Published: November 6th, 2021
Here, we provide a detailed protocol for the use of a rapid grid making device for both fast grid-making and for rapid mixing and freezing to conduct time-resolved experiments.
The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures and information on more challenging systems. Sample preparation for cryo-EM is undergoing a similar revolution with new approaches being developed to supersede the traditional blotting systems. These include the use of piezo-electric dispensers, pin printing and direct spraying. As a result of these developments, the speed of grid preparation is going from seconds to milliseconds, providing new opportunities, especially in the field of time-resolved cryo-EM where proteins and substrates can be rapidly mixed before plunge freezing, trapping short lived intermediate states. Here we describe, in detail, a standard protocol for making grids on our in-house time-resolved EM device both for standard fast grid preparation and also for time-resolved experiments. The protocol requires a minimum of about 50 µL sample at concentrations of ≥ 2 mg/mL for the preparation of 4 grids. The delay between sample application and freezing can be as low as 10 ms. One limitation is increased ice thickness at faster speeds and compared to the blotting method. We hope this protocol will aid others in designing their own grid making devices and those interested in designing time-resolved experiments.
Recent developments in cryo-electron microscopy (cryo-EM) have enabled structural studies of increasingly complex systems at high resolution. With few exceptions, such studies have been limited to biological macromolecules at equilibrium1 or relatively slow reactions2. Many processes in vivo occur on a faster timescale (milliseconds) and there is increasing interest in time-resolved cryo-EM (TrEM) on these timescales3. However, conventional cryo-EM sample preparation by the blotting method is too slow for millisecond TrEM.
1. Preparing the system
NOTE: The following protocol describes how to prepare grids of a single sample. Usually, a minimum of 2 replicate grids are prepared for each sample or condition. For faster plunge speeds (less than ~ 20 ms time delay), 3 or 4 replicate grids are typically prepared to account for a reduced number of thin ice areas.
Fast grid preparation with the TED
As a test specimen for fast grid preparation, we have used apoferritin from equine spleen at 20 µM in 30 mM HEPES, 150 mM NaCl, pH 7.5. A reconstruction at 3.5 Å resolution was obtained from 690 micrographs as described in ref.15 (Figure 7A). The defocus range was chosen so that particles can easily be identified in the raw images (Figure 7B). A typical grid prepared with.......
The protocols in this work can be used for fast grid preparation by direct spraying and TrEM experiments. Fast grid preparation can be used to reduce particle interactions with the air water interface5. The main limitations are the available sample concentration and ice thickness on the grid. Within these limits and provided that the sample quality is good, the protocol produces grids suitable for high resolution cryo-EM.
We would like to thank Molly S.C. Gravett for helpful discussions and the ABSL facility staff for help with cryo-EM data collection. David P. Klebl is a PhD student on the Wellcome Trust 4-year PhD program in The Astbury Centre funded by The University of Leeds. The FEI Titan Krios microscopes were funded by the University of Leeds (UoL ABSL award) and Wellcome Trust (108466/Z/15/Z). This work was funded by a BBSRC grant to Stephen P. Muench (BB/P026397/1) and supported by research grants to Howard D. White from the American Heart Association (AMR21-236078) and Howard D. White and Vitold Galkin from the U.S. National Institutes of Health (171261).....
|Time resolved device
|acrylic glass box
|digital humidity/temperature controller
|THE20 digital humidity/temperature controller
|dual rod pneumatic cylinder
|dual rod pneumatic cylinder TN 10x70
|Upchurch Scientific 1/16” O.D., 0.01'' I.D. FEP tubing
|Upchurch Scientific ETFE/ETFE flangeless fittings
|flexible reinforced PVC tubing
|12 mm OD. flexible reinforced PVC tubing
|Kloehn 250 µL zero-dead volume
|Interpret Aqua Air AP3
|liquid ethane container
|from Thermo/FEI VitrobotTM Mark IV
|GASARC class 3 multistage regulator
|negative pressure tweezers
|Dumont N5 Inox B negative pressure tweezers
|Hantek 6022BE oscilloscope
|Scientific Commodities Inc. 0.043” O.D., 0.015” I.D. PE tubing
|Mean Well GSM160A24-R7B
|Wanptek KPS305D power supply
|SMC TU0425 4 mm O.D., 2.5 mm I.D. PU tubing
|PS100 slide potentiometer
|SMC NVJ314M solenoid valve
|syringe drive pumps
|Kloehn V6 48K model
|Reagents & Materials
|apoferritin from equine spleen
|Quantifoil 300 mesh Cu, R 1.2/1.3
|Sigma Aldrich E3889
|Provided by H.D. White (for preparation procedure, see ref. 1)
|Cressington 208 carbon coater with a glow-discharge unit
|Skeletal muscle myosin S1
|Provided by H.D. White (for preparation procedure, see ref. 2)
|Spudich, J. A. & Watt, S. The regulation of rabbit skeletal muscle contraction I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. Journal of biological chemistry 246, 4866-4871 (1971).
|White, H. & Taylor, E. Energetics and mechanism of actomyosin adenosine triphosphatase. Biochemistry 15, 5818-5826 (1976).
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