Locating Label-free Molecules in Cells with High Precision and Sensitivity

The “coming of age” of cryo-EM has made structural analysis of macromolecular assemblies and their transformations in native cellular contexts a feasible goal for the coming decade and brought within reach a correspondingly complete picture of molecular mechanisms in cell biology. Molecules and complexes can be localized in cellular contexts by electron cryo-tomography (cryo-ET), without the need for labeling, but identifying molecular targets by 3D template matching (3DTM) lacks precision because of the attenuated high-resolution signal in tomograms that makes the distinction of targets with smaller differences difficult. At low resolution, targets are also easily missed in denser regions of a cell, such as the nucleus.

This team has been developing computational methods for 2D template matching (2DTM) that finds targets in images of untilted samples. The high-resolution signal in these images is better preserved than in tomograms, making 2DTM suitable for crowded environments and more specific to subtle details. However, without tilting targets must be detected against a background of overlapping cellular material, causing an increased false-negative rate. The group will develop computational methods and data collection protocols to combine 2DTM and 3DTM to achieve target detection with the best possible precision and sensitivity. Improved statistical methods and a new atomic coordinate scan (MOSAICS) will distinguish complexes with different composition or conformation, enabling the study of molecular mechanisms of entire assembly and reaction pathways inside cells. Together, these approaches will allow the creation of pseudo-atomic maps of localized targets within cells.