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Abstract

Introduction

Protocol

Representative Results

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Materials

References

Biochemistry

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

Published: November 6th, 2021

DOI:

10.3791/62199

1School of Biomedical Sciences, Faculty of Biological Sciences & Astbury Centre for Structural and Molecular Biology, University of Leeds, 2School of Molecular and Cellular Biology, Faculty of Biological Sciences & Astbury Centre for Structural and Molecular Biology, University of Leeds, 3Department of Physiological Sciences, Eastern Virginia Medical School

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.

Background
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.

The ....

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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.

  1. Dilute the protein sample to the target concentration in the desired buffer. Typically, final concentrations ≥ 2 mg/mL work well for grid p.......

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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.......

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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.

Troubleshooting
Liquid fl.......

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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).

....

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Name Company Catalog Number Comments
Time resolved device
acrylic glass box USA scientific
digital humidity/temperature controller THE20 digital humidity/temperature controller
dual rod pneumatic cylinder dual rod pneumatic cylinder TN 10x70
FEP tubing Upchurch Scientific 1/16” O.D., 0.01'' I.D. FEP tubing
flangeless fittings Upchurch Scientific ETFE/ETFE flangeless fittings
flexible reinforced PVC tubing 12 mm OD. flexible reinforced PVC tubing
glass syringes Kloehn 250 µL zero-dead volume
humidifier pump Interpret Aqua Air AP3
liquid ethane container from Thermo/FEI VitrobotTM Mark IV
multistage regulator GASARC class 3 multistage regulator
negative pressure tweezers Dumont N5 Inox B negative pressure tweezers
oscilloscope Hantek 6022BE oscilloscope
PE tubing Scientific Commodities Inc. 0.043” O.D., 0.015” I.D. PE tubing
power supply Mean Well GSM160A24-R7B
power supply Wanptek KPS305D power supply
PU tubing SMC TU0425 4 mm O.D., 2.5 mm I.D. PU tubing
regulator Norgren R72G-2GK-RMN
slide potentiometer PS100 slide potentiometer
solenoid valve SMC NVJ314M solenoid valve
syringe drive pumps Kloehn V6 48K model
Reagents & Materials
apoferritin from equine spleen Sigma-Aldrich, A3660
ATP Sigma-Aldrich, A2383
cryo-EM grids Quantifoil 300 mesh Cu, R 1.2/1.3
EGTA Sigma Aldrich E3889
F-actin Provided by H.D. White (for preparation procedure, see ref. 1)
glow-discharger Cressington 208 carbon coater with a glow-discharge unit
HEPES Sigma-Aldrich, H7006
KAc Sigma-Aldrich, P1190
MgCl2 Sigma-Aldrich, M8266
MOPS Sigma-Aldrich, M1254
NaCl Sigma-Aldrich, S9888
Skeletal muscle myosin S1 Provided by H.D. White (for preparation procedure, see ref. 2)
Ref 1 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).
Ref 2 White, H. & Taylor, E. Energetics and mechanism of actomyosin adenosine triphosphatase. Biochemistry 15, 5818-5826 (1976).

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