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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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slide1
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

slide2
Membranes offer native environment

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

slide3
Electrons have a very short wavelength (0.028 Å)

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

slide5
Solubilization

Purification

2D crystallization

General strategy to 2D crystallize membrane proteins

Expression

Imaging

slide6
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

slide7
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

slide8
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

slide9
Time course for removal of low and high CMC detergents

octylglucoside

docecylmaltoside

Temperature cycling (future)

Variomag thermoshake

slide10
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
slide11
macromolecule

Magnetic 96-format platform

Nickel EM grids

Final blot with filter paper 8 at-a-time

stain

support

slide12
Need to image 96 grids in the

electron microscope

Imaging

Scara = John

Cartesian = Henry

John Koss

Kevin D’Amico

Argonne Lab

slide13
laser

air

John loads the grid into the holder

slide14
Leginon software controls robot and 2DX imaging

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

slide15
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

slide16
Density histogram evaluated to select suitable areas for imaging

Metal grid square

Edge of grid square

Broken support film

Sample present

slide17
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
slide19
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

slide20
A HT screening for 2DX: An example

pH7

Object prevalence

pH8

Lipid type and LPR

slide21
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)

slide25
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

slide26
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

slide27
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)

slide29
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

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