scholarly journals Reducing effects of particle adsorption to the air-water interface in cryoEM

2018 ◽  
Author(s):  
Alex J. Noble ◽  
Hui Wei ◽  
Venkata P. Dandey ◽  
Zhening Zhang ◽  
Clinton S. Potter ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Dwell Time ◽  
Liquid Films ◽  
Thin Liquid Films ◽  
Water Interface ◽  
Cryo Electron Microscopy ◽  
Air Water Interface ◽  
Protein Particles ◽  
Particle Adsorption

AbstractMost protein particles prepared in vitreous ice for single particle cryo-electron microscopy are adsorbed to air-water or substrate-water interfaces, potentially causing particles to adopt preferred orientations. Using the Spotiton robot and nanowire grids, we can significantly reduce air-water interface issues by decreasing the dwell time of particles in thin liquid films. We demonstrate this by using single particle cryoEM and cryoET on three biological samples.

scholarly journals Amino and PEG-Amino Graphene Oxide Grids Enrich and Protect Samples for High-resolution Single Particle Cryo-electron Microscopy

2019 ◽  
Author(s):  
Feng Wang ◽  
Zanlin Yu ◽  
Miguel Betegon ◽  
Melody Campbell ◽  
Tural Aksel ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Graphene Oxide ◽  
High Resolution ◽  
Single Particle ◽  
Particle Distribution ◽  
Water Interface ◽  
Amino Groups ◽  
Cryo Electron Microscopy ◽  
Air Water Interface ◽  
Resolution Single

AbstractCryo-EM samples prepared using the traditional methods often suffer from too few particles, poor particle distribution, or strongly biased orientation, or damage from the air-water interface. Here we report that functionalization of graphene oxide (GO) coated grids with amino groups concentrates samples on the grid with improved distribution and orientation. By introducing a PEG spacer, particles are kept away from both the GO surface and the air-water interface, protecting them from potential denaturation.

scholarly journals Effect of charge on protein preferred orientation at the air-water interface in cryo-electron microscopy

2021 ◽  
Author(s):  
Bufan Li ◽  
Dongjie Zhu ◽  
Huigang Shi ◽  
Xinzheng Zhang
Keyword(s):  
Electron Microscopy ◽  
Preferred Orientation ◽  
Water Interface ◽  
Protein Orientation ◽  
Cryo Electron Microscopy ◽  
Charged Interface ◽  
Cationic Detergents ◽  
Air Water Interface ◽  
Orientation Distributions ◽  
Protein Particles

The air-water interface (AWI) tends to absorb proteins and frequently causes preferred orientation problems in cryo-electron microscopy (cryo-EM). Here, we examined cryo-EM data from protein samples frozen with different detergents and found that both anionic and cationic detergents promoted binding of proteins to the AWI. By contrast, nonionic and zwitterionic detergents tended to prevent proteins from attaching to the AWI. This ability was positively associated with the critical micelle concentration of the detergent. The protein orientation distributions with different anionic detergents were similar and resembled that obtained without detergent. By contrast, cationic detergents gave distinct orientation distributions. The AWI is negatively charged and the absorption of cationic detergents to the AWI alters its charge. Our results indicates that proteins absorb to charged interface and the negative charge of the AWI plays an important role in absorbing proteins in the conventional cryo-EM sample preparation. According to these findings, a new method was developed to modify the charge distribution of the AWI by adding a very low concentration of anionic detergent. Using this method, the protein particles exhibited a more evenly distributed orientations and still absorbed to the AWI enabling them embedding in a thin layer of ice, which will benefit the cryo-EM structural determination.

Tracking the interfacial dynamics of PNiPAM soft microgels particles adsorbed at the air–water interface and in thin liquid films

2013 ◽  
Vol 52 (5) ◽  
pp. 445-454 ◽  
Author(s):  
Yann Cohin ◽  
Maelle Fisson ◽  
Kévin Jourde ◽  
Gerald G. Fuller ◽  
Nicolas Sanson ◽  
...  
Keyword(s):  
Liquid Films ◽  
Thin Liquid Films ◽  
Water Interface ◽  
Interfacial Dynamics ◽  
Air Water Interface

scholarly journals Cryoet of Single Particle CryoEM Grids Reveals Widespread, but Reducible, Particle Adsorption to the Air-Water Interface

