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A refined atomic model for microsomal glutathione transferase 1 from electron crystallography
KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Microsomal glutathione transferase 1 (MGST1) is a detoxification enzyme belonging to the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. Here we have used electron crystallography of two-dimensional (2D) crystals in order to determine an atomic model of rat MGST1 in a lipid environment. The 2D crystals were of the p6 two-sided plane group symmetry. For the refinement, information to 3.5 Å resolution from 225 electron diffraction patterns recorded from specimens at tilt angles up to 66° was used. The model comprises 123 of the 155 amino acid residues, two structured phospholipid molecules, two hydrocarbon chains, and one glutathione (GSH) molecule. Interactions between subunits form trimers centered on the crystallographic three-fold axes of the unit cell. The GSH substrate binds in an extended conformation at the interface between two subunits of the trimer. The location of GSH is supported by mutagenesis data in vitro.

National Category
Structural Biology
Identifiers
URN: urn:nbn:se:kth:diva-161719OAI: oai:DiVA.org:kth-161719DiVA: diva2:795160
Funder
Swedish Research Council
Note

QS 2015

Available from: 2015-03-13 Created: 2015-03-13 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.
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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