Biology subjects

Pospich, S.

Publications and source records attributed to Pospich, S..

6 recordsLinked to original sources

bioRxiv2022

Structural basis of actin filament assembly and aging

The dynamic turnover of actin filaments (F-actin) controls cellular motility in eukaryotes and is coupled to changes in the F-actin nucleotide state. It remains unclear how F-actin hydrolyzes ATP and subsequently undergoes subtle conformational rearrangements that ultimately lead to filament depolymerization by actin-binding proteins. Here, we present cryo-EM structures of F-actin in all nucleotide states, polymerized in the presence of Mg2+ or Ca2+, at resolutions ([~]2.2 [A]) that allow for the visualization of hundreds of water molecules. The structures reveal that the G- to F-actin transition induces the relocation of water molecules in the nucleotide binding pocket, activating one of them for the nucleophilic attack of ATP. Unexpectedly, the back door for the subsequent release of inorganic phosphate (Pi) is closed in all structures, indicating that the F-actin conformation that allows for Pi release occurs transiently. The small changes in the nucleotide-binding pocket after ATP hydrolysis and Pi release are sensed by a key amino acid, amplified and transmitted to the filament periphery. Furthermore, differences in the positions of waters in the nucleotide binding pocket explain why Ca2+-actin exhibits slower polymerization rates than Mg2+-actin. Our work elucidates the solvent-driven rearrangements that govern actin filament assembly and aging and lays the foundation for the rational design of drugs and small molecules for imaging and therapeutic applications.

biochemistry↗

bioRxiv2021

Structure of the thin filament in native skeletal muscles reveals its interaction with nebulin and two distinct conformations of myosin

Nebulin is a major structural protein of skeletal sarcomeres and is essential for proper assembly and contraction of skeletal muscle1. It stabilises and regulates the length of thin filaments,2 but the structural mechanism remains nebulous. Using electron cryotomography and sub-tomogram averaging, we present the first structure of native nebulin bound to thin filaments within the A-band and I-band of intact sarcomeres. This in-situ reconstruction reveals unprecedented detail of interaction at pseudo-atomic resolution between nebulin and actin, providing the basis for understanding the structural and regulatory roles of nebulin. The position of nebulin on the thin filament indicates that there is no contact to tropomyosin or myosin, but an unexpected interaction with a troponin-T linker, possibly through two binding motifs on nebulin. In addition, our structure of myosin bound to the thin filaments reveals different conformations of the neck domain, both within the same sarcomere and when compared to purified structures, highlighting an inherent structural variability in muscle. We provide a complete description of cross-bridge formation on fully native, nebulin-containing thin filaments at near-atomic scale. Our structures establish the molecular basis for the role of nebulin as a thin filament "molecular ruler" and the impact of nemaline myopathies mutations that will aid future development of therapies.

biochemistry↗

bioRxiv2021

High-resolution structures of the actomyosin-V complex in three nucleotide states provide insights into the force generation mechanism

The molecular motor myosin undergoes a series of major structural transitions during its force-producing motor cycle. The underlying mechanism and its coupling to ATP hydrolysis and actin binding is only partially understood, mostly due to sparse structural data on actin-bound states of myosin. Here, we report 26 high-resolution cryo-EM structures of the actomyosin-V complex in the strong-ADP, rigor, and a previously unseen post-rigor transition state that binds the ATP analog AppNHp. The structures reveal a high flexibility of myosin in each state and provide valuable insights into the structural transitions of myosin-V upon ADP release and binding of AppNHp, as well as the actomyosin interface. In addition, they show how myosin is able to specifically alter the structure of F-actin. The unprecedented number of high-resolution structures of a single myosin finally enabled us to assemble a nearly complete structural model of the myosin-V motor cycle and describe the molecular principles of force production.

biochemistry↗

bioRxiv2020

TranSPHIRE: Automated and feedback-optimized on-the-fly processing for cryo-EM

Single particle electron cryomicroscopy (cryo-EM) requires full automation to allow high-throughput structure determination which is especially important for drug discovery research. Although several software packages exist where parts of the cryo-EM pipeline are automated, a complete solution that offers reliable, quality-optimized on-the-fly processing, resulting in a high-resolution three-dimensional reconstruction does not exist. Here we present TranSPHIRE: A software package for fully automated processing of cryo-EM data sets during data acquisition. TranSPHIRE transfers data from the microscope, automatically applies the common pre-processing steps, picks particles, performs 2D clustering, and 3D refinement parallel to image recording. Importantly, TranSPHIRE introduces a machine learning-based feedback loop to re-train its internally used picking model to adapt to any given data set live during processing. This elegant approach enables TranSPHIRE to process data more effectively, producing high-quality particle stacks. TranSPHIRE collects, and displays all microscope settings and metrics generated by its individual tools, in order to allow users to quickly evaluate data during acquisition. TranSPHIRE can run on a single work station and also includes the automated processing of filaments.

biophysics↗

bioRxiv2020

Two particle picking procedures for filamentous proteins: SPHIRE-crYOLO filament mode and SPHIRE-STRIPER

Structure determination of filamentous molecular complexes involves the selection of filaments from cryo-EM micrographs. The automatic selection of helical specimens is particularly difficult and thus many challenging samples with issues such as contamination or aggregation are still manually picked. Here we present two approaches for selecting filamentous complexes: one uses a trained deep neural network to identify the filaments and is integrated in SPHIRE-crYOLO, the other one, called SPHIRE-STRIPER, is based on a classical line detection approach. The advantage of the crYOLO based procedure is that it accurately performs on very challenging data sets and selects filaments with high accuracy. Although STRIPER is less precise, the user benefits from less intervention, since in contrast to crYOLO, STRIPER does not require training. We evaluate the performance of both procedures on tobacco mosaic virus and filamentous F-actin data sets to demonstrate the robustness of each method.

biophysics↗

bioRxiv2019

Structural effects and functional implications of phalloidin and jasplakinolide binding to actin filaments

Actin undergoes structural transitions during polymerization, ATP hydrolysis and subsequent release of inorganic phosphate. Several actin binding proteins sense specific states during this transition and can thus target different regions of the actin filament. Here we show in atomic detail that phalloidin, a mushroom toxin that is routinely used to stabilize and label actin filaments, suspends the structural changes in actin, likely influencing its interaction with actin binding proteins. Furthermore, high-resolution cryo-EM structures reveal structural rearrangements in F-actin upon inorganic phosphate release in phalloidin-stabilized filaments. We find that the effect of the sponge toxin jasplakinolide differs from the one of phalloidin, despite their overlapping binding site and similar interactions with the actin filament. Analysis of structural conformations of F-actin suggests that stabilizing agents trap states within the natural conformational space of actin.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC=\"FIGDIR/small/794495v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (47K):\norg.highwire.dtl.DTLVardef@1a81b98org.highwire.dtl.DTLVardef@1aba96borg.highwire.dtl.DTLVardef@ffc218org.highwire.dtl.DTLVardef@11fe454_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFive high-resolution cryo-EM structures of stabilized filamentous actin\nC_LIO_LIPhalloidin traps different structural states depending on when it is added\nC_LIO_LIThe effect of phalloidin and jasplakinolide on filamentous actin is not identical\nC_LIO_LIBoth toxins likely interfere with the binding of proteins sensing F-actins nucleotide state\nC_LI

biochemistry↗