A new statistical metric for robust target detection in cryo-EM using 2D template matching.

2D template matching (2DTM) can be used to detect molecules and their assemblies in cellular cryo-EM images with high positional and orientational accuracy. While 2DTM successfully detects spherical targets such as large ribosomal subunits, challenges remain in detecting smaller and more aspherical targets in various environments. In this work, a novel 2DTM metric, referred to as the 2DTM p-value, is developed to extend the 2DTM framework to more complex applications. The 2DTM p-value combines information from two previously used 2DTM metrics, namely the 2DTM signal-to-noise ratio (SNR) and z-score, which are derived from the cross-correlation coefficient between the target and the template. The 2DTM p-value demonstrates robust detection accuracies under various imaging and sample conditions and outperforms the 2DTM SNR and z-score alone. Specifically, the 2DTM p-value improves the detection of aspherical targets such as a modified artificial tubulin patch particle (500 kDa) and a much smaller clathrin monomer (193 kDa) in simulated data. It also accurately recovers mature 60S ribosomes in yeast lamellae samples, even under conditions of increased Gaussian noise. The new metric will enable the detection of a wider variety of targets in both purified and cellular samples through 2DTM.