Proceedings of the National Academy of Sciences · Publication/issue date: 2020-01-14
Authors: Yimo Han, Xiao Fan, Haozhe Wang, Fang Zhao, Christopher G. Tully, Jing Kong, Nan Yao, Nieng Yan
The support surface of a cryo-EM grid affects ice quality, protein adsorption, and the number of particles available for reconstruction. This paper presents a method for preparing monolayer graphene grids with up to 99% monolayer coverage. More than 70% of grid squares are suitable for productive data collection, with improvements in image quality and protein density. Using these grids, the authors determined a 2.6 Å structure of the 52 kDa protein streptavidin from only about 11,000 particles. Across the soluble protein, membrane protein, and lipoprotein samples tested, particle density increased at least fivefold, opening up opportunities for structural studies of samples that are available in limited quantities or difficult to concentrate. The method uses simple tools available in most structural biology laboratories and is compatible with a range of nanomaterials, laying the groundwork for designing support surfaces tailored to specific proteins.
Highlight · High-coverage monolayer graphene grids increase particle density and enable structure determination of small proteins with fewer particles.
02 · Application of Monolayer Graphene to Cryo-Electron Microscopy Grids for High-resolution Structure Determination
Journal of Visualized Experiments (JoVE) · Publication/issue date: 2023-11-10
Authors: Andrew V. Grassetti, Mira B. May, Joseph H. Davis
Sample preparation remains a major bottleneck in cryo-EM structure determination: particle distribution in the suspended ice layer and interactions with the air–water interface can limit data quality. This methods paper describes a workflow for adding a continuous monolayer graphene support to grids, aiming to increase particle density in imaging regions and reduce particle contact with the air–water interface. It also provides a rapid method for assessing the relative hydrophilicity of grids and an electron microscopy approach for confirming the presence of the support layer using graphene’s characteristic diffraction pattern, pairing surface treatment with quality checks. As a demonstration, the authors used purified Cas9 complexes at a relatively low concentration to rapidly obtain a 2.7 Å density map, illustrating the practical value of this support strategy for structure determination. The paper’s contribution is to bring grid treatment, surface assessment, and structural validation together in a workflow with clear checkpoints.
Highlight · Combining graphene support preparation with quality assessment enables high-resolution reconstruction of Cas9 complexes at low concentrations.
03 · Chemical disaggregation of alpha-synuclein fibrils as a therapy for synucleinopathies
Proceedings of the National Academy of Sciences · Publication/issue date: 2023-03-14
Authors: Shenjie Wu, Nancy C. Hernandez Villegas, Randy Schekman
This commentary discusses research by Murray and colleagues on the potential of small molecules to disaggregate α-synuclein fibrils as a treatment for synucleinopathies, rather than reporting an independent set of new experiments. The authors describe how the compounds can disaggregate both preformed fibrils and fibrils derived from patient brain tissue in vitro, while reducing their ability to seed further aggregation; a nematode model also showed fewer aggregate puncta. The article stresses that fibril breakdown alone does not establish therapeutic value, because small fragments produced by incomplete disaggregation may increase seeding activity. Structural heterogeneity among fibrils from different diseases, along with drug selectivity, toxicity, and brain exposure, poses further challenges. Although mouse experiments suggested that the compounds could enter the brain to some extent, the exposure levels measured at the time were insufficient to support disaggregation in vivo. The commentary frames these findings as early progress that still requires optimization and validation.
Summary based on the full text of the original article.
Highlight · For fibril disaggregation to become a viable treatment, it must also reduce seeding activity, and issues of brain exposure and safety must be addressed.
This issue features three articles. Two focus on monolayer graphene grids for cryo-electron microscopy, highlighting their ability to increase particle density, improve image quality, and enable high-resolution reconstruction from low-concentration samples. They also describe preparation and quality assessment workflows. The third, a commentary, explores the therapeutic potential of small molecules that disassemble α-synuclein fibrils. It stresses that disassembly must also reduce seeding activity, while challenges involving fibril heterogeneity, brain exposure, and safety remain unresolved. This research is still at an early stage.
