Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies

Summary

Lipid monolayers have been used as a foundation for forming two-dimensional (2D) protein crystals for structural studies for decades. They are stable at the air-water interface and can serve as a thin supporting material for electron imaging. Here we present the proven steps on preparing lipid monolayers for biological studies.

Abstract

Electron crystallography is a powerful tool for high-resolution structure determination. Macromolecules such as soluble or membrane proteins can be grown into highly ordered two-dimensional (2D) crystals under favorable conditions. The quality of the grown 2D crystals is crucial to the resolution of the final reconstruction via 2D image processing. Over the years, lipid monolayers have been used as a supporting layer to foster the 2D crystallization of peripheral membrane proteins as well as soluble proteins. This method can also be applied to 2D crystallization of integral membrane proteins but requires more extensive empirical investigation to determine detergent and dialysis conditions to promote partitioning to the monolayer. A lipid monolayer forms at the air-water interface such that the polar lipid head groups remain hydrated in the aqueous phase and the non-polar, acyl chains, tails partition into the air, breaking the surface tension and flattening the water surface. The charged nature or distinctive chemical moieties of the head groups provide affinity for proteins in solution, promoting binding for 2D array formation. A newly formed monolayer with the 2D array can be readily transfer into an electron microscope (EM) on a carbon-coated copper grid used to lift and support the crystalline array. In this work, we describe a lipid monolayer methodology for cryogenic electron microscopic (cryo-EM) imaging.

Introduction

Electron diffraction through 2D crystals or helical arrays of proteins can achieve sub-nanometer resolutions in favorable cases^1,2,3. Of particular interest are reconstituted 2D membrane protein arrays or crystals in their near-native environments¹. Because a crystal acts as a signal amplifier enhancing the intensities of the structural factors at specific spatial frequencies, electron crystallography allows probing a target with a smaller size at high resolutions, such as small molecules, than those for single-particle cryo-EM. The electron beam can b....

Protocol

1. Teflon block preparation

1. Prepare the Teflon block from chemical-resistant PTFE (polytetrafluoroethylene) resin. Make holes on the block using a general drill followed by the dimensions labeled in Figure 1.

2. Monolayer lipid preparation

NOTE: Estimated operating time: 30- 45 minutes

1. Lipid stock preparation
   1. Prepare a 0.01 mg/mL lipid mixture in 9:1 (v/v) chloroform/methanol using 8.91 mL of chloroform, 0.99 mL of methanol and 0.01 mL of 10 mg/mL lipids.
2. Teflon plate preparation

Results

A lipid monolayer deposited on the EM grid can be visualized under a transmission electron microscope (TEM) without staining. The monolayer presence can be recognized by the contrast difference from the area without any specimen in the beam path. Areas that have lipid monolayer coverage have lower local contrast than the ones with no coverage, since the electron beam through the empty holes has no scattering and shows a brighter illumination (Figure 3).

To screen .......

Discussion

A lipid monolayer is a powerful tool that facilitates the growth of large 2D crystals for structural studies of biological macromolecules. To successfully prepare an intact lipid monolayer at the air-water interface, it is strongly recommended that the lipids are prepared freshly on the day of the experiment, because oxidization of the lipid acyl chain could lead to packing disruption in the monolayer and adversely affect the resulting crystal formation. Purchased lipids in powder form should be dissolved using a mixture.......

Materials

Name	Company	Catalog Number	Comments
14:0 PC (DMPC)	Avanti Lipids	850345	1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1 x 25 mg, 10 mg/mL, 2.5 mL
Bulb for small pipets	Fisher Scientific	03-448-21
Chloroform	Sigma-Aldrich	C2432
Desiccator vacuum	Southern Labware	55207
EM grids	Electron Microscopy Sciences	CF413-50	CF-1.2/1.3-4C 1.2 µm hole, 1.3 µm space
Filter paper	GE Healthcare Life Sciences	1001-090	Diameter 90 mm
Glass Pasteur pipets	Fisher Scientific	13-678-20A
Hamilton syringe (25 µL)	Hamilton Company	80465
Hamilton syringe (250 µL)	Hamilton Company	81165
Hamilton syringe (5 µL)	Hamilton Company	87930
Hamilton syringe (500 µL)	Hamilton Company	203080
Methanol	Sigma-Aldrich	M1775-1GA
Petri dish	VWR	25384-342	100 mm × 15 mm
Teflon block	Grainger	55UK05	60 µL wells with side injection ports, manually made
Tweezers	Electron Microscopy Sciences	78325	Various styles
Ultra-pure water
Ultrasonic cleaner	VWR	97043-996