Biology subjects

Abrahams, J. P.

Publications and source records attributed to Abrahams, J. P..

13 recordsLinked to original sources

bioRxiv2026

Replacing H2O with D2O in cryo-EM reduces radiation damage in biological samples

Radiation damage limits the resolution achievable in electron microscopy of biological specimens. Hydrogen is the most abundant atom in these specimens, and its low mass maximizes the recoil energy it receives from incident electrons, making it the atom most readily lost under the beam. Substitution of hydrogen by deuterium halves this recoil energy and makes the bonds involved slightly harder to break, yet its protective effect on vitrified protein crystals has not been tested. Using cryogenic electron diffraction at 200 kV, we show that hen egg-white lysozyme crystals grown directly in D2O withstand a dose 1.7 times higher than crystals grown in H2O before their Bragg intensity at 10-7[A] falls to 1/e (13.2 {+/-} 1.0 against 7.7 {+/-} 0.4 e-/[A]2), with every D2O-grown crystal outlasting every H2O-grown crystal. The protection is achieved with deuteration confined mainly to the solvent and the exchangeable hydrogens at the protein surface, which form the hydrogen bonds that hold the crystal lattice together. The protein itself need not be deuterated by expression in deuterated media. Direct crystallization in D2O involves only a single substitution in the mother liquor and offers an accessible strategy to extend the usable dose in electron diffraction of protein crystals.

biophysics↗

bioRxiv2026

Diffractograph: single-particle reconstruction using electron-diffraction data

We recently showed that narrow-beam electron diffraction of single particles provides higher signal-to-noise ratios and lower radiation damage than imaging. Here we introduce Diffractograph, a suite for reconstructing three-dimensional structure from these data. Diffractograph selects mutually compatible diffraction frames, groups them into one or more models and discards incompatible frames. In the ab initio workflow, this is done without prior structural information. In the reference-guided workflow, a low-resolution electrostatic potential map from single-particle cryo-EM is used to select compatible frames and determine their orientations. The oriented frames are combined into a three-dimensional intensity distribution. The reference map supplies low-resolution phases for the oversampled structure factors, and density modification extends these phases to higher resolution. For synthetic 200-keV diffraction data extending to 2.2 A, Diffractograph retained all frames and recovered the correct orientation of every retained frame with a median plane-normal error of 0.27 degrees. These results establish a computational workflow for structure determination using single-particle electron-diffraction data.

biophysics↗

bioRxiv2026

CryoConvNeXt enables robust identification of low-abundance molecular species in experimental cryo-EM data

Rare but biologically important molecular species are easily lost when reconstruction-based cryo-EM classification is applied to heterogeneous samples dominated by more abundant particles. We developed CryoConvNeXt, a deep-learning classifier that combines cyclic equivariance with adaptive frequency filtering. It is trained on simulated projections and adapted to experimental data by self-training, an unsupervised domain adaptation technique where the model acts as its own annotator. We tested CryoConvNeXt on cryo-EM data collected for this study from controlled binary and ternary mixtures of Catalase, Apoferritin, and HSP60. Manual particle curation provided reference labels for benchmarks with class ratios from 1:1 to 1:16. CryoConvNeXt retained minority-species recall across this range. In contrast, cryoSPARC's recall collapsed in four of six pairwise conditions, as early as 1:4 when Catalase was the minority species. By recovering low-abundance species that conventional classification fails to recover reliably, CryoConvNeXt takes an important step towards solving a central problem in quantitative visual proteomics: measuring the molecular composition of complex biological samples.

biophysics↗

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

Mitochondrial Lon protease couples substrate translocation to proteolytic activation

