Research Talk Research Talk Tom Walz Tom Walz Department of Cell - - PowerPoint PPT Presentation

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

Research Talk Research Talk

Tom Walz Tom Walz Department of Cell Biology Department of Cell Biology Harvard Medical School Harvard Medical School

The aquaporin family of water pores The aquaporin family of water pores

AQP0 forms thin junctions AQP0 forms thin junctions in vivo in vivo

Adapted from: Paul & Goodenough (1983)

• J. Cell Biol. 96: 625-632

0.2 μm

Adapted from: Zampighi et al. (1982)

• J. Cell Biol. 93: 175-189

Purification of AQP0 from the lens Purification of AQP0 from the lens

Cortex Core

core cortex 97 67 45 31 21 14 full-length cleaved

Single- layered 2D crystals

Solubilization in 1% DM Anion exchange (MonoQ) Gel filtration (S12)

Double- layered 2D crystals

1 μm

Double Double-

• layered

layered 2D crystals 2D crystals

• f AQP0
• f AQP0

Electron Electron diffraction diffraction at liquid He at liquid He temperature temperature

The 1.9 The 1.9 Å Å density map density map

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 Protein-

• lipid interactions

lipid interactions

PC 1 PC 1 PC 5 PC 5 PC 6 PC 6

Protein Protein-

• lipid interactions

lipid interactions

Lipid dynamics Lipid dynamics

Acyl Acyl chains appear more constrained in membrane center chains appear more constrained in membrane center

Protein Protein-

• lipid interactions

lipid interactions

Lipid dynamics Lipid dynamics Effects of lipids on Effects of lipids on protein structure protein structure

Protein Protein-

• lipid interactions

lipid interactions

1.9 1.9 Å Å EM structure EM structure 2.2 2.2 Å Å X X-

• ray structure

ray structure

Protein Protein-

• lipid interactions

lipid interactions

B-factors – Electron diffraction versus X-ray diffraction

Lipid-contacting atoms

Number of atoms B-factor [Å2]

All atoms

Number of atoms B-factor [Å2]

Protein Protein-

• lipid interactions

lipid interactions

B-factors – Electron diffraction versus X-ray diffraction

B-factor [Å2] Position in z [Å]

EM structure

B-factor [Å2] Position in z [Å]

X-ray structure

Protein Protein-

• lipid interactions

lipid interactions

Lipid dynamics Lipid dynamics Effects of lipids on Effects of lipids on protein structure protein structure

Protein Protein-

• lipid interactions

lipid interactions

Lipid binding motifs Lipid binding motifs

Protein Protein-

• lipid interactions

lipid interactions

No obvious binding motif for PC No obvious binding motif for PC phosphodiester phosphodiester group group

Lipid dynamics Lipid dynamics Effects of lipids on Effects of lipids on protein structure protein structure

Protein Protein-

• lipid interactions

lipid interactions

Different lipids Different lipids Lipid binding motifs Lipid binding motifs

2D crystals in 2D crystals in E. coli

• E. coli polar lipids (67% PE, 23% PG, 10%

polar lipids (67% PE, 23% PG, 10% cardiolipin cardiolipin) )

Protein Protein-

• lipid interactions

lipid interactions

Biochemical purification Biochemical purification Specimen preparation Specimen preparation Low Low-

• dose imaging

dose imaging Image processing Image processing 3D reconstruction 3D reconstruction

Structure determination by Structure determination by single particle EM single particle EM

more or less automated more or less automated time time-

• efficient

efficient not automated not automated time time-

• consuming

consuming

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

Lichty Lichty et al et al., Protein Expression and Purification (2005) ., Protein Expression and Purification (2005)

Commonly used affinity tags Commonly used affinity tags – – His tag His tag – – FLAG tag FLAG tag – – TAP tag TAP tag

Ni2+ Ni2+ Ni2+ Ni2+ Ni2+ Ni2+ Ni2+ Ni2+ Ni2+Ni2+ Ni2+Ni2+ Ni2+Ni2+

2D crystallization of His 2D crystallization of His-

• tagged proteins

tagged proteins

• n lipid
• n lipid monolayers

monolayers

Kubalek Kubalek et al et al. (1994) . (1994) – – Ni Ni-

• NTA lipid

NTA lipid – – His His-

• tagged HIV1 RT

tagged HIV1 RT – – 2D, negative stain 2D, negative stain Kelly Kelly et al et al. (2006) . (2006) – – Ni Ni-

