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Strategies for 2D Crystallization for cryoEM

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  1. Strategies for 2D Crystallization for cryoEM Minghui Hu, Changki Kim NY Structural Biology Center Iban Ubarretxena-Belandia Dept of Structural and Chemical Biol Mt. Sinai School of Med James Love - NYCOMPS

  2. Membranes offer native environment Aquaporin: Gonen et al, 2005, Nature, 438, 633-638

  3. Electrons have a very short wavelength (0.028 Å) Aquaporin: Gonen et al, 2005, Nature, 438, 633-638

  4. Lack of high-throughput screening for 2D crystals is a major bottle-neck

  5. Solubilization Purification 2D crystallization General strategy to 2D crystallize membrane proteins Expression Imaging

  6. 1st steps in high-throughput screening for 2DX – same as 3DX Target selection Expression Solubilization Purification NYCOMPS Provide purified proteins that have not crystallized in 3D (rescue path) Provide pre-screened clones for scaling-up

  7. 2DX Relatively small crystallization space: Protein concentration: 0.4-1 mg/ml Lipid type: DMPC, DOPC, POPC, DOPG, E. coli lipids Lipid-to-protein-ratio (LPR, mg-mg): 0.2-1.5 Detergent type and concentration: DDM or OG No precipitants used Buffer: pH 6-8, monovalent/divalent cations, catalytically/conformationally active compounds Set up 2D crystallizations on a 96-well format

  8. 1050 ml buffer 50 ml sample Vink, M., Derr, KD, Love, J., Stokes, D.L., & Ubarretxena-Belandia, I., 2007 A high-throughput strategy to screen 2D crystallization trials of membrane proteins. JSB, 160, 294-304

  9. Time course for removal of low and high CMC detergents octylglucoside docecylmaltoside Temperature cycling (future) Variomag thermoshake

  10. Imaging Negatively staining of 96 specimens for evaluation by EM • Pipette 5ul sample • Blot with filter paper • Pipette 5ul 1% uranyl acetate • Repeat n times • Blot & dry

  11. macromolecule Magnetic 96-format platform Nickel EM grids Final blot with filter paper 8 at-a-time stain support

  12. Need to image 96 grids in the electron microscope Imaging Scara = John Cartesian = Henry John Koss Kevin D’Amico Argonne Lab

  13. laser air John loads the grid into the holder

  14. Leginon software controls robot and 2DX imaging LEGINON is a system designed for automated collection of images by TEM (NRAMM at Scripps)

  15. Define montage image area (e.g. 3x3) Take a images at low mag Pick target squares using square finder (histogram criteria) Take images of selected squares Pick targets from square images (histogram criteria) Take images at intermediate mag for evaluation

  16. Density histogram evaluated to select suitable areas for imaging Metal grid square Edge of grid square Broken support film Sample present

  17. Classification of imaged objects Class E Class D Class B Class C • Tubular Vesicle Strings of Lipid Protein aggregate • Vesicle • Physical growth • Lipid aggregate • with lipid thread Multi-lamella Lipid • Sheet

  18. Classification of imaged objects Class A 100 nm

  19. A 2DX screening example pH6 Sample: P2A3 33 kDa Detergent: DDM LPR 1.0 Object prevalence Lipid type and LPR A: Crystal lattice B: Tubular vesicle, sheets and physical growth C: Vesicle and vesicle cluster D: Protein aggregate and lipid aggregate E: Lipid string F: Failure, large precipitate (checked by light microcopy), bad staining, broken carbon, etc

  20. A HT screening for 2DX: An example pH7 Object prevalence pH8 Lipid type and LPR

  21. A HT screening for 2DX: An example Sample: P2A3 Detergent: DDM LPR 1.0 pH7 buffer solution (20 mM TES, 4mM NaN3, 5 mM MgCl2, 5 mM ZnCl2)

  22. Screening summary

  23. Picasa web site for screening results

  24. Picasa web site for screening results

  25. www.nysbc.org A HT screening for 2DX: Summary for the 1st year 1- Manual 2DX screen is fully functional 2- Automated grid imaging is “almost” functional 3- Started structure determination for P2A3 4- Continue screening

  26. Acknowledgements Changki Kim Minghui Hui Martin Vink Kumiko Sugawara Iban Ubarretxena James Love Filippo Mancia Ming Zhou Wayne Hendrickson JKD instruments John Koss Kevin D’amico Bill Rice KD Derr Ruben Diaz Bridget Carragher Clint Potter Jim Pulokos Anchi Cheng NIH R01 GM081817

  27. A HT screening for 2DX: An example Sample: MCV Lipid: DMPC Detergent: Foscholine-12 LPR 1.0 pH6 buffer solution (20 mM MES, 4mM NaN3, 5 mM MgCl2, 0 mM NaCl)

  28. Electron Microscopy of 2D Crystals ~

  29. 2DX Were Obtained With LH2 and CopA Purified LH2 (0.5 mg/ml) in LDAO Mixed with DOPC in OG Dialyzed against 10 mM Hepes, pH 7.5, 100 mM NaCl for 24-h at 30 oC Purified CopA (0.5 mg/ml) in 0.01% DDM Mixed with DOPC in C12E8 Dialyzed against buffer for 7-d at 20 oC