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Nans A.,New York University | Mohandas N.,New York Blood Center | Stokes D.L.,New York University | Stokes D.L.,New York Structural Biology Center
Biophysical Journal | Year: 2011

Erythrocytes possess a spectrin-based cytoskeleton that provides elasticity and mechanical stability necessary to survive the shear forces within the microvasculature. The architecture of this membrane skeleton and the nature of its intermolecular contacts determine the mechanical properties of the skeleton and confer the characteristic biconcave shape of red cells. We have used cryo-electron tomography to evaluate the three-dimensional topology in intact, unexpanded membrane skeletons from mouse erythrocytes frozen in physiological buffer. The tomograms reveal a complex network of spectrin filaments converging at actin-based nodes and a gradual decrease in both the density and the thickness of the network from the center to the edge of the cell. The average contour length of spectrin filaments connecting junctional complexes is 46 ± 15 nm, indicating that the spectrin heterotetramer in the native membrane skeleton is a fraction of its fully extended length (∼190 nm). Higher-order oligomers of spectrin were prevalent, with hexamers and octamers seen between virtually every junctional complex in the network. Based on comparisons with expanded skeletons, we propose that the oligomeric state of spectrin is in a dynamic equilibrium that facilitates remodeling of the network as the cell changes shape in response to shear stress. © 2011 Biophysical Society.

Mancia F.,Columbia University | Love J.,New York Structural Biology Center
Current Opinion in Structural Biology | Year: 2011

Structural genomics approaches on integral membrane proteins have been postulated for over a decade, yet specific efforts are lagging years behind their soluble counterparts. Indeed, high throughput methodologies for production and characterization of prokaryotic integral membrane proteins are only now emerging, while large-scale efforts for eukaryotic ones are still in their infancy. Presented here is a review of recent literature on actively ongoing structural genomics of membrane protein initiatives, with a focus on those aimed at implementing interesting techniques aimed at increasing our rate of success for this class of macromolecules. © 2011 Elsevier Ltd.

Hendrickson W.A.,Columbia University | Hendrickson W.A.,New York Structural Biology Center
Nature Structural and Molecular Biology | Year: 2016

Membrane proteins are substantially more challenging than natively soluble proteins as subjects for structural analysis. Thus, membrane proteins are greatly underrepresented in structural databases. Recently, focused consortium efforts and advances in methodology for protein production, crystallographic analysis and cryo-EM analysis have accelerated the pace of atomic-level structure determination of membrane proteins. © 2016 Nature America, Inc. All rights reserved.

Mancia F.,Columbia University | Love J.,New York Structural Biology Center
Journal of Structural Biology | Year: 2010

High-throughput (HT) methodologies have had a tremendous impact on structural biology of soluble proteins. High-resolution structure determination relies on the ability of the macromolecule to form ordered crystals that diffract X-rays. While crystallization remains somewhat empirical, for a given protein, success is proportional to the number of conditions screened and to the number of variants trialed. HT techniques have greatly increased the number of targets that can be trialed and the rate at which these can be produced. In terms of number of structures solved, membrane proteins appear to be lagging many years behind their soluble counterparts. Likewise, HT methodologies for production and characterization of these hydrophobic macromolecules are only now emerging. Presented here is an HT platform designed exclusively for membrane proteins that has processed over 5000 targets. © 2010 Elsevier Inc.

Ubarretxena-Belandia I.,The New School | Stokes D.L.,New York University | Stokes D.L.,New York Structural Biology Center
Current Opinion in Structural Biology | Year: 2012

During the past year, electron crystallography of membrane proteins has provided structural insights into the mechanism of several different transporters and into their interactions with lipid molecules within the bilayer. From a technical perspective there have been important advances in high-throughput screening of crystallization trials and in automated imaging of membrane crystals with the electron microscope. There have also been key developments in software, and in molecular replacement and phase extension methods designed to facilitate the process of structure determination. © 2012 Elsevier Ltd.

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