Difference: TwodxSoPIP (1 vs. 8)

Revision 803 Apr 2014 - Main.NicolasCoudray

Changed:
<
<
META TOPICPARENT name="2DCrystallizationExp"
>
>
META TOPICPARENT name="TwoDCrystallizationGroup"
 

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.

  • Set ALLOWTOPICVIEW =

-- NicolasCoudray - 23 May 2012

Deleted:
<
<
META FILEATTACHMENT attr="" autoattached="1" comment="images" date="1338313606" name="ralph_may28_X122_sopip.ppt" path="ralph_may28_X122_sopip.ppt" size="57863168" user="Main.RalphLasala" version="1"
 
META FILEATTACHMENT attr="" autoattached="1" comment="" date="1337781143" name="SOPIP-2012-05-18-Protein_acquisition_form.doc" path="SOPIP-2012-05-18-Protein_acquisition_form.doc" size="58880" user="Main.NicolasCoudray" version="1"
Added:
>
>
META FILEATTACHMENT attr="" autoattached="1" comment="images" date="1338313606" name="ralph_may28_X122_sopip.ppt" path="ralph_may28_X122_sopip.ppt" size="57863168" user="Main.RalphLasala" version="1"
 
META FILEATTACHMENT attr="" autoattached="1" comment="" date="1337781152" name="X122-SoPIP.xlsx" path="X122-SoPIP.xlsx" size="645102" user="Main.NicolasCoudray" version="1"

Revision 717 Aug 2012 - Main.NicolasCoudray

 
META TOPICPARENT name="2DCrystallizationExp"

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.

Changed:
<
<		Diffraction in cryo at LPR 0.3, DMPC at 28 deg in dialysis.
>
>		Diffraction in cryo at LPR 0.3, E.coli at 28 deg in dialysis.
 

  • Set ALLOWTOPICVIEW =

-- NicolasCoudray - 23 May 2012

META FILEATTACHMENT attr="" autoattached="1" comment="images" date="1338313606" name="ralph_may28_X122_sopip.ppt" path="ralph_may28_X122_sopip.ppt" size="57863168" user="Main.RalphLasala" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="" date="1337781143" name="SOPIP-2012-05-18-Protein_acquisition_form.doc" path="SOPIP-2012-05-18-Protein_acquisition_form.doc" size="58880" user="Main.NicolasCoudray" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="" date="1337781152" name="X122-SoPIP.xlsx" path="X122-SoPIP.xlsx" size="645102" user="Main.NicolasCoudray" version="1"

Revision 605 Jun 2012 - Main.NicolasCoudray

 
META TOPICPARENT name="2DCrystallizationExp"

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).
Changed:
<
<		Mainly small vesicles can be seen. Very few aggregates at lower LPR. LPR~0.4 seem better and Cryo-images have been acquired, but show no diffraction peaks.
>
>		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, DMPC at 28 deg in dialysis.

  • Set ALLOWTOPICVIEW =

-- NicolasCoudray - 23 May 2012

META FILEATTACHMENT attr="" autoattached="1" comment="images" date="1338313606" name="ralph_may28_X122_sopip.ppt" path="ralph_may28_X122_sopip.ppt" size="57863168" user="Main.RalphLasala" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="" date="1337781143" name="SOPIP-2012-05-18-Protein_acquisition_form.doc" path="SOPIP-2012-05-18-Protein_acquisition_form.doc" size="58880" user="Main.NicolasCoudray" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="" date="1337781152" name="X122-SoPIP.xlsx" path="X122-SoPIP.xlsx" size="645102" user="Main.NicolasCoudray" version="1"

Revision 501 Jun 2012 - Main.NicolasCoudray

 
META TOPICPARENT name="2DCrystallizationExp"

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, but show no diffraction peaks.

See image selection: ralph_may28_X122_sopip.ppt.

Added:
>
>		Diffraction in cryo at LPR 0.3, DMPC at 28 deg in dialysis.
 