• NTA lipid

NTA lipid – – ß ß1 1-

• integrin:

integrin:α α-

• actinin

actinin vinculin vinculinD1

D1 complex

complex – – 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 Establish whether Ni-

• NTA lipid

NTA lipid monolayers monolayers can be can be used as a tool to purify macromolecular complexes used as a tool to purify macromolecular complexes

The test system The test system

TfR Tf C N

Transferrin Transferrin – – transferrin transferrin receptor receptor ( (Tf Tf-

• TfR

TfR) complex ) complex

His6

Ni2+ Ni2+ Ni2+ Ni2+

Cheng Cheng et al et al. (2004) . (2004)

Tf Tf-

• TfR

TfR complex on Ni complex on Ni-

• NTA monolayer

NTA monolayer

0% Ni 0% Ni-

• NTA

NTA 2% Ni 2% Ni-

• NTA

NTA 20% Ni 20% Ni-

• NTA

NTA 40% Ni 40% Ni-

• NTA

NTA 2% Ni 2% Ni-

• NTA

NTA

Tf Tf-

• TfR

TfR complex on Ni complex on Ni-

• NTA monolayer

NTA monolayer

50:50 30:70 10:90 10:90 (no His tag) in solution 50:50 30:70 10:90 10:90 (no His tag) in solution

• n monolayer

Monolayer purification of the Monolayer purification of the Tf Tf-

• TfR

TfR complex from a defined protein mixture complex from a defined protein mixture

cell medium 273T extract SF9 extract in solution

Monolayer purification of the Monolayer purification of the Tf Tf-

• TfR

TfR complex from cell extracts complex from cell extracts

cell medium 273T extract SF9 extract in solution

• n monolayer

Monolayer purification of the Monolayer purification of the Tf Tf-

• TfR

TfR complex from cell extracts complex from cell extracts

cell medium 273T extract SF9 extract

• n monolayer

+ imidazole

• n monolayer

10 mM 10 mM 20 mM 20 mM 50 mM 50 mM

ML elution 150 100 75 50 37 25 ML ML

Characterization of purified complexes Characterization of purified complexes

300 mM imidazole Successively elute Successively elute 20 ML samples with 20 ML samples with 300 mM 300 mM imidazole imidazole

Ni2+ Ni2+ Ni2+ Ni2+

Ni-NTA column 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 Cryo-

• EM of the

EM of the Tf Tf-

• TfR

TfR complex complex from Sf9 cell extract from Sf9 cell extract

0% Ni 0% Ni-

• NTA

NTA 2% Ni 2% Ni-

• NTA

NTA 20% Ni 20% Ni-

• NTA

NTA 40% Ni 40% Ni-

• NTA

NTA 20% Ni 20% Ni-

• NTA

NTA

Cryo Cryo-

• EM of the

EM of the Tf Tf-

• TfR

TfR complex complex from Sf9 cell extract from Sf9 cell extract

Initial model from Initial model from pdb pdb-

• file

file (Cheng (Cheng et al. et al., 2004) , 2004) filtered to 30 filtered to 30 Å Å resolution resolution

3D reconstruction of the 3D reconstruction of the Tf Tf-

• TfR

TfR complex complex in vitrified ice on lipid monolayer in vitrified ice on lipid monolayer

FREALIGN (Grigorieff, 2007) FREALIGN (Grigorieff, 2007) – – refine orientation parameters refine orientation parameters – – correct for CTF correct for CTF – – calculate 3D reconstruction calculate 3D reconstruction

TfR Tf C-lobe Tf N-lobe

3D density map of the 3D density map of the Tf Tf-

• TfR

TfR complex complex

3D density map of the 3D density map of the Tf Tf-

• TfR

TfR complex complex

20 Å

3D density map of the 3D density map of the Tf Tf-

• TfR

TfR complex complex

20 Å Raw images Class averages Re-projections

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 used for structure determination by single particle EM Apply the method to a real system Apply the method to a real system

