exchange the buffer to 20 mM KMOPS, pH 7.2, 100 mM NaCl, 0.5 mM EDTA, 0.1 g‧L
À1
DDM. Pool the peak fractions and concentrate to ~100 μM.
2. Titrate with the following precipitant buffer: 100 mM KMES, pH 6.4, 0.1 g‧L
À1 DDM, 10%
w/w PEG 4 k, 0.5 mM EDTA. Turbidity appears after 0.3–0.5 volumes have been added.
Spin down, transfer the supernatant to a clean tube, and discard the first small pellet. This step
eliminates aggregated complexes. Under stirring, add more precipitant (~0.2 original
volumes) and incubate several hours at 4
C to let precipitate. Microscopic examination
reveals that the turbidity results from the presence of minute hexagonal crystals. Centrifuge
again so as to obtain a large pellet, while leaving some color in the supernatant (if the
supernatant is very red, add more precipitant and spin down again). Decant the pellet and
discard the supernatant.
3. Weigh the pellet, and resuspend in ~10 pellet volumes of 10 mM KMES, pH 6.4, 1% w/w
PEG 4 k, 0.5 mM EDTA, 0.1 g‧L
À1 DDM. Centrifuge and resuspend the pellet in about ten
volumes of cold distilled water. This preparation is then divided into aliquots and can be
stored for several weeks at 4
C or for longer periods at or below À20
C.
We suppose that resuspending in water before freezing minimizes damage due to peroxide and/or
free radicals from PEG, but have not documented this point.
11.4.2 Crystallization of Ternary Protein/Amphipol/Detergent Complexes
Procedure
1. The pre-crystallized bc 1 is resuspended at 10 g‧L
À1 in a solution of 100 mM Tris/HCl, pH
7.5, containing increasing concentrations of A8-35 (the best crystals are obtained at an A8-35/
bc 1 ratio of 0.3 g per g), and incubated on ice overnight.
2. On the following day, the sample is ultracentrifuged at 72,000 rpm for 12 min in the TLA
100.2 rotor of a Beckman TL100 centrifuge so as to pellet the aggregated material. The
supernatant is used directly to set up crystallization drops. The drops are set at room
temperature by combining the bc 1 sample, at a 1:1 v/v ratio, with increasing concentrations
of PEG, at low ionic strength. As a first screen, the concentrations of NaCl and PEG 4 k were
varied in the absence of added detergent: sucrose, 30 mM; KMES, pH 6.4, 100 mM; NaCl,
60–120 mM; PEG 4 k, 4–12% w/w.
Although hits were readily obtained in the absence of added detergent (but presence of
residual DDM), the size of the crystals increased upon including 5–15 mM n-octyl-β-Dglucoside (OG) in the precipitant solution (which is under the CMC of ~25 mM). Under these
conditions, it has been estimated that the ratio of detergent (OG + residual DDM) to A8-35 in
the crystals most enriched in APol is ~1:1 w/w (Charvolin et al. 2014).
3. Crystals were obtained after 10 days. They had a wedge shape and often grew head-to-tail.
They measured about 120 μm in length.
Protocol prepared by Martin Picard based on Charvolin et al. (2014), unpublished data, and
laboratory notes, with comments added.
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11 Amphipols and Membrane Protein Crystallization
À1
DDM. Pool the peak fractions and concentrate to ~100 μM.
2. Titrate with the following precipitant buffer: 100 mM KMES, pH 6.4, 0.1 g‧L
À1 DDM, 10%
w/w PEG 4 k, 0.5 mM EDTA. Turbidity appears after 0.3–0.5 volumes have been added.
Spin down, transfer the supernatant to a clean tube, and discard the first small pellet. This step
eliminates aggregated complexes. Under stirring, add more precipitant (~0.2 original
volumes) and incubate several hours at 4
C to let precipitate. Microscopic examination
reveals that the turbidity results from the presence of minute hexagonal crystals. Centrifuge
again so as to obtain a large pellet, while leaving some color in the supernatant (if the
supernatant is very red, add more precipitant and spin down again). Decant the pellet and
discard the supernatant.
3. Weigh the pellet, and resuspend in ~10 pellet volumes of 10 mM KMES, pH 6.4, 1% w/w
PEG 4 k, 0.5 mM EDTA, 0.1 g‧L
À1 DDM. Centrifuge and resuspend the pellet in about ten
volumes of cold distilled water. This preparation is then divided into aliquots and can be
stored for several weeks at 4
C or for longer periods at or below À20
C.
We suppose that resuspending in water before freezing minimizes damage due to peroxide and/or
free radicals from PEG, but have not documented this point.
11.4.2 Crystallization of Ternary Protein/Amphipol/Detergent Complexes
Procedure
1. The pre-crystallized bc 1 is resuspended at 10 g‧L
À1 in a solution of 100 mM Tris/HCl, pH
7.5, containing increasing concentrations of A8-35 (the best crystals are obtained at an A8-35/
bc 1 ratio of 0.3 g per g), and incubated on ice overnight.
2. On the following day, the sample is ultracentrifuged at 72,000 rpm for 12 min in the TLA
100.2 rotor of a Beckman TL100 centrifuge so as to pellet the aggregated material. The
supernatant is used directly to set up crystallization drops. The drops are set at room
temperature by combining the bc 1 sample, at a 1:1 v/v ratio, with increasing concentrations
of PEG, at low ionic strength. As a first screen, the concentrations of NaCl and PEG 4 k were
varied in the absence of added detergent: sucrose, 30 mM; KMES, pH 6.4, 100 mM; NaCl,
60–120 mM; PEG 4 k, 4–12% w/w.
Although hits were readily obtained in the absence of added detergent (but presence of
residual DDM), the size of the crystals increased upon including 5–15 mM n-octyl-β-Dglucoside (OG) in the precipitant solution (which is under the CMC of ~25 mM). Under these
conditions, it has been estimated that the ratio of detergent (OG + residual DDM) to A8-35 in
the crystals most enriched in APol is ~1:1 w/w (Charvolin et al. 2014).
3. Crystals were obtained after 10 days. They had a wedge shape and often grew head-to-tail.
They measured about 120 μm in length.
Protocol prepared by Martin Picard based on Charvolin et al. (2014), unpublished data, and
laboratory notes, with comments added.
524
11 Amphipols and Membrane Protein Crystallization