Biology subjects

Mohammed, I.

Publications and source records attributed to Mohammed, I..

14 recordsLinked to original sources

bioRxiv2025

Cryo-EM structure of alpha-Synuclein Fibrils Harboring the Dementia with Lewy Bodies - Associated E83Q Mutation

Aggregation of -synuclein (-Syn) into amyloid fibrils underlies the pathology of synucleinopathies. Rare familial mutations can modulate -Syn aggregation and may give rise to distinct fibril conformations linked to disease heterogeneity. The E83Q mutation, identified in a Dementia with Lewy Body disease (DLB) patient with atypical Lewy body distribution, has been shown to accelerate -Syn aggregation and enhance neuronal seeding, yet its structural basis remains unclear. Here, we determined the cryo-electron microscopy structure at 3.4 [A] resolution of recombinant full-length human -Syn fibrils harboring the E83Q mutation. The fibrils adopt a double-protofilament architecture with a conserved Greek-key-like core spanning residues 36-99, stabilized by intra- and inter-filament salt bridges. Despite this conserved fold, the E83Q mutation induces a distinct local rearrangement within the hydrophobic region: substitution of Glu83 with Gln reorients residue 83 inward, abolishing solvent exposure and altering interactions with the N-terminal region. This structural shift brings the hydrophobic region closer to the N-terminus, shielding residues implicated in post-translational modification and ligand binding. Our findings reveal how a single charge-neutralizing mutation reshapes -Syn fibril architecture, providing a structural framework to understand the enhanced aggregation and pathogenic properties associated with the E83Q variant.

neuroscience↗

bioRxiv2025

CryoWriter: A Robotic Solution for Improved Cryo-EM Grid Preparation

Cryo-electron microscopy (cryo-EM) structure determination relies on preparing thin, vitreous films of sample solution on EM grids. Cryo-EM is a mature technology, but preparing the grids remains a major bottleneck. Here, we evaluate the cryoWriter, a blotting-free, microfluidic grid-preparation robot that writes nanoliter volumes onto EM grids in a controlled environment. Using capillary-writing in spiral or line patterns, we prepared high-quality grids from minimal sample volumes and obtained near-atomic reconstructions for test specimens, including TMV, apoferritin, and the membrane protein TRPM4. We further demonstrate programmable deposition modes, such as writing the sample twice to boost particle density, or two-line writing for on-grid mixing to visualize time-resolved protein-ligand binding. In a challenging case (NrS-1 DNA polymerase), the cryoWriter grids exhibited reduced orientation bias relative to conventional blotting, enabling a more isotropic reconstruction. These results show that the cryoWriter provides a versatile platform for reproducible low volume cryo-EM grid preparation and for on-grid biochemical workflows.

biophysics↗

bioRxiv2025

Structural basis for a filamentous morpheein model of human cystathionine beta-synthase

Human cystathionine beta-synthase (CBS) is a vital enzyme that regulates sulfur amino acid metabolism, hydrogen sulfide production, and cellular redox balance. Using a multidisciplinary approach, we demonstrate that CBS functions as a filamentous morpheein, with its stability, turnover, and activity governed by dynamic quaternary structural transitions. Three distinct filamentous assemblies were resolved by cryo-EM and are mediated by the oligomerization loop (residues 516-525): (i) ligand-free trans-dimers that form trans-basal filaments with basal stability and activity, (ii) adenosylornithine-bound cis-dimers that assemble into stabilized cis-basal filaments and (iii) S-adenosylmethionine-bound allo-dimers, which, together with cis-dimers, form highly stable, allo-activated stacked filaments. These reversible filamentous assemblies redefine CBS biology by integrating oligomerization and allosteric regulation within a morpheein framework. These findings provide a transformative perspective on CBS function and open new avenues for pharmacological targeting of dysregulated CBS in various diseases including homocystinuria, cancer, and Down syndrome.

biochemistry↗

bioRxiv2025

Pulsed-electron illumination does not reduce beam damage for imaging biological macromolecules

Radiation damage remains a fundamental limitation in cryo-electron microscopy (cryo-EM), constraining the total electron dose that can be used and thus limiting high-resolution imaging of biological specimens. Recent studies have proposed that temporally structured or pulsed electron beams could reduce radiation damage by allowing time for energy dissipation between individual electron interactions. To evaluate this hypothesis, we conducted a systematic investigation using a radiofrequency (RF) driven 300 kV Titan Krios microscope equipped with cold field emission gun (c-FEG) to generate highly regular pulsed electron beams for specimens under cryogenic conditions. We compared radiation damage in three representative samples: paraffin 2D crystals, bacteriorhodopsin (purple membrane) 2D crystals, and plunge-frozen tobacco mosaic virus (TMV) in vitreous ice, under both pulsed and conventional (= random) illumination, while keeping all other imaging conditions constant. Radiation damage was quantified by tracking the decay of computed diffraction intensities to determine the critical dose (Ne). We observed no statistically significant difference in critical dose between pulsed and random illumination across all 3 samples. Our findings provide a critical reference point for future development and evaluation of temporally modulated electron sources in cryo-EM instrumentation.

