scholarly journals Eliminating effects of particle adsorption to the air/water interface in single-particle cryo-electron microscopy: Bacterial RNA polymerase and CHAPSO

2018 ◽  
Author(s):  
James Chen ◽  
Alex J. Noble ◽  
Jin Young Kang ◽  
Seth A. Darst
Keyword(s):  
Rna Polymerase ◽  
Single Particle ◽  
Particle Orientation ◽  
Water Interface ◽  
Bacterial Transcription ◽  
Ionic Detergent ◽  
Wide Range ◽  
Air Water Interface ◽  
Transcription Complexes ◽  
Orientation Bias

AbstractPreferred particle orientation presents a major challenge for many single particle cryoelectron microscopy (cryo-EM) samples. Orientation bias limits the angular information used to generate three-dimensional maps and thus affects the reliability and interpretability of the structural models. The primary cause of preferred orientation is presumed to be due to adsorption of the particles at the air/water interface during cryo-EM grid preparation. To ameliorate this problem, detergents are often added to cryo-EM samples to alter the properties of the air/water interface. We have found that many bacterial transcription complexes suffer severe orientation bias when examined by cryo-EM. The addition of non-ionic detergents, such as NP-40, does not remove the orientation bias but the Zwitter-ionic detergent CHAPSO significantly broadens the particle orientation distributions, yielding isotropically uniform maps. We used cryoelectron tomography to examine the particle distribution within the ice layer of cryo-EM grid preparations of Escherichia coli 6S RNA/RNA polymerase holoenzyme particles. In the absence of CHAPSO, essentially all of the particles are located at the ice surfaces. CHAPSO at the critical micelle concentration eliminates particle absorption at the air/water interface and allows particles to randomly orient in the vitreous ice layer. We find that CHAPSO eliminates orientation bias for a wide range of bacterial transcription complexes containing E. coli or Mycobacterium tuberculosis RNA polymerases. Findings of this study confirm the presumed basis for how detergents can help remove orientation bias in cryo-EM samples and establishes CHAPSO as a useful tool to facilitate cryo-EM studies of baterial transcription complexes.

scholarly journals Measurements of adsorption at the air-water interface by the microtome method

1936 ◽  
Vol 154 (883) ◽  
pp. 608-623 ◽  
Keyword(s):  
Dynamic Method ◽  
Fundamental Importance ◽  
Molecular Adsorption ◽  
Water Interface ◽  
Experimental Demonstration ◽  
Gibbs Equation ◽  
Experimental Conditions ◽  
Moving Surface ◽  
Wide Range ◽  
Air Water Interface

In spite of the wide use and fundamental importance of the classical Gibbs adsorption theorem, its validity has never been given adequate experimental demonstration. Until quite recently the principal means available for testing this theorem was the “moving bubble method”, developed by Donnan and Barker, and later by McBain, Davies, and DuBois. Almost without exception this method has given results many times greater than the values calculated from either the exact or the approximate Gibbs equation. A recent exhaustive investigation of this dynamic method by DuBois and Todd has shown, moreover, that the results for moving bubbles may be varied and controlled over a wide range by merely altering the size or speed of the bubbles or the amount of accompanying liquid. Thus the results, although definite and repro­ducible, vary greatly with the experimental conditions, and hence they bear no definite relation either to the Gibbs value or to that for mono-molecular adsorption. It is evident that a moving surface carries in general an amount of adsorbed material which is much greater than that predicted by the Gibbs theorem. Similar high results are reported by Seymour, Tartar, and Wright for moving droplets of benzene in water, which may carry with them as much soap as would correspond to twenty or more mono-layers.

