2dx - MDR 2D crystallization

General 2dx information

• Amino acid sequence:

MQRIIQFFSQRATTLFFPMALILYDFAAYLTTDLIQPGIINVVRDFNADVSLAPASVSLYLAGGMALQWLLGPLSDRIGRRPVLIAGALIFTLACAATLLTTSMTQFLVARFVQGTSICFIATVGYVTVQEAFGQTKAIKLMAIITSIVLVAPVIGPLSGAALMHFVHWKVLFGIIAVMGLLALCGLLLAMPETVQRGAVPFSAVSVLRDFRNVFRNPIFLTGAATLSLSYIPMMSWVAVSPVILIDAGGMSTSQFAWAQVPVFGAVIVANMIVVRLVKDPTRPRFIWRAVPIQLSGLATLLLGNLLLPHVWLWSVLGTSLYAFGIGMIFPTLFRFTLFSNNLPKGTVSASLNMVILTVMAVSVEVGRWLWFHGGRLPFHLLAAVAGVIVVFTLATLLQRVRQHEAAELAAEK

• 413 amino acids
• 44.8 kDa
• pI ~ 10.27
• 14 negatively charged residues
• 26 positively charged residues
• extinction coefficient: 58565
• instability index: 30.98
• alipathic index: 127.26
• GRAVY: 0.934
• TM segments: 12

Thermostability assay (Marisa)

Thermostability assay with different detergents:

• DDM the TM=77
• n-dodecyl-n,n-dimethylamine-n-oxide: TM=79
• n-decyl-b-d-maltopyranoside: TM= 80

Note: Andreas got crystals when solubilizing the lipid in OG (at LPR 0.2 and later at LPR 0.5)

X238-239, MDR - Before-After SEC

• Protein before SEC: 0.52 mg/ml
• Protein before SEC with Tag: 0.65 mg/ml
• Protein after SEC: Fraction 15: 1.04mg/ml; Fraction 16: 1.25mg/ml
• From X225 (frozen, to try to repeat the tubes with the "almost-same" prep): Fraction 14: 0.28mg/ml; Fraction 17: 0.76mg/ml.

We tried different lipids (mostly solubilized in Triton X-100) with the last 3 successful buffers (the one where we got diffraction, and the last 2 of the incomplete factorial where we got tubes). Details:

• X238-MDR_BeforeSEC.xlsx: X238-MDR_BeforeSEC.xlsx
• X239-MDR_AfterSEC.xlsx: X239-MDR_AfterSEC.xlsx

X225, MDR (Aug 15) - Incomplete Factorial Matrix

Matrix details: X225-MDR-IF.xlsx.

X225, Results:

• H12: HEPES pH 7.5 DMPC+10% brain extract in TritonX100 LPR 0.7 50 KCl + 5 MgCl2 Button @37C
• F5: HEPES pH7.5 SoyPC^? +10% brain extract in TritonX100 2% glycerol LPR 0.3 50 Na2SO4+5 CaCl2 Plate + 1 mM CD @temp.cycling

Details: X225_mdr-if.ppt.

X222, MDR - Lipids, LPR, time-course, rate

Conditions:

• Buffer: pH 6 100 mM NaCl, 50 mM MgCl2, 2 mM MBCD
• LPR 0.25 to 0.9 for E.coli-DDM
• E.coli, DOPG, DOPE, Cardiolipin (LPR 0.25-0.65)
• Time-course (LPR 0.35-0.55)
• Rate: udilaysis plate buffer changed 1x/day or 2x/day, buttons (E.coli OG or DDM)
• 27C

X222, results

Mostly aggregates/clusters of sheets and vesicles (or aggregates of vesicles only when buttons are used). Same results at higher LPRs or with other lipid, or solubilizing E.coli in OG instead of DDM. 1 tube with slight diffraction found.

X218, MDR - Repeat with 3 frozen MDRs (July 18 2013)

X218, results

It seems that there is much more 'material' on the grid than what we had when they were fresh (is it because most of the protein died and we have more lipid per protein alive, or is it because the protein like it...?). We have several non-diffracting sheets/vesicles, and for 2 conditions with very rare diffracting tubes (the best diffraction I've seen for tubes in neg stain so far). Note these 2 conditions (LPR 0.45 and 0.55) come from the same batch which gave MDR crystals the first time (but they are rare, so it may be a coincidence).

So either: ** this particular batch was somehow better ** the crystallization is sensitive to LPR, or to rate (buffer changed 1x/day instead of 2), or to when the conditions are harvested ** MDR likes only 1 component of the E.coli lipid (PG, PE or Ca) - so crystals are rare

X214, MDR - Detergent, Temperature, pH

• E.coli in DM, OG or DDM. All freshly prepared + 1 old frozen lipid/det prep
• 4C, 20C or Temp cycling
• pH 5, 6 (mostly), or 7 + 100 mM >nop>NaCl
• 50 mM MgCl2 or 5 mM EDTA
• microdialysis plate

X214, Results:

Image selection: X214-MDR-ImageSelection.pptx.

x205 - pH, MgCl2, lipids

• repeat pH6, 100 NaCl and 50 mM MgCl2, E.coli
• Try with EDTA instead of MgCl2
• Includes best and worst 2 lipids of thermostability assay (DMPC, DOPE, POPC, DOPG)
• E.coli:brain
• pH 5 and 7 for E.coli

See detailed matrix: X205-MDR.xlsx.

X205, Results

x202 - MDR - Sparse Matrix + other lipids

Note: 2 fractions from the peak (15: 0.8 mg/ml; 16: 0.75); Fraction 15 for rows A to D; and fraction 16 for rows E to G.

X202, Results

X191 - MDR - lipids

Protein: Protein_Acquisition_MDR.doc.

Objective: reeproduce Andreas' results. Since the robot does not work, cyclodextrin was added over 3 days at 27C manually (3 times to reach a 1:3 ratio), then placed at 37C. As a lot of protein has been received, so device and other lipids have also been tested:

• buffer: same as purification buffer (20 mM Tris-Cl pH 7.5, 100mM NaCl^?, EDTA)
• LPR 0.1-1.0
• E.coli in OG with MBCD, buttons, and plate
• E.coli-DDM, E.coli:brain extract-DDM, E.coli:cholesterol-DDM, DOPG-DDM, DOPC:cholesterol-DDM

Results, X191

Experiments from Andreas Engel

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-- NicolasCoudray - 22 Apr 2013