From Constructs to Crystals – Towards Structure Determination of β-barrel Outer Membrane Proteins

Summary

β-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 β-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

Abstract

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, α-helical and β-barrel. While α-helical membrane proteins can be found in nearly all cellular membranes, β-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 β-barrel outer membrane protein (OMP), general protocols are presented for the production of target β-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.

Introduction

β-barrel OMPs can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria^1-3. While they serve similar roles as α-helical proteins, they have a very different fold consisting of a central membrane-embedded β-barrel domain ranging from 8-26 anti-parallel β-strands with each strand being intimately connected to the two neighboring strands (Figures 1 and 2). The first and last strands of the β-barrel domain then interact with one another, almost exclusively in an anti-parallel fashion (except for mitochondrial VDAC), to close and seal the β-barrel domain from the surrounding membrane. All β-barrel OMPs have extracellular loops of varying sequence and length which play an important role in ligand interactions and/or protein-protein contacts, with these loops sometimes being as large as 75 residues, such as found in Neisserial transferrin binding protein A (TbpA)⁴. β-barrel OMPs can also have N-terminal or C-terminal periplasmic extensions which serve as additional domains for the protein's functional purpose (e.g., BamA^5-7, FimD^8,9, FadL¹⁰). While many types of β-barrel OMPs exist¹¹, two of the more common types are described below as examples for those less familiar with the field, (1) TonB-dependent transporters and (2) autotransporters.

TonB-dependent transporters (e.g., FepA, TbpA, BtuB, Cir, etc.) are essential for nutrient import and contain an N-terminal plug domain consisting of ~150 residues that is found tucked inside a C-terminal 22-stranded β-barrel domain embedded into the outer membrane¹² (Figure 3). While this plug domain prevents substrate from freely passing through the barrel domain, substrate binding induces a conformational change within the plug domain that leads to pore formation (either by plug rearrangement or by partial/full ejection of the plug) which can then facilitate substrate transport across the outer membrane into the periplasm. TonB-dependent transporters are especially important for the survival of some pathogenic strains of Gram-negative bacteria such as Neisseria meningitidis that have evolved specialized transporters that hijack nutrients such as iron directly from human host proteins^4,13,14.

Autotransporters belong to the type V secretion system of Gram-negative bacteria and are β-barrel OMPs that consist of a β-barrel domain (typically 12-strands as with EstA and EspP) and a passenger domain that is either secreted or presented at the surface of the cell^15,16 (Figure 3). These β-barrel OMPs often serve important roles in cell survival and virulence with the passenger domain serving either as a protease, adhesin, and/or other effector that mediates pathogenesis.

Structural methods such as X-ray crystallography, NMR spectroscopy, and electron microscopy (EM) allow us to determine models for the OMPs at atomic resolution which can in turn be used to decipher exactly how they function within the outer membrane. This invaluable information may then be used for drug and vaccine development if applicable. For example, transferrin binding protein A (TbpA) is found on the surface of Neisseria and is required for pathogenesis because it directly binds human transferrin and then extracts and imports the iron for its own survival. Without TbpA, Neisseria cannot scavenge iron from the human host and are rendered non-pathogenic. After the crystal structure of human transferrin bound to TbpA⁴ was solved, it became much clearer how the two proteins associated, what regions of TbpA mediated the interaction, what residues were important for iron extraction by TbpA, and how one might develop therapeutics against Neisseria targeting TbpA. Therefore, given the importance of β-barrel OMPs in Gram-negative bacteria for survival and pathogenesis, as well as in mitochondria and chloroplast function, and the need for additional structural information about this unique class of membrane proteins and the systems in which they function, general protocols are presented with the overall goal of expressing and purifying target OMPs at high levels for characterization by structural methods.

Protocol

1. Cloning and Expression

Note: To enable structural studies, sufficient quantities of highly purified protein must be prepared, and this generally starts with the cloning and overexpression of the target β-barrel outer membrane protein (OMP) in E. coli (Figure 4). To date, all β-barrel OMP structures, including those structures for mitochondrial VDAC, have been derived from bacterially expressed protein¹¹. Here, general protocols are presented for cloning and expressing β-barrel OMPs for (1) native expression directly into bacterial membranes and (2) expression into inclusions bodies for in vitro refolding¹⁷.

