2dx - 2D-crystallization trials of GtrB

Properties

• Amino acid sequence:

• 340 amino acids
• 39.1 kDa
• pI ~ 7.24
• 37 negatively charged residues
• 37 positively charged residues
• extinction coefficient: 69330
• instability index: 29.32
• alipathic index: 107.21
• GRAVY: -0.024
• TM helices: 2 (using TMHMM)

X305 (June 6, 2014) - "star-like" screening

• Lipids (Yeast, DMPC:DOPS 8.5:1.5 or 1:1, Cardiolipin:DOPC 8:2-C12E7, DMPC:DOPG 8.5:1.5, DOPC:DOPE:brain 8:1:1-DMNG
• salt: 300 mM NaCl^? + 2mM MgCl2, or 50 mM KCl + 5 mM CaCl2 or without 5mM EDTA
• pH: 5, 7 or 9
• 4 or 37C

Details: X305-GtrB.xlsx.

X305, results

Last-time, we had mix of vesicles, aggregates, and non-diffracting tubes at LPR 0.7-1.0 (in DMPC:DOPS 85:15, pH8). In the exact same condition this time, we still have aggregates and vesicles but also some helical tubes up to LPR 0.7. These tubes are also present in the protein only control (last time, protein control showed vesicles, so lipid content is probably different this time. Differences in prep underlined by John: a different machine used to lyse the cells [newer one this time], but this prep looked more contaminated by protein after washing). Those helical tubes are rare, though.

Filament do not show any pattern anymore when DOPS is replaced by DOPG, or when the proportion of DOPS is increased from 15 to 50% in the mixture.
Other lipids mixtures tried shows a few wider non-helical non-diffracting tubes.
When NaCl^? is increased from 50 to 300 mM or when MgCl2 is replaced by EDTA, there are more veiscles and rare non-helical tubes. When salts are replaced by KCl and CaCl2^?, there's mainly aggregates.
Vesicles, aggregates and non-diffracting tubes are seen at lower pH (5-7), but only vesicles at higher pH (9).
Vesicles, aggregates and non-diffracting non-helical tubes seen at 4C or 37C.

Next time, we should try to reproduce it by covering lower LPRs as well. We can try new lipid mixtures, a lower step-size of pHs.

Image selection: X305-GtrB-Summary.pptx.

X280 (March 11, 2014) - Sparse Matrix + 3dx buffers

Acquisition form: Protein_acquisition_form-Mar11-1_GtrB.doc.

Matrix:

• Sparse
• 3dx buffers @4C + lipids in DM (OG or Triton X100)

X280, results

• Some tubes @ pH8, 50 NaCl, 2 MgCl2 (DMPC:DOPS)
• Very rare tubes @ pH7, 150 NaCl, 40 MgCl2 (DMPC:DOPC)
• Few large vesicles (>1um) @pH9.5, 100 NaCl (DMPC:cholesterol)
• Striation (from lipids?) @ pH7, 300 NaCl (DMPC alone)

Protein control shows some vesicles as well.

Image selection: X280-GtrB-ImageSelection.pptx.

• Set ALLOWTOPICVIEW =

-- NicolasCoudray - 11 Mar 2014

2dx - 2D-crystallization trials of GtrB

Properties

• Amino acid sequence:

• 340 amino acids
• 39.1 kDa
• pI ~ 7.24
• 37 negatively charged residues
• 37 positively charged residues
• extinction coefficient: 69330
• instability index: 29.32
• alipathic index: 107.21
• GRAVY: -0.024
• TM helices: 2 (using TMHMM)

X305 (June 6, 2014) - "star-like" screening

• Lipids (Yeast, DMPC:DOPS 8.5:1.5 or 1:1, Cardiolipin:DOPC 8:2-C12E7, DMPC:DOPG 8.5:1.5, DOPC:DOPE:brain 8:1:1-DMNG
• salt: 300 mM NaCl^? + 2mM MgCl2, or 50 mM KCl + 5 mM CaCl2 or without 5mM EDTA
• pH: 5, 7 or 9
• 4 or 37C

Details: X305-GtrB.xlsx.

X280 (March 11, 2014) - Sparse Matrix + 3dx buffers

Acquisition form: Protein_acquisition_form-Mar11-1_GtrB.doc.

Matrix:

• Sparse
• 3dx buffers @4C + lipids in DM (OG or Triton X100)

X280, results

• Some tubes @ pH8, 50 NaCl, 2 MgCl2 (DMPC:DOPS)
• Very rare tubes @ pH7, 150 NaCl, 40 MgCl2 (DMPC:DOPC)
• Few large vesicles (>1um) @pH9.5, 100 NaCl (DMPC:cholesterol)
• Striation (from lipids?) @ pH7, 300 NaCl (DMPC alone)

Protein control shows some vesicles as well.

Image selection: X280-GtrB-ImageSelection.pptx.

• Set ALLOWTOPICVIEW =

-- NicolasCoudray - 11 Mar 2014

d97 2

2dx - 2D-crystallization trials of GtrB

Properties

• Amino acid sequence:

• 340 amino acids
• 39.1 kDa
• pI ~ 7.24
• 37 negatively charged residues
• 37 positively charged residues
• extinction coefficient: 69330
• instability index: 29.32
• alipathic index: 107.21
• GRAVY: -0.024
• TM helices: 2 (using TMHMM)

• Lipids (Yeast, DMPC:DOPS 8.5:1.5 or 1:1, Cardiolipin:DOPC 8:2-C12E7, DMPC:DOPG 8.5:1.5, DOPC:DOPE:brain 8:1:1-DMNG
• salt: 300 mM NaCl^? + 2mM MgCl2, or 50 mM KCl + 5 mM CaCl2 or without 5mM EDTA
• pH: 5, 7 or 9
• 4 or 37C

Details: X305-GtrB.xlsx.

