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Structure - Activities

Objectives

  • functional integration in space and time for formulating atomic models of migration organelles
  • determine atomic resolution structures of each major protein involved in the organelle
  • one example of each class of domain:domain interaction

Collaboration and Release Policy

Structures | Algorithm Development

Structures Being Studied

Structure Method Status / Publication Contact (Collaborators)
Talin (C-terminal-domain and Actin) EM, SAXS, docking and helical reconstruction Completed - CMC Update article Gingras et al., 2008 Hanein (Liddington, Critchley, Volkmann)
Alpha-actinin beta1-integrin complex 2D crystallization on lipid monolayers Completed - Kelly & Taylor, 2005 Taylor
Alpha-actinin2 (chicken smooth muscle) 2D crystallization on lipid monolayers Completed - Liu, et al., 2004 Taylor (J. Liu, D. Taylor)
Arp2/3 branch structure Cryo EM Completed - 2D structure completed Volkmann et al., 2001 Hanein (Volkmann, Li, Pollard)
Metavinculin C-terminal domain X-Ray crystallography, NMR Completed - Bakolitsa et al., 1999 Liddington
Lamellipodia leading edge (Ena/VASP) proteins Correlative light microscopy-electron microscopy Completed - Bear et al., 2002 Borisy (Gertler)
WASP and Scar - Drosophila actin nucleation Correlative light microscopy-electron microscopy Completed - Biyasheva et al., 2004 Borisy (Baum)
Actin comet tail Correlative light microscopy-electron microscopy Completed - Cameron et al., 2001 Borisy (Theriot)
PIPKIg-talin (196-400) X-Ray crystallography, NMR Completed - de Pereda et al., 2005 Liddington (Campbell)
Tropomyosin and the leading edge conventional EM Completed - Gupton et al., 2005 Hanein (Waterman-Storer)
Vinculin-tail actin EM, tomography and docking Completed - CMC Update article Janssen et al., 2006 Hanein (Volkmann, Liddington)
Rickettsia - structure of tail mediated by WASP-like protein Correlative light microscopy-electron microscopy Completed - Jeng et al., 2004 Borisy (Welch)
Alpha-actinin beta1-integrin-Vinculin1-258 complex 2D crystallization on lipid monolayers Completed - Kelly, et al., 2006 Taylor (Liddington)
Talin (Ferm-domain) and Actin EM and helical reconstruction Completed - Lee et al., 2004 Hanein (Liddington, Critchley, Volkmann)
Arp2/3 activation (mutants) EM, single particle reconstruction and docking Completed - Martin et al., 2005 Hanein (Drubin, Volkmann)
Tensin SH2 and PTB domains X-Ray crystallography, NMR Completed - CMC Update article McCleverty et al., 2007 Liddington
HIV-cytoskeletal association (microtubules) Correlative light microscopy-electron microscopy Completed - McDonald et al., 2002 Borisy (Hope)
Alpha-actinin cross-links 2D crystallization on lipid monolayers Completed - CMC Update article Hampton et al., 2007 Taylor
Actin filaments orientational order in vivo Correlative light microscopy-electron microscopy Completed - Verkhovsky et al., 2003 Borisy (Meister)
Myosin and Actin Cryo EM, helical reconstruction, docking Completed - Volkmann et al., 2000, Volkmann et al 2003, Volkmann & Hanein, 2000, Volkmann, et al., 2007, Trybus et al., 2007, Lowey et al., 2007 Hanein (Volkmann, Lowey, Trybus, DeRosier)
Actin fimbrin crosslink EM and bioinformatics Completed - completed Volkmann et al., 2001 Hanein (Volkmann)
Talin-beta3 integrin X-Ray crystallography, NMR Completed - crystallized and solved Garcia-Alvarez et al., 2003, Wegener et al., 2007 Liddington (Campbell, Ginsberg)
FAT domain of Focal Adhesion Kinase X-Ray crystallography, NMR Completed - completed Hayashi et al., 2002 Liddington
Vinculin (full length) X-Ray crystallography Completed - Structure determined CMC Update article Bakolitsa et al., 2004, Bakolitsa et al., 1999, Saunders et al., 2006 and Ziegler et al, 2006.. Liddington
Alpha-actinin2/F-actin rafts lipid monolayer, negative stain, single particle image processing Completed - in press - J. Mol. Biol. Taylor
Mapping of molecular sub-domains Multi color labeling; Clustering analysis Completed, publication in progress Geiger
Osteoclast structure Multi color labeling Completed, publication in progress Geiger
Cell-matrix interactions Multi color labeling Completed, publication in progress Geiger (Hanein)
Leading edge VMP, Cryo EM and conventional EM Completed, publication in progress Hanein
Fibronectin fibrillogenises EM, light and EM labeling, cell culture Completed, publication in progress Hanein (Geiger, Engel, Ruoslahti)
F-actin-villin (with David DeRosier) Electron Tomography Completed, publication in progress Taylor
alphaIIb-beta3 integrin structure in the membrane Electron Tomography Completed, publication in progress Taylor
Focal adhesion structure Correlative light microscopy-electron microscopy In progress Geiger (Hanein)
Organization of zyxin and alpha-actinin in focal adhesions and stress fibers Correlative light microscopy-electron microscopy In progress Geiger (Hanein, Taylor)
Podosome structure VMP and Cryo EM In progress Hanein (Geiger)
Focal adhesion structure in-situ VMP, light and EM, labeling, Cryo EM and tomography In progress Hanein (Geiger)
Vasp-actin EM, tomography, single particle reconstruction, and bioinformatics In progress Hanein (Gertler, Volkmann)
Formin-actin EM and biochemistry In progress Hanein (Higgs)
C-terminal fragment of Tensin and actin EM, helical reconstruction and bioinformatics In progress Hanein (Li, Li, Vuori, Volkmann, Liddington)
Talin-rod domain and actin EM and helical reconstruction In progress Hanein (Liddington)
Nanodisc Cryo EM In progress Hanein (Sligar, Volkmann)
Fimbrin -actin regulation EM and biochemistry In progress Hanein (Volkmann)
Fibronectin structure EM, Cryo EM and tomography In progress Hanein (Volkmann, Engel)
Full-length talin and actin EM and tomography In progress Hanein (Volkmann, Liddington)
Full-length Vinculin actin EM, tomography, single particle reconstructions, docking In progress Hanein (Volkmann, Liddington)
