Biology subjects

Mittal, S.

Publications and source records attributed to Mittal, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Computational simulations reproduce in vivo population receptive field mapping fMRI results

Population receptive field (pRF) mapping is widely used to characterize retinotopic organization based on functional magnetic resonance imaging (fMRI) data. Despite its broad adoption, the factors governing intra- and inter-subject variability in pRF estimates remain incompletely understood, limiting the ability to evaluate and optimize visual stimulation paradigms prior to data collection. Here, we investigate whether large-scale simulations can reproduce in vivo run-to-run variability patterns observed in pRF mapping and provide mechanistic insight into their origins. With GEMSim-pRF, our newly proposed computational framework for large-scale simulation and estimation of pRF responses, we generated millions of synthetic fMRI time courses across a wide range of receptive field parameters and noise conditions. We analyzed the variability of pRF estimation results derived from simulations and compared them with in vivo data from the publicly available NYU Retinotopy Dataset. Here we show that our simulation results matched the characteristic eccentricity-dependent variability observed in empirical pRF estimates. These findings show that key variability patterns observed in empirical pRF mapping can be successfully reproduced in large-scale simulations, establishing simulation-based analysis as a practical approach for understanding, predicting, evaluating and ultimately improving the behaviour of retinotopic mapping paradigms before empirical data collection.

neuroscience↗

bioRxiv2026

TAOK3 inhibition constrains invasion, potentiates paclitaxel, and reprograms the tumor microenvironment toward anti-tumor immunity in cervical cancer

TAOK3 is a lesser-studied MAPK family serine/threonine kinase our group has shown to be targeted by HPV integration, suggesting a potential role in driving invasive cervical cancer (ICC). Here, we profiled TAOK3 expression in patient tumors, metastases, and cervical cancer models and localized TAOK3 within a tumor epithelial subpopulation by integrating two single-cell RNA-seq datasets. Functional consequences of TAOK3 loss were assessed with siRNA and CRISPRi in cell lines and 3D spheroids. In vivo effects were evaluated in intracervical xenografts with species-specific RNA-seq to resolve tumor versus microenvironmental responses. TAOK3 mRNA/protein were elevated in primary and metastatic ICC and primarily localized to a keratin-positive epithelial subset (T3epi) enriched for cadherin/S100 binding, vesicle/endocytic pathways, and leading-edge programs. TAOK3 silencing reprogrammed transcriptomes and proteomes toward reduced WNT/cell-cycle and motility signaling, altered endocytosis and cytoskeleton organization, and reshaped phospho-networks linked to chromatin remodeling and ERBB2-ERBB3/cytoskeletal kinase activity. Functionally, TAOK3 inhibition prolonged G2/M, suppressed invasion, and enhanced sensitivity to low dose paclitaxel. Prolonged inactivation induced methuosis-like cell death with extracellular ATP release. In xenografts, TAOK3 knockdown reduced tumor burden, downregulated KRT14--a leader cell marker--within the human tumor compartment, and enriched microenvironmental pathways for immune activation, with a specific decrease in CD206+ M2 macrophages. TAOK3 delineates an invasion-competent epithelial state in ICC and coordinates cell-cycle control, cytoskeleton-membrane dynamics, and tumor-immune crosstalk. Genetic or pharmacologic TAOK3 inhibition constrains tumor growth, potentiates paclitaxel, and remodels the microenvironment toward anti-tumor immunity, supporting TAOK3 as a potential therapeutic target and biomarker in ICC. Statement of SignificanceTAOK3 marks an invasion-competent epithelial subpopulation in cervical cancer. TAOK3 inhibition slows tumor growth, enhances chemoresponse, and reduces M2 macrophages, revealing TAOK3 as a potential therapeutic target and biomarker for patient stratification.

cancer biology↗

bioRxiv2026

PhenoBIC: operator-free single-cell spatial phenotyping in multiplex imaging data using deep learning of cell staining patterns

