Biology subjects

Zhang, J. T.

Publications and source records attributed to Zhang, J. T..

3 recordsLinked to original sources

bioRxiv2024

Cryo-EM phase-plate images reveal unexpected levels of apparent specimen damage

Apoferritin (apoF) is commonly used as a test specimen in single-particle electron cryo-microscopy (cryo-EM), since it consistently produces density maps that go to 3 [A] resolution or higher. When we imaged apoF with a laser phase plate (LPP), however, we observed more severe particle-to-particle variation in the images than we had previously thought to exist. Similarly, we found that images of ribulose bisphosphate carboxylase/oxygenase (rubisco) also exhibited a much greater amount of heterogeneity than expected. By comparison to simulations of images, we verified that the heterogeneity is not explained by the known features of the LPP, shot noise, or differences in particle orientation. We also demonstrate that our specimens are comparable to those previously used in the literature, based on using the final-reconstruction resolution as the metric for evaluation. All of this leads us to the hypothesis that the heterogeneity is due to damage that has occurred either during purification of the specimen or during preparation of the grids. It is not, however, our goal to explain the causes of heterogeneity; rather, we report that using the LPP has made the apparent damage too obvious to be ignored. In hindsight, similar heterogeneity can be seen in images of apoF and the 20S proteasome which others had recorded with a Volta phase plate. We therefore conclude that the increased contrast of phase-plate images (at low spatial frequencies) should also make it possible to visualize, on a single-particle basis, various forms of biologically functional heterogeneity in structure that had previously gone unnoticed. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/606536v1_ufig1.gif" ALT="Figure 1"> View larger version (101K): org.highwire.dtl.DTLVardef@1c85263org.highwire.dtl.DTLVardef@990352org.highwire.dtl.DTLVardef@1a920b3org.highwire.dtl.DTLVardef@a1fcf0_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIPhase plates recover low-frequency information with significantly improved SNR C_LIO_LILaser phase-plate images reveal unexpected amounts of structural heterogeneity C_LIO_LIIn retrospect, similar heterogeneity can also be seen in Volta phase-plate images C_LIO_LIParticle heterogeneity produces "structural noise", which may diminish map quality C_LI

biophysics↗

bioRxiv2024

Host evolution improves genetic circuit function in complex growth environments

Genetically engineered bacteria have become an attractive platform for numerous biomedical and industrial applications. Despite genetic circuitry functioning predictably under favorable growth conditions in the lab, the same cannot be said when placed in more complex environments for eventual deployment. Here, we used a combination of evolutionary and rational engineering approaches to enhance E. coli for robust genetic circuit behavior in non-traditional growth environments. We utilized adaptive laboratory evolution (ALE) on E. coli MG1655 in a minimal media with a sole carbon source and saw improved dynamics of a population-lysis-based circuit after host evolution. Additionally, we improved lysis circuit tolerance of a more clinically relevant strain, the probiotic E. coli Nissle, using ALE of the host strain in a more complex media environment with added reactive oxygen species (ROS) stress. We observed improved recovery from circuit-induced lysis in the evolved Nissle strain, and in combination with directed mutagenesis, recovered circuit function in the complex media. These findings serve as a proof-of-concept that relevant strains of bacteria can be optimized for improved growth and performance in complex environments using ALE and that these changes can modify and improve synthetic gene circuit function for real-world applications.

synthetic biology↗

bioRxiv2023

Overcoming resolution loss due to thermal magnetic field fluctuations from phase plates in transmission electron microscopy

We identify thermal magnetic field fluctuations, caused by thermal electron motion ("Johnson noise") in electrically conductive materials, as a potential resolution limit in transmission electron microscopy with a phase plate. Specifically, resolution loss can occur if the electron diffraction pattern is magnified to extend phase contrast to lower spatial frequencies, and if conductive materials are placed too close to the electron beam. While our initial implementation of a laser phase plate (LPP) was significantly affected by these factors, a redesign eliminated the problem and brought the performance close to the expected level. The resolution now appears to be limited by residual Johnson noise arising from the electron beam liner tube in the region of the LPP, together with the chromatic aberration of the relay optics. These two factors can be addressed during future development of the LPP.

molecular biology↗