Full-length tensin and actin Cryo EM In progress Hanein (Vuori)
Calcium and integrin binding protein (CIB) complex with integrin X-Ray crystallography In progress Liddington
Calcium and integrin binding protein (CIB) complex with WASP X-Ray crystallography In progress Liddington
Paxillin -wt fragments 1-328 & 325-557 and full-length X-Ray crystallography In progress Liddington
Isolated membrane protrusions (with Klemke) Electron Tomography In progress Taylor
Crawling cells (with Condeelis) Electron Tomography In progress Taylor
Actin networks VMP, Cryo EM and tomography In progress Hanein (Volkmann)
Vinculin and Talin Atomic Force Microscope In progress - Atomic Force Microscope experiments begun to monitor force required to stretch-activate viniculin and talin in vitro in presence of ligands, and correlate with cell based studies (Schwartz). Liddington
Podosome structure Correlative light microscopy-electron microscopy In progress - Collaboration established, ongoing research visit Geiger (Hanein)
Vinculin N-terminal domain complex with alpha-actinin1 domain 3 X-Ray crystallography In progress - Complex purified by gel filtration. Crystallization trials in progress Liddington
Metavinculin (fragments and full length) X-Ray crystallography, NMR In progress - Crystalization trials. residues 879-1134; metavinculin residues 895-11; full-length metavinculin. All proteins expressed. Liddington
Talin-vinculin 2D crystallization on lipid monolayers In progress - Crystallization trials underway Taylor
Talin 2D crystallization on lipid monolayers In progress - Crystallization trials underway Taylor (Liddington)
Vinculin and Arp2/3 complex formation X-Ray crystallography In progress - Crystallization trials. Vinculin residues 715-1066 (domains 4 and 5, including proline-rich region) expressed. Arp2/3 subunit p.34Arc human Arp_C2 domain (34.3kDa) and p20Arc: human Arp_C4 domain (19.5kDa) expressed stably. Complex formation in progress. Liddington
Alpha-actinin1 (in EGTA) 2D crystallization on lipid monolayers In progress - Currently on hold pending expression and purification efforts Taylor
Alpha-actinin1 (plus calcium) 2D crystallization on lipid monolayers In progress - Currently on hold pending expression and purification efforts Taylor
Paxillin (1-328)-FAT domain of FAK NMR In progress - FAT resonances assigned, various paxillin fragments expressed Campbell
Alpha-actinin1//F-actin complexes Electron Tomography In progress - Focal adhesion complexes on lipid monolayers. Assemby of complexes of F-actin with talin, full length vinculin, alpha-actin-1 and beta1-integrin cytoplasmic domain. Exploring effects of different lipid composition. Taylor
Layilin-talin (196-400) X-Ray crystallography, NMR Completed - Structure was solved by NMR Wegener et al., 2008 Liddington
Alpha-actinin beta1-integrin-vinculin1-258-talin FERM domain complex 2D crystallization on lipid monolayers In progress - Large 2D arrays completed, 3D image underway. Identification of vinculin binding site on alpha-actinin; identification of beta1--integrin binding site on alpha-actinin. Nearing completion of ice embedded specimens Taylor (Liddington)
Organization of zyxin and alpha-actinin in focal adhesions and stress fibers Correlative light microscopy-electron microscopy In progress - Light microscopy aspect initiated Geiger (Hanein)
Mek1 X-Ray crystallography In progress - Protein expressed and purified from E. coli. Crystal trials in progress Liddington
Talin rod X-Ray crystallography In progress - Protein expressed and purified from E. coli. Crystal trials in progress. Complementary studies with Hanein in progress. Liddington
Talin (full length) X-Ray crystallography In progress - Protein expressed and purified from E.coli. Crystal trials in progress. Complementary EM studies with Hanein and Critchley in progress. Liddington
Talin fragments for protein splicing and chemical labeling Cell-based studies In progress - Protein expressed and purified from E.coli. Ligation in progress Liddington
Full-length FAK X-Ray crystallography In progress - Protein expressed and purified from insect cells. Crystal trials in progress Liddington
GIT-1 X-Ray crystallography In progress - Protein expressed and purified from insect cells. Crystal trials in progress Liddington
Beta 3-Src SH3 NMR In progress - Protein spectra assigned; currently being deuterated to study complex Campbell (Ginsberg)
Talin rod 655-911 NMR In progress - Resonance assignments of 8 helices ongoing Critchley
PIP3 binding DHR1 domain of DOCK180 X-Ray crystallography In progress - structural studies (with Kristiina Vuori and Jean-Francois Cote). Stable protein expression; crystalization trials in progress. Liddington
Talin FO/F1(1-200) NMR In progress - Structure of F0, F1, and F0F1 constructs have been solved. Model of the talin head based on the structure of F0F1 and F2F3 is being refined using SAXS data. Currently using NMR chemical shift experiments to map the Ha-Ras binding site in the head. Critchley
Talin C-terminal actin binding site (I/LWEQ motif) NMR In progress - Structure solved and submitted for publication Critchley
Talin C-terminal helix required for dimer formation X-Ray crystallography In progress - Structure solved to 2.5 Angstroms and submitted for publication Critchley
GIT-1 domains X-Ray crystallography In progress - Subcloning and expression trials in progress Liddington
Talin head for head-tail binding studies Calorimetric In progress - Talin head expressed in mg quantities. ITC and DSC studies in progress Liddington
alphaIIb-beta3 integrin structure in the membrane with Ligands and talin FERM domain Electron Tomography Under consideration Taylor
Beta tails as an interaction hub NMR In progress - Competition between PTB domains of talin, Dok1, ICap-1, effect of phosphorylation. Oxley et al., 2008 Campbell
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Algorithm Development and Refinement