Human LonP1 is an ATP-dependent mitochondrial protease that degrades damaged or redundant proteins. Indiscriminate proteolysis by LonP1 is limited through tight coordination of substrate recognition, unfolding, translocation and catalytic cleavage, yet the role of ATP hydrolysis in these individual steps remains unclear. Here, we show that LonP1 binds substrates and cleaves peptide bonds without ATP hydrolysis, whereas degradation of folded proteins strictly depends on ATP-driven unfolding and translocation. Initial substrate binding opens a closed ADP-bound resting state, enabling nucleotide exchange and stimulating ATPase activity. The opening also increases accessibility of the proteolytic chamber, modestly enhancing peptidase activity. Maximal peptidase activity is observed in a transition-state mimic stabilised by ADP{middle dot}AlF, in which substrate is engaged within the translocation channel. Cryo-EM analysis reveals that in this state the proteolytic active sites are no longer occluded, linking ATP-driven substrate translocation to full proteolytic activation. Together, these findings reveal how LonP1 prevents indiscriminate proteolysis during substrate selection by ensuring that efficient proteolysis occurs only in substrate-translocating states. Model of the conformational landscape and functional cycle of LonP1Schematic overview of LonP1 states and their inter-conversion. State transitions are modulated by substrate, nucleotide occupancy, temperature, and inhibitors. Key distinguishing features include the presence or absence of the lateral gap, nucleotide state, substrate engagement within the A-tunnel, and the handedness of the ATPase (A) domains. Additional indicators include the compactness of the proteolytic (P) domain and the presence of substrate density within the N-terminal (N) domain or at the coiled-coil domain (CCD) as well as the position of a loop within the catalytic centre. The depicted cryo-EM structures represent a model of a continuous conformational landscape and correspond to the closest matching biological states and positions within the reaction cycle, but may also capture transient intermediates or conformations stabilised by experimental conditions. The shown atomic models correspond to the states highlighted in larger font (R-state: PDB 7NGL; P1-state: PDB 7NFY; P2-state: PDB 7NGC; closed LonP1-ADP-substrate: PDB 9CC1). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/733973v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@62128forg.highwire.dtl.DTLVardef@b0a5faorg.highwire.dtl.DTLVardef@160ea7forg.highwire.dtl.DTLVardef@1c35e82_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

LonP1 chaperone-like activity is ATPase independent and is mediated by its N-domain

The mitochondrial Lon protease is essential for proteostasis through ATP-dependent proteolysis and suppression of protein aggregation through an unknown mechanism. Here we show in three independent aggregation systems that human Lon protease (LonP1) directly interacts with fibrillar aggregates to prevent further aggregation: LonP1 binds amyloid fibrils and inhibits their growth, independently of its protease and ATPase activities. This aggregation inhibition depends on hexamer stability, and even the N-domain hexamer of LonP1 lacking all catalytic domains inhibited aggregation, which localizes its fibril-binding interface. We propose that chaperone deficiencies in LonP1 mutants that are associated with genetic disease, are caused by reduced hexamer stability or increased turnover. Our results clarify the observed dual protease and chaperone function of LonP1 by localizing them to different domains and separating the catalytic activities, thereby facilitating targeting the specific functionalities. Further, we identify the structure of the chaperone substrate to be fibrillar aggregates, suggesting that LonP1 may protect against amyloid fibrils in healthy individuals. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/723147v3_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1715a1eorg.highwire.dtl.DTLVardef@d382fborg.highwire.dtl.DTLVardef@3c3fa0org.highwire.dtl.DTLVardef@1753f3e_HPS_FORMAT_FIGEXP M_FIG C_FIG SignificanceThe mitochondrial Lon protease has long been proposed to function both as a protease and as a chaperone, though the mechanism of its chaperone activity is debated. Here, we show that human Lon binds to fibrillar protein aggregates and inhibits their elongation, but do not find evidence for chaperoning unfolded chains. Further, our findings challenge the current view that ATPase activity is required for Lon chaperone function. Instead, our results suggest that chaperone deficiency of Lon variants can be explained by variant stability. Our results provide a mechanistic separation of the protease and chaperone-like function LonP1, thereby opening up for targeting one of the functions specifically, and provide new insight into how Lon dysfunction may contribute in multiple ways to age-related and proteostasis-related diseases.

