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 be diffracted by an ordered 2D array of proteins, generating a diffraction pattern or a lattice image depending on where the image plane is recorded on the detector⁴. The diffracted intensities can then be extracted and processed to reconstruct a 2D projection structure of the crystal. Electrons have a larger scattering cross-section than X-rays and its scattering mostly follows the Rutherford model based on the Coulomb interaction between the electrons and the charged atoms in the molecule⁵. The thicknesses of 2D membrane crystals are usually less than 100 nm, suitable for electron transmission without dynamical scattering occurring within specimen⁶. Electron crystallographic studies have been shown to be a powerful tool to probe high-resolution structural information of membrane proteins and lipid-protein interactions^{7,8,9,10,11,12,13,14,15,16,17}.

A lipid monolayer is one single lipid layer composed of phospholipids densely packed at an air-water interface⁶, which can assist the 2D array formation for soluble proteins or peripheral membrane proteins¹⁸. Depending on the density of the lipids and their lateral pressure, the lipid molecules can form an ordered 2D array on the air-water interface with their acyl chains extended to the air and hydrophilic headgroups exposed in the aqueous solution^1,6,19. The lipid headgroup can interact with proteins via electrostatic interaction or can be modified to provide an affinity tag to bind a specific protein domain. For example, the DOGS-NTA-Ni (1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl]^2- Ni²⁺) is often used in forming a lipid monolayer to bind the proteins with a poly-histidine tag^20,21,22. Also, the cholera toxin B can bind a particular pentasaccharide of ganglioside GM1 in a lipid monolayer for structural studies^23,24. By anchoring the proteins on the lipid headgroups, the lipid monolayer can assist the formation of the 2D arrays that are thin for high-resolution electron crystallographic studies. The lipid monolayer technique has been used in electron crystallography for structural studies of proteins, such as streptavidin^2,25, annexin V²⁶, cholera toxin²⁷, E. coli gyrase B subunit²⁸, E. coli RNA polymerase^25,29,30, carboxysome shell proteins³¹ and the capsid proteins of the HIV-1³² and Moloney murine leukemia virus³³. Due to the stability and chemical property of the lipid monolayer, different applications for sample preparation have been explored for cryo-EM imaging³⁴. However, optimization will be needed for protein array formation.

Here, we provide extensive details of the general preparation of lipid monolayers for cryo-EM imaging and some considerations that could affect the quality of the formed monolayers.

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
   1. Sonicate a Teflon plate in a methanol bath for 5 minutes.
   2. Wash with hot water for 15 minutes.
   3. Rinse with distilled water.
   4. Dry the Teflon plate in a desiccator for 30 minutes and keep it under vacuum when not in use (Figure 1).

3. Formation of lipid monolayer on buffer reservoir

NOTE: Estimated operating time: 1.5 hours

1. Freshly prepare a lipid mixture of 0.01 mg/mL in a 9:1 mixture of chloroform/methanol on the day of the experiment.
2. Place a Type-1 filter paper in a Petri dish and arrange the Teflon block on top of the filter paper.
3. Fill the wells of the Teflon plate with 60 µL of the buffer.
4. Carefully add lipid mixture on top of the buffer surface drop by drop (ideally 1 µL per drop) using a Hamilton syringe.
5. Wet the filter paper with distilled water to keep humidity in the Petri dish.
6. Incubate at room temperature for 60 minutes. The chloroform should evaporate, and a monolayer of lipid on the surface of the buffer should be formed.

4. Application of an EM grid on a lipid monolayer

NOTE: Estimated operating time: 1.5 hours

1. Gently place a holey carbon-coated EM grid without glow-discharging, carbon side down, onto the top of each buffer reservoir.
2. Carefully inject proteins into the side injection well. Use final protein concentrations in the well around 1 µM of protein as a starting point. Pre-optimize the protein concentration before the experiment.
3. Incubate for 60 minutes at room temperature.
4. Gently inject approximately 20-30 µL of buffer into the side injection port. This will raise the grid above the surface of the Teflon block.
5. Immediately pick up the grid with a tweezer and lift it vertically off the droplet.
6. Prepare monolayer samples for negative staining or cryo-TEM imaging (Figure 2). If the proteins form a 2D array, the image power spectrum or Fourier periodogram will show diffraction intensities at the spatial frequencies based on the crystal symmetry. If no diffraction spots are shown in the power spectrum, the proteins are unlikely to form a 2D crystal.
7. Check the coverage of the lipid monolayer on the EM grid using a transmission electron microscope (TEM). A lower-magnified electron image is helpful to identify the morphology of the covered monolayer. Usually, the image contrast of the monolayer-covered area will be slightly lower than that of the uncovered areas. In addition, the computed 2D image power spectrum will help identify the location of the 2D crystalline.

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