Automated screening of 2D crystallization trials using transmission electron microscopy: a high-throughput tool-chain for sample preparation and microscopic analysis.

We have built and extensively tested a tool-chain to prepare and screen two-dimensional crystals of membrane proteins by transmission electron microscopy (TEM) at room temperature. This automated process is an extension of a new procedure described recently that allows membrane protein 2D crystallization in parallel (Iacovache et al., 2010). The system includes a gantry robot that transfers and prepares the crystalline solutions on grids suitable for TEM analysis and an entirely automated microscope that can analyze 96 grids at once without human interference. The operation of the system at the user level is solely controlled within the MATLAB environment: the commands to perform sample handling (loading/unloading in the microscope), microscope steering (magnification, focus, image acquisition, etc.) as well as automatic crystal detection have been implemented. Different types of thin samples can efficiently be screened provided that the particular detection algorithm is adapted to the specific task. Hence, operating time can be shared between multiple users. This is a major step towards the integration of transmission electron microscopy into a high throughput work-flow.

The 2DX robot: a membrane protein 2D crystallization Swiss Army knife.

Among the state-of-the-art techniques that provide experimental information at atomic scale for membrane proteins, electron crystallography, atomic force microscopy and solid state NMR make use of two-dimensional crystals. We present a cyclodextrin-driven method for detergent removal implemented in a fully automated robot. The kinetics of the reconstitution processes is precisely controlled, because the detergent complexation by cyclodextrin is of stoichiometric nature. The method requires smaller volumes and lower protein concentrations than established 2D crystallization methods, making it possible to explore more conditions with the same amount of protein. The method yielded highly ordered 2D crystals diffracting to high resolution from the pore-forming toxin Aeromonas hydrophila aerolysin (2.9A), the plant aquaporin SoPIP2;1 (3.1A) and the human aquaporin-8 (hAQP8; 3.3A). This new method outperforms traditional 2D crystallization approaches in terms of accuracy, flexibility, throughput, and allows the usage of detergents having low critical micelle concentration (CMC), which stabilize the structure of membrane proteins in solution.

Projection maps of three members of the KdgM outer membrane protein family.

We present the projection structures of the three outer membrane porins KdgM and KdgN from Erwinia chrysanthemi and NanC from Escherichia coli, based on 2D electron crystallography. A wide screening of 2D crystallization conditions yielded tubular crystals of a suitable size and quality to perform high-resolution electron microscopy. Data processing of untilted samples allowed us to separate the information of the two crystalline layers and resulted in projection maps to a resolution of up to 7A. All three proteins exhibit a similar putative beta-barrel structure and the three crystal forms have the same symmetry. However, there are differences in the packing arrangements of the monomers as well as the densities of the projections. To interpret these projections, secondary structure prediction was performed using beta-barrel specific prediction algorithms. The predicted transmembrane beta-barrels have a high similarity in the arrangement of the putative beta-strands and the loops, but do not match those of OmpG, a related protein porin whose structure was solved.

Controlled 2D crystallization of membrane proteins using methyl-beta-cyclodextrin.

High-resolution structural data of membrane proteins can be obtained by studying 2D crystals by electron crystallography. Finding the right conditions to produce these crystals is one of the major bottlenecks encountered in 2D crystallography. Many reviews address 2D crystallization techniques in attempts to provide guidelines for crystallographers. Several techniques including new approaches to remove detergent like the biobeads technique and the development of dedicated devices have been described (dialysis and dilution machines). In addition, 2D crystallization at interfaces has been studied, the most prominent method being the 2D crystallization at the lipid monolayer. A new approach based on detergent complexation by cyclodextrins is presented in this paper. To prove the ability of cyclodextrins to remove detergent from ternary mixtures (lipid, detergent and protein) in order to get 2D crystals, this method has been tested with OmpF, a typical beta-barrel protein, and with SoPIP2;1, a typical alpha-helical protein. Experiments over different time ranges were performed to analyze the kinetic effects of detergent removal with cyclodextrins on the formation of 2D crystals. The quality of the produced crystals was assessed with negative stain electron microscopy, cryo-electron microscopy and diffraction. Both proteins yielded crystals comparable in quality to previous crystallization reports.

A novel method for detergent concentration determination.