1. Designing an Expression Construct
   1. Obtain or purchase the codon optimized target OMP gene.
   2. Purchase or obtain an T7 expression vector (i) containing a periplasmic localization signal sequence, an N-terminal 6x-Histidine tag, and a TEV protease site^4,18,19 for in vivo expression to the membrane or (ii) without a periplasmic localization signal sequence for expression to inclusion bodies for in vitro refolding.
      Note: An N-terminal protease cleavable 6x or 10x-Histidine tag would provide an easy method for purification. As with soluble proteins, vary the choice of affinity tag (Histidine, Strep, GST, etc.), the position of the affinity tag, and the inclusion of a protease site (TEV, enterokinase, thrombin, etc.) for subsequent tag removal.
   3. PCR amplify the target sequence with appropriate primers and subclone into the expression vector. Use ligation independent cloning (LIC)^20,21 or conventional cloning techniques (restriction enzymes/ligation) for subcloning. However, LIC can facilitate high throughput, allowing for a vast number of various constructs (truncations, variety of tags, variety of promoters) to be cloned in parallel more easily.
2. In Vivo Membrane-targeted Expression
   1. Use sequencing analysis to verify the target β-barrel OMP is cloned downstream and in frame with a signal sequence (i.e., pelB, OmpA) in the expression construct.
      Note: The signal sequence directs the nascent chain to the Sec translocon for secretion to the periplasm and subsequent assembly into the outer membrane.
   2. Transform the construct into an expression strain of bacteria for expression by pipetting 1.0 μl of plasmid into 50 μl of BL21(DE3) chemically competent cells and mix gently by pipetting up and down. Incubate on ice for 30 min.
   3. Heat pulse at 42 ºC for 30 sec using a water bath and then place back on ice for 1 min.
   4. Add 1 ml of pre-warmed SOC media and shake at 37 ºC for 1 hr at 1,000 rpm using a benchtop shaker incubator.
   5. Plate 100 μl of cells onto LB agar plates containing an appropriate antibiotic and incubate overnight at 37 ºC inverted.
   6. Perform small-scale expression tests by inoculating a 5 ml LB+antibiotic culture with a single colony. Repeat for 5-10 colonies.
      Note: Because of the potential toxicity of a β-barrel OMP and the reliance on basal expression levels, colony-to-colony variability is sometimes observed. Therefore, screening multiple colonies is recommended for each construct being tested. For small-scale expression tests, rather than conventional 5 ml cultures, growing 25 ml cultures in 125 ml baffled Erlenmeyer flasks is suggested. These conditions often better reflect what will happen in a large-scale growth. Additionally, it is a good idea to also screen various types of culture media (TB, LB, 2xYT, M9, etc.), since varying levels of success have been reported depending on the target protein.
      1. Grow at 37 ºC with shaking to an OD₆₀₀ of ~0.6.
      2. Induce expression of the target OMP by adding 5 μl of 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG) to each culture tube and allow to grow an additional 1-2 hr.
      3. Compare expression levels for all the colonies by centrifuging 1 ml of each culture (OD₆₀₀ of ~0.6) for 1 min at 15,000 x g using a microcentrifuge.
      4. Remove the supernatant and resuspend the cells in 200 μl of 1x SDS-PAGE loading buffer. Heat at 100 ºC for 5 min and then centrifuge again at 15,000 x g for 5 min.
      5. Analyze the samples using SDS-PAGE by pipetting 20 μl into each well of a 10% gel. Run the gel for 35 min at constant 200 V.
         Note: It would be beneficial at this point to be able to determine if the protein target is being properly folded or not. This can often be accomplished by doing small-scale purifications or by assaying for heat modifiability.
   7. Select the colony showing the best expression and perform large-scale expression using 12-24 L of medium.
      Note: Conventional methods typically grow the cultures at 37 ºC with shaking to an OD₆₀₀ of ~0.6 and then induce with an appropriate inducer (i.e., 0.1-1 mM for IPTG or ~0.2% for arabinose) for an additional 2-4 hr. If desired, prior to the induction, reduce the temperature to as low as 20 °C and extend the induction time.
      1. For leaky expression method, grow a 25 ml inoculate culture at 37 °C in LB medium containing the appropriate antibiotic(s) until OD₆₀₀ reaches ~0.6. Then, add 1 ml of inoculate to twelve 1 L flasks of TB medium plus antibiotic(s) and grow at 20 °C to saturation (~3 days).
   8. Harvest the cells by centrifugation at 6,000 x g for 10 min.
   9. Proceed to purification step Section 2.1 or freeze the cell pellet in liquid nitrogen for long-term storage at -80 ºC.
3.  Expression to Inclusion Bodies for In Vitro Refolding
   1. Use sequencing analysis to verify the expression construct does not contain a signal sequence. The absence of a signal sequence will direct the target protein to accumulate in the cytosol as inclusion bodies.
   2. Transform the expression construct into an expression strain of bacteria for expression. For inclusion body expression, it is best to control expression by induction (i.e., IPTG, arabinose, etc.) to minimize possible toxicity effects.
   3. Plate 100 μl of transformed cells onto LB agar plates containing an appropriate antibiotic and incubate overnight at 37 ºC inverted.
   4. Perform small-scale expression tests by inoculating a 5 ml LB+antibiotic culture with a single colony.
   5. Repeat step 1.3.4 for 2-4 additional colonies.
   6. Grow at 37 ºC with shaking to an OD₆₀₀ of ~0.6.
   7. Induce with an appropriate inducer for 1-2 hr.
   8. Compare expression levels for all the colonies by analysis using SDS-PAGE (see Step 1.2.6). Select the colony showing the best expression and perform large-scale expression using 12-24 L of medium.
      1. Grow the cells at 37 °C to an OD₆₀₀ of 0.6-0.8 and induce at 37 °C for 3-5 hr. While expression to inclusion bodies is more robust than other types of expression, as with all protein expression experiments, expression can be improved by varying growth medium, time of induction, temperature of induction, and concentration of the inducing agent.
   9. Harvest the cells by centrifugation at 6,000 x g for 10 min.
   10. Proceed to purification step Section 2.2 or freeze the cell pellet in liquid nitrogen for long-term storage at -80 ºC.

