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The projection structure of Kch, a putative potassium channel in Escherichia coli, by electron crystallography
KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.ORCID iD: 0000-0002-3220-9402
2014 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1838, no 1, 237-243 p.Article in journal (Refereed) Published
Abstract [en]

The kch gene, the only potassium channel gene in Escherichia coil, has the property to express both full-length Kch and its cytosolic domain (RCK) due to a methionine at position 240. The RCK domains are believed to form an octameric ring structure and regulate the gating of the potassium channels after having bound certain ligands. Several different gating ring structures have been reported for the soluble RCK domains, however, these were studied isolated from their transmembrane parts. We previously reported an octameric structure of Kch in solution by electron microscopy and single particle reconstruction, composed of two tetrameric full-length proteins through RCK interaction. To exclude the effect of the detergent, we have now performed an electron crystallographic study of the full-length Kch in membrane bound form. Well-ordered two-dimensional crystals were grown in a natural phospholipid environment. A projection map merged from the fifteen best images extended to 6 angstrom resolution. The c12 two-sided plane group of the two-dimensional crystals showed that Kch crystallized as two symmetrically related overlapping layers. The arrangement suggests that the two layers of RCK domains are shifted with respect to each other and the RCK octameric gating ring of Kch does not form under the crystallization condition.

Place, publisher, year, edition, pages
2014. Vol. 1838, no 1, 237-243 p.
Keyword [en]
Electron microscopy, Projection map, Potassium channel, RCK, Membrane protein
National Category
Biochemistry and Molecular Biology Biophysics
URN: urn:nbn:se:kth:diva-142509DOI: 10.1016/j.bbamem.2013.09.006ISI: 000330814000016ScopusID: 2-s2.0-84887960452OAI: diva2:703513
Swedish Research Council

QC 20140307

Available from: 2014-03-07 Created: 2014-03-06 Last updated: 2015-03-20Bibliographically approved
In thesis
1. Structural studies of membrane proteins using transmission electron microscopy
Open this publication in new window or tab >>Structural studies of membrane proteins using transmission electron microscopy
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Membrane proteins play important roles for living cells. They control transportation of ions, solutes, and nutrients across the membrane and catalyze metabolic reactions. Transmission electron microscopy has its advantages in convenient sample preparation, straightforward structural determination, and wide applications for diverse specimens. In this thesis, the structure of three membrane proteins are studied by this method.

Kch, a potassium channel in Escherichia coli, has a transmembrane part and a cytosolic domain. Large and well-ordered two dimensional crystals were obtained from both a functional mutant (KchM240L) and a modified protein possessing only the transmembrane part (KchTM). Both samples crystallize as two symmetry-related overlapping layers. Furthermore, the KchTM structure was reconstructed which showed that the transmembrane part of the two adjacent proteins are involved in forming the crystal contacts. Thus, the cytosolic domains of Kch in crystals are deduced to expose to the solvent and do not interact with each other.

MGST1 (microsomal glutathione transferase 1) is a detoxification enzyme. It was recombinantly over-expressed in the current study, instead of purified from rat liver as before. The crystallization condition was adjusted and isomorphic crystals were obtained. The refined model was built from a combined data set consisting of previous and new diffraction patterns. More residues at the C-terminus of the transmembrane helix 1 were assigned and the residues in the transmembrane helices 3 and 4 were remodeled. Several phospholipid molecules were observed and the ligand glutathione adopts an extended conformation in the refined model.

The structure of MelB (a sugar/sodium symporter in Escherichia coli) was determined using a refined single particle reconstruction method. This novel method is aimed for processing small or locally distorted crystals. In comparison with the previously published single particle reconstruction protocol, the current method is improved in several aspects. A more reliable reconstruction of MelB was obtained and the resolution was increased. The docking experiment indicates that MelB adopts an open conformation under the present two dimensional crystallization condition.

Electron microscopy has developed quickly recently with the help of modern instruments, techniques, and software. This method will without doubt play a more critical role in future structural biology.


Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2015. viii, 55 p.
TRITA-STH : report, ISSN 1653-3836 ; 2015:1
National Category
Structural Biology
urn:nbn:se:kth:diva-161721 (URN)978-91-7595-468-4 (ISBN)
Public defence
2015-04-13, Lecture hall 221, Alfred Nobels Allé 10, Flemingsberg, Huddinge, 09:00 (English)
Swedish Research Council

QC 20150320

Available from: 2015-03-20 Created: 2015-03-13 Last updated: 2015-09-11Bibliographically approved

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