Lamella micromachining by focused ion beam milling at cryogenic temperature (cryo-FIB) has matured into a preparation method widely used for cellular cryo-electron tomography. Due to the limited ablation rates of low Ga+ ion beam currents required to maintain the structural integrity of vitreous specimens, common preparation protocols are time-consuming and labor intensive. The improved stability of new generation cryo-FIB instruments now enables automated operations. Here, we present an open-source software tool, SerialFIB, for creating automated and customizable cryo-FIB preparation protocols. The software encompasses a graphical user interface for easy execution of routine lamellae preparations, a scripting module compatible with available Python packages, and interfaces with 3-dimensional correlative light and electron microscopy (CLEM) tools. SerialFIB enables the streamlining of advanced cryo-FIB protocols such as multi-modal imaging, CLEM-guided lamella preparation and in situ lamella lift-out procedures. Our software therefore provides a foundation for further development of advanced cryogenic imaging and sample preparation protocols.

Text 49Nuclear pore complexes (NPCs) are giant macromolecular assemblies with a very intricate 50 architecture. About 30 different genes termed nucleoporins (Nups) encode for components of 51NPCs. Scaffold Nups contain folded domains and form a cylindrical central channel. This channel 52 is lined with FG-Nups, which harbor intrinsically disordered FG-rich repeats that interact with 53 nuclear transport receptors. Nups assemble in multiple copies to form an eight-fold rotationally 54 symmetric complex, totaling in ~550 protein building blocks in yeast and ~1000 in mammals 1 . 55The NPC consists of two outer rings, also called nuclear (NR) and cytoplasmic rings (CR), which 56 are placed distally to the inner ring (IR) that resides at the fusion plane between the inner and outer 57 nuclear membranes. Within the three-ringed architecture, Nups are organized into subcomplexes 58 that form specific substructures. The Y-complex is the key scaffolding component of the outer 59 rings, whereas the IR scaffold is built by the inner ring complex 2 . Further, the yeast Nup159 60 complex (Nup214 complex in mammals) associates asymmetrically with the Y-complex at the CR 61 and facilitates the terminal steps of mRNA export. Its core consists of two Nup159-Nup82-Nsp1 62 heterotrimers that dimerize into a characteristic P-shaped structure 3,4 . 63Several studies have highlighted that the accurate spatial positioning of the Nup159 complex with 64 respect to the central channel at the cytoplasmic face of the NPC is critical for the spatial 65 organization and directionality of mRNA export 3,5,6 . While mRNAs are chaperoned through the 66 FG repeats within the central channel in a Mex67-dependent (but Ran independent) manner, 67 exported mRNPs encounter the ATPase activity of the DEAD-box RNA helicase Dpb5 at the 68 cytoplasmic face that removes Mex67 and ratchets the RNA into the cytoplasm 1,6,7 . Dbp5 69 recruitment and positioning is ensured by the N-terminal beta propeller of Nup159, which is 70 separated by a flexible linker from the C-terminal coiled coils that anchor Nup159 to the NPC 71 scaffold 7 . 72 However, understanding how Nup subcomplexes are positioned to each other within the context 73 of the nuclear membranes and the overall architecture of NPCs imposes a considerable challenge 74 to structural biologists and requires the convergence of in vitro and in situ approaches 8 . While 75 several high-resolution structures of Nups have been solved by X-ray crystallography, biochemical 76 analysis and cross-linking mass spectrometry revealed the interaction topology. Cryo-electron 77 microscopy (cryo-EM) maps provided an overall framework for the positioning of subcomplexes Systematic fitting of the Nup159 complex negative stain map 4 into our in situ structure confirmed 130 this fit (Fig. 2b, p-value 0.0027, Extended Data Fig. 4a, see Methods). Based on the top resulting 131 fit, we superimposed a previously published representative integrative model of the Nup159 132 complex 3,13 and we locally fit it into our cryo-EM m...

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Numerous chromatin-associated proteins have been linked to neurodevelopmental disorders, yet their molecular functions often remain elusive. PHF14, HMG20A, TCF20 and RAI1 are components of a putative chromatin-associated complex and have been implicated in neurological disorders. Here, we found that Phf14 knockout embryonic stem cells and neural progenitor cells exhibit impaired cell cycle progression and proliferation, inadequate protection of stalled replication forks, and decreased DNA repair. The PHF14 complex rapidly assembles at DNA damage sites and binds to DNA through HMG20A. The PHF14 complex forms DNA-containing phase separated droplets in vitro, where TCF20 facilitates droplet formation. Furthermore, TCF20 maintenance at DNA damage sites is destabilized upon pathological mutation. Our results suggest that the PHF14 complex contributes to DNA damage repair by sensing damaged sites and forming biomolecular condensates, thus supporting cell cycle progression, especially in neural progenitor cells whose spatiotemporal pool is critical for proper brain development.