We acknowledge our home institution, the &#201;cole Polytechnique F&#233;d&#233;rale de Lausanne (EPFL), for its generous support. This project has received funding from the European Union&#39;s Horizon 2020 Research and innovation program under the Marie Sk&#322;odowska-Curie grant agreement No. 665667. The authors thank Pascal Alexander Schouwink for his help with XRD.

CAUTION: Read carefully the material safety data sheets (MSDS) of the chemicals involved. Some of the chemicals used in the experiment are toxic. The present method involves the synthesis of nanoparticles. Therefore, take appropriate precautions. The entire synthesis procedure must be performed in a well-ventilated fume hood.
NOTE: The details of the instruments, the chemicals, and the materials involved in the synthesis of the MOF films are listed in Table 1.
1. Preparation of a Metal Salt Solution and Linker Solution for the Precursor Sol
NOTE: For the synthesis of ZIF-8 film, zinc nitrate hexahydrate [Zn(NO3)2.6H2O] is used as the metal salt and 2-methylimidazole (HmIm) is used as the linker.
<ol>
	<li>For the metal salt solution, dissolve 2.75 g of Zn(NO3)2.6H2O in 500 mL of water.</li>
	<li>For the linker solution, dissolve 56.75 g of HmIm in 500 mL of water and stir it continuously until a clear solution is obtained.</li>
</ol>
2. Preparation of Cu Electrodes
<ol>
	<li>Cut a high purity copper foil (with 99.9% purity, 127 &#181;m-thick) into 4 x 4 cm pieces.</li>
	<li>For the ease of clamping the cut foil to a set of Cu crocodile clips, draw a line at a distance of 0.5 cm from one of the edges of the square foil.</li>
	<li>Flatten the foil using a cylindrical roller on a clean surface.</li>
	<li>Clean the copper foils thoroughly by bath sonication in acetone for 15 min, followed by bath sonication in isopropanol for 15 min.</li>
	<li>Dry the copper foils in a clean atmosphere.</li>
</ol>
3. Attachment of Substrates to the Electrode (Cathode)
NOTE: Chose an appropriate substrate to deposit the MOF film. The method can be applied to AAO (Anodisc, 13 mm-diameter hosting pores with a pore diameter of 0.1 &#181;m), polyacrylonitrile (PAN, molecular weight cut-off: 100 kDa), Cu foil (99.9% purity, 25 &#181;m-thick), a chemical-vapor-deposition-based graphene film resting on a Cu foil11, and a homemade nanoporous carbon film resting on Cu foil12,13,14.
<ol>
	<li>Carefully position the desired substrate on the center of the Cu electrode using a tape.</li>
	<li>Rinse the substrate/electrode assembly with water, followed by isopropanol, and again with water for about 1 min.</li>
	<li>Attach a bare Cu electrode (4 x 4 cm) to the anode. Then attach the substrate/electrode assembly to the cathode.</li>
	<li>Adjust the separation between the two electrodes to 1 cm inside a 100 mL glass beaker.</li>
</ol>
4. The ENACT Procedure for the ZIF-8 Film
<ol>
	<li>Mix 31.6 g of the metal precursor solution and 35 g of the ligand solution in a 100 mL beaker and stir it for 30 s at room temperature to form the precursor sol. 
	NOTE: The induction time of ZIF-8 in these conditions is less than 60 s and, therefore, the EPD has to be carried out without delay.</li>
	<li>Transfer the precursor sol&#160;to the vessel hosting the electrodes.</li>
	<li>Immerse both the electrodes up to the 3.5 cm mark on the electrodes. 
	NOTE: From our experiments, we have observed that applying the electric field before 3 min of sol aging leads to the penetration of the smaller ZIF-8 nuclei into the pores of porous substrates (AAO in this particular case), which needs to be avoided. Therefore, the electric field is turned on only after 3 min, when the nuclei grow somewhat larger.</li>
	<li>Carry out the EPD with a deposition voltage of 1 V for 4 min. The current should be in the range of 2.5 - 3.5 mA.</li>
	<li>At the end of the EPD, lower the beaker slowly. As the adhesion between the freshly deposited nuclei and the substrate is weak, utmost care must be taken while handling the substrate.</li>
