PDBsum entry 1kme

Membrane protein

PDB id: 1kme

Contents

Protein chains

227 a.a. *

Ligands

RET ×2

SQU ×2

BGC ×2

Waters ×122

* Residue conservation analysis

PDB id:

1kme

Name:

Membrane protein

Title:

Crystal structure of bacteriorhodopsin crystallized from bicelles

Structure:

Bacteriorhodopsin. Chain: a, b. Fragment: residues 14-244. Synonym: br

Source:

Halobacterium salinarum. Organism_taxid: 2242

Resolution:

2.00Å R-factor: 0.263 R-free: 0.275

Authors:

S.Faham,J.U.Bowie

Key ref:

S.Faham and J.U.Bowie (2002). Bicelle crystallization: a new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure. J Mol Biol, 316, 1-6. PubMed id: 11829498 DOI: 10.1006/jmbi.2001.5295

Date:

14-Dec-01 Release date: 13-Feb-02

Protein chains

P02945  (BACR_HALSA) -  Bacteriorhodopsin from Halobacterium salinarum (strain ATCC 700922 / JCM 11081 / NRC-1)

Seq: 262 a.a.

Struc: 227 a.a.

DOI no: 10.1006/jmbi.2001.5295

J Mol Biol 316:1-6 (2002)

PubMed id: 11829498

Bicelle crystallization: a new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure.

S.Faham, J.U.Bowie.

ABSTRACT

Obtaining crystals of membrane proteins that diffract to high resolution remains a major stumbling block in structure determination. Here we present a new method for crystallizing membrane proteins from a bicelle forming lipid/detergent mixture. The method is flexible and simple to use. As a test case, bacteriorhodopsin (bR) from Halobacterium salinarum was crystallized from a bicellar solution, yielding a new bR crystal form. The crystals belong to space group P2(1) with unit cell dimensions of a=45.0 A, b=108.9 A, c=55.9 A, beta=113.58 degrees and a dimeric asymmetric unit. The structure was solved by molecular replacement and refined at 2.0 A resolution. In all previous bR structures the protein is organized as a parallel trimer, but in the crystals grown from bicelles, the individual bR subunits are arranged in an antiparallel fashion.

Selected figure(s)

Figure 1.
Figure 1. Bicelle crystallization method. (a) Outline of the method. (b) Crystals of bR grown from bicelle forming lipid/detergent mixture. Purple membrane was purified as described by Oesterhelt & Steckenius.[29] Purple membrane was suspended in water to a bR concentration of vert, similar 10 mg/ml and was mixed in a 4:1 ratio with a 40% (3:1) DMPC/Chapso bicellar solution, making a vert, similar 8.0 mg/ml bR/8% bicelles mixture. Reconstitution and homogenization of the protein into the bicelles was achieved simply by repeated pipeting of the solution. Crystals were grown using the hanging, or sitting drop method and all solutions were kept on ice prior to mixing to keep the bicelle mixtures fluid. The crystallization drops contained 6 µl of protein/bicelle solution with 2.5 µl of well solution and 1 µl of 2.5% b-octylglucoside (OG) solution. The well solution contained 3.2 M NaPO[4] (pH 3.5). The crystal trays were then placed in a 37°C incubator to allow the bicelles to gel. Diamond-shaped crystals grew within a few days to two weeks. The addition of OG was not required for crystal growth; however, larger crystals were obtained in its presence.

Figure 3.
Figure 3. The packing interfaces in the bicelle crystals. (a) Top view of a layer. (b) Side view of a layer. Helices A and B (red) and helices C and D (blue), provide the main contributions to interface I. Helices E and F (green) provide the main contributions to interface II. Figure 2 and Figure 3 were prepared using the program MOLSCRIPT. [30]

The above figures are reprinted by permission from Elsevier: J Mol Biol (2002, 316, 1-6) copyright 2002.

Figures were selected by the author.