2019 ◽  
Vol 116 (3) ◽  
pp. 11a ◽  
Author(s):  
Alex J. Noble ◽  
Venkata P. Dandey ◽  
Hui Wei ◽  
Julia Brasch ◽  
Jillian Chase ◽  
...  
Keyword(s):  
Single Particle ◽  
Water Interface ◽  
Air Water Interface ◽  
Particle Adsorption

scholarly journals CryoET of Single Particle CryoEM Grids Reveals Widespread Particle Adsorption to the Air-Water Interface, Which May be Reduced with New Plunging Techniques

2018 ◽  
Vol 24 (S1) ◽  
pp. 872-873
Author(s):  
Alex J. Noble ◽  
Venkata P. Dandey ◽  
Hui Wei ◽  
Julia Brasch ◽  
Jillian Chase ◽  
...  
Keyword(s):  
Single Particle ◽  
Water Interface ◽  
Air Water Interface ◽  
Particle Adsorption

scholarly journals A novel cryo-electron microscopy support film based on 2D crystal of HFBI protein

2021 ◽  
Author(s):  
Hongcheng Fan ◽  
Bo Wang ◽  
Yan Zhang ◽  
Yun Zhu ◽  
Bo Song ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Electrostatic Interactions ◽  
Preferred Orientation ◽  
Particle Orientation ◽  
Quality Data ◽  
Water Interface ◽  
Small Proteins ◽  
Cryo Electron Microscopy ◽  
Air Water Interface

Cryo-electron microscopy (cryo-EM) has become the most powerful tool to resolve the high-resolution structures of biomacromolecules in solution. However, the air-water interface induced preferred orientation, dissociation or denaturation of biomacromolecules during cryo-vitrification is still a major limitation factor for many specimens. To solve this bottleneck, we developed a new type of cryo-EM support film using the 2D crystal of hydrophobin I (HFBI) protein. The HFBI-film utilizes its hydrophilic side to adsorb protein particles via electrostatic interactions and keep air-water interface away, allowing thin-enough ice and high-quality data collection. The particle orientation distribution can be optimized by changing the buffer pH. We, for the first time, solved the cryo-EM structure of catalase (2.28 Å) that exhibited strong preferred orientation using conventional cryo-vitrification protocol. We further proved the HFBI-film is suitable to solve the high-resolution structures of small proteins including aldolase (150 kDa, 3.34 Å) and hemoglobin (64 kDa, 3.6 Å). Our work implied that the HFBI-film will have a wide application in the future to increase the successful rate and efficiency of cryo-EM.

scholarly journals A cryo-electron microscopy support film formed by 2D crystals of hydrophobin HFBI

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hongcheng Fan ◽  
Bo Wang ◽  
Yan Zhang ◽  
Yun Zhu ◽  
Bo Song ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Electrostatic Interactions ◽  
Particle Orientation ◽  
Quality Data ◽  
Water Interface ◽  
Small Proteins ◽  
Cryo Electron Microscopy ◽  
Air Water Interface ◽  
2D Crystals

AbstractCryo-electron microscopy (cryo-EM) has become a powerful tool to resolve high-resolution structures of biomacromolecules in solution. However, air-water interface induced preferred orientations, dissociation or denaturation of biomacromolecules during cryo-vitrification remains a limiting factor for many specimens. To solve this bottleneck, we developed a cryo-EM support film using 2D crystals of hydrophobin HFBI. The hydrophilic side of the HFBI film adsorbs protein particles via electrostatic interactions and sequesters them from the air-water interface, allowing the formation of sufficiently thin ice for high-quality data collection. The particle orientation distribution can be regulated by adjusting the buffer pH. Using this support, we determined the cryo-EM structures of catalase (2.29 Å) and influenza haemagglutinin trimer (2.56 Å), which exhibited strong preferred orientations using a conventional cryo-vitrification protocol. We further show that the HFBI film is suitable to obtain high-resolution structures of small proteins, including aldolase (150 kDa, 3.28 Å) and haemoglobin (64 kDa, 3.6 Å). Our work suggests that HFBI films may have broad future applications in increasing the success rate and efficiency of cryo-EM.

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