4BPD

Structure determination of an integral membrane kinase

Summary for 4BPD

• Entry DOI: 10.2210/pdb4bpd/pdb
• Descriptor: DIACYLGLYCEROL KINASE, (2S)-2,3-DIHYDROXYPROPYL(7Z)-PENTADEC-7-ENOATE, ZINC ION (3 entities in total)
• Functional Keywords: transferase, cell-free, dgka, in meso crystallization, in vitro expression, lipid metabolism, lipidic mesophase, lipidic cubic phase, lcp, membrane protein, microcrystal, 7.8 mag, thermostable mutant
• Biological source: ESCHERICHIA COLI
• Total number of polymer chains: 6
• Total formula weight: 86549.95

Authors

Li, D.,Boland, C.,Caffrey, M. (deposition date: 2013-05-24, release date: 2014-05-07, Last modification date: 2023-12-20)

Primary citation

Boland, C.,Li, D.,Shah, S.T.A.,Haberstock, S.,Dotsch, V.,Bernhard, F.,Caffrey, M.
Cell-Free Expression and in Meso Crystallisation of an Integral Membrane Kinase for Structure Determination.
Cell.Mol.Life Sci., 71:4895-, 2014

PubMed Abstract: Membrane proteins are key elements in cell physiology and drug targeting, but getting a high-resolution structure by crystallographic means is still enormously challenging. Novel strategies are in big demand to facilitate the structure determination process that will ultimately hasten the day when sequence information alone can provide a three-dimensional model. Cell-free or in vitro expression enables rapid access to large quantities of high-quality membrane proteins suitable for an array of applications. Despite its impressive efficiency, to date only two membrane proteins produced by the in vitro approach have yielded crystal structures. Here, we have analysed synergies of cell-free expression and crystallisation in lipid mesophases for generating an X-ray structure of the integral membrane enzyme diacylglycerol kinase to 2.28-Å resolution. The quality of cellular and cell-free-expressed kinase samples has been evaluated systematically by comparing (1) spectroscopic properties, (2) purity and oligomer formation, (3) lipid content and (4) functionality. DgkA is the first membrane enzyme crystallised based on cell-free expression. The study provides a basic standard for the crystallisation of cell-free-expressed membrane proteins and the methods detailed here should prove generally useful and contribute to accelerating the pace at which membrane protein structures are solved.

PubMed: 25012698
DOI: 10.1007/S00018-014-1655-7

Experimental method

X-RAY DIFFRACTION (3.3 Å)