Biology subjects

Maingi, V.

Publications and source records attributed to Maingi, V..

3 recordsLinked to original sources

bioRxiv2026

Depth-resolved analysis reveals target-dependent cryo-FIB damage profiles

Cryogenic focused ion beam (cryo-FIB) milling enables cellular cryo-electron tomography but can compromise high-resolution structural information near milled surfaces. Here we establish recombinant human apoferritin (apoF) as a reporter of depth-dependent structural preservation in cellular lamellae. ApoF yielded a 2.05 [A] in situ reconstruction. Apparent FIB damage was most pronounced within the first 15 nm for xenon, 30 nm for gallium, 45 nm for argon and remained detectable at least 75 nm from oxygen-milled surfaces under the workflows tested. Endogenous 70S ribosomes in the same xenon-milled tomograms showed a broader 30-45 nm profile, whereas {beta}-galactosidase showed a similar narrow xenon-associated profile in a different specimen. Xenon-milled lamellae 125-150 nm thick yielded the highest resolution among the thickness groups examined, with lower resolution in the thinner and thickest groups. These findings show structural preservation is constrained by both surface-associated damage and lamella thickness, with the apparent extent of damage varying by molecular reporter. Previous ribosome-based surface-exclusion estimates may therefore be overly conservative for some molecular targets.

molecular biology↗

bioRxiv2026

Transition Intermediates Encode Ligand Efficacy at the μ-Opioid Receptor-Gi Protein Complex

How ligands with different efficacies regulate G protein-coupled receptor signaling remains incompletely understood. Here, we combine Markov state modeling of nearly one millisecond of aggregate all-atom molecular dynamics simulations with time-resolved cryogenic-electron microscopy (cryo-EM) to reconstruct the most probable transition pathways connecting recently resolved structures of guanosine triphosphate (GTP)-bound -opioid receptor (MOR)-Gi1 complexes with the full agonist lofentanil (LFT) or partial agonist mitragynine pseudoindoxyl (MP). The models predict four previously unresolved intermediate conformations. Guided by these predictions, reanalysis of the cryo-EM particle ensemble identifies conformational populations consistent with all four intermediates. Remarkably, the largest ligand-dependent structural differences emerge within these intermediates rather than in the previously resolved states. LFT favors a predominantly sequential and kinetically efficient transition pathway, whereas MP stabilizes a broader intermediate ensemble along this pathway and slows progression toward later states. Together, these complementary approaches provide an enriched thermodynamic and kinetic description of MOR-mediated G protein activation, revealing that opioid efficacy is encoded, at least in part, in the differential stabilization and kinetics of transition intermediates extending beyond GTP binding but well before complete G protein dissociation.

biophysics↗

bioRxiv2022

Digital nanoreactors for control over absolute stoichiometry and spatiotemporal behavior of receptors within lipid bilayers

Interactions between membrane proteins are essential for cell survival and proper function, but the structural and mechanistic details of these interactions are often poorly understood. Even the biologically functional ratio of protein components within a multi-subunit membrane complex--the native stoichiometry--is difficult to establish. We have demonstrated digital nanoreactors that can control interactions between lipid-bound molecular receptors along three key dimensions: stoichiometric, spatial, and temporal. Each nanoreactor is based on a DNA origami ring, which both templates the synthesis of a liposome and provides tethering sites for DNA-based receptors. Receptors are released into the liposomal membrane using strand displacement and a DNA logic gate measures receptor heterodimer formation. High-efficiency tethering of receptors enables the kinetics of receptors in 1:1 and 2:2 absolute stoichiometries to be observed by bulk fluorescence in a plate reader which in principle is generalizable to any ratio. Similar single molecule in bulk experiments using DNA-linked membrane proteins could determine native stoichiometry and the kinetics of membrane protein interactions for applications ranging from signalling research to drug discovery.

bioengineering↗