	<li>Transfer the dried substrate to a microscopic glass slide. Use tape to hold the substrate in place.</li>
	<li>For crystal growth, mix 31.6 g of the metal salt solution and 35 g of the linker solution in a 100 mL beaker.</li>
	<li>Place the microscopic glass slide along with the substrate vertically in the precursor solution and leave it undisturbed for 10 h at 30 &#176;C.</li>
	<li>After up to 10 h of crystal growth, rinse the substrate with water for 30 min and dry it in a clean atmosphere.</li>
</ol>
5. The ENACT Procedure for the ZIF-7 Film
<ol>
	<li>Synthesize the ZIF-7 film on an AAO support by a method similar to the one used for the ZIF-8 film but with the following differences.</li>
	<li>After mixing 0.82 g of Zn(NO3)2&#183;6H2O in 30 mL of dimethylformamide (DMF) and 0.72 g of benzimidazole in 30 mL of methanol for 3 min, carry out the EPD for 1 min.</li>
	<li>Synthesize an intergrown ZIF-7 film by immersing the ZIF-7 nuclei film in a precursor sol containing 0.58 g of Zn(NO3)2&#183;6H2O, 0.3 g of benzimidazole, and 30 mL of DMF at 110 &#176;C for 4 h.</li>
	<li>After cooling down the solution, wash the ZIF-7/AAO membrane by soaking it in DMF, followed by drying it at 60 &#176;C for 12 h.</li>
</ol>
6. Membrane Preparation and Characterization
<ol>
	<li>Preparation of the membrane
	<ol>
		<li>For the preparation of a sealant, mix thoroughly an equal proportion of the resin and the hardener and leave the mixture for 1 h.</li>
		<li>Place the ZIF-8/support membranes on a 24 mm-wide steel disc with a 5 mm-diameter hole at the center.</li>
		<li>Apply the epoxy along the edges of the substrate at first and, subsequently, cover the substrate except for the 5 mm-diameter hole at the center.</li>
		<li>Allow the epoxy to dry overnight.</li>
		<li>Use a stereo microscope to scan the membrane along with a known reference scale.</li>
		<li>Use graphical software to calculate the exposed area of the membrane from the scanned image.</li>
	</ol>
	</li>
	<li>Gas permeation test 
	NOTE: In our experiments, the single-component gas permeation tests were carried out by the Wicke-Kallenbach technique in a homemade permeation cell. The mass flow controllers (MKS) and mass spectrometer used in the permeation set-up were calibrated within a range of 5% error. Mass flow controllers (MFC) regulated the feed and the sweep gas flow rate. Ar is used as the sweep gas which carried the permeate gas to a calibrated mass spectrometer (MS) for a real-time analysis of the permeate concentration.
	<ol>
		<li>Place the steel disc with the membrane in the stainless-steel permeation cell.</li>
		<li>Ensure a leak-tight fit by placing Viton O-rings above and below the steel disc and fasten the screws. To remove the adsorbed water during the synthesis, dry the membranes at 130 &#176;C under an H2/Ar atmosphere until a steady H2 permeance is reached.</li>
		<li>Place the set-up inside the oven and set the temperature to 130 &#176;C.</li>
		<li>Ensure the feed and the sweep lines are preheated.</li>
		<li>Set the gas flow rates on the feed and the sweep sides to 30 mL/min.</li>
		<li>Maintain a pressure of 0.1 MPa at the feed and the sweep side by adjusting the needle valves on the retentate and the permeate side, respectively.</li>
		<li>Calculate the permeance once a steady state is established.</li>
		<li>Slowly cool down the oven (in about 2 h) to 30 &#176;C and record the values again once a steady state is established.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The standout feature of the ENACT method with respect to the existing methods15 is that the ENACT method enables the synthesis of highly intergrown, ultrathin MOF films on a wide range of porous and nonporous substrates. Any substrate pretreatment is avoided, making this method quite straightforward for the synthesis of MOF films. Although EPD equipment has to be used for the deposition of a nuclei film, the equipment is composed of a power source, a metal electrode, and a beaker, which is quite s...

