3 Methods
The approach of 2D electron crystallography can be applied to both
soluble and membrane proteins. For 2D crystals of soluble proteins, the steps from the negative stain grid preparation and beyond
are applied. For 2D crystallization of membrane proteins, all of the
steps below are applied, unless 2D crystals already exist.
3.1 Crystallization
of Membrane Proteins
Through Detergent
Removal by Dialysis
Membrane protein 2D crystallization requires samples that are
purified and contain no or a minimal amount of co-purified lipids.
An absolutely minimal amount of co-purified lipids is critical since
the 2D crystallization of most membrane proteins to date has been
dependent on a carefully controlled lipid-to-protein ratio. At this
point, the lipid content of the purified, detergent-solubilized membrane protein sample has likely been characterized by thin layer
chromatography, mass spectrometry, and/or initial TEM analysis.
3.1.1 Preparation
of Solubilized Lipid Stock
1. If working with lipids in powder form, add buffer-detergent
solution for a final lipid concentration of 10 mg/mL (see Note
1), which will result in the required lipid stock solution. If
working with lipids in chloroform (steps 2–4 below apply to
these lipids), pipette 1 mL into a small round-bottom flask.
2. Evaporate the chloroform with a stream of nitrogen gas (see
Note 2).
3. The buffer-detergent solution is pipetted into the roundbottom flask for a final lipid concentration of 10 mg/mL.
4. The round-bottom flask is placed into a sonicator bath and
sonicated for several minutes until the solution becomes clear.
5. Aliquots of 10 μL of the solubilized lipid are pipetted into
Eppendorf tubes for storage at either À20
C or À80
C (see
Note 3).
3.1.2 Setup of 2D
Crystallization Trials
1. Soak dialysis tubing in 500–1000 mL of ultrapure water (see
Note 4).
2. Thaw membrane protein, if previously frozen and not freshly
purified, and lipid aliquots on ice.
3. Prepare 1 L of dialysis buffer based on the purification buffer,
omitting the detergent. In later rounds of crystallization trials,
up to 20% glycerol may be added, and the salt may be increased,
decreased, or altered.
4. Mix 75–100 μL of the membrane protein with stock solution
of lipid at molar lipid-to-protein ratios (LPRs) of 0, 3, 15, and
30 in Eppendorf tubes on ice (this membrane proteindetergent-lipid mixture will be referred to as dialysate in later
steps) (see Note 5).
2D Electron Crystallography of Membrane Proteins
231
The approach of 2D electron crystallography can be applied to both
soluble and membrane proteins. For 2D crystals of soluble proteins, the steps from the negative stain grid preparation and beyond
are applied. For 2D crystallization of membrane proteins, all of the
steps below are applied, unless 2D crystals already exist.
3.1 Crystallization
of Membrane Proteins
Through Detergent
Removal by Dialysis
Membrane protein 2D crystallization requires samples that are
purified and contain no or a minimal amount of co-purified lipids.
An absolutely minimal amount of co-purified lipids is critical since
the 2D crystallization of most membrane proteins to date has been
dependent on a carefully controlled lipid-to-protein ratio. At this
point, the lipid content of the purified, detergent-solubilized membrane protein sample has likely been characterized by thin layer
chromatography, mass spectrometry, and/or initial TEM analysis.
3.1.1 Preparation
of Solubilized Lipid Stock
1. If working with lipids in powder form, add buffer-detergent
solution for a final lipid concentration of 10 mg/mL (see Note
1), which will result in the required lipid stock solution. If
working with lipids in chloroform (steps 2–4 below apply to
these lipids), pipette 1 mL into a small round-bottom flask.
2. Evaporate the chloroform with a stream of nitrogen gas (see
Note 2).
3. The buffer-detergent solution is pipetted into the roundbottom flask for a final lipid concentration of 10 mg/mL.
4. The round-bottom flask is placed into a sonicator bath and
sonicated for several minutes until the solution becomes clear.
5. Aliquots of 10 μL of the solubilized lipid are pipetted into
Eppendorf tubes for storage at either À20
C or À80
C (see
Note 3).
3.1.2 Setup of 2D
Crystallization Trials
1. Soak dialysis tubing in 500–1000 mL of ultrapure water (see
Note 4).
2. Thaw membrane protein, if previously frozen and not freshly
purified, and lipid aliquots on ice.
3. Prepare 1 L of dialysis buffer based on the purification buffer,
omitting the detergent. In later rounds of crystallization trials,
up to 20% glycerol may be added, and the salt may be increased,
decreased, or altered.
4. Mix 75–100 μL of the membrane protein with stock solution
of lipid at molar lipid-to-protein ratios (LPRs) of 0, 3, 15, and
30 in Eppendorf tubes on ice (this membrane proteindetergent-lipid mixture will be referred to as dialysate in later
steps) (see Note 5).
2D Electron Crystallography of Membrane Proteins
231