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A helical processing pipeline for EM structure determination of membrane proteins.

ABSTRACT: Electron crystallography plays a key role in the structural biology of integral membrane proteins (IMPs) by offering one of the most direct means of providing insight into the functional state of these molecular machines in their lipid-associated forms, and also has the potential to facilitate examination of physiologically relevant transitional states and complexes. Helical or tubular crystals, which are the natural product of proteins crystallizing on the surface of a cylindrical vesicle, offer some unique advantages, such as three-dimensional (3D) information from a single view, compared to other crystalline forms. While a number of software packages are available for processing images of helical crystals to produce 3D electron density maps, widespread exploitation of helical image reconstruction is limited by a lack of standardized approaches and the initial effort and specialized expertise required. Our goal is to develop an integrated pipeline to enable structure determination by transmission electron microscopy (TEM) of IMPs in the form of tubular crystals. We describe here the integration of standard Fourier-Bessel helical analysis techniques into Appion, an integrated, database-driven pipeline.

SUBMITTER: Fisher LS

PROVIDER: S-EPMC3262078 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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A helical processing pipeline for EM structure determination of membrane proteins.

Fisher Lauren S LS Ward Andrew A Milligan Ronald A RA Unwin Nigel N Potter Clinton S CS Carragher Bridget B

Methods (San Diego, Calif.) 20110920 4

Electron crystallography plays a key role in the structural biology of integral membrane proteins (IMPs) by offering one of the most direct means of providing insight into the functional state of these molecular machines in their lipid-associated forms, and also has the potential to facilitate examination of physiologically relevant transitional states and complexes. Helical or tubular crystals, which are the natural product of proteins crystallizing on the surface of a cylindrical vesicle, offe ...[more]

PMID: 21964395

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Project description:Studies on four membrane protein systems, which combine information derived from crystal structures and biophysical studies have emphasized, as a precursor to crystallization, demonstration of functional activity. These assays have relied on sensitive spectrophotometric, electrophysiological, and microbiological assays of activity to select purification procedures that lead to functional complexes and with greater likelihood to successful crystallization: (I), Hetero-oligomeric proteins involved in electron transport/proton translocation. (1) Crystal structures of the eight subunit hetero-oligomeric trans-membrane dimeric cytochrome b(6)f complex were obtained from cyanobacteria using a protocol that allowed an analysis of the structure and function of internal lipids at specific intra-membrane, intra-protein sites. Proteolysis and monomerization that inactivated the complex and prevented crystallization was minimized through the use of filamentous cyanobacterial strains that seem to have a different set of membrane-active proteases. (2) An NADPH-quinone oxido-reductase isolated from cyanobacteria contains an expanded set of 17 monotopic and polytopic hetero-subunits. (II) ?-Barrel outer membrane proteins (OMPs). High resolution structures of the vitamin B(12) binding protein, BtuB, solved in meso and in surfo, provide the best example of the differences in such structures that were anticipated in the first application of the lipid cubic phase to membrane proteins [1]. A structure of the complex of BtuB with the colicin E3 and E2 receptor binding domain established a "fishing pole" model for outer membrane receptor function in cellular import of nuclease colicins. (III) A modified faster purification procedure contributed to significantly improved resolution (1.83Å) of the universal porin, OmpF, the first membrane protein for which meaningful 3D crystals have been obtained [2]. A crystal structure of the N-terminal translocation domain of colicin E3 complexed to OmpF established the role of OmpF as an import channel for colicin nuclease cytotoxins. (IV) ?-Synuclein, associated with the etiology of Parkinson's Disease, is an example of a protein, which is soluble and disordered in solution, but which can assume an ordered predominantly ?-helical conformation upon binding to membranes. When subjected in its membrane-bound form to a trans-membrane electrical potential, ?-synuclein can form voltage-gated ion channels. Summary of methods to assay functions/activities: (i) sensitive spectrophotometric assay to measure electron transfer activities; (ii) hydrophobic chromatography to deplete lipids, allowing reconstitution with specific lipids for studies on lipid-protein interactions; (iii) microbiological screen to assay high affinity binding of colicin receptor domains to Escherichia coli outer membrane receptors; (iv) electrophysiology/channel analysis (a) to select channel-occluding ligands for co-crystallization with ion channels of OmpF, and (b) to provide a unique description of voltage-gated ion channels of ?-synuclein.

Dataset Information

A helical processing pipeline for EM structure determination of membrane proteins.

Publications

A helical processing pipeline for EM structure determination of membrane proteins.

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