Thin molecular sieving membranes offer a high-energy efficiency in the separation of molecules and can reduce the overall cost of fuels, CO2 capture, water purification, solvent recovery, etc.1,2. MOFs are a promising class of material for the synthesis of molecular sieving membranes because of the involved isoreticular synthetic chemistry and relatively straightforward crystallization3. To date, MOF membranes comprising of diverse crystalline structures, including that of ZIF-4, -7, -8, -9, -11, -67, -90, and -93, and UiO-66, HKUST-1, and MIL-53 have been reported4,5. These membranes are synthesized by crystallizing high-quality polycrystalline MOF films on a porous support. Generally, to obtain a high separation selectivity, it is necessary to reduce the defects in the polycrystalline MOF film (such as pinholes and grain-boundary defects). A convenient approach to reduce the defects is to crystallize a thick film. Not surprisingly, several of the earlier reported on MOF membranes are extremely thick (over 5 &#181;m). Unfortunately, thick films lead to a long diffusion path, which limits the membrane permeance. Therefore, while selectivity is improved, permeance is sacrificed. To circumvent this trade-off, it is imperative to develop methods to crystallize ultrathin (&#60; 0.5 &#181;m-thick), defect-free MOF films.
ZIF-8 is the most intensively studied MOF for membrane synthesis, due to its exceptional chemical and thermal stability and a simple crystallization chemistry6,7. So far, the reported ultrathin ZIF-8 membranes have been realized by changing the surface chemistry or topology of the underlying porous substrate, favoring the heterogeneous nucleation of ZIF-8, which is essential for an intergrown polycrystalline film. For instance, Chen et al. reported the synthesis of 1 &#181;m-thick ZIF-8 film on (3-aminopropyl)triethoxysilane-modified TiO2-coated poly(vinylidene fluoride) (PVDF) hollow fibres8. They observed a high heterogeneous nucleation density and attributed it to the simultaneous modification of the surface chemistry and nanostructure. The Peinemann group reported an ultrathin ZIF-8 membrane on a metal-chelating, polythiosemicarbazide (PTSC) support9. This unique metal-chelating capability of PTSC led to the binding of zinc ions, promoting the heterogeneous nucleation of ZIF-8 which, subsequently, led to high-performance ZIF-8 membranes. In general, tuning the substrate chemistry and nanostructure facilitates the synthesis of high-performance MOF membranes; however, these methods are quite complex, and usually cannot be reapplied to synthesize MOF membranes from other attractive MOF structures.
Herein, we report the synthesis of ultrathin, highly intergrown ZIF-8 films using a simple and versatile crystallization approach that can be reapplied to form a thin intergrown film of several crystalline materials10. We show examples of ZIF-8 and ZIF-7 films prepared without any substrate pretreatment, which greatly simplifies the preparation process. The ZIF-8 films are prepared on a wide range of substrates (ceramic, polymer, metal, carbon, and graphene). The 500 nm-thick ZIF-8 film on an anodic aluminum oxide (AAO) support displays an attractive separation performance. A high H2 permeance of 8.3 x 10-6 mol m-2 s-1 Pa-1 and attractive ideal selectivities of 7.3 (H2/CO2), 15.5 (H2/N2), 16.2 (H2/CH4), and 2655 (H2/C3H8) are achieved.
The crystallization approach that enables the above-mentioned feat is ENACT. ENACT deposits ZIF-8 nuclei onto a substrate directly from the crystal&#39;s precursor sol. The approach utilizes EPD for a very short period of time (1 - 4 min) right after the induction time (the time when the nuclei appear in the precursor sol). The application of an electric field to the charged MOF nuclei drives them toward an electrode with a flux that is proportional to the strength of the applied electric field (E), the electrophoretic mobility of the colloid (&#956;), and the concentration of nuclei (Cn) as shown in the Equations 1 and 2.
<img alt="Equation 1" src="/files/ftp_upload/58301/58301eq1.jpg" /> 
(Equation 1)
<img alt="Equation 2" src="/files/ftp_upload/58301/58301eq2.jpg" /> 
(Equation 2)
Here, 
v = the drift velocity, 
&#950; = the zeta potential of the nuclei, 
&#949;o = the permittivity of vacuum, 
&#949;r = the dielectric constant, and 
&#951; = the viscosity of the precursor sol.
Therefore, by controlling E and the solution pH (which determines &#950;&#160;), the packing density of nuclei can be controlled. The subsequent growth of the densely-packed nuclei in the precursor sol allows researchers to obtain a highly intergrown polycrystalline film.

