Structure - Activities

Structures | Algorithm Development

Structures Being Studied

Structure	Method	Status / Publication	Contact (Collaborators)
Arp2/3 activation (mutants)	EM, single particle reconstruction and docking	Completed - Martin et al., 2005 (CMC Update Article)	Hanein (Drubin, Volkmann)
Arp2/3 branch structure	Cryo EM	Data available - 2D structure Volkmann et al., 2001	Hanein (Volkmann, Li, Pollard)
Alpha-actinin / beta1-integrin - Vinculin 1-258 complex	2D crystallization on lipid monolayers	Completed - Kelly et al., 2006	Taylor (Liddington)
Alpha-actinin beta1-integrin complex	2D crystallization on lipid monolayers	Completed - Kelly and Taylor, 2005 (CMC Update Article)	Taylor
Layilin-talin (196-400) PDB link	X-Ray crystallography, NMR	Completed - Structure was solved by NMR Wegener et al., 2008	Liddington
PIPKIg -talin (196-400) PDB link	X-Ray crystallography, NMR	Completed - de Pereda et al., 2005 (CMC Update Article)	Liddington (Campbell)
Myosin and Actin	Cryo EM, helical reconstruction, docking	Completed - Volkmann and Hanein, 2000; Volkmann et al., 2000; Volkmann et al., 2003; Lowey et al., 2007; Trybus et al., 2007; Volkmann et al., 2007	Hanein (Volkmann, Lowey, Trybus, DeRosier)
Actin fimbrin crosslink PDB link	EM and bioinformatics	Data available - Volkmann et al., 2001	Hanein (Volkmann)
Actin comet tail	Correlative light microscopy-electron microscopy	Completed - Cameron et al., 2001	Borisy (Theriot)
Actin filaments orientational order in vivo	Correlative light microscopy-electron microscopy	Completed - Verkhovsky et al., 2003	Borisy (Meister)
F-actin-villin (with David DeRosier)	Electron Tomography	Completed - Hampton et al., 2008 (CMC Update Article)	Taylor (DeRosier)
Talin -beta3 integrin PDB link	X-Ray crystallography, NMR	Data available - crystallized and solved García-Alvarez et al., 2003; Wegener et al., 2007 (CMC Update Article)	Liddington (Campbell, Ginsberg)
Talin (C-terminal Actin-binding domain) PDB link	EM, SAXS, docking and helical reconstruction	Completed - Gingras et al., 2008 (CMC Update Article)	Hanein (Liddington, Critchley, Volkmann)
Talin (Ferm-domain) and Actin	EM and helical reconstruction	Completed - Lee et al., 2004	Hanein (Liddington, Critchley, Volkmann)
Tensin SH2 and PTB domains PDB link	X-Ray crystallography, NMR	Completed - McCleverty et al., 2007 (CMC Update Article)	Liddington
Tropomyosin and the leading edge	conventional EM	Completed - Gupton et al., 2005 (CMC Update Article)	Hanein (Waterman-Storer)
Meta-vinculin C-terminal domain PDB link	X-Ray crystallography, NMR	Completed - Bakolitsa et al., 1999	Liddington
Vinculin (full length) PDB link	X-Ray crystallography	Data available - Bakolitsa et al., 1999; Bakolitsa et al., 2004 (CMC Update Article); Saunders et al., 2006; Ziegler et al., 2006 (CMC Update Article)	Liddington
Vinculin-tail actin	EM, tomography and docking	Completed - Janssen et al., 2006 (CMC Update Article)	Hanein (Volkmann, Liddington)
WASP and Scar - Drosophila actin nucleation	Correlative light microscopy-electron microscopy	Completed - Biyasheva et al., 2004	Borisy (Baum)
Alpha-actinin cross-links	2D crystallization on lipid monolayers	Completed - Hampton et al., 2007 (CMC Update Article)	Taylor
Alpha-actinin2/F-actin rafts	lipid monolayer, negative stain, single particle image processing	Completed - in press - J. Mol. Biol.	Taylor
Alpha-actinin2 (chicken smooth muscle) PDB link	2D crystallization on lipid monolayers	Completed - Liu et al., 2004	Taylor (Liu)
FAT domain of Focal Adhesion Kinase PDB link	X-Ray crystallography, NMR	Data available - Hayashi et al., 2002	Liddington
HIV-cytoskeletal association (microtubules)	Correlative light microscopy-electron microscopy	Completed - McDonald et al., 2002	Borisy (Hope)
Lamellipodia leading edge (Ena/VASP) proteins	Correlative light microscopy-electron microscopy	Completed - Bear et al., 2002	Borisy (Gertler)
Podosome structure	VMP and Cryo EM	Completed - Geblinger et al., 2009 (CMC Update Article)	Geiger (Hanein)
Rickettsia - structure of tail mediated by WASP-like protein	Correlative light microscopy-electron microscopy	Completed - Jeng et al., 2004	Borisy (Welch)
alphaIIb-beta3 integrin structure in the membrane	Electron Tomography	Completed, publication in progress	Taylor
Fibronectin fibrillogenises	EM, light and EM labeling, cell culture	Completed, publication in progress	Hanein (Geiger, Engel, Ruoslahti)
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
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
Alpha-actinin / beta1-integrin - vinculin 1-258 - 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)
Calcium and integrin binding protein (CIB) complex with WASP PDB link	X-Ray crystallography	In progress	Liddington
Calcium and integrin binding protein (CIB) complex with integrin PDB link	X-Ray crystallography	In progress	Liddington
Fibronectin structure	EM, Cryo EM and tomography	In progress	Hanein (Volkmann, Engel)
