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Free RCK Arrangement in Kch, a Putative Escherichia coli Potassium Channel, as Suggested by Electron Crystallography
KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.ORCID iD: 0000-0002-0569-3374
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2015 (English)In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 23, no 1, 199-205 p.Article in journal (Refereed) Published
Abstract [en]

The ligand-gated potassium channels are stimulated by various kinds of messengers. Previous studies showed that ligand-gated potassium channels containing RCK domains (the regulator of the conductance of potassium ion) form a dimer of tetramer structure through the RCK octameric gating ring in the presence of detergent. Here, we have analyzed the structure of Kch, a channel of this type from Escherichia coli, in a lipid environment using electron crystallography. By combining information from the 3D map of the transmembrane part of the protein and docking of an atomic model of a potassium channel, we conclude that the RCK domains face the solution and that an RCK octameric gating ring arrangement does not form under our crystallization condition. Our findings may be applied to other potassium channels that have an RCK gating ring arrangement.

Place, publisher, year, edition, pages
2015. Vol. 23, no 1, 199-205 p.
National Category
Structural Biology
Identifiers
URN: urn:nbn:se:kth:diva-160349DOI: 10.1016/j.str.2014.10.018ISI: 000347469500024Scopus ID: 2-s2.0-84920999760OAI: oai:DiVA.org:kth-160349DiVA: diva2:789438
Funder
Swedish Research Council
Note

QC 20150623

Available from: 2015-02-18 Created: 2015-02-18 Last updated: 2017-12-04Bibliographically 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.
Series
TRITA-STH : report, ISSN 1653-3836 ; 2015:1
National Category
Structural Biology
Identifiers
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)
Opponent
Supervisors
Funder
Swedish Research Council
Note

QC 20150320

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

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Hebert, Hans

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