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Biology subjects

Erdmann, P. S.

Publications and source records attributed to Erdmann, P. S..

7 recordsLinked to original sources

Serialized On-grid Lift-In Sectioning for Tomography (SOLIST)

Cryo-focused ion beam milling has enabled groundbreaking structural discoveries in native cells. Progress toward medically relevant applications, however, has been slow. We here present an adaptation of the cryo-lift out procedure for Serialized On-grid Lift-In Sectioning for Tomography (SOLIST), which increases throughput, reduces ice contamination, and enhances sample stability. With these improvements, new specimens, ranging from high-pressure frozen reconstituted LLPS droplets to human forebrain organoids, are accessible to cryo-electron tomography.

biophysics↗

Optimizing Cryo-FIB Lamellas for sub-5 Angstrom in situ Structural Biology

We here present a method based on metallic platinum sputtering that can substantially enhance the quality of subtomogram averages from lamellas and thereby reduce the number of particles needed for high-resolution subtomogram averaging. We provide evidence for the physical background of this improvement and demonstrate its usefulness by producing sub-5[A] ribosome averages from yeast.

molecular biology↗

In situ structural analysis reveals membrane shape transitions during autophagosome formation

Autophagosomes are unique organelles which form de novo as double-membrane vesicles engulfing cytosolic material for destruction. Their biogenesis involves a series of membrane transformations with distinctly shaped intermediates whose ultrastructure is poorly understood. Here, we combine cell biology, correlative cryo-electron tomography (ET) and novel data analysis to reveal the step-by-step structural progression of autophagosome biogenesis at high resolution directly within yeast cells. By mapping individual structures onto a timeline based on geometric features, we uncover dynamic changes in membrane shape and curvature. Moreover, we reveal the organelle interactome of growing autophagosomes, highlighting a polar organization of contact sites between the phagophore and organelles such as the vacuole and the ER. Collectively, these findings have important implications for the contribution of different membrane sources during autophagy and for the forces shaping and driving phagophores towards closure without a templating cargo.

cell biology↗

Multishot Tomography for High-Resolution In Situ Subtomogram Averaging.

Cryo-electron tomography (cryo-ET) and subtomogram averaging (STA) can resolve protein complexes at near atomic resolution, and when combined with focused ion beam (FIB) milling, macromolecules can be observed within their native context. Unlike single particle acquisition (SPA), cryo-ET can be slow, which may reduce overall project throughput. We here propose a fast, multi-position tomographic acquisition scheme based on beam-tilt corrected beam-shift imaging along the tilt axis, which yields sub-nanometer in situ STA averages.

molecular biology↗

In situ architecture of the ciliary base reveals the stepwise assembly of IFT trains

The cilium is an antenna-like organelle that performs numerous cellular functions, including motility, sensing, and signaling. The base of the cilium contains a selective barrier that regulates the entry of large intraflagellar transport (IFT) trains, which carry cargo proteins required for ciliary assembly and maintenance. However, the native architecture of the ciliary base and the process of IFT train assembly remain unresolved. Here, we use in situ cryo-electron tomography to reveal native structures of the transition zone region and assembling IFT trains at the ciliary base. We combine this direct cellular visualization with ultrastructure expansion microscopy to describe the front-to-back stepwise assembly of IFT trains: IFT-B forms the backbone, onto which IFT-A, then dynein-1b, and finally kinesin-2 sequentially bind before entry into the cilium. One Sentence SummaryNative molecular structure of the ciliary transition zone and hierarchical order of IFT assembly visualized within Chlamydomonas cells.

cell biology↗

A Modular Platform for Streamlining Automated Cryo-FIB Workflows

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, current 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. The software enables the streamlining of advanced cryo-FIB protocols such as multi-modal imaging, CLEM guided 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.

biophysics↗

The architecture of the centriole cartwheel-containing region revealed by cryo-electron tomography

Centrioles are evolutionarily conserved barrels of microtubule triplets that form the core of the centrosome and the base of the cilium. In the proximal region of the centriole, nine microtubule triplets attach to each other via A-C linkers and encircle a central cartwheel structure, which directs the early events of centriole assembly. While the crucial role of the proximal region in centriole biogenesis has been well documented in many species, its native architecture and evolutionary conservation remain relatively unexplored. Here, using cryo-electron tomography of centrioles from four evolutionarily distant species, including humans, we report on the architectural diversity of the centriolar proximal cartwheel-bearing region. Our work reveals that the cartwheel central hub, previously reported to have an 8.5 nm periodicity in Trichonympha, is constructed from a stack of paired rings with an average periodicity of [~]4 nm. In all four examined species, cartwheel inner densities are found inside the hubs ring-pairs. In both Paramecium and Chlamydomonas, the repeating structural unit of the cartwheel has a periodicity of 25 nm and consists of three ring-pairs with 6 radial spokes emanating and merging into a single bundle that connects to the triplet microtubule via the pinhead. Finally, we identified that the cartwheel is indirectly connected to the A-C linker through a flexible triplet-base structure extending from the pinhead. Together, our work provides unprecedented evolutionary insights into the architecture of the centriole proximal region, which underlies centriole biogenesis.

cell biology↗