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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. Membranes offer native environment.

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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


Membranes offer native environment

Aquaporin: Gonen et al, 2005, Nature, 438, 633-638


Electrons have a very short wavelength (0.028 Å)

Aquaporin: Gonen et al, 2005, Nature, 438, 633-638



Solubilization bottle-neck

Purification

2D crystallization

General strategy to 2D crystallize membrane proteins

Expression

Imaging


1 bottle-neckst 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


2DX bottle-neck

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


1050 bottle-neckml 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


Time course for removal of l bottle-neckow and high CMC detergents

octylglucoside

docecylmaltoside

Temperature cycling (future)

Variomag thermoshake


Imaging bottle-neck

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


macromolecule bottle-neck

Magnetic 96-format platform

Nickel EM grids

Final blot with filter paper 8 at-a-time

stain

support


Need to image 96 grids in the bottle-neck

electron microscope

Imaging

Scara = John

Cartesian = Henry

John Koss

Kevin D’Amico

Argonne Lab


laser bottle-neck

air

John loads the grid into the holder


Leginon software controls robot and 2DX imaging bottle-neck

LEGINON is a system designed for automated collection of images by TEM (NRAMM at Scripps)


Define montage image area (e.g. 3x3) bottle-neck

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


Density histogram evaluated to select suitable areas for imaging

Metal grid square

Edge of grid square

Broken support film

Sample present


Classification of imaged objects imaging

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


Classification of imaged objects imaging

Class A

100 nm


A 2DX screening example imaging

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


A HT screening for 2DX: An example imaging

pH7

Object prevalence

pH8

Lipid type and LPR


A HT screening for 2DX: An example imaging

Sample: P2A3

Detergent: DDM

LPR 1.0

pH7 buffer solution (20 mM TES, 4mM NaN3, 5 mM MgCl2, 5 mM ZnCl2)





www.nysbc.org imaging

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


Acknowledgements imaging

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


A HT screening for 2DX: An example imaging

Sample: MCV

Lipid: DMPC

Detergent: Foscholine-12

LPR 1.0

pH6 buffer solution (20 mM MES, 4mM NaN3, 5 mM MgCl2, 0 mM NaCl)



2DX Were Obtained With LH2 and CopA imaging

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


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