scholarly journals Need for speed: Examining protein behaviour during cryoEM grid preparation at different timescales

2020 ◽  
Author(s):  
David P. Klebl ◽  
Molly S. C. Gravett ◽  
Dimitrios Kontziampasis ◽  
David J. Wright ◽  
Robin S. Bon ◽  
...  
Keyword(s):  
Sample Preparation ◽  
New Technologies ◽  
Water Interface ◽  
Time Resolved ◽  
Macromolecular Complexes ◽  
Air Water Interface ◽  
Sample Application ◽  
Silver Bullet ◽  
Particle Behaviour

AbstractA host of new technologies are under development to improve the quality and reproducibility of cryoEM grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot™, chameleon and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms, (the fastest reported to date), to interrogate particle behaviour at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimise. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic ‘silver bullet’ for improving the quality of every cryoEM sample preparation.

scholarly journals Through-grid wicking enables high-speed cryoEM specimen preparation

2020 ◽  
Vol 76 (11) ◽  
pp. 1092-1103
Author(s):  
Yong Zi Tan ◽  
John L. Rubinstein
Keyword(s):  
Glass Fiber ◽  
High Speed ◽  
Protein Complexes ◽  
Absorption Capacity ◽  
Specimen Preparation ◽  
Water Interface ◽  
Time Resolved ◽  
Glass Fiber Filter ◽  
Liquid Absorption ◽  
Air Water Interface

Blotting times for conventional cryoEM specimen preparation complicate time-resolved studies and lead to some specimens adopting preferred orientations or denaturing at the air–water interface. Here, it is shown that solution sprayed onto one side of a holey cryoEM grid can be wicked through the grid by a glass-fiber filter held against the opposite side, often called the `back', of the grid, producing a film suitable for vitrification. This process can be completed in tens of milliseconds. Ultrasonic specimen application and through-grid wicking were combined in a high-speed specimen-preparation device that was named `Back-it-up' or BIU. The high liquid-absorption capacity of the glass fiber compared with self-wicking grids makes the method relatively insensitive to the amount of sample applied. Consequently, through-grid wicking produces large areas of ice that are suitable for cryoEM for both soluble and detergent-solubilized protein complexes. The speed of the device increases the number of views for a specimen that suffers from preferred orientations.

scholarly journals Molecular rotation in 3 dimensions at an air/water interface using femtosecond time resolved sum frequency generation

2019 ◽  
Vol 150 (9) ◽  
pp. 094709 ◽  
Author(s):  
Yi Rao ◽  
Yuqin Qian ◽  
Gang-Hua Deng ◽  
Ashlie Kinross ◽  
Nicholas J. Turro ◽  
...  
Keyword(s):  
Sum Frequency Generation ◽  
Molecular Rotation ◽  
Water Interface ◽  
Time Resolved ◽  
Sum Frequency ◽  
Air Water Interface ◽  
Frequency Generation

scholarly journals Time resolved study of hydroxyl radical oxidation of oleic acid at the air-water interface

2017 ◽  
Vol 683 ◽  
pp. 76-82 ◽  
Author(s):  
Xinxing Zhang ◽  
Kevin M. Barraza ◽  
Kathleen T. Upton ◽  
J.L. Beauchamp
Keyword(s):  
Oleic Acid ◽  
Hydroxyl Radical ◽  
Water Interface ◽  
Time Resolved ◽  
Radical Oxidation ◽  
Air Water Interface

scholarly journals Adsorption Kinetics of a Cationic Surfactant Bearing a Two-Charged Head at the Air-Water Interface

Coatings ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 95 ◽  
Author(s):  
Marcos Fernández Leyes ◽  
Santiago Gimenez Reyes ◽  
Ezequiel Cuenca ◽  
Jhon F. Sánchez Morales ◽  
Hernán Ritacco
Keyword(s):  
Cationic Surfactant ◽  
Adsorption Process ◽  
Surface Concentration ◽  
Interfacial Region ◽  
Water Interface ◽  
Surface Tension Data ◽  
Time Resolved ◽  
Initial Stage ◽  
Wilhelmy Plate ◽  
Air Water Interface

We studied the dynamics of adsorption at the air-water interface of a cationic surfactant bearing two charges, Gemini 12-2-12, at concentrations below and above the critical micelle concentration (cmc). We used maximum bubble pressure and Wilhelmy plate techniques in order to access all time scales in the adsorption process. We found that the adsorption dynamics are controlled by diffusion at the initial stage of the adsorption process (milliseconds) and it is kinetically controlled by an electrostatic barrier (minute) approaching the equilibrium surfactant surface concentration. Between these two extremes, we found several relaxation phenomena, all following exponential decays with characteristic times spanning from one to hundreds of seconds. By means of time-resolved surface potential measurements, we show that these processes involve charge redistribution within the interfacial region. The surface tension data are analyzed and interpreted in the framework of the free energy approach.

scholarly journals Time-resolved X-ray reflectivity investigation of lysozyme adsorption at the air–water interface

2008 ◽  
Vol 64 (a1) ◽  
pp. C551-C551
Author(s):  
Y.F. Yano ◽  
T. Uruga ◽  
T. Hajime ◽  
H. Toyokawa ◽  
Y. Terada ◽  
...  
Keyword(s):  
Water Interface ◽  
Time Resolved ◽  
X Ray ◽  
Air Water Interface ◽  
Lysozyme Adsorption

scholarly journals Shake-it-off: a simple ultrasonic cryo-EM specimen-preparation device

2019 ◽  
Vol 75 (12) ◽  
pp. 1063-1070 ◽  
Author(s):  
John L. Rubinstein ◽  
Hui Guo ◽  
Zev A. Ripstein ◽  
Ali Haydaroglu ◽  
Aaron Au ◽  
...  
Keyword(s):  
Thin Film ◽  
Raspberry Pi ◽  
Specimen Preparation ◽  
Analysis Software ◽  
Preparation Methods ◽  
Time Resolved ◽  
Transfer Protein ◽  
Air Water Interface ◽  
Sample Application ◽  
Ultrasonic Humidifier

Although microscopes and image-analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, specimen-preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air–water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow the use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here, a simple cryo-EM specimen-preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid is presented. The device is controlled by a Raspberry Pi single-board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM rather than instrument design. The simple open-source design permits the straightforward customization of the instrument for specialized experiments.

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