2. Purification

1. Isolation from Membrane Fractions
   Note: In contrast to soluble proteins, integral membrane proteins are embedded into the lipid bilayer and therefore require detergents to extract them for further purification and analysis (Figure 5). Following expression, the first step in the purification of a β-barrel OMP is extraction from the membrane fraction.
   1. Resuspend cells in Lysis buffer (50 mM Tris-HCl, pH 7.4, 200 mM NaCl, 10 mM MgCl₂, 50 µg/ml AEBSF, 5 µg/ml DNase I) at a ratio of 5 ml/g cell paste.
   2. Lyse the cells using a French press or cell homogenizer. Spin the lysed cells at 15,000 x g for 30 min at 4 ºC to remove unlysed cells and cell debris.
   3. Transfer the supernatant to a clean tube and centrifuge again at high speed (200,000 x g) for 1 hr at 4 ºC. The resulting pellet is the membrane fraction which contains the protein of interest.
   4. Using a dounce homogenizer, resuspend the membrane fraction, first transferring the membranes and then adding Solubilization buffer (50 mM KH₂PO₄ pH 7.5, 200 mM NaCl, 20 mM imidazole, pH 8.0) (50 ml per 20 g cells) at 2x concentration, without detergent.
   5. Pour the resuspended membranes into a small beaker and add detergent (i.e., n-dodecyl-β-D-maltoside (DDM), lauryldimethylamine-N-oxide (LDAO), n-octyl-β-D-glucopyranoside (OG), Triton X-100, etc.) slowly to a final concentration of ~10x critical micelle concentration (CMC).
   6. Fill with water until a final concentration of 1x Solubilization buffer. Stir for 0.5-16 hr at 4 ºC, depending on how easily the target protein is extracted from the membranes.
      Note: Detergent is used to slowly solubilize the membranes to extract the target protein. There are 3 types of detergents that can be used here: ionic (SDS, deoxycholic acid), non-ionic (Elugent, TWEEN, Triton X-100, OG, DDM), and zwitterionic (LDAO, CHAPS). A protein-detergent complex that is stable and monodisperse during the purification step will greatly aid in the success of membrane protein crystallization. More information about detergents, their properties, CMC information, and their use in the purification of membrane proteins and other applications can be found on commercial supplier websites.
   7. Centrifuge the solubilized sample at 300,000 x g for 1 hr at 4 ºC. The supernatant now contains the detergent-solubilized target β-barrel OMP.
   8. Proceed with Section 2.3 as outlined below.
2. Refolding from Inclusion Bodies
   Note: For in vitro refolding, the target β-barrel OMP is expressed directly into inclusion bodies. One advantage here is that these proteins can be produced at high levels. However the disadvantage is that refolding is often inefficient and varies from one refolding experiment to the next. Still, there are many examples of proteins that have been successfully refolded for structural studies. Following expression into inclusion bodies, one must now isolate the inclusion bodies for refolding experiments.
   1. Resuspend cells in Lysis buffer (50 mM Tris-HCl, pH 7.4, 200 mM NaCl, 10 mM MgCl₂, 50 µg/ml AEBSF, 5 µg/ml DNase I, 4 mM 2-mercaptoethanol (BME)) (5 ml per g cell paste). Lyse using either a French press, sonication, or cell homogenizer.
   2. Pellet the inclusion bodies by a low speed spin at 6,000 x g for 10 min at 4 ºC.
   3. Wash the inclusion bodies by resuspending in 25 ml of 1.0 M urea using a dounce homogenizer. Pellet again by a low speed spin at 6,000 x g for 10 min at 4 ºC.
   4. Repeat step 2.2.3 as necessary.
   5. Resuspend the washed inclusion bodies to a final concentration of 10 mg/ml using buffer containing 8.0 M urea (or 6.0 M guanidinium hydrochloride (GdCl)) plus detergent (i.e., DDM or LDAO) at 2x CMC or higher.
      Note: If desired, for membrane protein targets containing a Histidine tag, immobilized metal-affinity chromatography (IMAC) purification can be performed using denaturing conditions (in 8.0 M urea or 6.0 M GdCl) as an initial step similar to that outlined below in Section 2.3.
   6. Perform the refolding reaction by slowly (overnight) removing the denaturant by dialysis in buffer lacking the denaturant.
      Note: It may take several changes of the dialysis buffer to accomplish this. Alternatively, if the inclusion bodies are first bound to an IMAC column, one can do the refolding reaction while still bound to the column by slowly exchanging buffers to remove the denaturant. In either case, remember that this is a membrane protein, therefore, detergent must be present to stabilize the target. Also, if the detergent being used is dialyzable, it must be added to the dialysis buffer(s) as well.
   7. Spin the refolded samples at 200,000 x g for 1 hr at 4 ºC. The supernatant contains the refolded detergent-solubilized target β-barrel OMP.
