2dx - EmrE (Shewanella Oneidensis) 2D-crystallization trials

Properties

• Amino acid sequence:

M S W L L L I L A G L F E I G W A I G L K Y T D G F T R L W P S V F T V L S M A L S V L L L G L A V K Q L P I G T A Y G V W V G I G A M G T A I A G I I L L G E G V S L L K I A S L I L I L L G V L G L K L A H A E N L Y F Q G

• 112 amino acids
• 11.8 kDa, Expasy (10.9 kDa, Nanda)
• pI ~ 8.09
• 4 negatively charged residues
• 5 positively charged residues
• extinction coefficient: 26470
• instability index: 19.76
• alipathic index: 159.38
• GRAVY: 1.298
• TM helices: 4
• amino acids in the TM domain: 82 (LPR(m/m) expected: 1-40 ~ 0.03 to 1.4 in w/w; with LPR(m/m)=24>>LPR(w/w)=0.68; LPR(m/m)=5>>LPR(w/w)=0.14; LPR(m/m)=40>>LPR(w/w)=1.12;)

Experiment of December 2011 (X94)

• 5 Lipids in 5 detergent screening
• Buffer: pH8, 20mM sodium phosphate, 100mM NaCl^?. 2mM MgCl^?, 1mM EDTA, 4mM DTT, 4mM mercapto-ethanol, 5mM sodium azide.
• Temperature: 25 degrees
• microdialysis plate

Set Dec 15th.

Comments (Jan 2012)

Controls are ok, but no good results (aggregates, tiny vesicles, or grids mostly empty).

CD will be tried next time. If results are still bad, then the protein will be dropped.

Experiment of June 2011 (X72-EM.194)

Protocol

conditions to screen:

• Dialysis buffer: 20mM sodium phosphate, 100mM NaCl. 2mM MgCl, 1mM EDTA, 4mM DTT, 4mM mercapto-ethanol, 5mM sodium azide.
• pH: 7 and 8
• Lipids prepared in DDM and C12E8:
  • DMPC
  • E.Coli
  • POPC (C12E8 only)
• Temperature: 25 degrees
• LPR: 0.5 to 1
• dialysis block

See Protocol_EmrE_oneidensis_2011_June.xls for details.

2 weeks (Set 24th June; collected July 8th)

July 21th Meeting - comments

• All is aggregated.
• Controls of protein only and lipids only are OK
• Yiip control are bad -> do experiments to find out why...

• Set ALLOWTOPICVIEW =

-- NicolasCoudray - 23 Jun 2011