<table><tbody><tr><td>Zinc nitrate hexahydrate</td><td>Sigma-Aldrich</td><td>96482-500G</td><td>98% purity</td></tr><tr><td>2-Methylimidazole</td><td>Sigma-Aldrich</td><td>M50850-500G</td><td>99% purity</td></tr><tr><td>Benzimidazole</td><td>TCI</td><td>B0054-500G</td><td>98% purity</td></tr><tr><td>Tape</td><td>DuPont</td><td>KPT-1/8</td><td></td></tr><tr><td>Epoxy</td><td>GC Electronics</td><td>19-823</td><td></td></tr><tr><td>Copper foil</td><td>Alfa Aesar</td><td>13380.CV</td><td>99.9% purity</td></tr><tr><td>Power source for EPD</td><td>Gamry Instruments</td><td>Interface 1000E Potentiostat</td><td></td></tr><tr><td>Ultrasonic cleaner</td><td>MTI corporation</td><td>VGT-1860QTD</td><td></td></tr><tr><td>AAO</td><td>GE Healthcare Life Sciences&amp;lrm;</td><td>6809-7013</td><td></td></tr><tr><td>PAN</td><td>Shandong MegaVision</td><td></td><td>The molecular weight cut-off is 100 kDa</td></tr></tbody></table>

electrophoretic crystallization of ultrathin high-performance metal-organic framework membranes

We report the synthesis of thin, highly intergrown, polycrystalline metal-organic framework (MOF) membranes on a wide range of unmodified porous and non-porous supports (polymer, ceramic, metal, carbon, and graphene). We developed a novel crystallization technique, which is termed the ENACT approach: the electrophoretic nuclei assembly for the crystallization of highly intergrown thin films (ENACT). This approach allows for a high density of heterogeneous nucleation of MOFs on a chosen substrate via the electrophoretic deposition (EPD) directly from the precursor sol. The growth of well-packed MOF nuclei leads to a highly intergrown polycrystalline MOF film. We show that this simple approach can be used for the synthesis of thin, intergrown zeolite imidazole framework (ZIF)-7 and ZIF-8 films. The resulting 500 nm-thick ZIF-8 membranes show a considerably high H2 permeance (8.3 x 10-6 mol m-2 s-1 Pa-1) and ideal gas selectivities (7.3 for H2/CO2, 15.5 for H2/N2, 16.2 for H2/CH4, and 2655 for H2/C3H8). An attractive performance for C3H6/C3H8 separation is also achieved (a C3H6 permeance of 9.9 x 10-8 mol m-2 s-1 Pa-1 and a C3H6/C3H8 ideal selectivity of 31.6 at 25 &#176;C). Overall, the ENACT process, owing to its simplicity, can be extended to synthesize intergrown thin films of a wide range of nanoporous crystalline materials.