   8. Proceed with Section 2.3 as outlined below.
3. Purification Using IMAC, Ion-Exchange, and Gel Filtration
   Note: Assuming the protein has been engineered with a Histidine tag, whether natively expressed or refolded using in vitro methods, then immobilized metal-affinity chromatography (IMAC) is performed for purification much like for Histidine tagged soluble proteins, except detergent must be kept in all subsequent buffers to keep the target β-barrel OMP solubilized and stable.
   1. Prepare a 1-5 ml IMAC column or use a prepacked column. Perform subsequent chromatography steps at 4 °C. Flush with water to remove any traces of preservatives. If using an automated purification system, install the column according to manufacturer's instructions.
   2. Prepare 500 ml of IMAC Buffer A (50 mM K₂HPO₄, pH 7.5, 200 mM NaCl, 0.1% DDM) and 250 ml of IMAC Buffer B (50 mM K₂HPO₄, pH 7.5, 200 mM NaCl, 0.1% DDM, and 1.0 M imidazole).
      Note: Other detergents that may be used include Cymal-6 (0.05%), Triton X-100 (0.03%), and LDAO (0.05%).
   3. Equilibrate the IMAC column using 10 column volumes of IMAC Buffer A.
   4. Add imidazole to the protein sample to a final concentration of 25 mM and mix. Load the sample onto the equilibrated IMAC column at 2 ml/min. Collect the flow through.
   5. Wash the IMAC column with 5 column volumes each of increasing concentrations of imidazole using Buffer B (i.e., 25 mM, 50 mM, 100 mM). Collect the washes in 2 ml fractions.
   6. Elute the sample with a final concentration of 250 mM for 5 column volumes. Collect the eluted sample in 2 ml fractions.
   7. Analyze the flow through, the wash fractions and the elution fractions using SDS-PAGE analysis (see Step 1.2.6) based on their absorbance at 280 nm. Pool the fractions containing the target β-barrel OMP as verified by SDS-PAGE analysis.
   8. Remove the 6x-Histidine tag by adding TEV protease to the pooled sample (according to manufacturer's protocol) and incubating overnight at 4 ºC with gentle rocking.
   9. Load the sample/protease solution onto an IMAC column again to separate the target β-barrel OMP (flow through) from the cleaved tags and any uncleaved sample. The flow through will contain the cleaved sample lacking the tag.
      Note: This would also remove any protease like TEV-His, which also has a non-cleavable 6x-Histidine tag itself. Otherwise, other methods will be needed to remove the protease after digestion.
   10. Optionally, perform ion-exchange chromatography to further purify the sample. Follow the manufacturer's instructions for the column to be used, being sure to include detergent in all buffers.
   11. To prepare for crystallization, load the sample onto a gel filtration column into a buffer containing the detergent to be used for crystallization, such as 25 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1% OG.
   12. Collect 1 ml fractions and analyze using SDS-PAGE analysis (see Step 1.2.6) based on their absorbance at 280 nm. Pool fractions with absorbances at 280 nm as they contain the target protein. Concentrate to ~10 mg/ml.
       Note: If desired, proper folding may be assessed using a heat modifiability assay as outlined below.
4. Heat Modifiability Assay
   Note: One method to monitor proper folding of purified β-barrel OMPs is to perform heat modifiability assays using a semi-native SDS-PAGE²². Here, unheated samples that are folded properly will typically migrate differently from those that are heat denatured. This property is unique to β-barrel OMPs and widely used to study this family of membrane proteins.
   1. Prior to preparing samples, assemble the gel apparatus. To begin, place the buffer tank into an ice bucket and surround completely with ice. Insert an in-house cast or commercial native gradient gel into the holder and place into the tank. Fill the tank completely with cold 1x MES Running Buffer (50 mM 2-[N-morpholino]ethanesulfonic acid, 50 mM Tris base, 1 mM EDTA, 0.1% SDS, pH 7.3).
   2. Pipette 0.25 µl of the sample (10 mg/ml) into two 1.5 ml microcentrifuge tubes. Label one as 'Boiled' and the other as 'RT'.
   3. Add 9.75 µl of sample buffer to each and mix by pipetting. To both samples, add 10 µl of 2x SDS loading buffer (100 mM Tris-HCl, pH 6.8, 2% SDS, 0.2% bromophenol blue, and 20% glycerol) and mix gently by pipetting.
   4. Boil the 'Boiled' sample at 95 ºC for 5 min while leaving the 'RT' sample at RT. Spin the 'Boiled' sample briefly.
   5. Load 20 μl of both samples onto the preassembled native gel. Run for 60 min at constant 150 V. Remove the gel and soak in staining solution to visualize the results.

