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22Cryo-EM now commonly generates close-to-atomic resolution as well as intermediate 23 resolution maps from macromolecules observed in isolation and in situ. Interpreting 24 these maps remains a challenging task due to poor signal in the highest resolution 25 shells and the necessity to select a threshold for density analysis. In order to facilitate 26 this process, we developed a statistical framework for the generation of confidence 27 maps by multiple hypothesis testing and false discovery rate (FDR) control. In this 28 way, 3D confidence maps contain separated signal from background noise in the form 29 of local detection rates of EM density values. We demonstrate that confidence maps 30 and FDR-based thresholding can be used for the interpretation of near-atomic 31 resolution single-particle structures as well as lower resolution maps determined by 32 subtomogram averaging. Confidence maps represent a conservative way of 33 interpreting molecular structures due to minimized noise. At the same time they 34 provide a detection error with respect to background noise, which is associated with 35 the density and particularly beneficial for the interpretation of weaker cryo-EM 36 densities in cases of conformational flexibility and lower occupancy of bound 37 molecules and ions to the structure. 38

Growing microtubule ends organize end-tracking proteins into comets of mixed composition. Here using a reconstituted fission yeast system consisting of end-binding protein Mal3, kinesin Tea2 and cargo Tip1, we found that these proteins can be driven into liquid-phase droplets both in solution and at microtubule ends under crowding conditions. In the absence of crowding agents, cryo-electron tomography revealed that motor-dependent comets consist of disordered networks where multivalent interactions may facilitate non-stoichiometric accumulation of cargo Tip1. We found that two disordered protein regions in Mal3 are required for the formation of droplets and motor-dependent accumulation of Tip1, while autonomous Mal3 comet formation requires only one of them. Using theoretical modelling, we explore possible mechanisms by which motor activity and multivalent interactions may lead to the observed enrichment of Tip1 at microtubule ends. We conclude that microtubule ends may act as platforms where multivalent interactions condense microtubule-associated proteins into large multi-protein complexes.

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