Metal-mediated crystallization of the xylose transporter XylE from Escherichia coli in three different crystal forms.

XylE is a major facilitator (MFS) xylose transporter, which is homologous to the mammalian glucose transporters (GLUT family). We have previously reported the structure of XylE in fully inward open and partially occluded inward open conformations in space groups P61 and C2, respectively. Here we present the crystallization of a third crystal form, P212121 (~4 Å resolution), also representing an inward facing conformation, and analyze all three forms in terms of crystallization conditions and packing. The crystallization conditions were generally very similar with only slight changes needed to favor one form over another, e.g. the presence of lanthanide ions greatly favors C2 over P212121 under otherwise identical conditions. Cadmium was essential for crystallization of all three forms, which indeed all contain a Cd(2+) ion in a crystal packing interface, though surprisingly in different positions. Cadmium was also found to bind to XylE in solution. The diffraction data were highly anisotropic for all forms, reflecting a lack of ordered crystal contacts along one or two of the cell axes. The best diffracting directions thus consistently correlate with the presence of ordered contacts, most of which are metal-mediated. The data presented here highlight the utility of metal ions in membrane protein crystallization and suggest that metal site engineering may be a productive path towards obtaining additional crystal forms of XylE and other membrane proteins.

Lipidic cubic phase serial femtosecond crystallography structure of a photosynthetic reaction centre.

Serial crystallography is a rapidly growing method that can yield structural insights from microcrystals that were previously considered to be too small to be useful in conventional X-ray crystallography. Here, conditions for growing microcrystals of the photosynthetic reaction centre of Blastochloris viridis within a lipidic cubic phase (LCP) crystallization matrix that employ a seeding protocol utilizing detergent-grown crystals with a different crystal packing are described. LCP microcrystals diffracted to 2.25 Šresolution when exposed to XFEL radiation, which is an improvement of 0.15 Šover previous microcrystal forms. Ubiquinone was incorporated into the LCP crystallization media and the resulting electron density within the mobile QB pocket is comparable to that of other cofactors within the structure. As such, LCP microcrystallization conditions will facilitate time-resolved diffraction studies of electron-transfer reactions to the mobile quinone, potentially allowing the observation of structural changes associated with the two electron-transfer reactions leading to complete reduction of the ubiquinone ligand.

Membrane-protein crystals for neutron diffraction.

Neutron macromolecular crystallography (NMX) has the potential to provide the experimental input to address unresolved aspects of transport mechanisms and protonation in membrane proteins. However, despite this clear scientific motivation, the practical challenges of obtaining crystals that are large enough to make NMX feasible have so far been prohibitive. Here, the potential impact on feasibility of a more powerful neutron source is reviewed and a strategy for obtaining larger crystals is formulated, exemplified by the calcium-transporting ATPase SERCA1. The challenges encountered at the various steps in the process from crystal nucleation and growth to crystal mounting are explored, and it is demonstrated that NMX-compatible membrane-protein crystals can indeed be obtained.

A novel microseeding method for the crystallization of membrane proteins in lipidic cubic phase.

Random microseed matrix screening (rMMS), in which seed crystals are added to random crystallization screens, is an important breakthrough in soluble protein crystallization that increases the number of crystallization hits that are available for optimization. This greatly increases the number of soluble protein structures generated every year by typical structural biology laboratories. Inspired by this success, rMMS has been adapted to the crystallization of membrane proteins, making LCP seed stock by scaling up LCP crystallization conditions without changing the physical and chemical parameters that are critical for crystallization. Seed crystals are grown directly in LCP and, as with conventional rMMS, a seeding experiment is combined with an additive experiment. The new method was used with the bacterial integral membrane protein OmpF, and it was found that it increased the number of crystallization hits by almost an order of magnitude: without microseeding one new hit was found, whereas with LCP-rMMS eight new hits were found. It is anticipated that this new method will lead to better diffracting crystals of membrane proteins. A method of generating seed gradients, which allows the LCP seed stock to be diluted and the number of crystals in each LCP bolus to be reduced, if required for optimization, is also demonstrated.