molecular biology↗

bioRxiv2024

Uncovering the Neural Correlates of the Urge-to-Blink: A Study Utilising Subjective Urge Ratings and Paradigm Free Mapping

Neuroimaging plays a significant role in understanding the neurophysiology of Tourette syndrome (TS), in particular the main symptom, tics, and the urges associated with them. Premonitory urge is thought to be a negative reinforcer of tic expression in TS. Tic expression during neuroimaging is most often required as an overt marker of increased urge-to-tic, which can lead to considerable head movement, and thus data loss. This study aims to identify the brain regions involved in urge in healthy subjects using multi-echo functional MRI and a timing- free approach to localise the BOLD response associated with the urge-to-act without information of when these events occur. Blink suppression is an analogous behaviour that can be expressed overtly in the MRI scanner which gives rise to an urge like those described by individuals with TS. We examined the urge-to-blink in 20 healthy volunteers with an experimental paradigm including two conditions, "Okay to blink" and "Suppress blinking", to identify brain regions involved in blink suppression. Multi-echo functional MRI data was analysed using a novel approach to investigate the BOLD signal correlated with the build-up of the urge-to-blink that participants continuously reported using a rollerball device. In addition, we used the method of multi-echo paradigm free mapping (MESPFM) to identify these regions without prior specification of task timings. Subjective urge scores were correlated with activity in the right posterior and ventral-anterior insula as well as the mid-cingulate and occipital cortices. Furthermore, blink suppression was associated with activation in the dorsolateral prefrontal cortex, cerebellum, right dorsal-anterior insula, mid-cingulate cortex and thalamus. These findings illustrate that different insula subregions contribute to the urge-for-action and suppression networks. The MESPFM approach showed co-activation of the right insula and cingulate cortex. The MESPFM activation maps showed the highest overlap with activation associated with blink suppression, as identified using general linear model analysis, demonstrating that activity associated with suppression can be determined without prior knowledge of task timings.

neuroscience↗

bioRxiv2024

1.94 Angstrom structure of synthetic alpha-synuclein fibrils seeding MSA neuropathology

Summary ParagraphMultiple system atrophy (MSA) is a rapidly progressive neurodegenerative disease of unknown cause, typically affecting individuals aged 50-60 and leading to death within a decade1-3. It is characterized by glial cytoplasmic inclusions (GCIs) composed of fibrillar alpha-synuclein (aSyn)4-8, whose formation shows parallels with prion propagation9,10. While fibrils extracted from MSA brains have been structurally characterized11, their ability to replicate in a "protein-only" manner has been questioned12, and their capacity to induce GCIs in vivo remains unexplored. By contrast, the synthetic fibril strain 1B13,14, assembled from recombinant human aSyn, self-replicates in vitro and induces GCIs in mice15 - suggesting direct relevance to MSA - but awaited scrutiny at an atomic scale. Here, we report high-resolution structural analyses of 1B fibrils and of fibrils extracted from diseased mice injected with 1B that developed GCIs (1BP). We show in vivo that conformational templating enables fibril strain replication, resulting in MSA-like inclusion pathology. Remarkably, the structures of 1B and 1BP are highly similar and mimic the fold of aSyn observed in one protofilament of fibrils isolated from MSA patients11. Moreover, reinjection of crude mouse brain homogenates containing 1BP into new mice reproduces the same MSA-like pathology induced by the parent synthetic seed 1B. Our findings identify 1B as a synthetic pathogen capable of self-replication in vivo and reveal structural features of 1B/1BP that may underlie MSA pathology, offering insights for therapeutic strategies.

