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Technical development efforts at UCSF

Technical development efforts at UCSF. development with Gatan of high-resolution CCD camera UCSF Tomo: accurate, predictive automated cryo tomography collection (±100nm focus, 60-100kX) incorporated into Leginon automated collection of aligned tilt pairs-

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Technical development efforts at UCSF

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  1. Technical development efforts at UCSF • development with Gatan of high-resolution CCD camera • UCSF Tomo: accurate, predictive automated cryo • tomography collection (±100nm focus, 60-100kX) • incorporated into Leginon • automated collection of aligned tilt pairs- • Random Conical Tilt (±50nm focus, 60-150kX) • tilted image CTF and MTF correction • Real-Time EM Tomography (512x512xN) for inspection • automated marker-free iterative refinement (2kx2kxN) • averaging structures within tomograms

  2. Technical development efforts at UCSF • development with Gatan of high-resolution CCD camera • UCSF Tomo: accurate, predictive automated cryo • tomography collection (±100nm focus, 60-100kX) • incorporated into Leginon • automated collection of aligned tilt pairs- • Random Conical Tilt (±50nm focus, 60-150kX) • tilted image CTF and MTF correction • Real-Time EM Tomography (512x512xN) for inspection • automated marker-free iterative refinement (2kx2kxN) • averaging structures within tomograms

  3. UltraCam: improve resolution via lens coupling scattering from fiber optic limits resolution of normal CCD camera solution: remove fiber optic Detector Lens Scintillator and mirror GIF

  4. 40% nyquist W. Chiu 70% nyquist film better than CCD CCD better than film (based on DQE) UltraCam: the first serious film challenger MTF at 300kV fiber optic new lens & new camera (predicted) MTF old lens new lens nyquist

  5. Real-time cryoEMT on TMV raw data as collected • collected at -4µm focus, 3.4Å/pixel(2kx2k) • reconstructed at 13.6Å/pixel(512x512) Zheng, Keszthelyi,Braunfeld,Lyle

  6. Real-time cryoEMT on TMV raw data as collected instant reconstruction • collected at -4µm focus, 3.4Å/pixel(2kx2k) • reconstructed at 13.6Å/pixel(512x512) Zheng, Keszthelyi,Braunfeld,Lyle

  7. Understanding centrosome architecture and microtubule nucleation 1011 Da, 1 mm3 (mitchison lab images) • multi-resolution structural analysis • atomic to whole centrosome • kinetic mechanism of nucleation • role of nucleators

  8. EMT of isolated Drosophila centrosomes • g-tubulin ring complexes in PCM • g-tubulin at minus ends of MTs 0.7 µm thick plastic section M. Moritz, M. Braunfeld

  9. gTuSC Tub4 gTuRC in progress: ~30 Å resolution 2.2MDa 280KDa gTuSC g-tubulin complexes are directly responsible for MT nucleation g-Tubulin Ring Complex g-tubulin Dgrips 84 91 Dgrips 163 128 75s J. Kollman, L. Rice, J. Lyle, V. Guenebaut M. Moritz, T. Davis Y. Zheng, M. Moritz

  10. (+) end (+) end b b a a gTuRC g a g (-) end gTuRC (-) end Models of MT nucleation by gTuRC ‘Template’ Model (Zheng & Alberts) ‘Protofilament’ Model (Erikson & Stoffler) cryoEMT to determine structure of MT minus ends in centrosomes

  11. Examining gTuRC-nucleated MTs in situ: Cryo-EM Tomography of intact centrosomes J.Lyle tilt data : 300kv, FEG, energy filter, new CCD

  12. Examining gTuRC-nucleated MTs in situ: Cryo-EM Tomography of intact centrosomes J.Lyle 3D reconstruction

  13. g-TuRC functions as a minus-end template in vitro centrosomes J. Lyle, M. Moritz

  14. Is the data of sufficient quality to average? MT search model P. Koenig, J.Lyle

  15. Searching for structures in cryo-EMT MT model averages of real density show improved resolution limited by defocus (now correcting) P. Koenig, J.Lyle

  16. How do g-tubulin complexes nucleate MTs? what is the mechanism?? • kinetic studies of MT nucleation • implications of 3D structures of g-tubulin

  17. b Löwe et al., JMB 313, 2001 Growing a Mandelkow et al., JCB 114, 1991 GDP b Shrinking Knossow et al., Nature 428, 2004 a Mandelkow et al., JCB 114, 1991 GTP Structural changes and nucleotide state GTP hydrolysis drives dynamic instability Current model: ab-tubulin conformation dictated by GTP/GDP

  18. D D T T T T T T T T T T T T T T T T T T T T T T + GTP Current model:nucleotide dictates ab-tubulin conformation GDP conformation: curved GTP conformation: straight Rationalizes GTP dependence of microtubule assembly

  19. 2.3Å structure of g-tubulin:GDP2.7Å structure of g-tubulin:GTPS first GTP/GDP pair for any eukaryotic tubulin and highest resolution L. Rice, H. Aldaz, L. Montabana

