Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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.......

Discussion

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.......

Materials

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).