neuroscience↗

bioRxiv2024

Low-dose cryo-electron ptychography of proteins at sub-nanometer resolution

Cryo-transmission electron microscopy (cryo-EM) of frozen hydrated specimens is an efficient method for the structural analysis of purified biological molecules. However, cryo-EM and cryo-electron tomography are limited by the low signal-to-noise ratio (SNR) of recorded images, making detection of smaller particles challenging. For dose-resilient samples often studied in the physical sciences, electron ptychography - a coherent diffractive imaging technique using 4D scanning transmission electron microscopy (4D-STEM) - has recently demonstrated excellent SNR and resolution down to tens of picometers for thin specimens imaged at room temperature. Here we applied 4D-STEM and ptychographic data analysis to frozen hydrated proteins, reaching sub-nanometer resolution 3D reconstructions. We employed low-dose cryo-EM with an aberration-corrected, convergent electron beam to collect 4D-STEM data for our reconstructions. The high frame rate of the electron detector allowed us to record large datasets of electron diffraction patterns with substantial overlaps between the interaction volumes of adjacent scan positions, from which the scattering potentials of the samples were iteratively reconstructed. The reconstructed micrographs show strong SNR enabling the reconstruction of the structure of apoferritin protein at up to 5.8 [A] resolution. We also show structural analysis of the Phi92 capsid and sheath, tobacco mosaic virus, and bacteriorhodopsin at slightly lower resolutions.

biophysics↗

bioRxiv2021

Cryo-EM structure of a single-chain β1-adrenoceptor - AmpC β-lactamase fusion protein

The insertion of fusion proteins has enabled the crystallization of a wide range of G-protein-coupled receptors. Here, we adapted this engineering strategy to cryo-electron microscopy (cryo-EM). We inserted the soluble protein AmpC {beta}-lactamase into the third intracellular loop (ICL3) of ultra-thermostable {beta}1-adrenoceptor ({beta}1AR) via chimeric helix fusions. Biochemical and biophysical characterization showed that the resulting fusion protein after expression, solubilization and purification was monodisperse and able to bind the known {beta}1AR weak partial agonist cyanopindolol, and the antagonist propranolol. The protein particles comprised sufficient mass and discernable structural features to elucidate its cryo-EM structure in complex with cyanopindolol without any natural (G-proteins, arrestins) or artificial (Nanobodies, DARPins) binding partners, to an overall resolution of 4.2 [A]. The seven-helix architecture and helix eight, as well as both GPCR - AmpC {beta}-lactamase connections are clearly resolved. {beta}1AR is in its inactive conformation. 3D variability analysis revealed significant flexibility between the two protein domains and within the GPCR helices, offering insights into conformational dynamics. The map contains clear density for the cyanopindolol. The fusion protein geometry theoretically fits a wide range of class A GPCRs, presenting a powerful platform for structure elucidation of a diverse array of class A GPCR - ligand complexes by cryo-EM in the inactive receptor state. The approach furthermore holds potential for structure elucidation of GPCRs in the absence of ligands.

molecular biology↗

bioRxiv2021

Structural basis of adenylyl cyclase 9 activation

Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPS, (iv) bound to Gs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four new conformations show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation. One Sentence SummaryCryo-EM reveals activator-induced conformational changes in adenylyl cyclase AC9

biochemistry↗

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↗

bioRxiv2021

Evaluation of Host Defense Peptide (CaD23)-Antibiotic Interaction and Mechanism of Action: Insights from Experimental and Molecular Dynamics Simulations Studies

Background/aimHost defense peptides (HDPs) have the potential to provide a novel solution to antimicrobial resistance (AMR) in view of their unique and broad-spectrum antimicrobial activities. We had recently developed a novel hybrid HDP based on LL-37 and human beta-defensin-2, named CaD23, which was shown to exhibit good in vivo antimicrobial efficacy against Staphylococcus aureus in a bacterial keratitis murine model. This study aimed to examine the potential CaD23-antibiotic synergism and to evaluate the underlying mechanism of action of CaD23. MethodsAntimicrobial efficacy was determined using minimum inhibitory concentration (MIC) assay with broth microdilution method. Peptide-antibiotic interaction was evaluated against S. aureus, methicillin-resistant S. aureus (MRSA), and Pseudomonas aeruginosa using established checkerboard assay and time-kill kinetics assay. Fractional inhibitory concentration index (FICI) was calculated and interpreted as synergistic (FICI<0.5), additive (FICI between 0.5-1.0), indifferent (FICI between >1.0 and [≤]4), or antagonistic (FICI>4). SYTOX green uptake assay was performed to determine the membrane-permeabilising action of CaD23. Molecular dynamics (MD) simulations were performed to evaluate the interaction of CaD23 with bacterial and mammalian mimetic membranes. ResultsCaD23-amikacin and CaD23-levofloxacin combination treatment exhibited a strong additive effect against S. aureus SH1000 (FICI=0.56) and MRSA43300 (FICI=0.56) but a borderline additive-to-indifferent effect against P. aeruginosa (FIC=1.0-2.0). CaD23 (at 25 g/ml; 2x MIC) was able to achieve complete killing of S. aureus within 30 mins. When used at sub-MIC concentration (3.1 g/ml; 0.25x MIC), it was able to expedite the antimicrobial action of amikacin against S. aureus by 50%. The rapid antimicrobial action of CaD23 was attributed to the underlying membrane-permeabilising mechanism of action, evidenced by the SYTOX green uptake assay and MD simulations studies. MD simulations revealed that cationicity, alpha-helicity, amphiphilicity and hydrophobicity (related to the Trp residue at C-terminal) play important roles in the antimicrobial action of CaD23. ConclusionsCaD23 is a novel membrane-active synthetic HDP that can enhance and expedite the antimicrobial action of antibiotics against Gram-positive bacteria when used in combination. MD simulation serves as a useful tool in dissecting the mechanism of action and guiding the design and optimisation of HDPs.

