Difference: DavidStokes (31 vs. 32)

• Name: David Stokes
• Email: stokes@nysbc.org
• Company Name: NYSBC
• Company URL: http://www.nysbc.org/facilities/CEM
• Location: ParkBuildingOffice
• Country: USA
• Comment: 212-263-1580

NYU Users Request Page for EM Use

NyuMailingLists

StokesLabProtocols

TwoDxSuppl

HeiDualBeamGrant

My Project Logbook-CaATPase

• OReilly_Learning_Python_3rd.pdf
• NyuGraduateProgram
• StokesKissenaBoard
• StokesKissenaNominations2010

Personal Preferences (details in TWikiVariables)

• Horizontal size of text edit box:
  • Set EDITBOXWIDTH = 70
• Vertical size of text edit box:
  • Set EDITBOXHEIGHT = 25
• Style of text edit box. width: 99% for full window width (default), width: auto to disable.
  • Set EDITBOXSTYLE = width: 99%
• Optionally write protect your home page: (set it to your WikiName)
  • Set ALLOWTOPICCHANGE =

Related topics

• TWikiPreferences has site-level preferences of TWiki.
• WebPreferences has preferences of the TWiki.Main web.
• TWikiUsers has a list of other TWiki users.
• David Stokes:
  

Public Information on Grants Associated with NYSBC Grant Number: 5R01GM081817-03 Project Title: Membrane Protein Structure by Electron Crystallography PI Information: Name Email Title STOKES, DAVID L. stokes@nyu.edu ASSOCIATE PROFESSOR

Abstract: DESCRIPTION (provided by applicant): The overall goal of this proposal is to promote structure determination of integral membrane proteins by cryoelectron microscopy (cryo-EM) of 2D crystals. Membrane proteins are essential to all cells and favored drug targets, yet their 3D structures have proven difficult to ascertain. Cryo-EM has a demonstrated capability for structure determination at atomic resolution and the important advantage of maintaining integral membrane proteins within their native membrane environment. Nevertheless, the proliferation of high- throughput screening for 3D crystallization trials has given a distinct advantage to X-ray crystallography. We propose to implement analogous technologies for screening 2D crystallization trials of integral membrane proteins within lipid bilayers in order to reestablish cryo-EM as a viable alternative in the high-throughput, post-genomic era. We will form a partnership with the New York Consortium on Membrane Protein Structure (NYCOMPS), which is a Specialized Center of the NIH Protein Structure Initiative operating out of the New York Structural Biology Center. NYCOMPS will provide requested expression vectors from a large database of membrane proteins that they are screening for expression levels and homogeneity. After scaling up expression, we will use a robotic liquid handler to set up 2D crystallization trials by dialysis and prepare EM samples by negative stain in a 96-well format. Imaging currently represents a critical bottleneck for screening 2D crystal trials and we will implement technologies to automatically acquire images from these samples in the electron microscope. Thus, we expect to assess -50 different protein targets per year. The resulting information will be used to establish general principles governing the 2D crystallization process and, importantly, to produce 2D crystals that are suitable for structure determination at atomic resolution. Given a workable method to systematically search for and optimize 2D crystals, cryo-EM will become generally viable for high resolution structure determination and offer an important alternative to X-ray crystallography and NMR spectroscopy. In particular, the modest requirements for quantity and purity of proteins, as well as the natural environment provided by the lipid membrane, make cryo-EM an attractive alternative, especially as we move from bacterial proteomes towards large membrane protein complexes from eukaryotic cells that have proven more difficult to express and to maintain in a detergent solubilized state.

Public Health Relevance: This Public Health Relevance is not available.

Thesaurus Terms:

There are no thesaurus terms on file for this project.

