Furthering our understanding of microtubule dynamic instability by - - PowerPoint PPT Presentation

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Furthering our understanding of microtubule dynamic instability by - - PowerPoint PPT Presentation

Feb 22, 2024 264 likes •787 views

Furthering our understanding of microtubule dynamic instability by CryoEM Gabriel Lander Postdoc, Eva Nogales Lab UC Berkeley/Lawrence Berkeley National Lab Greg Alushin The Microtubule Microtubules are among most important components of

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Furthering our understanding of microtubule dynamic instability by CryoEM

Gabriel Lander

Postdoc, Eva Nogales Lab UC Berkeley/Lawrence Berkeley National Lab

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Greg Alushin

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The Microtubule

  • Microtubules are among most important

components of the cytoskeleton

  • Fundamental part of many physiological

processes:

  • intracellular transport
  • cell motility
  • cell polarization
  • cell division
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Beta Subunit Alpha Subunit The Tubulin Dimer - The Microtubule Building Block

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Tubulin dimers assemble longitudinally Protofilament

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Microtubule

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Microtubule Seam Breaks Helical Symmetry

Microtubules are not static structures - their ability to assemble & depolymerize is essential to cellular function.

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(GDP) Alpha Subunit (GTP) GTP Beta Subunit The Nucleotide Binding Pocket

Free tubulin can exchange GDP for GTP Non-exchangeable site (N-site) Exchangeable site (E-site)

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Alpha Subunit (GTP) (GTP) Beta Subunit

GTP is required at beta subunit for MT polymerization, creating strong inter- tubulin contacts

The Nucleotide Binding Pocket

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The Nucleotide Binding Pocket Alpha Subunit (GTP) (GDP) Beta Subunit

GTP hydrolysis to GDP weakens the inter-tubulin contacts

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Microtubule Dynamic Instability GTP “Cap”{

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Microtubule Dynamic Instability

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Microtubule Dynamic Instability Mechanism relating GTP hydrolysis to dynamic instability still unknown

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Atomic-Resolution Structures

Electron Crystallography (1TUB,1JFF,1TVK) Zinc-induced antiparallel tubulin sheets + stabilizing ligand X-ray Crystallography (4DRX,4F6R) DARPin-bound dimer X-ray Crystallography (4FFB) TOG-bound dimer X-ray Crystallography (1FFX,1SA0,1Z2B, 3DU7,3HKB,3N2G,3RYC, 4F61,4UT5) Polymers bound to stathmin-like domains

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CryoEM of Microtubules

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Subnanometer-Resolution CryoEM Structures

Li et al. Structure 2002 (9Å resolution) Maurer et al. Cell 2012 (8Å resolution) Yajima et al. JCB 2012 (9Å resolution) Sindelar and Downing PNAS 2010 (8.5Å resolution) Alushin et al. Nature 2010 (8.6Å resolution) Kikkawa and Hirokawa EMBO J 2006 (9.5Å resolution) Fourniol et al. JCB 2010 (8Å resolution)

Are microtubules only ordered to 8Å resolution?

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FEI Titan EM (aka “The Beast”)

  • C3 active, parallel illumination
  • 300keV
  • 2K CCD, no DD = film collection
  • No Leginon = Tecnai Low Dose
  • Side-entry holder
  • “Weird State” feature!
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Bending Flattening

Microtubule Distortions

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Distinguishing Alpha from Beta

In an EM micrograph, alpha tubulin is indistinguishable from beta tubulin

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Rice et al. Nature, Dec 1999

Human Kinesin Monomer

Mutation in switch II region inhibits ATP hydrolysis, stably binds to microtubules (plasmid from Vale lab, UCSF)

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Heterogeneous Protofilament Symmetries & Seam 12pf 13pf 14pf 15pf

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8Å

72000X (0.87Å/pixel) 25e-/Å2

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Ice ring at ~3.6Å

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Remove images with drift/ beam induced motion

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Pick MT Filaments Box out at every 80Å 768x768 pixels, binned by 2 for processing

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80Å 40Å 20Å 10Å 5Å

Layer lines visible out to ~5Å resolution

80Å 40Å 20Å 10Å 5Å

2D classification (IMAGIC MSA/MRA)

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80Å 40Å 20Å 10Å 5Å

Remove low resolution particles 2D classification (IMAGIC MSA/MRA)

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Remove particles missing kinesin, also 12pf & 15pf

80Å 40Å 20Å 10Å 5Å

2D classification (IMAGIC MSA/MRA)

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Masked particle segments with mixed protofilament numbers (13 & 14pfs)

Refinement Scheme (EMAN2/SPARX Libs)

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Masked particle segments with mixed protofilament numbers (13 & 14pfs)

Refinement Scheme (EMAN2/SPARX Libs)

13 & 14pf initial models

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Masked particle segments with mixed protofilament numbers (13 & 14pfs)

Particles are sorted by multi- model projection matching using 13pf and 14pf models Asymmetric back projection

  • f each pf symmetry

Refinement Scheme (EMAN2/SPARX Libs)

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Low-resolution asymmetric densities

Determine helical symmetry of each pf number using only monomer density (Egelman’s hsearch_lorentz)

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14 protofilament turn = -25.75º rise = 8.89Å 13 protofilament turn = -27.67º rise = 9.51Å

Applying Pseudo-Symmetry

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Applying Pseudo-Symmetry Average symmetry mates in Fourier space during back projection

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1x

For each protofilament density, use the helical parameters to symmetrize the density with pf-1 symmetry mates

Applying Pseudo-Symmetry (14pf example)

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2x

For each protofilament density, use the helical parameters to symmetrize the density with pf-1 symmetry mates

Applying Pseudo-Symmetry (14pf example)

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3x

For each protofilament density, use the helical parameters to symmetrize the density with pf-1 symmetry mates

Applying Pseudo-Symmetry (14pf example)

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13x

For each protofilament density, use the helical parameters to symmetrize the density with pf-1 symmetry mates

Applying Pseudo-Symmetry (14pf example)

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Extract protofilament containing symmetrized tubulin dimers

Applying Pseudo-Symmetry

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Generating Seamed Density

Regenerate 13 or 14-fold microtubule with seam

14 protofilament turn = -25.75º rise = 8.89Å

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Projection matching & back projection using multiple pf models Particle segments with mixed protofilament #’s For each, find helical parameters (Ed Egelman’s hsearch_lorentz) Over symmetrize using helical parameters (real space) Extract the “good” protofilaments & create new models using helical params iterate Final refinement in FREALIGN with same averaging & pf extraction

Pseudo-Helical Microtubule Reconstruction

iterate

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X X X X X X

Assessing alignment with the seam

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Removing “bad” microtubules

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FREALIGN refinement

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FREALIGN refinement

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GMPCPP MTs + Kinesin

1.4-3.5um underfocus 25e-/Å2 (1sec exposure) 311 Films acquired 252 used for processing 92,581 segments (40:60 ratio 13:14pfs)

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Microtubule at 4.7Å Resolution (FSC=0.143)

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Microtubule at 4.7Å Resolution (FSC=0.143)

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Eva Nogales (UCB/LBNL)

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Greg Alushin (UCB) Atomic modeling

Paul Adams & Jeff Head (LBNL)

Acknowledgements