle the problem of imaging and classifying extremely heterogeneous macromolecular assemblies such as intact HIV particles. The central motivation for the Living Lab collaboration is to explore whether these recent advances, which have so far been restricted to specialized systems, can become broadly applicable to various other protein complexes of interest to NIH. The scientific teams from NIH and FEI bring complementary strengths to the collaboration, and the research will be specifically structured to identify the methods and procedures that will enable extension of these recent advances to classes of protein complexes that are currently intractable to analysis at high resolution by present-day methodologies in structural biology.
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