Biology subjects

Filipcik, P.

Publications and source records attributed to Filipcik, P..

8 recordsLinked to original sources

bioRxiv2026

Narrow-beam geometry improves the efficiency of cryo-EM

Cryo-electron microscopy (cryo-EM) of biological specimens is limited by radiation damage and a low signal-to-noise ratio (SNR). Here, we show that reducing the illuminated area substantially slows the observed diffraction decay in protein microcrystals. We further show that narrow parallel-beam electron diffraction from thin non-crystalline biological specimens provides substantially higher reciprocal-space SNR than conventional cryo-EM imaging. We developed a multimodal scanning workflow, 4D-para-STEM, that records narrow-beam diffraction patterns together with corresponding images. Using viruses, peptide assemblies, and microtubules, we demonstrate interpretable diffraction signals from both crystalline and non-crystalline biological specimens. Together, these results show that narrow parallel-beam scanning reduces observed radiation damage and improves the SNR in cryo-EM.

biophysics↗

bioRxiv2026

Structural basis of microtubule destabilization by GTP hydrolysis

Microtubules are cytoskeletal filaments that dynamically grow and shrink to support vital biological functions. Their behavior is regulated by GTP hydrolysis in tubulin incorporated into the lattice, but the underlying chemical transitions and their impact on microtubule stability remain unclear. Using cryo-electron microscopy, we resolved microtubule structures at 1.9-2.2 [A] in four different nucleotide states, revealing the roles of amino acids, ions, and hundreds of water molecules in the hydrolysis process. We found that compaction of the GTP-bound lattice into a more stable pre-hydrolysis state activates a catalytic water molecule for {gamma}-phosphate cleavage. Hydrolysis intermediates are stabilized by strengthened lattice contacts, but in the final GDP state, release of reaction products disrupts these contacts and destabilizes the lattice. Our results explain how chemical changes in tubulins nucleotide site drive lattice transitions and offer an atomistic framework for understanding microtubule dynamic instability.

biochemistry↗

bioRxiv2026

Human mitochondrial Lon protease initiates unidirectional degradation from either substrate terminus

The ATP-dependent Lon protease (LonP1) unfolds and progressively degrades damaged or redundant proteins in the mitochondrial matrix. However, how LonP1 recognizes substrates remains unclear. Here, we investigated LonP1 degradation of the mitochondrial transcription factor TFAM, a physiological substrate. Using engineered TFAM variants carrying bulky moieties at either terminus, we show that degradation can initiate from either the N- or the C-terminus, with no evidence for internal initiation. Once engaged, substrate processing proceeds unidirectionally. Nanogold labeling experiments trapped TFAM termini within the N-domain cavity of LonP1, providing direct evidence that substrates enter through the N-domain assembly. A high-resolution cryo-EM structure of LonP1 in a transition-state-like complex with Mg2+, ADP, and AlF3 revealed substrate density within the axial channel with resolved main-chain carbonyl and C moieties, yet the peptide can be modelled equally well in either direction. Mass spectrometry of degradation products further supports initiation from either substrate terminus while confirming unidirectional processive translocation. These observations explain how LonP1 combines broad substrate specificity with selective recognition of flexible termini, avoiding initiation at internal flexible loops while enabling efficient ATP-dependent unfolding and degradation.

biochemistry↗

bioRxiv2026

A cryo-EM processing pipeline for microtubules using CryoSPARC

Microtubules are cytoskeletal filaments typically characterized by a discontinuous helical lattice of /{beta}-tubulin heterodimers. Microtubules can also adopt variable lattice architectures both in vitro and in cellular contexts. Pseudo-helical averaging processing strategies have been developed to generate cryo-EM reconstructions of microtubules with and without decorating protein-binding partners, but these pipelines can be difficult to implement for the average user, especially for undecorated filaments. Here, we describe MiCSPARC, a cryo-EM processing pipeline developed around CryoSPARC (Punjani et al., 2017), which leverages automated particle picking and fast 3D refinement times in CryoSPARC to determine structures of both decorated and undecorated microtubules. We generated reconstructions of undecorated GDP microtubules, as well as kinesin-1 motor domain-decorated GMPCPP filaments at resolutions of up to 2.8 [A], demonstrating the robustness of the pipeline. Based on its convenient implementation and ability to routinely generate high-resolution, seam-corrected microtubule reconstructions, MiCSPARC should provide a valuable tool for understanding microtubule dynamics, microtubule-associated proteins, and microtubule-targeting agents.