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↗

bioRxiv2023

TERSE: Efficient compression of the diffraction data

High-throughput data collection in crystallography poses significant challenges in handling massive amounts of data. Here, we present TERSE, a novel lossless compression algorithm specifically designed for diffraction data. We compare TERSE with the established lossless compression algorithms implemented in gzip, CBF, and HDF5, in terms of compression efficiency and speed, using continuous rotation electron diffraction data of an inorganic compound. Our results show that TERSE outperforms these algorithms by achieving a higher data compression at a speed that is at least an order of magnitude faster. TERSE files are byte-order independent and the algorithm can be readily implemented in hardware. By providing a tailored solution for diffraction data, TERSE facilitates more efficient data analysis and interpretation while mitigating storage and transmission concerns. TERSE C++20 compression/decompression code and an ImageJ/Fiji java plugin for reading TERSE files are open-sourced on GitHub under the permissive MIT license. SynopsisWe present a fast and lossless algorithm for compressing diffraction data, achieving up to 85% reduction in file size while processing up to 2000 512x512 frames per second. This breakthrough in compression technology is a significant step towards more efficient analysis and storage of large diffraction datasets.

biophysics↗

bioRxiv2021

Catalytic cycling of human mitochondrial Lon protease

The mitochondrial Lon protease homolog (LonP1) hexamer controls mitochondrial health by digesting proteins from the mitochondrial matrix that are damaged or must be removed. Understanding how it is regulated requires characterizing its mechanism. Here, we show how human LonP1 functions, based on eight different conformational states that we determined by cryo-EM with a resolution locally extending to 3.6 [A] for the best ordered states. LonP1 has a poorly ordered N-terminal part with apparent threefold symmetry, which apparently binds substrate protein and feeds it into its AAA+ unfoldase core. This translocates the extended substrate protein into a proteolytic cavity, in which we report an additional, previously unidentified Thr-type proteolytic center. Threefold rocking movements of the flexible N-terminal assembly likely assist thermal unfolding of the substrate protein. Our data suggest LonP1 may function as a sixfold cyclical Brownian ratchet controlled by ATP hydrolysis.

biochemistry↗

bioRxiv2020

Cryptosporulation in Kurthia spp. forces a rethinking of asporogenesis in Firmicutes

Sporulation is a complex morphophysiological process resulting in a cellular structure that is more resistant than the vegetative form. In Firmicutes, this structure is produced within the mother cell, and is called an endospore. Endospore formation is thought to have evolved in the common ancestor of Firmicutes. However, sporulation has apparently been lost in some extant lineages that are defined as asporogenic. We isolated strain 11kri321, a representative of the genus Kurthia, from an oligotrophic geothermal reservoir. While Kurthia spp. is considered to comprise only asporogenic species, strain 11kri321 produced spores. Genomic reconstruction of the sporulation pathway shows elements typical of sporulation in Bacilli, including the signaling for sporulation onset. However, key genes were missing, including those involved in engulfment and dipicolinic acid synthesis. Based on the results for strain 11kri321, sporulation was investigated in other Kurthia strains. Genes involved in signaling, cell division and spore coat formation were detected in three available Kurthia genomes. Moreover, endosporulation was clearly visualized in at least two of the four strains tested. These results show that Kurthia is an endospore-forming Firmicute lineage. However, the genetic background of sporulation in this genus deviates from the known sporulation pathway in Firmicutes and even within Bacilli, suggesting that a revision of the minimal set of genes used for genomic detection of sporulation is required. Based on our findings we propose the term cryptosporulant to refer to putative asporogenic Firmicutes for which a detailed genomic and physiological characterization of sporulating is missing. ImportanceEndospore-forming Firmicutes include many environmental and medical relevant bacterial clades. In these microorganisms, the ability to produce endospores is essential for survival in the environment and even for pathogenesis. The minimum core of genes required to produce a viable and resistant spore, the distinction between endospore-forming and asporogenic groups, as well as the evolution of sporulation have been a subject of investigation and debate for decades. Here, we demonstrate endosporulation in the genus Kurthia, considered as asporogenic. Morphological, physiological and genomic analyses were undertaken to demonstrate that sporulation is not lost within this lineage. Based on our results we propose a re-examination of the minimal genetic requirements of sporulation and the use of the term cryptosporulant to describe lineages of Firmicutes that have not previously been observed to sporulate, but for which a detailed analysis is still missing.