Monolayer purification of ribosomal Monolayer purification of ribosomal complexes from complexes from E. coli

• E. coli extract

extract

His His-

• tagged hEx1 clone (from RZPD library)

tagged hEx1 clone (from RZPD library) for rpl3 (60S ribosomal protein) for rpl3 (60S ribosomal protein) (has 47 additional residues at N (has 47 additional residues at N-

• terminus

terminus compared to compared to E. coli

• E. coli homolog)

homolog)

90 90° °

Ni2+ Ni2+ Ni2+ Ni2+

Monolayer purification of ribosomal Monolayer purification of ribosomal complexes from complexes from E. coli

• E. coli extract

extract

Express His Express His-

• tagged

tagged human rpl3 in human rpl3 in E. coli

• E. coli

to purify 50S ribosome to purify 50S ribosome from extract from extract

Monolayer purification of ribosomal Monolayer purification of ribosomal complexes from complexes from E. coli

• E. coli extract

extract

cell extract cell extract Ni Ni-

• NTA column

NTA column Ni Ni-

• NTA monolayer

NTA monolayer

100 100 75 75 50 50 37 37 25 25

SDS SDS-

• PAGE gel

PAGE gel

100 100 75 75 50 50 37 37 25 25

Western blot Western blot

Monolayer purification of ribosomal Monolayer purification of ribosomal complexes from complexes from E. coli

• E. coli extract

extract

Vitrified specimen Vitrified specimen

Monolayer purification of ribosomal Monolayer purification of ribosomal complexes from complexes from E. coli

• E. coli extract

extract

45,444 particles in 200 classes 45,444 particles in 200 classes

Monolayer purification of ribosomal Monolayer purification of ribosomal complexes from complexes from E. coli

• E. coli extract

extract

50S 50S 70S 70S

Monolayer purification of ribosomal Monolayer purification of ribosomal complexes from complexes from E. coli

• E. coli extract

extract

Monolayer purification of ribosomal Monolayer purification of ribosomal complexes from complexes from E. coli

• E. coli extract

extract

Angular distribution FSC curve

22 Å

Comparison with re-projections

Advantages of monolayer purification Advantages of monolayer purification

• fast

fast → → ideal for unstable and transient complexes ideal for unstable and transient complexes

• one
• ne-
• step procedure that needs little material

step procedure that needs little material → → ideal for low ideal for low-

• abundance and low

abundance and low-

• yield complexes

yield complexes

• easy to vitrify because of lipid monolayer

easy to vitrify because of lipid monolayer easy to adjust particle concentration easy to adjust particle concentration

• purification of all complexes that contain tagged subunit

purification of all complexes that contain tagged subunit (assembly intermediates, alternative complexes etc.) (assembly intermediates, alternative complexes etc.)

• tagging of transient subunits, activators or substrates

tagging of transient subunits, activators or substrates would allow imaging of specific complexes would allow imaging of specific complexes

• combination with libraries of tagged constructs

combination with libraries of tagged constructs → → potential for high potential for high-

• throughput studies

throughput studies

Future directions Future directions

• use of His

use of His-

• tagged

tagged calmodulin calmodulin → → suitable for TAP suitable for TAP-

• tagged constructs

tagged constructs

• use of His

use of His-

• tagged protein A

tagged protein A → → suitable for any tagged construct in combination with suitable for any tagged construct in combination with antibodies against the tag antibodies against the tag

• use of fluorinated lipids

use of fluorinated lipids → → suitable for membrane proteins suitable for membrane proteins

Harvard Harvard Medical School Medical School

Tamir Tamir Gonen Gonen Yifan Yifan Cheng Cheng Richard Hite Richard Hite Stephen Harrison Stephen Harrison Piotr Piotr Sliz Sliz Deborah Kelly Deborah Kelly Danijela Danijela Dukovski Dukovski

University of Auckland University of Auckland

Joerg Joerg Kistler Kistler

Kyoto University Kyoto University

Yoshinori Yoshinori Fujiyoshi Fujiyoshi Yoko Hiroaki Yoko Hiroaki