Multiplex imaging is a valuable tool for spatially examining tissue microenvironments at the single-cell level to uncover biological and clinical insights. However, most multiplex image analysis workflows currently require manual intervention for cell phenotyping, which slows progress, demands human effort, and yields operator-dependent outputs. Here, we developed PhenoBIC, a pre-trained deep learning model for image classification of the multiplexed biomarker signals in a cell (Biomarker Imprint of a Cell) to classify cell phenotypes. We show that PhenoBIC (F1-score [~]0.88) outperforms manual gating (widely used) and other machine learning-based computational approaches for cell marker expression classification. We validated this across multiple biomarkers, tissue sampling strategies (whole biopsies and tissue microarrays), multiplex panels, imaging platforms, and tissue types. We have released our in-house training and validation datasets of [~]1.4 million manually curated cell expression ground truth labels. We have also open-sourced PhenoBIC and enabled its community-wide deployment via the QuPath interface.

bioinformatics↗

bioRxiv2025

Non-apoptotic death of the C. elegans linker cell is primed by MYRF-1 activation of pqn-41/polyQ

Linker cell-type death (LCD) is a morphologically conserved non-apoptotic cell-death process with features resembling polyglutamine-dependent neurodegeneration. In C. elegans development, LCD eliminates the male-specific linker cell following its long-range migration. Using single-cell mRNA sequencing of migrating and dying linker cells, we identify myrf-1, encoding a membrane-bound transcription factor implicated in human developmental disorders, as a key LCD regulator. MYRF-1 translocates to the linker cell nucleus during early migration and, surprisingly, its auxin-inducible degradation then, but not later, blocks LCD. MYRF-1 directly binds known LCD genes, including pqn-41, encoding an aggregation-prone polyglutamine protein. Deleting a bona fide MYRF-1-binding site within pqn-41 promotes linker cell survival. Our findings reveal that linker cell death is primed well before cell demise takes place, temporally uncoupling death commitment and execution.

cell biology↗

bioRxiv2025

GEM-pRF: GPU-Empowered Mapping of Population Receptive Fields for Large-Scale fMRI Analysis

Population receptive field (pRF) mapping is a fundamental technique for understanding retinotopic organization of the human visual system. Since its introduction in 2008, however, its scalability has been severely hindered by the computational bottleneck of iterative parameter refinement. Current state-of-the-art implementations either sacrifice precision for speed or rely on slow iterative parameter updates, limiting their applicability to large-scale datasets. Here, we present a novel mathematical reformulation of the General Linear Model (GLM), wrapped in a GPU-Empowered Mapping of population Receptive Fields (GEM-pRF) software implementation. By orthogonalizing the design matrix, our approach enables the direct and fast computation of the objective functions derivatives, which are used to eliminate the iterative refinement process. This approach dramatically accelerates pRF estimation while maintaining full accuracy. Validation using empirical and simulated data confirms GEM-pRFs accuracy, and benchmarking against established tools demonstrates an order-of-magnitude reduction in computation time. With its modular and extensible design, GEM-pRF provides a critical advancement for large-scale fMRI retinotopic mapping. Furthermore, our reformulated GLM approach in combination with GPU-based implementation offer a broadly applicable solution that may extend beyond visual neuroscience, accelerating computational modelling across various domains in neuroimaging and beyond.

neuroscience↗

bioRxiv2024

Eukaryotic Translation Initiation Factor Loaded Extracellular Vesicles Promotes Macrophage Cholesterol Metabolism in ovarian cancer

Tumor-driven immune suppression poses a significant impediment to the success of immunotherapy in ovarian cancer. Among the various mechanisms contributing to immune suppression, intracellular communication facilitated by tumor-derived extracellular vesicles (EVs) within the tumor microenvironment (TME) emerges as a pivotal factor influencing tumor growth. We discovered that EVs from both ovarian tumor cell lines and the plasma of ovarian cancer patients are encapsulated with eukaryotic translation initiation factor 4E (eIF4E). Our study revealed a new mechanism showing how these EVs are loaded with eIF4E and its impact on ovarian cancer progression. We also demonstrated that eIF4E-containing EVs (eIF4E-EVs) alter protein translation in macrophages, contributing to anti-tumor immune response. Treatment of macrophages with eIF4E-EVs induces an immunosuppressive phenotype marked by the release of cytokines such as IL-6 and an elevated expression of Programmed death-ligand 1 (PD-L1). Notably, eIF4E-packaged EVs enhance the expression of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) a pivotal enzyme in cholesterol biosynthesis, resulting in increased cholesterol levels within macrophages. Inhibition of HMGCR or reduction of cholesterol in macrophages effectively restores their antitumor activity by decreasing PD-L1 on macrophages. Analysis of tumor tissue from ovarian cancer patients revealed a positive correlation between HMGCR and TAM in ovarian cancer. In summary, we have characterized the mechanism of how eIF4E loaded EVs induced cholesterol synthesis, creating an immunosuppressive environment by upregulating PD-L1 expression in macrophages.