This issue features three papers covering graphene cryo-EM grids and the disaggregation of α-synuclein fibrils.
Graphene cryo-EM grids (2 papers)
01 · High-yield monolayer graphene grids for near-atomic resolution cryoelectron microscopy
Proceedings of the National Academy of Sciences · Publication/issue date: 2020-01-14
Authors: Yimo Han, Xiao Fan, Haozhe Wang, Fang Zhao, Christopher G. Tully, Jing Kong, Nan Yao, Nieng Yan
The support surface of a cryo-EM grid affects ice quality, protein adsorption, and the number of particles available for reconstruction. This paper presents a method for preparing monolayer graphene grids with up to 99% monolayer coverage. More than 70% of grid squares are suitable for productive data collection, with improvements in image quality and protein density. Using these grids, the authors determined a 2.6 Å structure of the 52 kDa protein streptavidin from only about 11,000 particles. Across the soluble protein, membrane protein, and lipoprotein samples tested, particle density increased at least fivefold, opening up opportunities for structural studies of samples that are available in limited quantities or difficult to concentrate. The method uses simple tools available in most structural biology laboratories and is compatible with a range of nanomaterials, laying the groundwork for designing support surfaces tailored to specific proteins.
Highlight · High-coverage monolayer graphene grids increase particle density and enable structure determination of small proteins with fewer particles.
DOI: 10.1073/pnas.1919114117
02 · Application of Monolayer Graphene to Cryo-Electron Microscopy Grids for High-resolution Structure Determination
Journal of Visualized Experiments (JoVE) · Publication/issue date: 2023-11-10
Authors: Andrew V. Grassetti, Mira B. May, Joseph H. Davis
Sample preparation remains a major bottleneck in cryo-EM structure determination: particle distribution in the suspended ice layer and interactions with the air–water interface can limit data quality. This methods paper describes a workflow for adding a continuous monolayer graphene support to grids, aiming to increase particle density in imaging regions and reduce particle contact with the air–water interface. It also provides a rapid method for assessing the relative hydrophilicity of grids and an electron microscopy approach for confirming the presence of the support layer using graphene’s characteristic diffraction pattern, pairing surface treatment with quality checks. As a demonstration, the authors used purified Cas9 complexes at a relatively low concentration to rapidly obtain a 2.7 Å density map, illustrating the practical value of this support strategy for structure determination. The paper’s contribution is to bring grid treatment, surface assessment, and structural validation together in a workflow with clear checkpoints.
Highlight · Combining graphene support preparation with quality assessment enables high-resolution reconstruction of Cas9 complexes at low concentrations.
DOI: 10.3791/66023
Other literature (1 paper)
03 · Chemical disaggregation of alpha-synuclein fibrils as a therapy for synucleinopathies
Proceedings of the National Academy of Sciences · Publication/issue date: 2023-03-14
Authors: Shenjie Wu, Nancy C. Hernandez Villegas, Randy Schekman
This commentary discusses research by Murray and colleagues on the potential of small molecules to disaggregate α-synuclein fibrils as a treatment for synucleinopathies, rather than reporting an independent set of new experiments. The authors describe how the compounds can disaggregate both preformed fibrils and fibrils derived from patient brain tissue in vitro, while reducing their ability to seed further aggregation; a nematode model also showed fewer aggregate puncta. The article stresses that fibril breakdown alone does not establish therapeutic value, because small fragments produced by incomplete disaggregation may increase seeding activity. Structural heterogeneity among fibrils from different diseases, along with drug selectivity, toxicity, and brain exposure, poses further challenges. Although mouse experiments suggested that the compounds could enter the brain to some extent, the exposure levels measured at the time were insufficient to support disaggregation in vivo. The commentary frames these findings as early progress that still requires optimization and validation.
Summary based on the full text of the original article.
Highlight · For fibril disaggregation to become a viable treatment, it must also reduce seeding activity, and issues of brain exposure and safety must be addressed.
DOI: 10.1073/pnas.2300965120