Biology subjects

Redecke, L.

Publications and source records attributed to Redecke, L..

5 recordsLinked to original sources

bioRxiv2025

In situ protein crystallography: a single-crystal electron diffraction pipeline for structure determination inside living cells.

Intracellular crystallization is an emerging approach in structural biology that bypasses the need for protein purification. An InCellCryst pipeline was recently established for structure determination by serial X-ray crystallography. Serial crystallography requires the exposure of tens of thousands of cells containing intracellular crystals, precluding high-resolution structural studies on proteins with low numbers of crystals. To overcome this limitation, we combined the InCellCryst approach with the advantages of 3D electron diffraction and established the new IncellED method. Using microcrystals of the HEX-1 protein from Magnaporthe grisea, grown inside High Five insect cells, we demonstrate that electron diffraction data collected from a single crystal yields a high-resolution structure at 1.9 [A] resolution, comparable to 1.8 [A] achieved by serial X-ray crystallography. The IncellED methodology opens structure determination for proteins that crystallize with low efficiency and use widely available electron cryo-microscope infrastructure, paving the way for intracellular macromolecular electron crystallography at high resolution.

biophysics↗

bioRxiv2024

The PAZ domain of Aedes aegypti Dicer 2 is critical for accurate and high-fidelity size determination of virus-derived small interfering RNAs.

The exogenous siRNA (exo-siRNA) pathway is a critical RNA interference response involved in controlling arbovirus replication in mosquito cells. It is initiated by the detection of viral long double-stranded RNA (dsRNA) by the RNase III enzyme Dicer 2 (Dcr2), which is processed into predominantly 21 nucleotide (nt) virus-derived small interfering RNAs, or vsiRNAs that are taken up by the Argonaute 2 (Ago2) protein to target viral single-stranded RNAs. The detailed understanding of Dicer structure, function and domains owes much to studies outside the context of viral infection, and how Dcr2 domains contribute to detecting viral dsRNA to mount antiviral responses in infected mosquito cells remains much less understood. Here, we used a Dcr2 reconstitution system in Aedes aegypti derived Dcr2 KO cells to assess the contribution of the PAZ domain to induction of the exo-siRNA pathway following infection with Semliki Forest virus (SFV; Togaviridae, Alphavirus). Amino acids critical for PAZ activity were identified, and loss of PAZ function affected the production of 21 nt vsiRNAs -though not the overall ability of Dcr2 to process viral dsRNA- and silencing activity. This study establishes the importance of correct vsiRNA size in mosquito exo-siRNA antiviral responses, as well as the PAZ domains functional contribution to Dcr2 processing of viral dsRNA to 21 nt vsiRNAs.

microbiology↗

bioRxiv2023

InCellCryst - A streamlined approach to structure elucidation using in cellulo crystallized recombinant proteins

With the advent of serial X-ray crystallography on microfocus beamlines at free-electron laser and synchrotron facilities, the demand for protein microcrystals has significantly risen in recent years. However, by conventional methods extensive efforts are usually required to purify proteins and produce sufficiently homogeneous microcrystals. Here, we present InCellCryst, an advanced pipeline for producing homogeneous microcrystals directly within living insect cells. Our baculovirus-based cloning system enables the production of crystals from completely unmodified proteins as well as the screening of different cellular compartments to maximize chances for protein crystallization. By optimizing cloning procedures, recombinant virus production, crystallization and crystal detection, X-ray diffraction data can be collected 24 days after the start of target gene cloning. Furthermore, new strategies for serial synchrotron diffraction data collection directly from crystals within living cells abolish the need to purify the recombinant protein or the associated microcrystals.

biophysics↗

bioRxiv2023

Convolutional neural network approach for the automated identification of in cellulo crystals

In cellulo crystallization is a rarely occurring event in nature. Recent advances, making use of heterologous overexpression, can promote the intracellular formation of protein crystals, but new tools are required to detect and to characterize these targets in the complex cell environment. In the present work we make use of Mask R-CNN, a Convolutional Neural Network (CNN) based instance segmentation method, for the identification of either single or multi-shaped crystals growing in living insect cells, using conventional bright field images. The algorithm can be rapidly adapted to recognize different targets, with the aim to extract relevant information to support a semi-automated screening pipeline, with the purpose to aid in the development of the intracellular protein crystallization approach.

biophysics↗

bioRxiv2019

Processing of the SARS-CoV pp1a/ab nsp7-10 region

1.1.Severe acute respiratory syndrome coronavirus (SARS-CoV) is the causative agent of a respiratory disease with a high case fatality rate. During the formation of the coronaviral replication/transcription complex (RTC), essential steps include processing of the conserved polyprotein nsp7-10 region by the main protease Mpro and subsequent complex formation of the released nsps. Here, we analyzed processing of the coronavirus nsp7-10 region using native mass spectrometry showing consumption of substrate, rise and fall of intermediate products and complexation. Importantly, there is a clear order of cleavage efficiencies, which is influenced by the polyprotein tertiary structure. Furthermore, the predominant product is an nsp7+8(2:2) hetero-tetramer with nsp8 scaffold. In conclusion, native MS, opposed to other methods, can expose the processing dynamics of viral polyproteins and the landscape of protein interactions in one set of experiments. Thereby, new insights into protein interactions, essential for generation of viral progeny, were provided, with relevance for development of antivirals.

biophysics↗