The European Membrane Protein Consortium (E-MeP)
By integrating the activities of many of the world leaders in membrane protein structural biology and relevant related areas, the E-MeP consortium has solved 5 crystal structures, produced 22 diffracting crystals and made progress in elucidating the parameters responsible for success and failure at each key stage of heterologous production, purification and crystallisation. At the same time E-MeP is building a sustainable European membrane protein structural genomics research area through its research activities, dissemination of expertise, and by directly addressing the current shortage of appropriately-trained young scientists in cooperation with the training programme E-MeP-Lab.
E-MeP Structures
The following membrane protein structures have been determined as part of E-MeP's research.
Crystal Structure of AcrB in Complex with a Single Transmembrane Subunit Reveals Another TwistHere, we present the X-ray structure of AcrB in complex with a single transmembrane protein, assigned by mass spectrometry as YajC. A specific rotation of the periplasmic porter domain of AcrB is also revealed, consistent with the hypothesized “twist-to-open” mechanism for TolC activation. Growth experiments with yajc-deleted E. coli reveal a modest increase in the organism's susceptibility to β-lactam antibiotics, but this effect could not conclusively be attributed to the loss of interactions between YajC and AcrB.
"Crystal Structure of a Divalent Metal Ion Transporter CorA at 2.9 Angstrom Resolution"We have determined a 2.9 angstrom resolution structure of CorA from Thermotoga maritima that reveals a pentameric cone-shaped protein.
Structural mechanism of plant aquaporin gatingHere we report the X-ray structure of the spinach plasma membrane aquaporin SoPIP2;1 in its closed conformation at 2.1 Å resolution and in its open conformation at 3.9 Å resolution, and molecular dynamics simulations of the initial events governing gating. In the closed conformation loop D caps the channel from the cytoplasm and thereby occludes the pore.
"Structural basis for synthesis of inflammatory mediators by human leukotriene C4 synthase"Here we present the crystal structure of the human LTC4 synthase in its apo and GSH-complexed forms to 1.00 and 2.15 Å resolution, respectively. The structure reveals a homotrimer, where each monomer is composed of four transmembrane segments.
Structural evidence for induced fit and a mechanism for sugar/H+ symport in LacYHere, we present two novel ligand-free X-ray structures of the lactose permease (LacY) of Escherichia coli determined at acidic and neutral pH, and propose a model for the mechanism of coupling between lactose and H+ translocation.
E-MeP Technologies
Cell membranes suspended across nanoaperture arrays for functional analysis of membrane proteinsWe present a method for spreading large (>100 μm2) cell membrane fragments across nanoapertures in planar supports. Electron-beam and focused-ion-beam lithography were used to fabricate arrays of 50-600 nm diameter holes in free-standing silicon nitride (SiN) solid films 100-500 nm thick. By pressing adhering live cells onto the nanostructured SiN surface and then removing them, planar cell membrane sheets (CMSs) were transferred in a well-defined orientation onto the SiN support.
High-throughput cloning and expression in recalcitrant bacteria.Presented here is a generic method for high-throughput cloning in bacteria less amenable to conventional DNA manipulations. The method employs ligation-independent cloning in an intermediary Escherichia coli vector, which is rapidly converted via vector backbone exchange (VBEx) into a bona fide, organism-specific plasmid ready for high-efficiency transformation. Here, we describe the VBEx procedure for Lactococcus lactis.
E-MeP Lab
The E-MeP-Lab project provides funds to exploit this confluence of talent.
For the first time Europe's membrane protein structural biology community will converge as teachers and demonstrators in a Master Class and five Advanced Practical Courses in the best equipped laboratories in their fields in Europe. This research field is important because membrane proteins comprise the major target group of modern structural genomics, with approximately 70% of human targets for therapeutic intervention being from this class.E-MeP-Lab's objectives are to:
- increase the pool of appropriately-skilled young researchers in membrane protein structural genomics;
- provide access for all European researchers to training programmes.
In addition to working with researchers from European Member States, the project aims to harness the considerable scientific talent in the Accession countries to ensure their full participation within the European Structural Genomics community, through the provision of ring-fenced funding.
Together with an analysis provided by an expert in gender and mobility issues, this will provide an excellent opportunity to evaluate the potential impact of enlargement on scientific mobility and gender participation.
Thus, achievement of balanced growth in the wider ERA will be achieved with a particular focus on the transfer of knowledge in the structural genomics of membrane proteins.