Research Talk Research Talk Tom Walz Tom Walz Department of Cell Biology Department of Cell Biology Harvard Medical School Harvard Medical School Workshop on Advanced Topics Workshop on Advanced Topics in EM Structure Determination in EM Structure Determination The Scripps Research Institute The Scripps Research Institute La Jolla, November 2007 La Jolla, November 2007
The aquaporin family of water pores The aquaporin family of water pores
AQP0 forms thin junctions in vivo AQP0 forms thin junctions in vivo 0.2 μ m Adapted from: Adapted from: Paul & Goodenough (1983) Zampighi et al. (1982) J. Cell Biol. 96: 625-632 J. Cell Biol. 93: 175-189
Purification of AQP0 from the lens Purification of AQP0 from the lens cortex core Cortex Single- 97 layered 67 2D crystals Core 45 31 full-length cleaved 21 Solubilization in 1% DM Anion exchange (MonoQ) Double- 14 Gel filtration (S12) layered 2D crystals
1 μ m 2D crystals 2D crystals of AQP0 of AQP0 Double- - layered layered Double
Electron Electron diffraction diffraction at liquid He at liquid He temperature temperature
density map density map The 1.9 Å Å The 1.9
The packing of AQP0 in the 2D crystals The packing of AQP0 in the 2D crystals
The packing of AQP0 in the 2D crystals The packing of AQP0 in the 2D crystals
The packing of AQP0 in the 2D crystals The packing of AQP0 in the 2D crystals
The lipids surrounding an AQP0 monomer The lipids surrounding an AQP0 monomer
Protein- -lipid interactions lipid interactions Protein PC 1 PC 5 PC 6 PC 1 PC 5 PC 6
Protein- -lipid interactions lipid interactions Protein Lipid dynamics Lipid dynamics
Protein- -lipid interactions lipid interactions Protein Acyl chains appear more constrained in membrane center chains appear more constrained in membrane center Acyl
Protein- -lipid interactions lipid interactions Protein Lipid dynamics Lipid dynamics Effects of lipids on Effects of lipids on protein structure protein structure
Protein- -lipid interactions lipid interactions Protein 1.9 Å Å EM structure EM structure 2.2 Å Å X X- -ray structure ray structure 1.9 2.2
Protein- -lipid interactions lipid interactions Protein B-factors – Electron diffraction versus X-ray diffraction Lipid-contacting atoms All atoms Number of atoms Number of atoms B-factor [Å 2 ] B-factor [Å 2 ]
Protein- -lipid interactions lipid interactions Protein B-factors – Electron diffraction versus X-ray diffraction EM structure X-ray structure B-factor [Å 2 ] B-factor [Å 2 ] Position in z [Å] Position in z [Å]
Protein- -lipid interactions lipid interactions Protein Lipid dynamics Lipid dynamics Effects of lipids on Effects of lipids on protein structure protein structure Lipid binding motifs Lipid binding motifs
Protein- -lipid interactions lipid interactions Protein No obvious binding motif for PC phosphodiester phosphodiester group group No obvious binding motif for PC
Protein- -lipid interactions lipid interactions Protein Lipid dynamics Lipid dynamics Effects of lipids on Effects of lipids on protein structure protein structure Lipid binding motifs Lipid binding motifs Different lipids Different lipids
Protein- -lipid interactions lipid interactions Protein 2D crystals in E. coli E. coli polar lipids (67% PE, 23% PG, 10% polar lipids (67% PE, 23% PG, 10% cardiolipin cardiolipin) ) 2D crystals in
Structure determination by Structure determination by single particle EM single particle EM Biochemical purification Biochemical purification not automated not automated time- -consuming consuming time Specimen preparation Specimen preparation Low- -dose imaging dose imaging Low more or less automated more or less automated Image processing Image processing time- -efficient efficient time 3D reconstruction 3D reconstruction
Purification of macromolecular complexes Purification of macromolecular complexes Challenges Challenges – unstable, heterogeneous unstable, heterogeneous – – low expression, low yield low expression, low yield – – high purity high purity – Commonly used affinity tags Commonly used affinity tags – His tag His tag – – FLAG tag FLAG tag – – TAP tag TAP tag – Lichty et al ., Protein Expression and Purification (2005) Lichty et al ., Protein Expression and Purification (2005)