biophysics↗

bioRxiv2024

DET1 dynamics underlie co-operative ubiquitination by CRL4DET1 COP1 complexes

Ubiquitin ligases regulate core cellular processes through diverse mechanisms. The ubiquitin ligase COP1 is conserved from plants to humans and is particularly important for targeting developmental transcription factors for ubiquitination. COP1 can function independently, but can also be recruited to Cullin-4 ubiquitin ligase complexes via the DET1 adaptor protein. However, the mechanism of action of complexes containing COP1 and DET1 is not well understood. Here we report the cryo-electron microscopy structure of human DET1, bound to proteins that enable Cullin-4 recruitment (DDB1-DDA1) and an additional ubiquitin ligase enzyme (Ube2e2). We observe that DDA1 stabilises a closed conformation of DET1, binding adjacent to a unique Ube2e2 binding-insert in DET1. Moreover, we demonstrate that closure of DET1 underlies COP1 recruitment, which binds in an antiparallel dimeric state. Disrupting either the Ube2e2-binding insertion of DET1, or distinct recruitment sites on COP1, abolish DET1-COP1 binding and DET1-mediated modulation of COP1 levels. The multifaceted architecture provides an efficient platform for ubiquitination of substrates, or COP1 itself, by Cullin-4DET1 and offers multiple opportunities for physiological regulation.

biochemistry↗

bioRxiv2024

DiffGAN: a conditional generative adversarial network for phasing single molecule diffraction data to atomic resolution

IntroductionProteins that adopt multiple conformations pose significant challenges in structural biology research and pharmaceutical development, as structure determination via single particle cryo-electron microscopy (cryo-EM) is often impeded by data heterogeneity. In this context, the enhanced signal-to-noise ratio of single molecule cryo-electron diffraction (simED) offers a promising alternative. However, a significant challenge in diffraction methods is the loss of phase information, which is crucial for accurate structure determination. MethodsHere, we present DiffGAN, a conditional generative adversarial network (cGAN) that estimates the missing phases at high resolution from a combination of high-resolution single particle diffraction data and low-resolution image data. ResultsFor simulated datasets, DiffGAN allows effectively determine protein structures at atomic resolution from diffraction patterns and noisy low-resolution images. DiscussionOur findings suggest that combining single particle cryo-electron diffraction with advanced generative modeling, as in DiffGAN, could revolutionize the way protein structures are determined, offering a more accurate and efficient alternative to existing methods.

biophysics↗

bioRxiv2021

Second Generation Antibodies Neutralize Emerging SARS-CoV-2 Variants of Concern

Recently emerged SARS-CoV-2 variants show resistance to some antibodies that were authorized for emergency use. We employed hybridoma technology combined with authentic virus assays to develop second-generation antibodies, which were specifically selected for their ability to neutralize new variants of SARS-CoV-2. AX290 and AX677, two monoclonal antibodies with non-overlapping epitopes, exhibit subnanomolar or nanomolar affinities to the receptor binding domain of the viral Spike protein carrying amino acid substitutions N501Y, N439K, E484K, K417N, and a combination N501Y/E484K/K417N found in the circulating virus variants. The antibodies showed excellent neutralization of an authentic SARS-CoV-2 virus representing strains circulating in Europe in spring 2020 and also the variants of concern B.1.1.7 and B.1.351. Finally, the combination of the two antibodies prevented the appearance of escape mutations of the authentic SARS-CoV-2 virus. The neutralizing properties were fully reproduced in chimeric mouse-human versions, which may represent a promising tool for COVID-19 therapy.

microbiology↗

bioRxiv2020

A cryptic tubulin-binding domain links MEKK1 to microtubule remodelling

The MEKK1 protein is a pivotal kinase activator of responses to cellular stress. Activation of MEKK1 can trigger various responses, including mitogen activated protein (MAP) kinases, NF-{kappa}B signalling, or cell migration. Notably, MEKK1 activity is triggered by microtubule-targeting chemotherapies, amongst other stressors. Here we show that MEKK1 contains a previously unidentified tumour overexpressed gene (TOG) domain. The MEKK1 TOG domain binds to tubulin heterodimers--a canonical function of TOG domains--but is unusual in that it appears alone rather than as part of a multi-TOG array, and has structural features distinct from previously characterised TOG domains. MEKK1 TOG demonstrates a clear preference for binding curved tubulin heterodimers, which exist in soluble tubulin and at sites of microtubule polymerisation and depolymerisation. Mutations disrupting tubulin-binding lead to destabilisation of the MEKK1 protein in cells, and ultimately a decrease in microtubule density at the leading edge of polarised cells. We also show that MEKK1 mutations at the tubulin-binding interface of the TOG domain recur in patient derived tumour sequences, suggesting selective enrichment of tumour cells with disrupted MEKK1-microtubule association. Together, these findings provide a direct link between the MEKK1 protein and tubulin, which is likely to be relevant to cancer cell migration and response to microtubule-modulating therapies. SIGNIFICANCE STATEMENTThe protein kinase MEKK1 activates stress response pathways in response to various cellular stressors, including chemotherapies that disrupt dynamics of the tubulin cytoskeleton. Filip[c]ik et al., show that MEKK1 contains a previously uncharacterised domain that can preferentially bind to the curved tubulin heterodimer--which is found at sites of microtubule assembly and disassembly. Mutations that interfere with MEKK1-tubulin binding disrupt microtubule networks in migrating cells and are enriched in patient-derived tumour sequences. These results suggest that MEKK1-tubulin binding may be relevant to cancer progression, and the efficacy of microtubule-disrupting chemotherapies that require the activity of MEKK1.

biochemistry↗