A fast and precise method for detergent concentration determination is presented. (Patent applications for the method described here have been submitted (EP05011904 and US60/702,261). Depending on the interest of the scientific community, the system will be commercialized. (For further information contact Hervé-W. Rémigy at the e-mail address below.) A small droplet of the detergent solution is deposited on a piece of Parafilm M and side views are recorded by two orthogonally arranged TV cameras. The droplet contours are then approximated by ellipses to determine the contact angles. Comparison of the observed contact angle values to calibrated standard curves of known detergent concentrations gives the concentration of the detergent assessed. A range of commonly used detergents was studied to demonstrate the reproducibility and precision of this simple method. As a first application, the detergent binding capacity of the Escherichia coli galactose/proton symporter (GalP) was assessed. Aggregation of GalP was observed when <260 +/- 5 dodecyl-beta,D-maltoside molecules were bound to one GalP molecule. These measurements document the efficacy of the drop-shape based detergent concentration determination described.

Structural insights into the secretin PulD and its trypsin-resistant core.

Limited proteolysis, secondary structure and biochemical analyses, mass spectrometry, and mass measurements by scanning transmission electron microscopy were combined with cryo-electron microscopy to generate a three-dimensional model of the homomultimeric complex formed by the outer membrane secretin PulD, an essential channel-forming component of the type II secretion system from Klebsiella oxytoca. The complex is a dodecameric structure composed of two rings that sandwich a closed disc. The two rings form chambers on either side of a central plug that is part of the middle disc. The PulD polypeptide comprises two major, structurally quite distinct domains; an N domain, which forms the walls of one of the chambers, and a trypsin-resistant C domain, which contributes to the outer chamber, the central disc, and the plug. The C domain contains a lower proportion of potentially transmembrane beta-structure than classical outer membrane proteins, suggesting that only a small part of it is embedded within the outer membrane. Indeed, the C domain probably extends well beyond the confines of the outer membrane bilayer, forming a centrally plugged channel that penetrates both the peptidoglycan on the periplasmic side and the lipopolysaccharide and capsule layers on the cell surface. The inner chamber is proposed to constitute a docking site for the secreted exoprotein pullulanase, whereas the outer chamber could allow displacement of the plug to open the channel and permit the exoprotein to escape.

Isolation and Characterization of an Outer Membrane Protein of Chlorobium tepidum.

A protein was isolated from membranes of the green sulfur bacterium Chlorobium tepidum. This protein was characterized by gel electrophoresis, gel filtration, analytical ultracentrifugation and amino acid sequencing. The molecular weight of the purified protein was shown to be 26 kDa by SDS-PAGE. HPLC gelfiltration, SDS-PAGE and analytical ultracentrifugation are consistent with the presence of a homogenous protein in the preparations. Amino acid analysis was obtained from the isolated protein after fragmentation with Lys-C, trypsin and cyanogen bromide. The cleavage pattern resulting from these treatments combined with Edman sequencing yield a sequence allowing the identification of an integral membrane agglutinin in Chl. tepidum.

Characterization by Mass Spectroscopy of a 10 kDa c-554 Cytochrome from the Green Sulfur Bacterium Chlorobium tepidum.

Preparative isoelectric focusing was used to isolate a type c cytochrome from photosynthetic membranes of the green sulfur bacterium Chlorobium tepidum. The purified protein showed a molecular weight of 10 kDa according to SDS-PAGE and ESI mass spectrometry. The absorption spectrum in the visible range is typical of a cytochrome with peaks at 420, 525.2 and 554.4 nm. Cleavage by either trypsin or endoproteinase lys-C of the isolated cytochrome combined with tandem mass spectrometry and Edman sequencing yielded a sequence perfectly matching parts of the recently sequenced genome of C. tepidum.

The reaction centre from green sulphur bacteria: progress towards structural elucidation.

The reaction centre (RC) of green sulphur bacteria is a FeS-type RC, as are the RCs of Photosystems I (PS I) of oxygenic photosynthetic organisms and of heliobacteria. The core domains of both green sulphur bacterial and heliobacterial RCs are considered to be homodimeric, in contrast to those of purple bacteria, PS I and Photosystem II (PS II). This paper briefly describes the techniques of electron microscopy and image processing suited to investigate the structure of these proteins. Recent advances in the study of the structure of the green sulphur bacterial RC, primarily achieved by the application of scanning transmission electron microscopy, are reviewed.