Biology subjects

Bachochin, M. J.

Publications and source records attributed to Bachochin, M. J..

4 recordsLinked to original sources

bioRxiv2026

A Surfactant Cocktail Overcomes Air-Water Interface Artifacts in Single-Particle CryoEM

Single-particle cryogenic electron microscopy (cryoEM) is a widely used technique for structure determination of biomacromolecules to near-atomic resolution. Random distributions of these molecules in vitrified ice are necessary to accumulate enough two-dimensional views to generate a complete three-dimensional (3-D) reconstruction. However, interactions between the sample and the air-water interface (AWI) that occur during vitrification often bias the views of the sample, a phenomenon termed preferred orientation, limiting our ability to obtain 3-D reconstructions. Surfactants are often used as sample additives to prevent AWI-induced deterioration, but no general strategy exists for surfactant choice, requiring laborious screening for each sample. To circumvent these issues, we developed SurfACT, a cocktail of diverse surfactants with distinct physicochemical properties that limits AWI-dependent sample denaturation and orientation bias, while mitigating individual surfactant-specific drawbacks. Here we demonstrate SurfACTs effectiveness with four proteins plagued by AWI-induced issues, including two species of hemagglutinin (HA), molybdenum-iron protein (MoFeP) from the nitrogenase enzyme, and aldolase. All four samples show drastically improved viewing distribution and map completeness when SurfACT is applied. Cryogenic electron tomography demonstrates that SurfACT redistributes particles from the AWI into the bulk ice, driving signal recovery and inhibiting denaturation. This versatile sample additive minimizes sample-specific screening and expands the capabilities and range of suitable samples for cryoEM.

biophysics↗

bioRxiv2025

ANTIDOTE: A Metadata-Driven Neural Network for Improving CryoEM 3-D Particle Sorting

Despite the maturation of cryogenic electron microscopy (cryoEM) methodologies, generating high-resolution three-dimensional (3-D) reconstructions from micrographs is a time-intensive process involving iterative rounds of subjective data curation and hyperparameter optimization. Current approaches to particle classification are often unable to remove all low-quality particles from particle stacks, largely due to the low signal-to-noise ratio, the high dimensionality of particle images, and the multiple degrees of freedom associated with each particles unknown rotation, orientation, and class assignment. The retention of low-quality particles negatively affects the overall quality of the final EM density and continued efforts to eliminate their deleterious contributions are warranted. Here, we present ANTIDOTE (A Neural network Trained In Deleterious Object deTection and Elimination), a neural network framework that discriminates between constructive and deleterious particles using per-particle metadata generated during 3-D classification in RELION. Using benchmark and real-world cryoEM datasets, we demonstrate that ANTIDOTE paired with RELION 3-D classification achieves higher particle classification accuracy than conventional data processing approaches alone, yielding improvements in reconstruction quality, global and local resolution, and map interpretability while reducing time-consuming hyperparameter optimization. We additionally detail practical use-case scenarios for ANTIDOTE and demonstrate its versatility in increasing particle curation accuracy for high-quality cryoEM reconstruction.

biophysics↗

bioRxiv2025

An allosteric network governs Tom70 conformational dynamics to coordinate mitochondrial import

Tom70 mediates mitochondrial protein import by coordinating the transfer of cytosolic preproteins from Hsp70/Hsp90 to the translocase of the outer membrane (TOM) complex. In humans, the cytosolic domain of Tom70 (HsTom70c) is entirely -helical and comprises modular TPR motifs divided into an N-terminal chaperone-binding domain (NTD) and a C-terminal preprotein-binding domain (CTD). However, the mechanisms linking these functional regions remain poorly understood. Here, we present the 2.04 [A] crystal structure of unliganded HsTom70c, revealing two distinct conformations - open and closed - within the asymmetric unit. These states are stabilized in part by interdomain crystal contacts and are supported in solution by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations. Principal component and dynamical network analyses reveal a continuum of motion linking the NTD and CTD via key structural elements, notably residues in helices 7, 8, and 25. Engagement of the CTD by the viral protein Orf9b interrupts this network, stabilizing a partially-closed intermediate conformation and dampening dynamics at distal NTD sites. Collectively, our findings lay the groundwork for understanding Tom70 allostery and provide a framework for dissecting its mechanistic roles in chaperone engagement, mitochondrial import, and viral subversion.

biophysics↗

bioRxiv2022

An intrinsically disordered transcription activation domain alters the DNA binding affinity and specificity of NFκB p50/RelA

Many transcription factors contain intrinsically disordered transcription activation domains (TADs), which mediate interactions with co-activators to activate transcription. Historically, DNA-binding domains and TADs have been considered as modular units, but recent studies have shown that TADs can influence DNA binding. We biophysically characterized the NF{kappa}B p50/RelA heterodimer including the RelA TAD and investigated the TADs influence on NF{kappa}B-DNA interactions. In solution the RelA TAD is disordered but compact, with helical tendency in two regions that interact with co-activators. The presence of the TAD increased the stoichiometry of NF{kappa}B-DNA complexes containing promoter DNA sequences with tandem {kappa}B recognition motifs by promoting the binding of NF{kappa}B dimers in excess of the number of {kappa}B sites. We measured the binding affinity of p50/RelA for DNA containing tandem {kappa}B sites and single {kappa}B sites. While the presence of the TAD enhanced the binding affinity of p50/RelA for all {kappa}B sequences tested, it increased the affinity for non-specific DNA sequences by over 10-fold, leading to an overall decrease in specificity for {kappa}B DNA sequences. Our results reveal a novel function of the RelA TAD in promoting binding to non-consensus DNA previously observed by in vivo studies of NF{kappa}B-DNA binding in response to strong inflammatory signals.

biochemistry↗