cancer biology↗

bioRxiv2024

Biomechanical Evaluation of Femoral Neck Fracture Fixation Based on Finite Element Analysis

Pauwels type III fracture, wherein the fracture plane makes an angle of 50{degrees}-70{degrees} with the transverse plane, is the most unstable femoral neck fracture. Although there are various types of internal fixation devices, including Cannulated cancellous screws (CCS), dynamic hip screw (DHS), dynamic hip screw along with anti-rotation screws (DHS+ARS), and a newly introduced femoral neck system (FNS), patients with Pauwels type III fracture often experience implant failure. There was no study investigating the performance of these internal fixators under physiological musculoskeletal loading. The objective of the present study was to compare the biomechanical performance of CCS, DHS, DHS+ARS and FNS under daily physiological activities like normal walking and stair climbing. The unstable Pauwels type III fracture was virtually induced in a composite femur bone model and was fixed with all four internal fixators. Second-order tetrahedral elements were used to develop the FE models. The stresses, strains, and deformations of intact and reconstructed femur and implant components were compared to understand the biomechanical efficacy. FNS showed the highest inferior axial displacement of the femoral head while DHS+ARS exhibited the least inferior axial displacement. Among the four fracture fixation systems, DHS+ARS offered the highest post-fixation stability in terms of fracture gap opening (normal walking: 0.82mm and stair climbing: 0.95mm) and contact sliding, while the performance of single hole locking plated FNS was the worst (normal walking: 2.65mm, stair climbing: 3.72mm). Based on this computational study, DHS+ARS seems to be a better performing system in Pauwels type III fracture fixation.

bioengineering↗

bioRxiv2024

Microarray Integrated Spatial Transcriptomics (MIST) for Affordable, Robust, and Comprehensive Digital Pathology

10X Visium, a popular Spatial transcriptomics (ST) method, faces limited adoption due to its high cost and restricted sample usage per slide. To address these issues, we propose Microarray Integrated Spatial Transcriptomics (MIST), combining conventional tissue microarray (TMA) with Visium, using laser-cutting and 3D printing to enhance slide throughput. Our design facilitates independent replication and customization in individual labs to suit specific experimental needs. We provide a step-by-step guide from designing TMAs to the library preparation step. We demonstrate MISTs cost-effectiveness and technical benefits over Visium and GeoMx Nanostring. We also introduce AnnotateMap, a novel computational tool for efficient analysis of multiple ROIs processed through MIST.

bioinformatics↗

bioRxiv2023

Cell-type-resolved somatic mosaicism reveals clonal dynamics of the human forebrain

Debate remains around anatomic origins of specific brain cell subtypes and lineage relationships within the human forebrain. Thus, direct observation in the mature human brain is critical for a complete understanding of the structural organization and cellular origins. Here, we utilize brain mosaic variation within specific cell types as distinct indicators for clonal dynamics, denoted as cell-type-specific Mosaic Variant Barcode Analysis. From four hemispheres from two different human neurotypical donors, we identified 287 and 780 mosaic variants (MVs), respectively that were used to deconvolve clonal dynamics. Clonal spread and allelic fractions within the brain reveal that local hippocampal excitatory neurons are more lineage-restricted compared with resident neocortical excitatory neurons or resident basal ganglia GABAergic inhibitory neurons. Furthermore, simultaneous genome-transcriptome analysis at both a cell-type-specific and single-cell level suggests a dorsal neocortical origin for a subgroup of DLX1+ inhibitory neurons that disperse radially from an origin shared with excitatory neurons. Finally, the distribution of MVs across 17 locations within one parietal lobe reveals restrictions of clonal spread in the anterior-posterior axis precedes that of the dorsal-ventral axis for both excitatory and inhibitory neurons. Thus cell-type resolved somatic mosaicism can uncover lineage relationships governing the development of the human forebrain.

neuroscience↗

bioRxiv2023

RNA conformation and metal ion coordination in the nonenzymatic primer extension reaction center