  20. Straight Curved H6-H7: ‘piston’ movement b-sheet: ‘pivot’ movement g-tubulin:GDP on straightb-tubulin g-tubulin:GDP on curvedb-tubulin g-tubulin:GDP is curved All alignments performed using the N-terminal domain (grey)

  21. G144 N207 T145 E70 F225 C13 D68 Q12 N229 g-tubulin:GTP structure conserved binding site identical residues (b,g) contact nucleotide g- and b-tubulin have identical GTP/GDP binding affinities = similar functional energetics therefore expect -tubulin:GTP to be straight L. Rice H. Aldaz

  22. g-tubulin on straightb-tubulin g-tubulin on curvedb-tubulin g-tubulin:GTP is unambiguously curved b b-tubulin conformations b a a Straight Curved b b a a H6-H7 b-sheet and H10

  23. Current Model: cis-acting GTP Alternate Model: trans-acting GTP GTP GDP GTP GDP D D T T T T T T T T T D T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T Lattice effects: The GTP that matters is the one on the microtubule Is ab-tubulin:GTP curved too? If so, what is role of GTP?

  24. GDP:ab-tub GTP:ab-tub No measurable linkage between GTP binding and curvature Curved conformation dominates independent of nucleotide Kd (mM) GTP:ab-tub 0.79±0.15 GDP:ab-tub 0.74±0.15 Support for the new model:Differentiating curved & straight: allocolchicine Curved: binds allo. Straight: cannot bind allo. Model Knossow et al. L. Rice

  25. Direct Measurement of ab-tubulin conformation: Small Angle X-ray Scattering (SAXS) Calculated SAXS profiles show differences between curved andstraight conformations Scattering Intensity (log scale, arb.y units) Q (Å-1) Measured SAXS profiles show identical (curved) conformations GTP/GDP GTP/GDP+colchicine GMPCPP/GMPCP Scattering Intensity (log scale, arb.y units) Q (Å-1) Q (Å-1) Q (Å-1) 20 mM HEPES pH 7.5, 1 mM MgCl2, 1 mM EGTA, 1 mM nucleotide L. Rice

  26. Elongation 1 curved to straight + + Old Model: cis-acting GTP New Model: trans-acting GTP 2 curved to straight Initiation T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T + + Consequences for spontaneous assembly New model predicts important additional barrier to initiation

  27. g M-loop M-loop H10 H10 g g g g • lateral interface fully functional in curved conformation • architecture of ab-tubulin heterodimer limits lateral association g-tubulin forms MT-like lateral interactions g-tubulin crystal packing b-tubulin microtubule packing

  28. T T T T T T b b b b b b a a a a a a g g g Longitudinal: Kd ~ mM Composite: Kd ~ mM Lateral: Kd ~ M High affinity lateral sites drive MT assembly & reduce nucleus size Model for g-TuRC function Weak lateral associations limit de novo microtubule assembly

  29. DEspring bent (free) straight (MT) Energy storage drives MT dynamics • microtubules are kinetically trapped structures • undergo rapid disassembly (catastrophe) • GTP provides energy for MT assembly • spring constant, NOT GTP determines • energy storage in MT • uncouples assembly from disassembly Rogers et al., JCB 158, 2002

  30. New ideas about microtubule assembly • Curved conformation is default for ab-tubulin outside lattice • Builds up “strain energy” on entering lattice • New model: assembly is the key • GTP matters on the microtubule, not on free ab-tubulin • GTP enhances longitudinal interactions • Strain in lattice from GTP hydrolysis => MT instability • Unanticipated additional barriers to spontaneous assembly • Unique propensity of monomeric g-tubulin for lateral association • g-tubulin polymers reduce nucleus size, accelerate nucleation

  31. Centrosome Studies Hector Aldaz* Michael Braunfeld John Lyle* Justin Kollman Liz Montabana Michelle Moritz Luke Rice Mariano Tablos Matt Trammell EM Methods Development (with John Sedat) Michael Braunfeld Mel Jones Bettina Keszthelyi Peter Koenig* Winnie Ling Siddharth Shah Shawn Zheng Collaborators Tim Stearns, Stanford Trisha Davis, U. Washington Ron Vale, Nichole Mahoney, UCSF Supported by HHMI, NIH and the Keck Foundation * left lab

  32. automated tilt-pair data collection 60°tilt 120 kV 62,000 x mag. -2 mm defocus, focus error < 50nm

  33. the solution: • Single Particle Tomography • search from cryo-EMT maps in 3D for structures • average density • re-align to average, repeat • correct for focus changes with tilt How can we get better resolution from cryo-EMT? the problem: • • must average to overcome beam damage • but structures only visible in tomograms

  34. gTuSC Tub4 gTuRC 2.2MDa 280KDa gTuSC g-tubulin complexes are directly responsible for MT nucleation g-Tubulin Ring Complex g-tubulin Tub4 complex, 25Å res Dgrips 84 91 Dgrips 163 128 75s J. Kollman, L. Rice, J. Lyle, V. Guenebaut M. Moritz, T. Davis Y. Zheng, M. Moritz

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