3. Crystallization

Note: For crystallization of both soluble and membrane protein targets, it is standard protocol to maximize sample purity and stability (i.e., best detergent, ligands, cofactors, etc.). Current methodology for crystallizing membrane protein targets in general encompasses three major approaches that satisfy the amphiphilic requirements of bilayer-embedded proteins: (1) detergent, (2) bicelle, and (3) lipidic cubic phase (LCP) (Figure 6)²³. Use of a nanoliter crystallization robot is strongly recommended when possible in order to increase the number of conditions that can be screened for a given sample volume, as well as, utilizing recent advances in tools aimed to aid structure determination (Figure 7).

1. Crystallization using Detergents
   1. Obtain a detergent solubilized protein sample that is at ~10 mg/ml concentration. Optionally, add the additive 1,2,3-heptanetriol directly to the sample to a final concentration of 3% to reduce detergent micelle size.
   2. Filter the sample using a 0.22 µm centrifugal filter to remove particulates and precipitation.
   3. Using a crystallization robot, perform broad matrix crystallization screening using commercially available 96 well screens by either hanging or sitting drop vaporization method with drops composed of ~200 nl protein sample and ~200 nl crystallization buffer.
   4. Incubate the crystallization plates at ~21 °C. Inspect the plates weekly for crystal growth using a stereomicroscope.
   5. Optimize crystallization leads by varying components of the crystallization condition in a systematic fashion (increasing/decreasing salt or precipitant concentrations, buffer pH, incubation temperature, and/or protein concentration).
2. Crystallization Using Bicelles
   Note: A more detailed demonstration of bicelle techniques has previously been described by Ujwal and Abramson²⁴.
   1. Prepare or purchase a 35% bicelle mixture consisting of DMPC:CHAPSO at a ratio of 2.8:1 according to published protocols²⁵. Other concentrations may also be assayed, as well as, other lipids and detergents, as necessary.
   2. Obtain a detergent solubilized protein sample that is at ~10 mg/ml concentration.
   3. Add the bicelle mixture, on ice, at a starting ratio of 4:1 v/v (protein:bicelle) (e.g., add 10 µl of bicelle mixture to 40 µl of protein and mix rapidly by pipetting).
   4. Incubate on ice 30-60 min.
      Note: The protein:bicelle solution should remain mostly clear but can often turn slightly opaque. However, if the protein:bicelle solution turns milky white, indicating precipitation, then other variables should be assayed (i.e., detergent, buffer, etc.). For new samples, doing small scale tests (8 µl protein and 2 µl bicelles) is recommended to verify stability prior to scaling up to prevent unnecessary sample loss.
   5. Using a crystallization robot, perform broad matrix crystallization screening using commercially available 96 well screens by either hanging or sitting drop vaporization method with drops composed of ~200 nl protein sample and ~200 nl crystallization buffer.
   6. Incubate the crystallization plates at ~21 °C. Inspect the plates weekly for crystal growth using a stereomicroscope.
   7. Optimize crystallization leads by varying components of the crystallization condition in a systematic fashion (increasing/decreasing salt or precipitant concentrations, buffer pH, incubation temperature, and/or protein concentration).
3. Crystallization Using Lipidic Cubic Phase (LCP)
   Note: A more detailed demonstration of LCP techniques has previously been described by Liu and Cherezov^26,27.