The mechanism by which genetic information was copied prior to the evolution of ribozymes is of great interest because of its importance to the origin of life. The most effective known process for the nonenzymatic copying of an RNA template is primer extension by a two-step pathway in which 2-aminoimidazole activated nucleotides first react with each other to form an imidazolium-bridged intermediate that subsequently reacts with the primer. Reaction kinetics, structure-activity relationships, and X-ray crystallography have provided insight into the overall reaction mechanism, but many puzzles remain. In particular, high concentrations of Mg2+ are required for efficient primer extension, but the mechanism by which Mg2+ accelerates primer extension remains unknown. By analogy with the mechanism of DNA and RNA polymerases, a role for Mg2+ in facilitating the deprotonation of the primer 3'-hydroxyl is often assumed, but no catalytic metal ion is seen in crystal structures of the primer extension complex. To explore the potential effects of Mg2+ binding in the reaction center, we performed atomistic molecular dynamics simulations of a series of modeled complexes in which a Mg2+ ion was placed in the reaction center with inner sphere coordination to different sets of functional groups. Our simulations suggest that coordination of a Mg2+ ion to both O3' of the terminal primer nucleotide and the pro-Sp non- bridging oxygen of the reactive phosphate of an imidazolium-bridged dinucleotide would help to preorganize the structure of the primer/template substrate complex to favor the primer-extension reaction. Our results suggest that the catalytic metal ion may play an important role in overcoming electrostatic repulsion between a deprotonated O3' and the reactive phosphate of the bridged dinucleotide. Our simulations lead to testable predictions of the mode of Mg2+ binding that is most relevant to catalysis of primer extension. STATEMENT OF SIGNIFICANCEPrior to the evolution of complex enzymes, the replication of genetic material must have relied on nonenzymatic mechanisms. Nonenzymatic RNA template copying can be achieved through the extension of a primer by reaction with a 2-aminoimidazole (2AI) bridged dinucleotide in the presence of Mg2+. Despite progress in understanding the mechanism of this reaction, the catalytic role of Mg2+ remains poorly understood. Here, we present a series of molecular dynamics simulations of a model RNA primer-extension complex in different potential reactive conformations. We find that one configuration of both the 2AI moiety and coordination state of the Mg2+ promotes a geometry that is most favorable to reaction, suggesting a potential structural role for Mg2+ and providing insights to guide future experiments.

biophysics↗

bioRxiv2023

Experimental Tests of the Virtual Circular Genome Model for Non-enzymatic RNA Replication

The virtual circular genome (VCG) model was proposed as a means of going beyond template copying to indefinite cycles of nonenzymatic RNA replication during the origin of life. In the VCG model the protocellular genome is a collection of short oligonucleotides that map to both strands of a virtual circular sequence. Replication is driven by templated nonenzymatic primer extension on a subset of kinetically trapped partially base-paired configurations, followed by shuffling of these configurations to enable continued oligonucleotide elongation. Here we describe initial experimental studies of the feasibility of the VCG model for replication. We designed a small 12-nucleotide model VCG and synthesized all 247 oligonucleotides of length 2 to 12 corresponding to this genome. We experimentally monitored the fate of individual labeled primers in the pool of VCG oligonucleotides following the addition of activated nucleotides, and investigated factors such as oligonucleotide length, concentration, composition, and temperature on the extent of primer extension. We observe a surprisingly prolonged equilibration process in the VCG system that enables a considerable extent of reaction. We find that environmental fluctuations would be essential for continuous templated extension of the entire VCG system, since the shortest oligonucleotides can only bind to templates at low temperatures, while the longest oligonucleotides require high temperature spikes to escape from inactive configurations. Finally, we demonstrate that primer extension is significantly enhanced when the mix of VCG oligonucleotides is pre-activated. We discuss the necessity of ongoing in-situ activation chemistry for continuous and accurate VCG replication.

biochemistry↗

bioRxiv2023

Integrated ACMG approved genes and ICD codes for the translational research and precision medicine