Cell-Free Production of Membrane Proteins in Escherichia coli Lysates for Functional and Structural Studies.

The complexity of membrane protein synthesis is largely reduced in cell-free systems and it results into high success rates of target expression. Protocols for the preparation of bacterial lysates have been optimized in order to ensure reliable efficiencies in membrane protein production that are even sufficient for structural applications. The open accessibility of the semisynthetic cell-free expression reactions allows to adjust membrane protein solubilization conditions according to the optimal folding requirements of individual targets. Two basic strategies will be exemplified. The post-translational solubilization of membrane proteins in detergent micelles is most straightforward for crystallization approaches. The co-translational integration of membrane proteins into preformed nanodiscs will enable their functional characterization in a variety of natural lipid environments.

Fusion-protein-assisted protein crystallization.

Fusion proteins can be used directly in protein crystallization to assist crystallization in at least two different ways. In one approach, the `heterologous fusion-protein approach', the fusion partner can provide additional surface area to promote crystal contact formation. In another approach, the `fusion of interacting proteins approach', protein assemblies can be stabilized by covalently linking the interacting partners. The linker connecting the proteins plays different roles in the two applications: in the first approach a rigid linker is required to reduce conformational heterogeneity; in the second, conversely, a flexible linker is required that allows the native interaction between the fused proteins. The two approaches can also be combined. The recent applications of fusion-protein technology in protein crystallization from the work of our own and other laboratories are briefly reviewed.

Crystallization of membrane proteins by vapor diffusion.

X-ray crystallography remains the most robust method to determine protein structure at the atomic level. However, the bottlenecks of protein expression and purification often discourage further study. In this chapter, we address the most common problems encountered at these stages. Based on our experiences in expressing and purifying antimicrobial efflux proteins, we explain how a pure and homogenous protein sample can be successfully crystallized by the vapor diffusion method. We present our current protocols and methodologies for this technique. Case studies show step-by-step how we have overcome problems related to expression and diffraction, eventually producing high-quality membrane protein crystals for structural determinations. It is our hope that a rational approach can be made of the often anecdotal process of membrane protein crystallization.

An unconventional anaerobic membrane protein production system based on Wolinella succinogenes.

In cases where membrane protein production attempts in more conventional Escherichia coli-based systems have failed, a solution is to resort to a system based on the nonpathogenic epsilon-proteobacterium Wolinella succinogenes. This approach has been demonstrated to be successful for structural and mechanistic analyses not only for homologous production of W. succinogenes membrane proteins but also for the heterologous production of membrane protein complexes from the human pathogens Helicobacter pylori and Campylobacter jejuni. The procedure to establish a system for the production of native and variant enzymes in W. succinogenes is presented in detail for the examples of the quinol:fumarate reductase and the SdhABE complexes of W. succinogenes. Subsequently, further projects using W. succinogenes as expression host are covered.

The lipid cubic phase or in meso method for crystallizing proteins. Bushings for better manual dispensing.

The lipid cubic phase or in meso method can be used to set up crystallization trials of soluble and membrane proteins. The cubic phase is noted for being a sticky and viscous mesophase. Dispensing the protein-laden mesophase by hand into wells on crystallization plates has been facilitated by the use of an inexpensive repeat dispenser. However, the assembled dispensing device is prone to damage. Specifically, the delicate plunger used to dispense the viscous mesophase by positive displacement can be bent and scarred when the locking nut that fixes the plunger to the ratchet-driven dispensing mechanism is inadvertently overtightened. A defective plunger can render the device useless as a dispensing tool. More importantly, it can lead to catastrophic loss of valuable protein and lipid due to leakage when the dispensing syringe is being recharged with fresh mesophase. This note describes two types of bushings designed to protect the plunger from mechanical damage, which provide facile and reliable dispenser performance. One is a split bushing in brass and is a highly durable solution. The other is a small sleeve made from readily available plastic tubing. While it lacks durability, the plastic sleeve is simple yet highly effective, and can be replaced as the need arises.