   1. Obtain a detergent solubilized protein sample that is at ~20 mg/ml concentration.
   2. Pre-warm the monoolein to ~40 ºC. Load 60 µl monoolein into one gas-tight 100 µl syringe and 40 µl protein sample into another.
   3. Using a mixing coupler, connect the syringes, being careful not to introduce air.
   4. Mix gently by applying alternating pressure on the syringe plungers pushing the mixture from one syringe to the other completely until the sample becomes translucent and homogenous.
   5. Using a crystallization robot designed for LCP methods, perform broad matrix crystallization screening using commercially available 96 well screens with sandwich-style crystallization plates (0.1 mm well thickness) with drops composed of 100 nl protein sample and 750 nl crystallization buffer.
      Note: Drop ratios may be adjusted if needed. Also, solid covers (i.e., glass or thick plastic) are recommended which support the drops nicely rather than thin films, which tend to allow the drops to move about the well as the plates are handled.
   6. Incubate the crystallization plates at ~21 °C. Inspect the plates weekly for crystal growth using a stereomicroscope.
   7. Optimize crystallization leads by varying components of the crystallization condition in a systematic fashion (increasing/decreasing salt or precipitant concentrations, buffer pH, incubation temperature, and/or protein concentration).

Results

YiuR is a TonB dependent iron transporter that is a putative vaccine target against Yersinia pestis. It was originally identified using a microarray assay. Here, the steps that were taken to determine the structure of YiuR using X-ray crystallography are outlined (Figure 9). For cloning, the DNA sequence of YiuR (minus the N-terminal signal sequence) was PCR amplified from genomic DNA and subcloned into a vector containing an N-terminal pelB signal sequence and 1...

Discussion

β-barrel OMPs serve essential roles in Gram-negative bacteria, mitochondria and chloroplasts and are important targets for structural analysis that offer a wealth of information about essential molecular mechanisms at the outer membranes of these respective organelles. However, producing enough sample for structural analysis is not always straightforward and therefore, a general pipeline is presented for the production of sufficient quantities of target β-barrel OMPs for structure determination, explaining in d...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Crystallization Robot	TTP Labtech, Art Robbins	-	Any should work here, except for LCP crystallization
PCR thermocycler	Eppendorf, BioRad	-
Media Shaker	New Brunswick, Infors HT	-
UV-vis spectrometer	Eppendorf	-
SDS-PAGE apparatus	BioRad	1645050, 1658005
SDS-PAGE and native gels	BioRad, Life Technologies	4561084, EC6035BOX (BN1002BOX)
AkTA Prime	GE Healthcare	-
AkTA Purifier	GE Healthcare	-
Microcentrifuge	Eppendorf	-
Centrifuge (low-medium speed)	Beckman-Coulter	-
Ultracentrifuge (high speed)	Beckman-Coulter	-
SS34 rotor	Sorvall	-
Type 45 Ti rotor	Beckman-Coulter	-
Type 70 Ti rotor	Beckman-Coulter	-
Dounce homogenizer	Fisher Scientific	06 435C
Emulsiflex	Avestin	-
Dialysis tubing	Sigma	D9652
LCP tools	Hamilton, TTP Labtech	-
VDX 24 well plates	Hampton Research	HR3-172
Sandwich plates	Hampton Research, Molecular Dimensions	HR3-151, MD11-50 (MD11-53)
Grace Crystallization sheets	Grace Bio-Labs	875238
HiPrep S300 HR column	GE Healthcare	17-1167-01
Q-Sepharose column	GE Healthcare	17-0510-01
Crystallization screens	Hampton Research, Qiagen, Molecular Dimensions	-
Gas-tight syringe (100 ml)	Hamilton