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 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 Current limitations to high-resolution structure determination by single-particle cryoEM

2021 ◽  
Vol 54 ◽  
Author(s):  
Edoardo D'Imprima ◽  
Werner Kühlbrandt
Keyword(s):  
Structure Determination ◽  
Single Particle ◽  
Specimen Preparation ◽  
Water Interface ◽  
New Techniques ◽  
Persistent Problem ◽  
Air Water Interface ◽  
High Resolution Structure ◽  
Future Potential ◽  
Multiple Conformations

Abstract CryoEM has become the method of choice for determining the structure of large macromolecular complexes in multiple conformations, at resolutions where unambiguous atomic models can be built. Two effects that have limited progress in single-particle cryoEM are (i) beam-induced movement during image acquisition and (ii) protein adsorption and denaturation at the air-water interface during specimen preparation. While beam-induced movement now appears to have been resolved by all-gold specimen support grids with very small holes, surface effects at the air-water interface are a persistent problem. Strategies to overcome these effects include the use of alternative support films and new techniques for specimen deposition. We examine the future potential of recording perfect images of biological samples for routine structure determination at atomic resolution.

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

Pressure fluctuations and interfacial robustness in turbulent flows over superhydrophobic surfaces

2015 ◽  
Vol 783 ◽  
pp. 448-473 ◽  
Author(s):  
J. Seo ◽  
R. García-Mayoral ◽  
A. Mani
Keyword(s):  
Drag Reduction ◽  
Numerical Simulations ◽  
Superhydrophobic Surface ◽  
Stagnation Pressure ◽  
Direct Numerical Simulations ◽  
Superhydrophobic Surfaces ◽  
Water Interface ◽  
System Parameters ◽  
Wide Range ◽  
Air Water Interface

Superhydrophobic surfaces can entrap gas pockets within their grooves when submerged in water. Such a mixed-phase boundary is shown to result in an effective slip velocity on the surface, and has promising potential for drag reduction and energy-saving in hydrodynamic applications. The target flow regime, in most such applications, is a turbulent flow. Previous analyses of this problem involved direct numerical simulations of turbulence with the superhydrophobic surface modelled as a flat boundary, but with a heterogeneous mix of slip and no-slip boundary conditions corresponding to the surface texture. Analysis of the kinematic data from these simulations has helped to establish the magnitude of drag reduction for various texture topologies. The present work is the first investigation that, alongside a kinematic investigation, addresses the robustness of superhydrophobic surfaces by studying the load fields obtain from data from direct numerical simulations (DNS). The key questions at the focus of this work are: does a superhydrophobic surface induce a different pressure field compared to a flat surface? If so, how does this difference scale with system parameters, and when does it become significant that it can deform the air–water interface and potentially rapture the entrapped gas pockets? To this end, we have performed DNS of turbulent channel flows subject to superhydrophobic surfaces over a wide range of texture sizes spanning values from $L^{+}=6$ to $L^{+}=155$ when expressed in terms of viscous units. The pressure statistics at the wall are decomposed into two contributions: one coherent, caused by the stagnation of slipping flow hitting solid posts, and one time-dependent, caused by overlying turbulence. The results show that the larger texture size intensifies the contribution of stagnation pressure, while the contribution from turbulence is essentially insensitive to $L^{+}$. The two-dimensional stagnation pressure distribution at the wall and the pressure statistics in the wall-normal direction are found to be self-similar for different $L^{+}$. The scaling of the induced pressure and the consequent deformations of the air–water interface are analysed. Based on our results, an upper bound on the texture wavelength is quantified that limits the range of robust operation of superhydrophobic surfaces when exposed to high-speed flows. Our results indicate that when the system parameters are expressed in terms of viscous units, the main parameters controlling the problem are $L^{+}$ and a Weber number based on inner dimensions; We obtain good collapse when all our results are expressed in wall units, independently of the Reynolds number.

scholarly journals Assembly of iron oxide nanosheets at the air–water interface by leucine–histidine peptides

RSC Advances ◽  
2021 ◽  
Vol 11 (45) ◽  
pp. 27965-27968
Author(s):  
Nina Hoinkis ◽  
Helmut Lutz ◽  
Hao Lu ◽  
Thaddeus W. Golbek ◽  
Mikkel Bregnhøj ◽  
...  
Keyword(s):  
Iron Oxide ◽  
Water Interface ◽  
Wide Range ◽  
Air Water Interface ◽  
Inorganic Nanomaterials

The fabrication of inorganic nanomaterials is important for a wide range of disciplines.

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.

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