microbiology↗

bioRxiv2020

A lipocalin mediates unidirectional haem biomineralization in malaria parasites

During blood stage development, malaria parasites are challenged with the detoxification of enormous amounts of haem released during the proteolytic catabolism of erythrocytic haemoglobin. They tackle this problem by sequestering haem into bioinert crystals known as haemozoin. The mechanisms underlying this biomineralization process remain enigmatic. Here, we demonstrate that both rodent and human malaria parasite species secrete and internalize a lipocalin-like protein, PV5, to control haem crystallization. Transcriptional deregulation of PV5 in the rodent parasite Plasmodium berghei results in inordinate elongation of haemozoin crystals, while conditional PV5 inactivation in the human malaria agent Plasmodium falciparum causes excessive multi-directional crystal branching. Although haemoglobin processing remains unaffected, PV5-deficient parasites generate less haemozoin. Electron diffraction analysis indicates that despite the distinct changes in crystal morphology neither the crystalline order nor unit cell of haemozoin are affected by impaired PV5 function. Deregulation of PV5 expression renders P. berghei hypersensitive to the antimalarial drugs artesunate, chloroquine, and atovaquone, resulting in accelerated parasite clearance following drug treatment in vivo. Together, our findings demonstrate the Plasmodium-tailored role of a lipocalin family member in haemozoin formation and underscore the haem biomineralization pathway as an attractive target for therapeutic exploitation. SIGNIFICANCEDuring blood stage development, the malaria parasite replicates inside erythrocytes of the vertebrate host, where it engulfs and digests most of the available haemoglobin. This results in release of the oxygen-binding prosthetic group haem, which is highly toxic in its unbound form. The parasite crystallizes the haem into an insoluble pigment called haemozoin, a process that is vital for parasite survival and which is exploited in antimalarial therapy. We demonstrate that the parasite uses a protein called PV5 in haemozoin formation and that interfering with PV5 expression can increase the parasites sensitivity to antimalarial drugs during blood infection. An improved understanding of the mechanisms underlying haem sequestration will provide valuable insights for future drug development efforts.

microbiology↗

bioRxiv2019

Longitudinal cell division is associated with single mutations in the FtsZ-recruiting SsgB in Streptomyces

In most bacteria, cell division begins with the polymerization of the GTPase FtsZ at the mid-cell, which recruits the division machinery to initiate cell constriction. In the filamentous bacterium Streptomyces, cell division is positively controlled by SsgB, which recruits FtsZ to the future septum sites and promotes Z-ring formation. Here we show via site-saturated mutagenesis that various amino acid substitutions in the highly conserved SsgB protein result in the production of ectopically placed septa, that sever spores diagonally or along the long axis, perpendicular to the division plane. Ectopic septa were especially prominent when cells expressed SsgB variants with substitutions in residue E120. Biochemical analysis of SsgB variant E120G revealed that its interaction with - and polymerization of - FtsZ had been maintained. The crystal structure of S. coelicolor SsgB was resolved and the position of residue E120 suggests its requirement for maintaining the proper angle of helix 3, thus providing a likely explanation for the aberrant septa formed in SsgB E120 substitution mutants. Taken together, our work presents the first example of longitudinal division in a free living bacterium, which is explained entirely by changes in the FtsZ-recruiting protein SsgB.

developmental biology↗