Algorithm & Application Status / Publication Contact (Collaborators)
Refinement of docking algorithms for high-resolution interpretation of cryoEM maps Completed - Volkmann & Hanein, 2003 Shacham et al., 2007 Volkmann
Test and refinement of noise reduction algorithms for tomographic reconstructions Completed - CMC Update article Van der Heide et al., 2007 Volkmann (Hanein, Taylor, Marsh)
Use of reduced-representation templates for particle selection in cryoEM Completed - Volkmann, 2004 , Zhu et al., 2004 Volkmann
Application of docking algorithms for high-resolution interpretation of cryoEM maps Completed - CMC Update article 1, 2 and 3 Rouiller et al., 2008, Lowey et al., 2007, Trybus et al., 2007, Gingras et al., 2008, Janssen et al., 2006, Lee et al., 2004 Volkmann (Hanein, Liddington, Critchley)
Refinement of docking algorithms for use with tomograms Data available, CMC Update article first application published in Rouiller et al., 2008. Technical publication in preparation. Software available Volkmann (Hanein)
Refinement of reconstruction algorithms for helical structures Completed - CMC Update article 1 and 2 Gingras et al., 2008, Lowey et al., 2007, Volkmann, et al., 2007, Trybus et al., 2007, Janssen et al., 2006, Craig et al., 2006. Initial version completed; more detailed studies in progress.Technical publication in preparation. Volkmann
Application of filament tracking approaches to interpretation of worm sperm tomograms In progress Volkmann (Taylor, Roberts)
Refinement of watershed segmentation for use with electron tomograms In progress - Initial test completed, publication: Volkmann, 2002 more detailed studies in progress Volkmann (Hanein, Taylor, Marsh)
Improved single-particle reconstruction algorithms for small particles and mixed populations In progress - initial tests completed Volkmann
Test and refinement of reduced-representation templates for filament detection in electron tomograms In progress - Initial tests completed, refinement of parameters in progress Volkmann (Hanein, Taylor)
Adaptation of watershed transform for use with light microscopy In progress - Initial tests successfully completed, more detailed studies in progress Volkmann (Hanein, Danuser, Waterman, Geiger)
Averaging of motifs extracted from tomographic reconstructions Initial version completed, first application published in Rouiller et al., 2008. Technical publication in preparation Volkmann

The 3D watershed transform (Volkmann, 2002) has been successfully adapted and applied to time series data from light microscopy (with Hanein, Danuser and Waterman). The computer programs and parameter sets developed and refined in connection with this project are available upon request.


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