The preparation of this manuscript was partially supported by US Army Research Office (W911NF2010321) and Arizona State University startup funds to P.-L.C.

1. Teflon block preparation
<ol>
	<li>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.</li>
</ol>
2. Monolayer lipid preparation
NOTE: Estimated operating time: 30- 45 minutes
<ol>
	<li>Lipid stock preparation
	<ol>
		<li>Prepare a 0.01 mg/mL lipid mixture in 9:1 (v/v) chlo...

The authors have no conflict of interest to declare.

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...

Electron diffraction through 2D crystals or helical arrays of proteins can achieve sub-nanometer resolutions in favorable cases1,2,3. 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 interactions7,8,9,10,11,12,13,14,15,16,17.
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 solution1,6,19. 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 tag20,21,22. Also, the cholera toxin B can bind a particular pentasaccharide of ganglioside GM1 in a lipid monolayer for structural studies23,24. 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 streptavidin2,25, annexin V26, cholera toxin27, E. coli gyrase B subunit28, E. coli RNA polymerase25,29,30, 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>14:0 PC (DMPC)</td>
			<td>Avanti Lipids</td>
			<td>850345</td>
			<td>1,2-dimyristoyl-sn-glycero-3-phosphocholine, 
			1 x 25 mg, 10 mg/mL, 2.5 mL</td>
		</tr>
		<tr>
			<td>Bulb for small pipets</td>
			<td>Fisher Scientific</td>
			<td>03-448-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>C2432</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator vacuum</td>
			<td>Southern Labware</td>
			<td>55207</td>
			<td></td>
		</tr>
		<tr>
			<td>EM grids</td>
			<td>Electron Microscopy Sciences</td>
			<td>CF413-50</td>
			<td>CF-1.2/1.3-4C 1.2 &#181;m hole, 1.3 &#181;m space</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>GE Healthcare Life Sciences</td>
			<td>1001-090</td>
			<td>Diameter 90 mm</td>
		</tr>
		<tr>
			<td>Glass Pasteur pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe (25 &#181;L)</td>
			<td>Hamilton Company</td>
			<td>80465</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe (250 &#181;L)</td>
			<td>Hamilton Company</td>
			<td>81165</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe (5 &#181;L)</td>
			<td>Hamilton Company</td>
			<td>87930</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe (500 &#181;L)</td>
			<td>Hamilton Company</td>
			<td>203080</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>M1775-1GA</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>VWR</td>
			<td>25384-342</td>
			<td>100 mm &#215; 15 mm</td>
		</tr>
		<tr>
			<td>Teflon block</td>
			<td>Grainger</td>
			<td>55UK05</td>
			<td>60 &#181;L wells with side injection ports, manually made</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Electron Microscopy Sciences</td>
			<td>78325</td>
			<td>Various styles</td>
		</tr>
		<tr>
			<td>Ultra-pure water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td>VWR</td>
			<td>97043-996</td>
			<td></td>
		</tr>
	</tbody>
</table>

sample preparation using a lipid monolayer method for electron crystallographic studies

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.

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 ...

Watch this Scientific Journal Video about Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies at JoVE.com

Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies

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.

Electron crystallography is a powerful tool for high-resolution structure determination. Macromolecules such as soluble or membrane proteins can be grown ...

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, as some integral membrane proteins. But requires more extensive empirical investigation to determine detergent and dialysis conditions to promote partitioning to the lipid monolayer.A lipid monolayer forms at the air water interface, such that the polar head groups of the lipid remain hydrated in the aqueous phase. And the non-polar acyl tails of the lipid partition to the air water interface, which breaks the surface tension and flattens the water surface. The charge nature the distinctive chemical moieties of the lipid head groups provide the affinity for proteins in solution.Promoting binding, and in principle 2D array formation. Any 2D crystals formed on a lipid monolayer can be readily transferred into the electron microscope on a carbon coated EM grid that is used to lift and support the crystalline array on the lipid layer. We described in this manuscript, a lipid monolayer methodology for cryogenic electron microscopy imaging.Lipid monolayer preparation. Lipid stock preparation, prepare a 0.01 mg per ml lipid mixture in 9:1 volume to volume chloroform, methanol. Using 8.91 ml of chloroform and 0.99 ml of methanol and 0.1 mls of a 10 mg per ml lipid solution.Teflon plate preparation. Sonicate a Teflon plate in a methanol bath for five minutes. Wash with hot water for 15 minutes and then rinse with distilled water.Dry the Teflon plate in a desiccated for 30 minutes and keep it under vacuum until use. Formation of lipid monolayer on buffer reservoir. Estimated operating time will be one and a half hours.Place a type one filter paper in a Petri dish and arrange the Teflon block on top of the filter paper. Fill the wells of the Teflon plate with 60 microliters of buffer. Carefully add liquid mixture on top of the buffer surface drop by drop.Ideally one microliter per drop using a Hamilton syringe. Wet the filter paper with distilled water and keep humidity in the Petri dish. Incubate at room temperature for 60 minutes, the chloroform should evaporate and a monolayer of lipid on the surface of the buffers should be formed.Application of an EM grid on a lipid monolayer. Estimated operating time one and a half hours. Gently place a carbon coated EM grid without glow discharging carbon side down onto the surface of each buffer reservoir.Carefully inject proteins into the side injection well, use final protein concentrations in the well around one micromolar. Incubate 60 minutes at room temperature. Gently inject approximately 20 to 30 microliters of buffer in the site injection port to raise the grid above the surface of the Teflon block.This allows you to pick up the grid with a pair of tweezers and lift it vertically off the droplet. Representative results. A lipid monolayer deposited on an EM grid can be visualized under the transmission electron microscope without contrast staining.The monolayer presence can be recognized, by the contrast difference from the area without any specimen in that beam path. Areas that have lipid monolayer coverage have lower contrast than ones with no coverage. Since the electron beam through the empty holes has no scattering it shows a brighter illumination.In conclusion, the lipid monolayer method offers an opportunity for studying protein structures due to its simplicity. It may provide an alternative approach to facilitate the process of 2D crystallization.

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Biochemistry

3.6K Views.  Arizona State University. 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, as some integral membrane proteins. But requires more extensive empirical investigation to determine detergent and dialysis conditions to promote partitioning to the lipid monolayer.A lipid monolayer forms at the air water interface, such that the polar head groups of t...