Monolayer Purification for for Monolayer Purification Single - - PowerPoint PPT Presentation

Monolayer Purification Monolayer Purification for for Single Particle Single Particle EM EM

Debbie Kelly Debbie Kelly Walz Walz Laboratory Laboratory Department of Cell Biology Department of Cell Biology Harvard Medical School Harvard Medical School kelly@crystal kelly@crystal. .harvard harvard. .edu edu

Historical Background Historical Background

• Uzgiris

Uzgiris and and Kornberg Kornberg (1983) (1983)

2D 2D xtals xtals of

• f Ab

Ab on lipid

• n lipid

Antigen Antigen

• Darst

Darst et al et al., 1988 ., 1988

RNA polymerase RNA polymerase

• Avila

Avila-

• Sakar

Sakar et al et al., 1994 ., 1994

50S ribosome subunit 50S ribosome subunit

• Kubalek

Kubalek et al et al., (1994) ., (1994)

His His-

• tagged HIV1 RT

tagged HIV1 RT Dietrich and Dietrich and Venien Venien-

• Bryan

Bryan (20 (20

Monolayer Structures Monolayer Structures

~ ~ 90

90 projection projection maps maps 15 15 Cryo Cryo-

• EM

EM Only 7 Only 7 Cryo Cryo-

• EM

EM 3D 3D Reconstructions Reconstructions

Current Specimen Limitations

• Screening conditions

Screening conditions

• Fragile transfer step

Fragile transfer step

• Specimen Flatness

Specimen Flatness

• Alternative approach =

Alternative approach = Single Single particle EM particle EM

A combinatorial approach for A combinatorial approach for protein purification / EM protein purification / EM structural studies structural studies

• Develop the Ni

Develop the Ni-

• NTA monolayer

NTA monolayer technique as technique as a novel purification method a novel purification method

• Single Particle

Single Particle Cryo Cryo-

• EM

EM for 3D for 3D reconstruction reconstruction

• Apply the methodology to a real

Apply the methodology to a real system system

Monolayer Purification Setup

1) Add protein sample well 1) Add protein sample well

Cell Cell lysate w/ lysate w/ target (25 target (25 μ μl) l) 50mM 50mM Hepes Hepes+150 +150 mM NaCl mM NaCl+ +imidazole imidazole

2) Cast monolayer 2) Cast monolayer

Filler / Ni Filler / Ni-

• NTA lipid (1mg/ml

NTA lipid (1mg/ml in CHCl in CHCl3

3)

) Apply 1 Apply 1μ μl l Mix Mix w/ w/ Hamilton Hamilton syringe syringe Incubate 15 Incubate 15 -

• 30 min., 4

30 min., 4° °C C

Ni2+ Ni2+ Ni2+ Ni2+

3) Sample with EM 3) Sample with EM grid grid

Apply Apply clean EM grid clean EM grid Grid bar side Grid bar side on

• n

monolayer monolayer

Ni2+ Ni2+ Ni2+ Ni2+

Basic Basic Considerations Considerations

• Biological sample

Biological sample preparation preparation

• Grid preparation

Grid preparation

• Transfer Step

Transfer Step

• Neg
• Neg. stain screening

. stain screening

• Vitrification

Vitrification

• Low

Low-

• dose Imaging

dose Imaging

Preparation of Cell Extracts Preparation of Cell Extracts

• Grow His

Grow His-

• tagged construct

tagged construct

• Lyse

Lyse cells with cells with lysozyme lysozyme, , sonication sonication

• Obtained cleared

Obtained cleared lysate lysate; ML input ; ML input Insect cells Insect cells

(Sf9) (Sf9)

Bacteria Bacteria

(E.coli, BL21) (E.coli, BL21)

Mammalian cells Mammalian cells

(293T) (293T)

QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.

Quantifoil Quantifoil Grid Preparation Grid Preparation

Whatman Whatman #1 paper #1 paper Saturated w/ Saturated w/ Ethyl Acetate o/n Ethyl Acetate o/n in hood in hood Bake for 1 hr Bake for 1 hr At least 100 At least 100o

• C

C

Transfer Methods

Direct Direct Transfer Transfer Loop Loop Transfer Transfer

Direct Transfer vs. Loop transfer

Quantifoil Quantifoil 2/1 Holey Grids 2/1 Holey Grids Direct Direct Loop Loop

7Å Projection Map of SbpA

Norville Norville et al., JSB (in et al., JSB (in

Transfer of 2D crystal Transfer of 2D crystal vs vs Single Single particles particles

• Crystals = Loop transfer /

Crystals = Loop transfer / 5% Trehalose embedding 5% Trehalose embedding /ethane /ethane

