Monolayer Purification for for Monolayer Purification Single - PowerPoint PPT Presentation

Monolayer Purification for for Monolayer Purification Single Particle EM EM Single Particle Debbie Kelly Debbie Kelly Walz Laboratory Laboratory Walz Department of Cell Biology Department of Cell Biology Harvard Medical School Harvard Medical School kelly@crystal. .harvard harvard. .edu edu kelly@crystal

Historical Background Historical Background Uzgiris and and Kornberg Kornberg Uzgiris • • (1983) (1983) 2D xtals xtals of of Ab Ab on lipid on lipid 2D Antigen Antigen Darst et al ., 1988 Darst et al ., 1988 • • RNA polymerase RNA polymerase Avila- -Sakar Sakar et al ., 1994 Avila et al ., 1994 • • 50S ribosome subunit 50S ribosome subunit Kubalek et al ., (1994) Kubalek et al ., (1994) • • His- -tagged HIV1 RT tagged HIV1 RT His Dietrich and Venien Venien- -Bryan Bryan (20 (20 Dietrich and

Monolayer Structures Monolayer Structures 90 ~ 90 ~ projection projection maps maps 15 Cryo Cryo- -EM EM 15 Only 7 Cryo Cryo- -EM EM Only 7 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 μ l) target (25 μ Cell lysate w/ Cell lysate w/ target (25 l) 50mM Hepes Hepes+150 +150 mM NaCl mM NaCl+ +imidazole imidazole 50mM 2) Cast monolayer 2) Cast monolayer Filler / Ni- -NTA lipid (1mg/ml NTA lipid (1mg/ml Filler / Ni Ni 2+ Ni 2+ Ni 2+ Ni 2+ in CHCl 3 ) in CHCl 3 ) Apply 1 μ μ l Apply 1 l Mix Mix w/ w/ Hamilton Hamilton syringe syringe Incubate 15 - - 30 min., 4 30 min., 4° °C C Incubate 15 3) Sample with EM 3) Sample with EM grid grid Ni 2+ Ni 2+ Ni 2+ Ni 2+ Apply clean EM grid clean EM grid Apply Grid bar side on on Grid bar side monolayer monolayer

Basic Considerations Considerations Basic • 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 QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Insect cells Insect cells Bacteria Bacteria Mammalian cells Mammalian cells (Sf9) (Sf9) (E.coli, BL21) (E.coli, BL21) (293T) (293T) Grow His- -tagged construct tagged construct Grow His • • Lyse cells with cells with lysozyme lysozyme, , sonication sonication Lyse • • Obtained cleared lysate lysate; ML input ; ML input Obtained cleared • •

Quantifoil Grid Preparation Grid Preparation Quantifoil Whatman #1 paper #1 paper Whatman 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 o o C C At least 100

Transfer Methods Direct Loop Direct Loop Transfer Transfer Transfer Transfer

Direct Transfer vs. Loop transfer Loop Direct Loop Direct Quantifoil 2/1 Holey Grids 2/1 Holey Grids Quantifoil

7Å Projection Map of SbpA Norville et al., JSB (in et al., JSB (in Norville

Transfer of 2D crystal vs vs Single Single Transfer of 2D crystal 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- -dose Imaging dose Imaging Low • •

Negative Stain Screening Test specimen: Tf-TfR complex TfR TfR Tf Tf Ni 2+ Ni 2+ Ni 2+ Ni 2+ C C His N N His 6 6 Transferrin- -Transferrin Transferrin Transferrin Receptor Complex Complex Receptor Ni 2+ Ni 2+ Ni 2+ Ni 2+

Negative Stain Screening Negative Stain Screening 0% Ni-NTA 2% Ni-NTA 20% Ni-NTA 40% Ni-NTA DLPC Room Temp DLPC 4 o C (T m -1 o C) DMPC 4 o C (T m 23 o C)

Screening Monolayer Purified Screening Monolayer Purified Tf- -TfR TfR complex from Sf9 extracts complex from Sf9 extracts Tf In Soln In Soln. . 2% ML ML 2% 2% ML 2% ML + + 50 50 mM mM Imid Imid. .

Neg. stain reconstruction using . stain reconstruction using Neg RCT RCT TfR TfR C- -lobe lobe C N- N -lobe lobe

Vitrification of ML specimens of ML specimens Vitrification • Place Place “ “Grid bar Grid bar” ” • on top of ML ML on top of • Remove grid Remove grid w/ w/ • forceps forceps μ l Blot 3 μ • Blot 3 l sub sub- - • phase phase • Plunge into Plunge into • ethane ethane

Manual vs vs. Automated Freezing . Automated Freezing Manual • Vitrobot Vitrobot (FEI) (FEI) • Manual Manual • • Uncontrolled Uncontrolled QuickTime™ and a Environment Environment • • TIFF (Uncompressed) decompressor • • are needed to see this picture. environment environment (22 o o C, 65% C, 65% rh rh) ) (22 Calibrate blotting blotting Calibrate • • Blotting time ∝ ∝ Blotting time • • μ l) volume ( μ l) volume ( Consistent ice over Consistent ice over • • entire grid entire grid Gradient of vitreous Gradient of vitreous • • ice ice

Low- -dose imaging dose imaging Low Tecnai F F- -20 operating at 200 kV 20 operating at 200 kV Tecnai • • μ m holes, 1 μ m 2/1 (2 μ m holes, 1 μ Quantifoil 2/1 (2 m Quantifoil • • spacing) spacing) Magnification = 50,000x Magnification = 50,000x • • μ m 5 μ Defocus = - - 2 to 2 to - - 5 m Defocus = • • - / 10 e e - / Å Å 2 2 , 1 sec exposure , 1 sec exposure 10 • • μ m 7 μ Images on Film, scanned w/ w/ 7 m Images on Film, scanned • • step (1.4 Å Å / pixel, / pixel, 3 x 3 3 x 3 step (1.4 sub- -sampling) sampling) sub

Imaging Monolayer Imaging Monolayer Purified Tf Tf- -TfR TfR from Sf9 from Sf9 Purified extracts extracts 2% 2% ML ML In Soln In Soln. . 20% ML 20% ML

Leginon for Screening for Screening Leginon ML Cryo Cryo- -EM EM specimen specimen ML 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 + + 6 parts. 6 5 - 5 - 8 x 10 8 x 10 parts.

High- -throughput Potential throughput Potential High 2) Prep. grid 2) Prep. grid 3) 3) Direct Direct Ni 2+ Ni 2+ Ni 2+ Ni 2+ 4) 4) Neg Neg. . Transfe Transfe Stain Stain r r screen screen 1) Grow 1) Grow Construct Construct 5) Vitrification Vitrification 5) 6) Cryo Cryo- -EM EM 6)

Current interests interests Current • 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