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 GIF Scintillator and mirror Lens Detector scattering from fiber optic limits resolution of normal CCD camera solution: remove fiber optic

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

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

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

7. Understanding centrosome architecture and microtubule nucleation multi-resolution structural analysis atomic to whole centrosome kinetic mechanism of nucleation role of nucleators 10 11 Da, 1  m 3 (mitchison lab images)

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

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

10. Models of MT nucleation by  TuRC ‘Template’ Model (Zheng & Alberts) ‘Protofilament’ Model (Erikson & Stoffler) (-) end (+) end    TuRC  (-) end (+) end     cryoEMT to determine structure of MT minus ends in centrosomes

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

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

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

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

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

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

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

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

19. 2.3Å structure of  -tubulin:GDP 2.7Å structure of  -tubulin:GTP  S L. Rice, H. Aldaz, L. Montabana first GTP/GDP pair for any eukaryotic tubulin and highest resolution

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

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

22. Straight Curved H6-H7  -sheet and H10  -tubulin:GTP is unambiguously curved  -tubulin conformations          -tubulin on straight  -tubulin  -tubulin on curved  -tubulin

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

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

25. 20 mM HEPES pH 7.5, 1 mM MgCl 2, 1 mM EGTA, 1 mM nucleotide Calculated SAXS profiles show differences between curved and straight conformations Scattering Intensity (log scale, arb.y units) Q (Å -1 ) Measured SAXS profiles show identical (curved) conformations GTP/GDPGTP/GDP+colchicineGMPCPP/GMPCP Q (Å -1 ) Scattering Intensity (log scale, arb.y units) Direct Measurement of  -tubulin conformation: Small Angle X-ray Scattering (SAXS) L. Rice

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

27.  -tubulin forms MT-like lateral interactions M-loop H10   lateral interface fully functional in curved conformation architecture of  -tubulin heterodimer limits lateral association     -tubulin crystal packing  -tubulin microtubule packing

28. Model for  -TuRC function Weak lateral associations limit de novo microtubule assembly Longitudinal: K d ~ mM T   Lateral: K d ~ M T   Composite: K d ~  M T T T       T      High affinity lateral sites drive MT assembly & reduce nucleus size

29. Energy storage drives MT dynamics Rogers et al., JCB 158, 2002 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  E spring bent (free)straight (MT)

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

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

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

33. must average to overcome beam damage but structures only visible in tomograms How can we get better resolution from cryo-EMT? the problem: 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

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