Biology subjects

Fromm, S. A.

Publications and source records attributed to Fromm, S. A..

5 recordsLinked to original sources

bioRxiv2026

LowDoseWizard - rapid and standardised setup of low-dose cryo-TEM imaging in SerialEM

Structure elucidation of biological macromolecules by single particle cryogenic electron microscopy (SPA cryo-EM) or cryogenic electron tomography (cryo-ET) relies on low-dose imaging on cryogenic transmission electron microscopes (cryo-TEMs). Routine microscope setup remains technically demanding and can be time-consuming, particularly for inexperienced or infrequent users. We present LowDoseWizard, a guided workflow implemented in SerialEM that enables rapid and standardised setup of cryo-TEM imaging conditions. From minimal user input, the workflow configures microscope optics, camera parameters and image shift settings for all low-dose imaging states, and guides the user through key daily alignment procedures including beam shift offset calibration, objective lens astigmatism correction and coma-free alignment. The workflow is organised into modular routines that can be executed sequentially or independently, while microscope-specific acquisition parameters are defined in editable configuration files, allowing flexible adaptation to different instruments without modification of the core scripts. Across user sessions on three microscopes at EMBL Heidelberg, the complete setup required on average less than 15 minutes. To assess whether predefined imaging conditions generated by the workflow are compatible with high-resolution data collection, we acquired apoferritin data on a 200 kV Glacios and a 300 kV Titan Krios. These datasets yielded reconstructions at 1.62 [A] and 1.09 [A] resolution, respectively, demonstrating that rapid, guided setup can support near-atomic and atomic-resolution single particle cryo-EM. LowDoseWizard lowers the barrier to robust cryo-TEM setup, reduces the time spent on routine parameter selection and alignment, and helps users focus on sample-specific aspects of data acquisition such as target selection. The workflow should be particularly valuable in shared instrumentation environments, where accessibility, reproducibility and efficient microscope use are critical.

molecular biology↗

bioRxiv2023

GSK3β phosphorylation catalyzes the aggregation of Tau into Alzheimer's disease-like amyloid strain

The pathological deposition of proteins is a hallmark of several devastating neurodegenerative diseases. These pathological deposits comprise aggregates of proteins that adopt distinct structures named strains. However, the molecular factors responsible for the formation of distinct aggregate strains are unknown. Here we show that the serine/threonine kinase GSK3{beta} catalyzes the aggregation of the protein tau into an Alzheimers disease-like amyloid strain. We demonstrate that phosphorylation by GSK3{beta}, but not by several other kinases, promotes the aggregation of full-length tau through enhanced phase separation into gel-like condensate structures. Cryo-electron microscopy further reveals that the amyloid fibrils formed by GSK3{beta}-phosphorylated tau adopt a fold comparable to that of paired helical filaments isolated from the brains of AD patients. Our results elucidate the intricate relationship between post-translational modification and the formation of tau strains in neurodegenerative diseases.

biophysics↗

bioRxiv2022

The translating bacterial ribosome at 1.55 A resolution by open access cryo-EM

Our understanding of protein synthesis has been conceptualised around the structure and function of the bacterial ribosome1-4. This complex macromolecular machine is the target of important antimicrobial drugs5, an integral line of defence against infectious diseases. Here, we describe how open access to state-of-the-art cryogenic electron microscopy facilities combined with bespoke user support offered by the newly established EMBL Imaging Centre enabled structural determination of the translating ribosome from Escherichia coli at 1.55 [A] resolution. The obtained structures allow for direct determination of the rRNA sequence to identify ribosome polymorphism sites in the E. coli strain used in this study and enables interpretation of the ribosomal active and peripheral sites at unprecedented resolution. This includes scarcely populated chimeric hybrid states of the ribosome engaged in several tRNA translocation steps resolved at ~2 [A] resolution. The current map not only improves our understanding of protein synthesis but also allows for more precise structure-based drug design of antibiotics to tackle rising bacterial resistance.

molecular biology↗

bioRxiv2022

Structural basis for FLCN RagC GAP activation in MiT-TFE substrate-selective mTORC1 regulation

mTORC1 regulates cell growth and catabolism in response to fluctuations in nutrients through phosphorylation of key substrates. The tumor suppressor FLCN is a RagC GTPase activating protein (GAP) that regulates mTORC1 phosphorylation of TFEB, controlling lysosome biogenesis and autophagy. Here, we determined the cryo-EM structure of the active FLCN complex (AFC) containing FLCN, FNIP2, the N-terminal tail of SLC38A9, the RagAGDP:RagCGDP.BeFx- GTPase dimer, and the Ragulator scaffold. Relative to the inactive lysosomal FLCN complex (LFC) structure, FLCN reorients by 90{degrees}, breaks its contacts with RagA, and makes new contacts with RagC that position its Arg164 finger for catalysis. Disruption of the AFC-specific interfaces of FLCN and FNIP2 with RagC eliminated GAP activity in vitro and led to nuclear retention of TFE3, with no effect on mTORC1 phosphorylation of S6K or 4E-BP1. The structure thus provides a roadmap to discover TFEB-selective mTORC1 antagonists. One-Sentence SummaryThe cryo-EM structure of the active FLCN RagC GAP complex provides a structural basis for TFEB/TFE3 substrate-selective targeting of mTORC1.

biochemistry↗

bioRxiv2022

Mechanism of RNA Polymerase I selection by transcription factor UAF

Pre-ribosomal RNA is selectively transcribed by RNA Polymerase (Pol) I in eukaryotes. The yeast transcription factor Upstream Activating Factor (UAF) represses Pol II transcription and mediates Pol I preinitiation complex (PIC) formation during the early stages of transcription initiation at the 35S ribosomal RNA gene. To unravel the DNA recognition and Pol I selection mechanisms of UAF, we determined the structure of UAF bound to native promoter DNA and transcription factor TBP. We found that UAF recognizes DNA using a hexameric histone-like scaffold with markedly different interactions than the nucleosome and the histone-fold-rich TFIID. UAF strategically sequesters TBP from DNA and Pol II/III-specific factors, and positions it for Core Factor binding, supporting Pol I recruitment. Our findings therefore reveal the molecular basis of Pol I selection for ribosome biogenesis. As well, they reveal an unexpected potential within the histone fold as a motif for specific protein-DNA interactions inside the cell.

molecular biology↗