David L. Stokes

Professor, Skirball Institute of Biomolecular Medicine, Structural Biology. Department of Cell Biology Scientific Director - NYCSBE CEM

Ph.D., 1986 Brandeis University

Lab Website: http://saturn.med.nyu.edu/research/sb/stokeslab

Keywords:Membrane Transport, Electron Microscopy, Molecular Structure, Macromolecular Complexes, Cellular Dynamics

 

Contact Information: 

Skirball Institute of Biomolecular Medicine
540 First Avenue 3rd floor, Lab 13
New York, N.Y. 10016
Office Tel: (212) 263-1580
Lab Tel: (212) 263-1599
Fax: (212) 263-8951
E-mail: david.stokes@med.nyu.edu

Administrative Contact:

Zachary Klush
Tel: (212) 263-3261
Email: zachary.klush@med.nyu.edu


Electron Microscopy of Macromolecular Complexes

Our lab studies the structure of a variety of macromolecular complexes using methods of electron microscopy. One general theme is the transport of ions across membranes by ATP-dependent pumps and by secondary transporters. Specifically, we are studying Ca2+-ATPase and CopA, which are members of the P-type ATPases family, and YiiP, which is a secondary Zn2+ transporter from the Cation Diffusion Facilitator family. In all cases, we have produced so-called two-dimensional crystals of these proteins and used methods of electron crystallography to generate three-dimensional structures. Although these structures are not at atomic resolution, we have used existing X-ray crystallographic structures to interpret our density maps. In all three cases, we have thus characterized conformational changes that we believe are relevant to the transport mechanism. This approach is generally complementary to X-ray crystallography and offers the advantage of providing the more natural environment of a lipid membrane. Indeed, we have documented that the crystal packing and conformation of CopA (a Cu+ pump) is sensitive to the lipid composition of the membrane, and most likely also affected by the detergent micelle used to mimic this bilayer for X-ray crystallography.

Electron tomography is an alternative approach that we use to determine the 3D structure of individual cells, organelles and macromolecular assemblies. We are applying this methodology to T-cells as they form an immunological synapse with antigen presenting cells, to mitochondria in order to learn about the role of cardiolipin in stabilizing cristae and their arrays of ATP synthase, and to the red blood cell cytoskeleton to elucidate the architecture of wild-type and mutant spectrin networks. We have developed methods for correlative microscopy, with which these samples are viewed by both light microscopy and electron microscopy, thus combining dynamic, functional information with structure. For the red blood cell cytoskeleton, we are collaborating with other labs to combine super-resolution fluorescence microscopy and mathematical modeling with the architecture observed by cryo-electron tomography in order to understand the biophysical principles that confer strength and flexibility to these cells. These studies use a wide range of different transmission electron microscopes, both at NYU and at the New York Structural Biology Center, where a dual-beam scanning EM is also being used to create large-scale serial section reconstructions of whole cells and to produce thin samples for cryo-tomography.

Selected Publications:

Acehan D, Malhotra A, Xu Y, Ren M, Stokes DL, Schlame M. 2011. Cardiolipin Affects the Supramolecular Organization of ATP Synthase in Mitochondria. Biophys. J. 100:2184-2192. PMID: 21539786

Leo-Macias, A., Katz, Wei H, Alimova A, Katz A, Rice WJ, Diaz-Avalos R, Hu GB, Stokes DL, Gottlieb P., 2011. Toroidal surface complexes of bacteriophage ϕ12 are responsible for host-cell attachment. Virology. 414:103-109. PMID: 21489589

Allen, G.S. Wu, C., Cardozo, T, Stokes, D.L. 2011. The Architecture of CopA from Archeaoglobus fulgidus Studied by Cryo-Electron Microscopy and Computational Docking. Structure. 19:1219-1232. PMID: 21820315

Nans, A., Mohandas, N., Stokes, D.L. 2011. Native ultrastructure of the red cell cytoskeleton by cryo-electron tomography. Biophys. J. 101:2341-2350. PMID: 22098732

Ubarretxena-Belandia, I., Stokes, D.L. 2012. Membrane protein structure determination by electron microscopy. Curr. Opin. Struct. Biol. 22:520-528. PMID: 22572457

Click here to see all publications in PubMed