A homemade EPD set-up was used to synthesize the MOF films (Figure 1). Scanning electron microscopy (SEM) images and X-ray diffraction (XRD) patterns were collected for the ZIF-8 nuclei film (Figure 2). SEM was used to image the surface and cross-sectional morphologies of the AAO support, ZIF-8/AAO membrane, PAN support, ZIF-8/PAN membrane, ZIF-8/graphene film, and ZIF-7/AAO membrane (Figure 3). The ...

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Electrophoretic Crystallization of Ultrathin High-performance Metal-organic Framework Membranes

A simple, reproducible, and versatile approach for the synthesis of intergrown, polycrystalline metal-organic framework membranes on a wide range of unmodified porous and non-porous supports is presented.

We report the synthesis of thin, highly intergrown, polycrystalline metal-organic framework (MOF) membranes on a wide range of unmodified porous and ...

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9.9K Views.  École Polytechnique Fédérale de Lausanne (EPFL). Een eenvoudige, reproduceerbare en veelzijdige aanpak voor de synthese van intergrown, polykristallijne metaal-organische kader membranen op een breed scala van ongewijzigde poreuze en niet-poreuze ondersteunt wordt gepresenteerd.

Video: Electrophoretic Crystallization of Ultrathin High-performance Metal-organic Framework Membranes

Preparation of Hydrophobic Metal-Organic Frameworks via Plasma Enhanced Chemical Vapor Deposition of Perfluoroalkanes for the Removal of Ammonia

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high surface area microporous materials, such as metal-organic frameworks (MOFs), unique challenges present themselves for PECVD treatments. Herein the protocol for development of a MOF that was previously unstable to humid conditions is presented. The protocol describes the synthesis of Cu-BTC (also known as HKUST-1), the treatment of Cu-BTC with PECVD of perfluoroalkanes, the aging of materials under humid conditions, and the subsequent ammonia microbreakthrough experiments on milligram quantities of microporous materials. Cu-BTC has an extremely high surface area (~1,800 m2/g) when compared to most materials or surfaces that have been previously treated by PECVD methods. Parameters such as chamber pressure and treatment time are extremely important to ensure the perfluoroalkane plasma penetrates to and reacts with the inner MOF surfaces. Furthermore, the protocol for ammonia microbreakthrough experiments set forth here can be utilized for a variety of test gases and microporous materials.

Herein the procedures for plasma enhanced chemical vapor deposition of perfluoroalkanes on microporous materials such as metal-organic frameworks to enhance their stability and hydrophobicity are described. Furthermore, breakthrough testing of milligram quantities of samples is described in detail.

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high ...

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g., other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2 drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.

Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges.

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

Preparation of Highly Porous Coordination Polymer Coatings on Macroporous Polymer Monoliths for Enhanced Enrichment of Phosphopeptides

We describe a protocol for the preparation of hybrid materials based on highly porous coordination polymer coatings on the internal surface of macroporous polymer monoliths. The developed approach is based on the preparation of a macroporous polymer containing carboxylic acid functional groups and the subsequent step-by-step solution-based controlled growth of a layer of a porous coordination polymer on the surface of the pores of the polymer monolith. The prepared metal-organic polymer hybrid has a high specific micropore surface area. The amount of iron(III) sites is enhanced through metal-organic coordination on the surface of the pores of the functional polymer support. The increase of metal sites is related to the number of iterations of the coating process.
The developed preparation scheme is easily adapted to a capillary column format. The functional porous polymer is prepared as a self-contained single-block porous monolith within the capillary, yielding a flow-through separation device with excellent flow permeability and modest back-pressure. The metal-organic polymer hybrid column showed excellent performance for the enrichment of phosphopeptides from digested proteins and their subsequent detection using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The presented experimental protocol is highly versatile, and can be easily implemented to different organic polymer supports and coatings with a plethora of porous coordination polymers and metal-organic frameworks for multiple purification and/or separation applications.

A procedure for the preparation of porous hybrid separation media composed of a macroporous polymer monolith internally coated by a high surface area microporous coordination polymer is presented.