Timely understanding of biological secrets of complex diseases will ultimately benefit millions of individuals by reducing the high risks for mortality and improving the quality of life with personalized diagnoses and treatments. Due to the advancements in sequencing technologies and reduced cost, genomics data is developing at an unmatched pace and levels to foster translational research and precision medicine. Over ten million genomics datasets have been produced and publicly shared in the year 2022. Diverse and high-volume genomics and clinical data have the potential to broaden the scope of biological discoveries and insights by extracting, analyzing, and interpreting the hidden information. However, the current and still unresolved challenges include the integration of genomic profiles of the patients with their medical records. The disease definition in genomics medicine is simplified, when in the clinical world, diseases are classified, identified, and adopted with their International Classification of Diseases (ICD) codes, which are maintained by the World Health Organization (WHO). Several biological databases have been produced, which includes information about human genes and related diseases. However, still, there is no database exists, which can precisely link clinical codes with relevant genes and variants to support genomic and clinical data integration for clinical and translation medicine. In this project, we are focused on the development of an annotated gene-disease-code database, which is accessible through an online, cross-platform, and user-friendly application i.e., PAS-GDC. However, our scope is limited to the integration of ICD-9 and ICD-10 codes with the list of genes approved by the American College of Medical Genetics and Genomics (ACMG). Results include over seventeen thousand diseases and four thousand ICD codes, and over eleven thousand gene-disease-code combinations.

bioinformatics↗

bioRxiv2022

An efficient method to generate fluorescent amyloid fibrils

Studies on fluorophore-tagged peptides help in elucidating the molecular mechanism of amyloidogenesis including their cellular internalization and crosstalk potential. Despite several advantages, unavoidable difficulties including expensive and tedious synthesis-protocols exist in fluorophore-based tools. Importantly, covalently-tagged fluorophores could introduce structural constraints which may influence the conformation of the monomeric and aggregated forms of protein. To resolve this problem, we describe a robust yet simple method to make fluorescent amyloid fibrils through non-covalent incorporation of fluorophores into amyloid fibrils. We used aggregation protocol in which a small amount of fluorophore is incorporated into the amyloids, and this protocol does not alter the aggregation kinetics and the characteristic {beta}-sheet-conformers of the generated amyloid fibrils. We have successfully prepared fluorescent amyloid fibrils of Insulin, Lysozyme and A{beta}1-42, and the noncovalently incorporated fluorophores remained intact in the amyloid fibrils without leaching, even after serial-dilutions and prolonged-storage. Further, this method enables successful monitoring of cellular-internalization of the fluorescent amyloids into SH-SY5Y and A549 cells, and it also detects FRET-signals during interfibrillar interactions. The findings establish a simple and affordable protocol to prepare fluorescent amyloid structures, which may significantly help amyloid researchers working on both in vitro and animal model systems.

biophysics↗

bioRxiv2022

Characterization of sub-tropical maize (Zea Mays L.) inbred lines for the variation in kernel row numbers (KRN)

Genetic dissection of high KRN trait and cob length has been undertaken by several researchers resulting in identification of loci controlling the traits. Further fine mapping of QTLs controlling KRN and cob length and TILLING strategies identified the underlying genes. All these studies are used temperate maize genotypes which are of limited use to researcher in sub-tropical region, and the sub-tropical maize production systems. Present investigation explores the availability of genetic regions responsible for KRN in sub-tropical maize germplasm. A total of 280 subtropical maize germplasm was analyzed for their KRN variation and selected 45 stable lines were subjected to molecular characterization using genes linked to KRN in maize. Diversity analysis was also performed to understand the possible association of character with the KRN gene in deciding its variation in the given population. It was revealed that four genes showed highest probability of influencing KRN traits in these tropical maize inbred lines. The remaining genes not establishing specific pattern of association with high and low KRN genotypes may need further study on its allelic variation.

plant biology↗

bioRxiv2022

Comprehensive multiomic profiling of somatic mutations in malformations of cortical development

Malformations of cortical development (MCD) are neurological conditions displaying focal disruption of cortical architecture and cellular organization arising during embryogenesis, largely from somatic mosaic mutations. Identifying the genetic causes of MCD has been a challenge, as mutations remain at low allelic fractions in brain tissue resected to treat epilepsy. Here, we report a genetic atlas from 317 brain resections, identifying 69 mutated genes through intensive profiling of somatic mutations, combining whole-exome and targeted-amplicon sequencing with functional validation and single-cell sequencing. Genotype-phenotype correlation analysis elucidated specific MCD gene sets associating distinct pathophysiological and clinical phenotypes. The unique spatiotemporal expression patterns identified by comparing single-nucleus transcriptional sequences of mutated genes in control and patient brains implicate critical roles in excitatory neurogenic pools during brain development, and in promoting neuronal hyperexcitability after birth.

