An automated pipeline to screen membrane protein 2D crystallization

Abstract

Electron crystallography relies on electron cryomicroscopy of two-dimensional (2D) crystals and is particularly well suited for studying the structure of membrane proteins in their native lipid bilayer environment. To obtain 2D crystals from purified membrane proteins, the detergent in a protein-lipid-detergent ternary mixture must be removed, generally by dialysis, under conditions favoring reconstitution into proteoliposomes and formation of well-ordered lattices. To identify these conditions a wide range of parameters such as pH, lipid composition, lipid-to-protein ratio, ionic strength and ligands must be screened in a procedure involving four steps: crystallization, specimen preparation for electron microscopy, image acquisition, and evaluation. Traditionally, these steps have been carried out manually and, as a result, the scope of 2D crystallization trials has been limited. We have therefore developed an automated pipeline to screen the formation of 2D crystals. We employed a 96-well dialysis block for reconstitution of the target protein over a wide range of conditions designed to promote crystallization. A 96-position magnetic platform and a liquid handling robot were used to prepare negatively stained specimens in parallel. Robotic grid insertion into the electron microscope and computerized image acquisition ensures rapid evaluation of the crystallization screen. To date, 38 2D crystallization screens have been conducted for 15 different membrane proteins, totaling over 3000 individual crystallization experiments. Three of these proteins have yielded diffracting 2D crystals. Our automated pipeline outperforms traditional 2D crystallization methods in terms of throughput and reproducibility.

Figure 1. Pipeline to screen 2D crystallization trials of membrane proteins

Target membrane proteins are purified in detergent micelles in a stable and monodisperse form. Following the addition of lipids to form protein-lipid-detergent ternary mixtures, excess detergent is removed by dialysis using the 96-well crystallization block. Upon completion of a 2D crystallization experiment the 96 conditions are harvested, transferred to EM grids and negative stained specimens made in parallel using a liquid-handling robot. The EM grids are robotically inserted into the electron microscope, and images of the crystallization outcomes are recorded automatically by image-acquisition software. Finally, an experienced microscopist performs data analysis and evaluation. A negatively stained image of a 2D crystal of YkgB-D36, in which the lattice is clearly visible, is shown as an example of a successful outcome.

Figure 2. Production of target membrane proteins and lipid solubilization

a) Membrane protein stability screen in different detergents. In this case, the protein YkgB-D332 was purified in DDM and run on a silica based size exclusion chromatography column equilibrated with an aqueous mobile phase containing the detergents DM, DDM, OG and NG at a concentration of twice the CMC. The elution profiles (rate of 0.25 ml/min) are shown. The color codes for the traces are red=DM, blue=DDM, green=OG and pink=NG. b) As evaluated by SDS-PAGE the proteins used in the 2D crystallization trials were at least 90% pure. Note that the acryl amide concentration used in the gels varies. The Kdp protein is composed of four different subunits, of which one is only 3kDa and appears as a smear at the bottom of the gel. 1: E2P1, 2: E2P2, 3: E2P3, 4: E2P4, 5: P2A3, 6: Rhomboid PA3086, 7: YkgB-D332, 8: YkgB-D36, 9: β1-adrenergic receptor, 10: Rhomboid GlpG, 11: Cytochrome b561, 12: E1 protein, 13: P40B7, 14: Kdp-ATPase, 15: P39H10. c) Turbidity measurements were employed to assess the solubilization of lipid by detergent. The initial turbidity of the solution was normalized to 100%. The lipid concentration was kept constant at 1.5mg/ml throughout the experiment. Increasing amounts of highly concentrated OG were added to the mixture and the UV absorbance was recorded at 500nm following equilibration. As detergent is added, and the lipid vesicles become solubilized as lipid-detergent mixed micelles, the OD₅₀₀ decreases, eventually falling to a stable baseline corresponding to fully solubilized lipid. All lipids became fully solubilized at an OG concentration of 10mg/ml.

Figure 3. Setup for preparation of EM specimens by negative staining

a) The magnetic plate used for the automated negative staining protocol. Each post on the plate is made from an individual rare earth magnet. A carbon coated, glow-discharged Ni-grid is manually transferred to the plate by tweezers. b) Following completion of the staining protocol, the last aliquot of stain is left on the grids and the magnetic plate is removed from the liquid-handling robot. Long strips of filter paper are used to blot away the remaining stain from the grids, one row of grids at a time.

Figure 4. Scoring system and outcome evaluation

a) Representative images of the six-grade quality scoring system of the 2D crystallization outcomes. b) Outcome from a 2D crystallization screen of the protein P2A3 is displayed in the form of a histogram. For clarity, the histogram displays only results obtained at pH 7.0. The depth axis corresponds to the quality score described in a. In addition, the y-axis reflects the abundance of the observed objects; 1: up to 2 objects/grid square, 2: 3–10 objects/grid square, 3: 11–20 objects/grid square, and 4: more than 20 objects/grid square.

Figure 5. Gallery of negatively stained images of diffracting crystals obtained using the 2D crystallization pipeline

a) P2A3 and b) YkgB-D36 yielded narrow tubular crystals, whereas c) SFV fusion protein produced flat, sheet-like crystals. The scale bars are 50nm.