The Multiscale Molecular Microscope

Project  2 – Analysis of Complexes

It is the dynamic and regulated interactions of macromolecular interaction hierarchies that breathe life into a cell – and it is these kinds of dynamic data that we propose to gather and interpret to elucidate cellular function. The first three approaches below provide a highly detailed but static picture of macromolecular hierarchies. The fourth approach gathers two kinds of data that inform on the dynamics of macromolecular complexes: (i)“snapshots” of the dynamic process mediated by a complex, and (ii) comparisons of ensembles of complexes in different states, where the states are defined by differences in the composition, connectivity, and morphology.

Determining the Composition and Stoichiometry of Macromolecular Complexes and their Vicinal Interactomes.

Currently, the major limitations of existing proteomic approaches include difficulties in:

  1. differentiating between specific in vivo associations and non-specific associations;
  2. robustly determining the stoichiometry and amounts of specific interactors;
  3. differentiating interactions that are direct from those that occur through more than one degree of separation; and (iv) isolating and recognizing transient and/or low affinity specific interactors. We address the specificity problem through: (a) automated multi-well affinity optimization in combination with pattern recognition algorithms, (b) Isotopic Differentiation of Interactions as Random or Targeted (I‑DIRT), (c) ultrafast complex isolation, and (d) by trapping the dynamic interactome with chemical stabilization.

Determining the Connectivities and Spatial Relationships of the Components of Macromolecular Complexes.

Our major goal is to develop robust methods that provide an accurate description in space and time of macromolecular interactions over a wide range of distance scales, ranging from the macromolecular neighborhood of cellular machines and organelles to atomic resolution contacts between pairs of macromolecules. We pursue in-cell chemical stabilization technologies (chemical cross-linking) that utilize cell-permeable reagents, such as glutaraldehyde, to stabilize the most transient of proximal and vicinal macromolecules in vivo, reading out the relative position and composition of any macromolecular interaction no matter how fleeting.

To read more about chemical cross-linking, click here…

Obtaining Multiscale Morphological Characterizations of Macromolecular Complexes.

Critical information, such as the handedness, symmetry, mass envelope distribution, and relative position of components are needed to reveal how macromolecular complexes are constructed and how they function. Our tools to label and isolate macromolecular complexes place us in the unique position of being able to generate molecular and atomic resolution structures for a variety of important macromolecular complexes, as well as visualize them in the context of intact cells. Our methods span spatial resolutions from microns to Angstroms and provide increasingly detailed morphological maps of the isolated complex. Moreover, the resolution of each technique overlaps significantly with that of another, providing independent confirmation of results from more than one approach (see figure).

Methodologies used to study the macromolecular interactions in the cell.