X280 (March 11, 2014) - Sparse Matrix + 3dx buffers

Acquisition form: Protein_acquisition_form-Mar11-1_GtrB.doc.

Matrix:

• Sparse
• 3dx buffers @4C + lipids in DM (OG or Triton X100)

X280, results

• Some tubes @ pH8, 50 NaCl, 2 MgCl2 (DMPC:DOPS)
• Very rare tubes @ pH7, 150 NaCl, 40 MgCl2 (DMPC:DOPC)
• Few large vesicles (>1um) @pH9.5, 100 NaCl (DMPC:cholesterol)
• Striation (from lipids?) @ pH7, 300 NaCl (DMPC alone)

Protein control shows some vesicles as well.

Image selection: X280-GtrB-ImageSelection.pptx.

• Set ALLOWTOPICVIEW =

-- NicolasCoudray - 11 Mar 2014

d97 2

2dx - 2D-crystallization trials of GtrB

Properties

• Amino acid sequence:

• 340 amino acids
• 39.1 kDa
• pI ~ 7.24
• 37 negatively charged residues
• 37 positively charged residues
• extinction coefficient: 69330
• instability index: 29.32
• alipathic index: 107.21
• GRAVY: -0.024
• TM helices: 2 (using TMHMM)

X280 (March 11, 2014) - Sparse Matrix + 3dx buffers

Acquisition form: Protein_acquisition_form-Mar11-1_GtrB.doc.

Matrix:

• Sparse
• 3dx buffers @4C + lipids in DM (OG or Triton X100)

X280, results

• Some tubes @ pH8, 50 NaCl, 2 MgCl2 (DMPC:DOPS)
• Very rare tubes @ pH7, 150 NaCl, 40 MgCl2 (DMPC:DOPC)
• Few large vesicles (>1um) @pH9.5, 100 NaCl (DMPC:cholesterol)
• Striation (from lipids?) @ pH7, 300 NaCl (DMPC alone)

Protein control shows some vesicles as well.

Image selection: X280-GtrB-ImageSelection.pptx.

• Set ALLOWTOPICVIEW =

-- NicolasCoudray - 11 Mar 2014

d97 2

2dx - 2D-crystallization trials of GtrB

Properties

• Amino acid sequence:

• 340 amino acids
• 39.1 kDa
• pI ~ 7.24
• 37 negatively charged residues
• 37 positively charged residues
• extinction coefficient: 69330
• instability index: 29.32
• alipathic index: 107.21
• GRAVY: -0.024
• TM helices: 2 (using TMHMM)

X280 (March 11, 2014) - Sparse Matrix + 3dx buffers

Acquisition form: Protein_acquisition_form-Mar11-1_GtrB.doc.

Matrix:

• Sparse
• 3dx buffers @4C + lipids in DM (OG or Triton X100)

X280, results

• Some tubes @ pH8, 50 NaCl, 2 MgCl2 (DMPC:DOPS)
• Very rare tubes @ pH7, 150 NaCl, 40 MgCl2 (DMPC:DOPC)
• Few large vesicles (>1um) @pH9.5, 100 NaCl (DMPC:cholesterol)
• Striation (from lipids?) @ pH7, 300 NaCl (DMPC alone)

Protein control shows some vesicles as well.

Image selection: X280-GtrB-ImageSelection.pptx.

• Set ALLOWTOPICVIEW =

-- NicolasCoudray - 11 Mar 2014

d97 2

2dx - 2D-crystallization trials of GtrB

Properties

• Amino acid sequence:

• 340 amino acids
• 39.1 kDa
• pI ~ 7.24
• 37 negatively charged residues
• 37 positively charged residues
• extinction coefficient: 69330
• instability index: 29.32
• alipathic index: 107.21
• GRAVY: -0.024
• TM helices: 2 (using TMHMM)

X280 (March 11, 2014) - Sparse Matrix + 3dx buffers

Acquisition form: Protein_acquisition_form-Mar11-1_GtrB.doc.

Matrix:

• Sparse
• 3dx buffers @4C + lipids in DM (OG or Triton X100)

• Set ALLOWTOPICVIEW =

-- NicolasCoudray - 11 Mar 2014

2dx - 2D-crystallization trials of GtrB

Properties

• Amino acid sequence:

X280 (March 11, 2014) - Sparse Matrix + 3dx buffers

• Sparse

• Set ALLOWTOPICVIEW =

-- NicolasCoudray - 11 Mar 2014

2dx - 2D-crystallization trials of GtrB

Properties

• Amino acid sequence:

• amino acids
• kDa
• pI ~
• negatively charged residues
• positively charged residues
• extinction coefficient:
• instability index:
• alipathic index:
• GRAVY:
• TM helices: (using TMHMM)

X280 (March 11, 2014) - Sparse Matrix + 3dx buffers

Matrix:

• Sparse
• 3dx buffers @4C + lipids in DM (OG or TritonX^?)

• Set ALLOWTOPICVIEW =

-- NicolasCoudray - 11 Mar 2014

* Protein_acquisition_form-Mar11-1_GtrB.doc: Protein_acquisition_form-Mar11-1_GtrB.doc