• Particles = Direct transfer

Particles = Direct transfer /ethane /ethane

Basic Considerations Basic Considerations

• Biological sample

Biological sample preparation preparation

• Grid preparation

Grid preparation

• Transfer Step

Transfer Step

• Neg. stain screening
• Neg. stain screening
• Vitrification

Vitrification

• Low

Low-

• dose Imaging

dose Imaging

Negative Stain Screening Test specimen: Tf-TfR complex

Ni2+ Ni2+ Ni2+ Ni2+

His His 6 6

TfR TfR Tf Tf C C N N

Transferrin Transferrin-

• Transferrin

Transferrin Receptor Receptor Complex Complex

Ni2+ Ni2+ Ni2+ Ni2+

DLPC Room Temp DLPC 4oC

(Tm-1oC)

DMPC 4oC

(Tm23oC)

0% Ni-NTA 2% Ni-NTA 20% Ni-NTA 40% Ni-NTA

Negative Stain Screening Negative Stain Screening

Screening Monolayer Purified Screening Monolayer Purified Tf Tf-

• TfR

TfR complex from Sf9 extracts complex from Sf9 extracts

In In Soln Soln. . 2% 2% ML ML + + 50 50 mM mM Imid Imid. . 2% 2% ML ML

Neg

• Neg. stain reconstruction using

. stain reconstruction using RCT RCT

TfR TfR C C-

• lobe

lobe N N-

• lobe

lobe

Vitrification Vitrification of ML specimens

• f ML specimens
• Place

Place “ “Grid bar Grid bar” ”

• n top of
• n top of ML

ML

• Remove grid

Remove grid w/ w/ forceps forceps

• Blot 3

Blot 3μ μl l sub sub-

• phase

phase

• Plunge into

Plunge into ethane ethane

• Manual

Manual

• Uncontrolled

Uncontrolled environment environment

• Calibrate

Calibrate blotting blotting

• Consistent ice over

Consistent ice over entire grid entire grid

Manual Manual vs

• vs. Automated Freezing

. Automated Freezing

• Vitrobot

Vitrobot (FEI) (FEI)

• Environment

Environment

(22 (22o

• C, 65%

C, 65% rh rh) )

• Blotting time

Blotting time ∝ ∝ volume ( volume (μ μl) l)

• Gradient of vitreous

Gradient of vitreous ice ice

QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.

• Tecnai

Tecnai F F-

• 20 operating at 200 kV

20 operating at 200 kV

• Quantifoil

Quantifoil 2/1 (2 2/1 (2μ μm holes, 1 m holes, 1μ μm m spacing) spacing)

• Magnification = 50,000x

Magnification = 50,000x

• Defocus =

Defocus = -

• 2 to

2 to -

• 5

5 μ μm m

• 10

10 e e-

• /

/ Å Å2

2, 1 sec exposure

, 1 sec exposure

• Images on Film, scanned

Images on Film, scanned w/ w/ 7 7μ μm m step step (1.4 (1.4 Å Å / pixel, / pixel, 3 x 3 3 x 3 sub sub-

• sampling)

sampling)

Low Low-

• dose imaging

dose imaging

Imaging Monolayer Imaging Monolayer Purified Purified Tf Tf-

• TfR

TfR from Sf9 from Sf9 extracts extracts

In In Soln Soln. . 2% 2% ML ML 20% 20% ML ML

Leginon Leginon for Screening for Screening ML ML Cryo Cryo-

• EM

EM specimen specimen

Taylor et al., JSB (in press) Taylor et al., JSB (in press)

Hole selection based on Hole selection based on radial density function radial density function

+ + + +

*

* * 28,000 holes

28,000 holes w/ crystals; w/ crystals; 200 parts./hole 200 parts./hole 5 5 -

• 8 x 10

8 x 10

6 6 parts.

parts.

High High-

• throughput Potential

throughput Potential

1) Grow 1) Grow Construct Construct

Ni2+ Ni2+ Ni2+ Ni2+

2) Prep. grid 2) Prep. grid 3) 3) Direct Direct Transfe Transfe r r 4) 4) Neg Neg. . Stain Stain screen screen 5) 5) Vitrification Vitrification 6) 6) Cryo Cryo-

• EM

EM

Current Current interests interests

• Adapt ML method for use with

Adapt ML method for use with membrane proteins and other membrane proteins and other tags tags

• 3D reconstructions of native

3D reconstructions of native complexes complexes large data sets and automated large data sets and automated routines routines

Acknowledgements Acknowledgements

• Danijela Dukovski

Danijela Dukovski

• Tom

Tom Walz Walz

• Dept. of Cell
• Dept. of Cell

Biology Biology Harvard Medical Harvard Medical School School