| | 2dx - SoPIP 2D-crystallization
General Information
- Amino acid-chain (A-mutant)
MGHHHHHHSSGVDLGTENLYFQSSMWELRSIAFSRAVFAEFLATLLFVFFGLGSALNWP
QALPSVLQIAMAFGLGIGTLVQALGHISGAHINPAVTVACLVGCHVSVLRAAFYVAAQL
LGAVAGAALLHEITPADIRGDLAVNALSNSTTAGQAVTVELFLTLQLVLCIFASTDERR
GENPGTPALSIGFSVALGHLLGIHYTGCSMNPARSLAPAVVTGKFDDHWVFWIGPLV
GAILGSLLYNYVLFPPAKSLSERLAVLKGLEPDTDWEEREVRRRQAVELHSPQSLPRG
TKA
- 295 amino acids
- 31518 Da
- pI 6.38
- 21 negatively charged ans 17 positively charges residues
- 35200 ext coefficient
- instability index: 42.65
- aliphatic index: 108.17
- GRAVY: 0.403
From Kukulski et al., 2005:
- Protein in OG, E.coli polar lipids at LPR 0.3, pH8 buffer (20mM Tris-HCl,100mM NaCl, 50mM MgCl2, 2mM DTT, 0.03% NaN3?), 3 days dialysis
From Signorell et al., 2007:
- crystallization:
- dialyze the protein only, overnight at 4 degrees against 20mM Hepes, pH 7.5, 100mM NaCl, 50mM MgCl2, 2mM dithiothreitol, 3mM NaN3 and 1% OG.
- MBCD added over 2 hours leads to vesicles and small sheets with no significant diffraction. MBCD added over 72-144 hours leads to larger sheets as the Kukulski's ones.
From Iacovache et al., 2010:
- Protein S115A mutant in DDM, E.coli polar extracts, LPR 0.5, pH8 buffer (20mM Tris-HCl,2mM DTT, 0.03% NaN3?)
- crystallization:
- dialyze 2 hours the protein only to adjust the pH and salt concentration: no regular array at 100mM NaCl? but at 500 mM.
- MBCD added to neutralize detergent in 40hours (7.5-15 nl/hour), room temperature for 15h, increase to 37 degrees then.
Experiment of March 2012 - X122; CD robot and dialysis at different temperatures
Protein acquisition form: SOPIP-2012-05-18-Protein_acquisition_form.doc.
Conditions:
- E.coli in DDM
- LPR 0.1 to 0.8 for MBCD, 0.3 to 0.6 for dialysis
- 28 degrees (buttons), 25 (~20h)-ramp (12h)-37(24h)-ramp down (12h)-20 degrees (12h) (dialysis plate), 28 degrees (MBCD robot)
- MBCD: protein + lipids + water up to 20 ul
- 1mM MBCD in the buffer of the dialysis experiments (pH8, 20mM Tris-HCl, 2mM DTT, 500 mM NaCl?, 50 mM MgCl?, 5mM NaN3?)
See details of matrix: X122-SoPIP.xlsx: X122-SoPIP.xlsx
Comments (May 2012)
The MBCD robot did not behave as expected: amount and rate of MBCD was not as expected (Ok for the first few hours, then, no MBCD for a while, then default amount for the rest of the experiment), which make difficult to estimate the exact amount of MBCD added, though, it should be at least 1:2 (Detergent:MBCD).
Mainly small vesicles can be seen. Very few aggregates at lower LPR. LPR~0.4 seem better and Cryo-images have been acquired. No diffraction peaks for the samples from the MBCD robot, but diffraction peaks for LPR 0.4 from the dialysis at 28 degrees.
See image selection: ralph_may28_X122_sopip.ppt.
Diffraction in cryo at LPR 0.3, E.coli at 28 deg in dialysis.
-- NicolasCoudray - 23 May 2012
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