genetics↗

bioRxiv2022

Wireless closed-loop smart bandage for chronic wound management and accelerated tissue regeneration

Chronic non-healing wounds represent a major source of morbidity for patients and a significant economic burden. Current wound care treatments are generally passive and are unable to adapt to changes in the wound environment in real time. By integrating multimodal sensors and adding stimulators in a bandage, real-time physiological monitoring is possible and provides an opportunity for active intervention into the complex wound environment. Here, we develop a battery-free flexible bioelectronic system consisting of wirelessly powered, closed-loop sensing and stimulation circuits with tissue-interfacing tough conducting hydrogel electrodes for robust signal transduction, on-demand adhesion, and detachment. Using multiple pre-clinical models, we demonstrate the capability of our wound care system to continuously monitor skin impedance and temperature, to trigger directional electrical stimulation. The accelerated wound closure was confirmed to be due to the activation of pro-regenerative genes linked to accelerated wound closure, increased neovascularization, and enhanced dermal recovery.

bioengineering↗

bioRxiv2022

Evaluating Human Mutation Databases for 'Treatability' Using Personalized Antisense Oligonucleotides

Genome sequencing in the clinic often allows patients to receive a molecular diagnosis. However, variants are most often evaluated for pathogenicity, neglecting potential "treatability", and thus often yielding limited clinical benefit. Several collaborative efforts now aim to provide a therapy based upon the genetic variants, even if the drug will benefit only a single patient. Antisense oligonucleotide (ASO) therapies, among others, offer attractive "programmable" and relatively safe platforms for individualized therapy. The landscape of "ASO-treatable" variants is largely uncharted, with new developments emerging for loss-of-function (LOF), haploinsufficient, and gain-of-function (GOF) variants. ASOs can access the genome to target splice-gain variants, poison exons, untranslated/regulatory regions, and naturally-occurring antisense transcripts. Many of these approaches have yet to be proven clinically beneficial, and it is unclear if disease in some patients has progressed past the point where benefit could reasonably be expected. Here we mine public variant databases to identify potential future therapeutic targets. We found that the majority of human pathogenic genetic variants have one or more approaches that could be targeted therapeutically, advantaging the many ways that ASOs can regulate gene expression. The future might see medical teams considering "treatability" when interpreting genome sequencing results, to fully realize benefits for patients.

genetics↗

bioRxiv2020

Reconciling Membrane Protein Simulations with Experimental DEER Spectroscopy Data

Spectroscopy experiments are crucial to study membrane proteins for which traditional structure determination methods still prove challenging. Double electron-electron resonance (DEER) spectroscopy experiments provide protein residue-pair distance distributions that are indicative of their conformational heterogeneity. Atomistic molecular dynamics (MD) simulations are another tool that have proved vital to study the structural dynamics of membrane proteins such as to identify inward-open, occluded, and outward-open conformations of transporter membrane proteins, among other partially open or closed states of the protein. Yet, studies have reported that there is no direct consensus between distributional data from DEER experiments and MD simulations, which has challenged validation of structures obtained from long-timescale simulations and using simulations to design experiments. Current coping strategies for comparisons rely on heuristics, such as mapping nearest matching peaks between two ensembles or biased simulations. Here we examine the differences in residue-pair distance distributions arising due to choice of membrane around the protein and covalent modification of a pair of residues to nitroxide spin labels in DEER experiments. Through comparing MD simulations of two proteins, PepTSo and LeuT - both of which have been characterized using DEER experiments previously - we show that the proteins dynamics are similar despite the choice of the detergent micelle as a membrane mimetic in DEER experiments. On the other hand, covalently modified residues show slight local differences in their dynamics and a huge divergence when the spin labels anointed oxygen atom pair distances are measured rather than protein backbone distances. Given the computational expense associated with pairwise MTSSL labeled MD simulations, we examine the use of biased simulations to explore the conformational dynamics of the spin labels only to reveal that such simulations alter the underlying protein dynamics. Our study identifies the main cause for the mismatch between DEER experiments and MD simulations and will accelerate developing potential mitigation strategies to improve simulation observables match with DEER spectroscopy experiments.

biophysics↗