2D crystallization of His- -tagged proteins tagged proteins 2D crystallization of His on lipid monolayers monolayers on lipid Kubalek et al . (1994) Kubalek et al . (1994) – Ni Ni- -NTA lipid NTA lipid – – His His- -tagged HIV1 RT tagged HIV1 RT – – 2D, negative stain 2D, negative stain – Ni 2+ Ni 2+ Ni 2+ Ni 2+ Kelly et al . (2006) Kelly et al . (2006) – Ni Ni- -NTA lipid NTA lipid – α - integrin: α – ß ß1 1- -integrin: -actinin actinin – vinculin D1 complex vinculin D1 complex Ni 2+ Ni 2+ Ni 2+ Ni 2+ Ni 2+ Ni 2+ Ni 2+ Ni 2+ Ni 2+ Ni 2+ – 3D, cryo 3D, cryo- -EM EM –
A combinatorial approach for A combinatorial approach for protein purification and sample preparation protein purification and sample preparation for single particle EM studies for single particle EM studies Establish whether Ni- -NTA lipid NTA lipid monolayers monolayers can be can be Establish whether Ni used as a tool to purify macromolecular complexes used as a tool to purify macromolecular complexes
The test system The test system TfR Transferrin – – Transferrin Tf C transferrin receptor receptor transferrin (Tf Tf- -TfR TfR) complex ) complex ( N His 6 Ni 2+ Ni 2+ Ni 2+ Ni 2+ Cheng et al . (2004) Cheng et al . (2004)
Tf- -TfR TfR complex on Ni complex on Ni- -NTA monolayer NTA monolayer Tf 0% Ni- -NTA NTA 2% Ni- 2% Ni- -NTA -NTA NTA NTA 0% Ni 2% Ni 2% Ni 20% Ni- -NTA NTA 40% Ni- -NTA NTA 20% Ni 40% Ni
Tf- -TfR TfR complex on Ni complex on Ni- -NTA monolayer NTA monolayer Tf
Monolayer purification of the Tf Tf- -TfR TfR Monolayer purification of the complex from a defined protein mixture complex from a defined protein mixture 10:90 (no His tag) 10:90 (no His tag) 50:50 50:50 10:90 10:90 30:70 30:70 in solution in solution on monolayer
Monolayer purification of the Tf Tf- -TfR TfR Monolayer purification of the complex from cell extracts complex from cell extracts SF9 extract SF9 extract 273T extract 273T extract cell medium cell medium in solution in solution on monolayer
Monolayer purification of the Tf Tf- -TfR TfR Monolayer purification of the complex from cell extracts complex from cell extracts SF9 extract 273T extract cell medium on monolayer + imidazole 10 mM 10 mM 20 mM 20 mM 50 mM 50 mM on monolayer
Characterization of purified complexes Characterization of purified complexes Successively elute Successively elute 20 ML samples with 20 ML samples with Ni 2+ Ni 2+ Ni 2+ Ni 2+ 300 mM 300 mM imidazole imidazole 300 mM imidazole ML ML 150 100 75 50 37 25 Ni-NTA column ML elution ML elution
A combinatorial approach for A combinatorial approach for protein purification and sample preparation protein purification and sample preparation for single particle EM studies for single particle EM studies Establish whether Ni-NTA lipid monolayers can be used as a tool to purify macromolecular complexes Establish whether lipid monolayer samples can be Establish whether lipid monolayer samples can be used for structure determination by single particle EM used for structure determination by single particle EM
Cryo- -EM of the EM of the Tf Tf- -TfR TfR complex complex Cryo from Sf9 cell extract from Sf9 cell extract 0% Ni- -NTA NTA 2% Ni- -NTA NTA 0% Ni 2% Ni 20% Ni- 20% Ni- -NTA -NTA NTA NTA 40% Ni- -NTA NTA 20% Ni 20% Ni 40% Ni
Cryo- -EM of the EM of the Tf Tf- -TfR TfR complex complex Cryo from Sf9 cell extract from Sf9 cell extract
3D reconstruction of the Tf Tf- -TfR TfR complex complex 3D reconstruction of the in vitrified ice on lipid monolayer in vitrified ice on lipid monolayer Initial model from pdb pdb- -file file Initial model from (Cheng et al. , 2004) (Cheng et al. , 2004) filtered to 30 Å Å resolution resolution filtered to 30 FREALIGN (Grigorieff, 2007) FREALIGN (Grigorieff, 2007) – refine orientation parameters refine orientation parameters – – correct for CTF correct for CTF – – calculate 3D reconstruction calculate 3D reconstruction –
3D density map of the Tf Tf- -TfR TfR complex complex 3D density map of the TfR Tf C-lobe Tf N-lobe
3D density map of the Tf Tf- -TfR TfR complex complex 3D density map of the 20 Å
3D density map of the Tf Tf- -TfR TfR complex complex 3D density map of the 20 Å Raw images Class averages Re-projections
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