Shah, P. N. M. | Search bioRxiv

bioRxiv2026

Rapid femtosecond laser trenching accelerates cryo-electron tomography lamella workflows

Transmission electron microscopy requires electron-transparent specimens, typically below 200 nm thick. For cellular and tissue samples this is typically achieved by focused ion-beam (FIB) thinning to form lamellae. Milling times scale with specimen thickness, and high-pressure-frozen specimens more than 10 m thick can take up to 2 hours per lamella. We present a pre-processing approach in which bulk material is removed by ultrashort-pulse laser ablation prior to milling, rapidly generating large trenches in vitrified grids under cryogenic conditions and leaving material to be finished by conventional milling to yield electron-transparent lamellae. Laser ablation completes in seconds (4 seconds per pattern), compared with minutes to tens of minutes for FIB trench milling, and introduces negligible damage to the final lamellae. By replacing the most time-intensive, contamination-sensitive bulk-removal steps with seconds-long laser trenching, this open-source approach reduces time-to-lamellae, increasing throughput and preserves compatibility with standard cryo-FIB/SEM polishing and cryo-ET data-collection pipelines.

biophysics↗

bioRxiv2023

A robust normalized local filter to estimate occupancy directly from cryo-EM maps

Cryo electron microscopy (cryo-EM) is used by biological research to visualize biomolecular complexes in 3D, but the heterogeneity of cryo-EM reconstructions is not easily estimated. Current processing paradigms nevertheless exert great effort to reduce flexibility and heterogeneity to improve the quality of the reconstruction. Clustering algorithms are typically employed to identify populations of data with reduced variability, but lack assessment of remaining heterogeneity. We have developed a fast and simple algorithm based on spatial filtering to estimate the heterogeneity of a reconstruction. In the absence of flexibility, this estimate approximates macromolecular component occupancy. We show that our implementation can derive reliable input parameters automatically, that the resulting estimate is accurate, and the reconstruction can be modified accordingly to emulate altered constituent occupancy, which may benefit conventionally employed maximum-likelihood classification methods. Here, we demonstrate the utility of this method for cryo-EM map interpretation, quantification, and particle-image signal subtraction.

bioinformatics↗

bioRxiv2020

Cryo-EM structures reveal two distinct conformational states in a picornavirus cell entry intermediate.

The virions of enteroviruses such as poliovirus undergo a global conformational change after binding to the cellular receptor, characterized by a 4% expansion, and opening of holes at the two and quasi-three-fold symmetry axes of the capsid. The resultant particle is called a 135S particle or A-particle and is thought to be on the pathway to a productive infection. Previously published studies have concluded that the membrane interactive peptides, namely VP4 and the N-terminus of VP1, are irreversibly externalized in the 135S particle. However, using established protocols to produce the 135S particle, and single particle cryo-electron microscopy methods, we have identified at least two unique states that we call the early and late 135S particle. Surprisingly, only in the "late" 135S particles have detectable levels of the VP1 N-terminus trapped outside the capsid. Moreover, we observe a distinct density inside the capsid that can be accounted for by VP4 that remains associated with the genome. Taken together our results conclusively demonstrate that the 135S particle is not a unique conformation, but rather a family of conformations that could exist simultaneously. AUTHOR SUMMARYNonenveloped viruses need to provide mechanisms that allow their genomes to be delivered across membrane. This process remains poorly understood. For enterovirus such as poliovirus, genome delivery involves a program of conformational changes that include expansion of the particle and externalization of two normal internal peptides, VP4 and the VP1 N-terminus, which then insert into the cell membrane, triggering endocytosis and the creation of pores that facilitate the transfer of the viral RNA genome across the endosomal membrane. This manuscript describes five high-resolution cryo-EM structures of altered poliovirus particles that represent a number of intermediates along this pathway. The structures reveal several surprising findings, including the discovery of a new intermediate that is expanded but has not yet externalized the membrane interactive peptides, the clear identification of a unique exit site VP1 N-terminus, the demonstration that the externalized VP1 N-terminus partitions between two different sites in a temperature-dependent fashion, direct visualization of an amphipathic helix at the N-terminus of VP1 in an ideal position for interaction with cellular membranes, and the observation that a significant portion of VP4 remains inside the particle and accounts for a feature that had been previously ascribed to part of the viral RNA. These findings represent significant additions to our understanding of the cell entry process of an important class of human pathogens.

biophysics↗