grid, and blotted with the torn side of the filter paper (see
Note 13).
4. The grid is dried and then either screened by TEM immediately
or kept in a grid box in a desiccator cabinet until the scheduled
TEM session (see Note 14).
3.2.2 Screening by TEM
1. Grids are screened with a TEM at 120 kV (see Note 15).
2. The grid is viewed at a low magnification of approximately
2000–10,000Â for a first assessment of sample concentration.
For efficient and thorough screening, most grid squares should
contain a minimum of 5–10 membranes/proteoliposomes.
3. For samples with low concentrations on the grid, steps 1–4 in
Subheading 3.2.1 are repeated after the sample has been concentrated (see Note 16).
4. Sample membrane morphology (vesicle, planar-tubular, tubular/helical, membrane sheet, stacked sheets/vesicles), average
membrane size and range of sizes, as well as aggregation of
either membranes and/or protein are noted.
5. Suitable membranes of a diameter of at least 50–100 nm are
selected for identification of ordered arrays at magnifications of
~30,000–50,000Â (see Note 17).
6. Images of membranes are collected for immediate evaluation
by Fast Fourier Transform (FFT) (see Note 18).
7. Each image is assessed by FFT for identification of ordered
arrays, size of ordered arrays, initial evaluation of symmetry,
and potential crystal mosaicity (see Note 19).
3.2.3 Refinement
of Crystallization
Conditions
1. Crystallization conditions are considered in terms of crystal
order, size, and morphology.
2. Order is of highest priority and will require consideration of
changes in LPR.
3. Once 2D crystals are obtained and the entire membrane is
ordered and does not contain significant mosaicity, the 2D
crystal size is considered. Dialysis buffer salt concentration,
salt mixtures, glycerol, and/or temperature are modified,
while carefully considering the activity of the protein.
4. The steps in Subheadings 3.1.2–3.2.2 are repeated with modified conditions until the largest possible 2D crystals are
obtained. The 2D crystals will be at least 200 nm and ideally
1 μm or larger in size.
5. Crystal morphology is considered for the cryo-EM approach in
terms of methods selection below.
2D Electron Crystallography of Membrane Proteins
233
Note 13).
4. The grid is dried and then either screened by TEM immediately
or kept in a grid box in a desiccator cabinet until the scheduled
TEM session (see Note 14).
3.2.2 Screening by TEM
1. Grids are screened with a TEM at 120 kV (see Note 15).
2. The grid is viewed at a low magnification of approximately
2000–10,000Â for a first assessment of sample concentration.
For efficient and thorough screening, most grid squares should
contain a minimum of 5–10 membranes/proteoliposomes.
3. For samples with low concentrations on the grid, steps 1–4 in
Subheading 3.2.1 are repeated after the sample has been concentrated (see Note 16).
4. Sample membrane morphology (vesicle, planar-tubular, tubular/helical, membrane sheet, stacked sheets/vesicles), average
membrane size and range of sizes, as well as aggregation of
either membranes and/or protein are noted.
5. Suitable membranes of a diameter of at least 50–100 nm are
selected for identification of ordered arrays at magnifications of
~30,000–50,000Â (see Note 17).
6. Images of membranes are collected for immediate evaluation
by Fast Fourier Transform (FFT) (see Note 18).
7. Each image is assessed by FFT for identification of ordered
arrays, size of ordered arrays, initial evaluation of symmetry,
and potential crystal mosaicity (see Note 19).
3.2.3 Refinement
of Crystallization
Conditions
1. Crystallization conditions are considered in terms of crystal
order, size, and morphology.
2. Order is of highest priority and will require consideration of
changes in LPR.
3. Once 2D crystals are obtained and the entire membrane is
ordered and does not contain significant mosaicity, the 2D
crystal size is considered. Dialysis buffer salt concentration,
salt mixtures, glycerol, and/or temperature are modified,
while carefully considering the activity of the protein.
4. The steps in Subheadings 3.1.2–3.2.2 are repeated with modified conditions until the largest possible 2D crystals are
obtained. The 2D crystals will be at least 200 nm and ideally
1 μm or larger in size.
5. Crystal morphology is considered for the cryo-EM approach in
terms of methods selection below.
2D Electron Crystallography of Membrane Proteins
233