  • Set ALLOWTOPICVIEW =

-- NicolasCoudray - 23 May 2012

META FILEATTACHMENT attr="" autoattached="1" comment="images" date="1338313606" name="ralph_may28_X122_sopip.ppt" path="ralph_may28_X122_sopip.ppt" size="57863168" user="Main.RalphLasala" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="" date="1337781143" name="SOPIP-2012-05-18-Protein_acquisition_form.doc" path="SOPIP-2012-05-18-Protein_acquisition_form.doc" size="58880" user="Main.NicolasCoudray" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="" date="1337781152" name="X122-SoPIP.xlsx" path="X122-SoPIP.xlsx" size="645102" user="Main.NicolasCoudray" version="1"

Revision 430 May 2012 - Main.NicolasCoudray

 
META TOPICPARENT name="2DCrystallizationExp"

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, but show no diffraction peaks.

Added:
>
>		See image selection: ralph_may28_X122_sopip.ppt.
 
Deleted:
<
<		a59 1
 
  • Set ALLOWTOPICVIEW =

-- NicolasCoudray - 23 May 2012

META FILEATTACHMENT attr="" autoattached="1" comment="images" date="1338313606" name="ralph_may28_X122_sopip.ppt" path="ralph_may28_X122_sopip.ppt" size="57863168" user="Main.RalphLasala" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="" date="1337781143" name="SOPIP-2012-05-18-Protein_acquisition_form.doc" path="SOPIP-2012-05-18-Protein_acquisition_form.doc" size="58880" user="Main.NicolasCoudray" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="" date="1337781152" name="X122-SoPIP.xlsx" path="X122-SoPIP.xlsx" size="645102" user="Main.NicolasCoudray" version="1"

Revision 329 May 2012 - Main.RalphLasala

 
META TOPICPARENT name="2DCrystallizationExp"

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, but show no diffraction peaks.

a59 1

  • Set ALLOWTOPICVIEW =

-- NicolasCoudray - 23 May 2012

Added:
>
>
 
META FILEATTACHMENT attr="" autoattached="1" comment="images" date="1338313606" name="ralph_may28_X122_sopip.ppt" path="ralph_may28_X122_sopip.ppt" size="57863168" user="Main.RalphLasala" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="" date="1337781143" name="SOPIP-2012-05-18-Protein_acquisition_form.doc" path="SOPIP-2012-05-18-Protein_acquisition_form.doc" size="58880" user="Main.NicolasCoudray" version="1"
Added:
>
>
META FILEATTACHMENT attr="" autoattached="1" comment="" date="1337781152" name="X122-SoPIP.xlsx" path="X122-SoPIP.xlsx" size="645102" user="Main.NicolasCoudray" version="1"
 

Revision 229 May 2012 - Main.NicolasCoudray

 
META TOPICPARENT name="2DCrystallizationExp"

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).
Changed:
<
<		LPR 0.4 on MBCD robot seems to show slight diffraction.
>
>		Mainly small vesicles can be seen. Very few aggregates at lower LPR. LPR~0.4 seem better and Cryo-images have been acquired, but show no diffraction peaks.
Added:
>
>
 a59 1
  • Set ALLOWTOPICVIEW =

-- NicolasCoudray - 23 May 2012

META FILEATTACHMENT attr="" autoattached="1" comment="images" date="1338313606" name="ralph_may28_X122_sopip.ppt" path="ralph_may28_X122_sopip.ppt" size="57863168" user="Main.RalphLasala" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="" date="1337781143" name="SOPIP-2012-05-18-Protein_acquisition_form.doc" path="SOPIP-2012-05-18-Protein_acquisition_form.doc" size="58880" user="Main.NicolasCoudray" version="1"

Revision 123 May 2012 - Main.NicolasCoudray

 
META TOPICPARENT name="2DCrystallizationExp"

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).

LPR 0.4 on MBCD robot seems to show slight diffraction.

a59 1

  • Set ALLOWTOPICVIEW =

-- NicolasCoudray - 23 May 2012

META FILEATTACHMENT attr="" autoattached="1" comment="images" date="1338313606" name="ralph_may28_X122_sopip.ppt" path="ralph_may28_X122_sopip.ppt" size="57863168" user="Main.RalphLasala" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="" date="1337781143" name="SOPIP-2012-05-18-Protein_acquisition_form.doc" path="SOPIP-2012-05-18-Protein_acquisition_form.doc" size="58880" user="Main.NicolasCoudray" version="1"
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