GIT-1	X-Ray crystallography	In progress - Protein expressed and purified from insect cells. Crystal trials in progress	Liddington
GIT-1 domains	X-Ray crystallography	In progress - Subcloning and expression trials in progress	Liddington
Formin-actin	EM and biochemistry	In progress	Hanein (Higgs)
Beta 3-Src SH3	NMR	In progress - Protein spectra assigned; currently being deuterated to study complex	Campbell (Ginsberg)
Fimbrin - actin regulation	EM and biochemistry	In progress	Hanein (Volkmann)
Mek1	X-Ray crystallography	In progress - Protein expressed and purified from E. coli. Crystal trials in progress	Liddington
Paxillin (1-328)-FAT domain of FAK	NMR	In progress - FAT resonances assigned, various paxillin fragments expressed	Campbell
Paxillin -wt fragments 1-328 & 325-557 and full-length	X-Ray crystallography	In progress	Liddington
Actin networks	VMP, Cryo EM and tomography	In progress	Hanein (Volkmann)
Talin	2D crystallization on lipid monolayers	In progress - Crystallization trials underway	Taylor (Liddington)
Talin-vinculin	2D crystallization on lipid monolayers	In progress - Crystallization trials underway	Taylor
Talin C-terminal actin binding site (I/LWEQ motif) PDB link	NMR	In progress - Structure solved and submitted for publication	Critchley
Talin C-terminal helix required for dimer formation PDB link	X-Ray crystallography	In progress - Structure solved to 2.5 Angstroms and submitted for publication	Critchley
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 fragments for protein splicing and chemical labeling	Cell-based studies	In progress - Protein expressed and purified from E.coli. Ligation 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 head for head-tail binding studies	Calorimetric	In progress - Talin head expressed in mg quantities. ITC and DSC studies in progress	Liddington
Talin rod PDB link	X-Ray crystallography	In progress - Protein expressed and purified from E. coli. Crystal trials in progress. Complementary studies with Hanein in progress.	Liddington
Talin rod 655-911	NMR	In progress - Resonance assignments of 8 helices ongoing	Critchley
Talin-rod domain and actin	EM and helical reconstruction	In progress	Hanein (Liddington)
Vasp-actin	EM, tomography, single particle reconstruction, and bioinformatics	In progress	Hanein (Gertler, Volkmann)
Metavinculin (fragments and full length) PDB link	X-Ray crystallography, NMR	In progress - Crystalization trials. residues 879-1134; metavinculin residues 895-11; full-length metavinculin. All proteins expressed.	Liddington
Vinculin and Arp2/3 complex formation PDB link	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
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
Vinculin N-terminal domain complex with alpha-actinin1 domain 3 PDB link	X-Ray crystallography	In progress - Complex purified by gel filtration. Crystallization trials in progress	Liddington
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
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
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 (CMC Update Article)	Campbell
Crawling cells (with Condeelis)	Electron Tomography	In progress	Taylor (Condeelis)
C-terminal fragment of Tensin and actin	EM, helical reconstruction and bioinformatics	In progress	Hanein (Li, Li, Vuori, Volkmann, Liddington)
Focal adhesion structure	Correlative light microscopy-electron microscopy	In progress	Geiger (Hanein)
Focal adhesion structure in-situ	VMP, light and EM, labeling, Cryo EM and tomography	In progress	Hanein (Geiger)
Full-length FAK PDB link	X-Ray crystallography	In progress - Protein expressed and purified from insect cells. Crystal trials in progress	Liddington
Full-length talin and actin	EM and tomography	In progress	Hanein (Volkmann, Liddington)
Full-length tensin and actin	Cryo EM	In progress	Hanein (Vuori)
Full-length Vinculin actin	EM, tomography, single particle reconstructions, docking	In progress	Hanein (Volkmann, Liddington)
Isolated membrane protrusions (with Klemke)	Electron Tomography	In progress	Taylor (Klemke)
Nanodisc	Cryo EM	In progress	Hanein (Sligar, Volkmann)
Organization of zyxin and alpha-actinin in focal adhesions and stress fibers	Correlative light microscopy-electron microscopy	In progress	Geiger (Hanein, Taylor)
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)
PIP3 binding DHR1 domain of DOCK180	X-Ray crystallography	In progress - structural studies. Stable protein expression; crystalization trials in progress.	Liddington (Vuori, Cote)
Podosome structure	Correlative light microscopy-electron microscopy	In progress - Collaboration established, ongoing research visit	Geiger (Hanein)
alphaIIb-beta3 integrin structure in the membrane with Ligands and talin FERM domain	Electron Tomography	Under consideration	Taylor

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Algorithm Development and Refinement

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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