microbiology↗

bioRxiv2021

Hybrid Derivative of Cathelicidin and Human Beta Defensin-2 Against Gram-Positive Bacteria: A Novel Approach for the Treatment of Bacterial Keratitis

Bacterial keratitis (BK) is a major cause of corneal blindness globally. This study aimed to develop a novel class of antimicrobial therapy, based on human-derived hybrid host defense peptides (HyHDPs), for treating BK. HyHDPs were rationally designed through combination of functional amino acids in parent HDPs, including LL-37 and human beta-defensin (HBD)-1 to -3. Minimal inhibitory concentrations (MICs) and time-kill kinetics assay were performed to determine the concentration- and time-dependent antimicrobial activity and cytotoxicity was evaluated against human corneal epithelial cells and erythrocytes. In vivo safety and efficacy of the most promising peptide was examined in the corneal wound healing and Staphylococcus aureus (ATCC SA29213) keratitis murine models, respectively. A second-generation HyHDP (CaD23), based on rational hybridization of the middle residues of LL-37 and C-terminal of HBD-2, was developed and was shown to demonstrate good efficacy against methicillin-sensitive and methicillin-resistant S. aureus [MIC=12.5-25.0g/ml (5.2-10.4M)] and S. epidermidis [MIC=12.5g/ml (5.2M)], and moderate efficacy against P. aeruginosa [MIC=25-50g/ml (10.4-20.8M)]. CaD23 (at 25g/ml or 2x MIC) killed all the bacteria within 30 mins, which was 8 times faster than amikacin (25g/ml or 20x MIC). After 10 consecutive passages, CaD23 did not develop any antimicrobial resistance (AMR) whereas amikacin, a commonly used treatment for BK, developed significant AMR (i.e. a 32-fold increase in MIC). Pre-clinical murine studies showed that CaD23 (0.5mg/ml) achieved a median reduction of S. aureus bioburden by 94% (or 1.2 log10 CFU/ml) while not impeding corneal epithelial wound healing. In conclusion, rational hybridization of human-derived HDPs has led to generation of a potentially efficacious and safe topical antimicrobial agent for treating Gram-positive BK, with no/minimal risk of developing AMR.

microbiology↗

bioRxiv2019

Microfluidic protein isolation and sample preparation for high resolution cryo-EM

High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousands to a few million protein particles required for structure-determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a microfluidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than 1 L of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens new opportunities for the isolation of sensitive, endogenous protein complexes.

biophysics↗

bioRxiv2019

Cryo-EM structure of the rhodopsin-Gαi-βγ complex reveals binding of the rhodopsin C-terminal tail to the Gβ subunit

G protein-coupled receptors (GPCRs) are the largest class of integral membrane proteins and represent key targets for pharmacological research. GPCRs modulate cell physiology by engaging and activating a diversity of intracellular transducers, prominently heterotrimeric G proteins, but also G protein-receptor kinases (GRKs) and arrestins. The recent surge in the number of structures of GPCR-G protein complexes has expanded our understanding of G protein recognition and GPCR-mediated signal transduction. However, many aspects of these mechanisms, including the existence of transient interactions with transducers, have remained elusive. Here, we present the cryo-EM structure of the light-sensitive GPCR rhodopsin in complex with heterotrimeric Gi. In contrast to all reported structures, our density map reveals the receptor C-terminal tail bound to the G{beta} subunit of the G protein heterotrimer. This observation provides a structural foundation for the role of the C-terminal tail in GPCR signaling, and of G{beta} as scaffold for recruiting G subunits and GRKs. By comparing all available complex structures, we found a small set of common anchoring points that are G protein-subtype specific. Taken together, our structure and analysis provide new structural basis for the molecular events of the GPCR signaling pathway.

biochemistry↗