Institution: NEW YORK STRUCTURAL BIOLOGY CENTER

1. CONVENT AVE NEW YORK, NY 10027

Grant Number: 5R01GM071044-02 PI Name: STOKES, DAVID L. PI Email: stokes@saturn.med.nyu.edu PI Title: ASSOCIATE PROFESSOR Project Title: Electron Tomography of Adhesive Junctions

Abstract: DESCRIPTION (provided by applicant): Adhesive junctions are involved in a wide range of cellular processes ranging from embryogenesis and tissue formation to cell transformation. They participate in cell-cell recognition, provide tensile strength to epithelial sheets and participate in intracellular signaling that direct cell growth and migration; several component proteins function either as oncogenes or as tumor suppressors. There are two types of adhesive junctions, desmosomes and adherens junction, both of which are well characterized from a biochemical and cell biological perspective and several x-ray crystal structures exist for important domains of several key proteins. Nevertheless, the architectural principles by which these components form a live junction and mediate adhesion remains largely guesswork. This proposal seeks to elucidate this architecture using the method of electron tomography to determine 3D structures of intact junctions in their native cellular environment. In preliminary work, we have studied intact desmosomes from after freeze-substitution and plastic embedding of newborn mouse epidermis. From the resulting tomograms, we have described the organization of desmosomal cadherins within the intercellular space and proposed structural mechanisms for adhesion. For our first aim, frozen-hydrated specimens and methods of cryotomography will be used to eliminate specimen preparation artifacts as a potential factor and to establish this methodology as an alternative to plastic sections. In our second aim, the architecture of adherens junctions will be studied either in epidermis, in cultured keratinocytes, or in lens tissue. Although adherens junctions have an analogous architecture to desmosomes, they have different molecular components, different morphology, and have a more dynamic role in cellular behavior. In our third aim, we will study the structure and function of the dense cytoplasmic plaque, by isolating epidermis from transgenic mice with knockout of several key desmosomal components: plakoglobin, desmoplakin, desmocollin, and keratin 5. In addition, we will explore immunolabelling as a technology for identifying plaque component in tomograms. In our fourth aim, we will study the assembly of both adherens junctions and desmosomes in cultured keratinocytes, using calcium to initiate junction assembly between confluent cell cultures.

Thesaurus Terms: cell adhesion, intercellular connection, membrane structure cadherin, cell cycle, cell morphology, cryoscience, keratin, keratinocyte, skin cryoelectron microscopy, genetically modified animal, laboratory mouse, three dimensional imaging /topography, tissue /cell culture, tomography

Institution: NEW YORK UNIVERSITY SCHOOL OF MEDICINE 550 1ST AVE NEW YORK, NY 10016 Fiscal Year: 2005 Department: CELL BIOLOGY Project Start: 01-MAR-2004 Project End: 29-FEB-2008 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: BBCB

Grant Number: 1S10RR017291-01 PI Name: STOKES, DAVID L. PI Email: stokes@nysbc.org PI Title: Project Title: 300 kV Electron Microscope for Structural Biology

Abstract: DESCRIPTION (provided by applicant):A cryoelectron microscope (cryoEM) is being requested for a diverse group of users from New York City. The user projects fall into three categories: tomography, single-particle analysis and crystallography. Features of this microscope have been selected to benefit all three applications and include a 300 kV accelerating voltage, a field emission gun, a tilting, liquid-helium specimen stage, an energy filter and a 4k x 4k CCD camera. These features provide superior imaging optics, minimal noise contribution from inelastically scattered electrons, maximal specimen stability, reduced radiation sensitivity, and a state-of-the-art digital imaging capacity. The microscope will be housed at a new cryoEM facility soon to be initiated at the New York Structural Biology Center at the Manhattan Campus of City University of New York. This facility will include two support microscopes and associated equipment for specimen preparation. The Center will recruit two cryoelectron microscopists at the faculty level to assure implementation of the latest technologies and to provide a resource of local expertise.

Thesaurus Terms: biomedical equipment purchase, cryoelectron microscopy, structural biology charge coupled device camera, tomography

Institution: NEW YORK STRUCTURAL BIOLOGY CENTER 89 CONVENT AVE NEW YORK, NY 10027 Fiscal Year: 2002 Department: Project Start: 30-SEP-2002 Project End: 29-SEP-2005 ICD: NATIONAL CENTER FOR RESEARCH RESOURCES IRG: ZRG1