We describe a protocol for the preparation of hybrid materials based on highly porous coordination polymer coatings on the internal surface of macroporous ...

From Constructs to Crystals &#8211; Towards Structure Determination of &#946;-barrel Outer Membrane Proteins

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, &#945;-helical and &#946;-barrel. While &#945;-helical membrane proteins can be found in nearly all cellular membranes, &#946;-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite &#946;-barrel outer membrane protein (OMP), general protocols are presented for the production of target &#946;-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.

&#946;-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of &#946;-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% ...

Layer-by-layer Synthesis and Transfer of Freestanding Conjugated Microporous Polymer Nanomembranes

CMP as large surface area materials have attracted growing interest recently, due to their high variability in the incorporation of functional groups in combination with their outstanding thermal and chemical stability, and low densities. However, their insoluble nature causes problems in their processing since usually applied techniques such as spin coating are not available. Especially for membrane applications, where the processing of CMP as thin films is desirable, the processing problems have hindered their commercial application.
Here we describe the interfacial synthesis of CMP thin films on functionalized substrates via molecular layer-by-layer (l-b-l) synthesis. This process allows the preparation of films with desired thickness and composition and even desired composition gradients.
The use of sacrificial supports allows the preparation of freestanding membranes by dissolution of the support after the synthesis. To handle such ultra-thin freestanding membranes the protection with sacrificial coatings showed great promise, to avoid rupture of the nanomembranes. To transfer the nanomembranes to the desired substrate, the coated membranes are upfloated at the air-liquid interface and then transferred via dip coating.

In this paper we describe the interfacial synthesis of conjugated microporous polymers (CMP) on sacrificial substrates, and the dissolution of the substrate for the preparation of freestanding CMP nanomembranes. In addition, we will describe how the fragile nanomembranes can be transferred to other substrates.

CMP as large surface area materials have attracted growing interest recently, due to their high variability in the incorporation of functional groups in ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Biology

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Self-Assembly of Polymer-Grafted Metal Organic Framework Monolayers

Synthesis and Characterization of Self-Assembled Metal-Organic Framework Monolayers Using Polymer-Coated Particles

Metal-organic frameworks (MOFs) are materials with potential applications in fields such as gas adsorption and separation, catalysis, and biomedicine. Attempts to enhance the utility of MOFs have involved the preparation of various composites, including polymer-grafted MOFs. By directly grafting polymers to the external surface of MOFs, issues of incompatibility between polymers and MOFs can be overcome. Polymer brushes grafted from the surface of MOFs can serve to stabilize the MOF while enabling particle assembly into self-assembled metal-organic framework monolayers (SAMMs) via polymer-polymer interactions.
Control over the chemical composition and molecular weight of the grafted polymer can allow for tuning of the SAMM characteristics. In this work, instructions are provided on how to immobilize a chain transfer agent (CTA) onto the surface of the MOF UiO-66 (UiO = Universitetet i Oslo). The CTA&#160;serves as initiation sites for the growth of polymers. Once polymer chains are grown from the MOF surface, the formation of SAMMs is achieved through self-assembly at an air-water interface. The resulting SAMMs are characterized and shown to be freestanding by scanning electron microscopy imaging. The methods presented in this paper are expected to make the preparation of SAMMs more accessible to the research community and thereby expand their potential use as a&#160;MOF-polymer composite.

Author Spotlight: Exploring Self-Assembled MOF-Polymer Composites

A protocol for the synthesis and characterization of self-assembled metal-organic framework monolayers is provided using polymer-grafted, metal-organic framework (MOF) crystals. The procedure shows that polymer-grafted MOF particles can be self-assembled at an air-water interface resulting in well-formed, free-standing, monolayer structures as evidenced by scanning electron microscopy imaging.

Metal-organic frameworks (MOFs) are materials with potential applications in fields such as gas adsorption and separation, catalysis, and biomedicine. ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Electrophoretic Crystallization of Ultrathin High-performance Metal-organic Framework Membranes

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