Hi Yibin,
I cannot tell the actual scale of your crystals but judging by the curve of the drop outline, these crystals look to be plenty big enough to mount in a loop to stick in the beam and test diffraction. If indeed they are too small to mount a single crystal, and you are mainly interested in testing to see if they are salt, you can gather up several of them in your loop and shoot the group - this won't be useful for data collection if it turns out to be protein but will be sufficient to tell you if your crystals are salt.
good luck,
Eric
__________________________
Eric Larson, PhD
Biomolecular Structure Center
Department of Biochemistry
Box 357742
University of Washington
Seattle, WA 98195
On Tue, 16 Nov 2010, yybbll wrote:
| Hi, everybody,
|
| I try to crystallize one membrane protein. All crystals were grown by handing-drop vapor diffusion at 20 degree. A protein solution containing
| about 8-10mg/ml protein in 20mM Tris (pH7.5), 0.017% DDM, 100mM NaCl, 10% glycerol, 2mM DDT was mixed with an equal volume of a reservoir solution
| containing 45% PEG200, 0.1 M phosphate/citrate (pH4.2). First crystal appeared in the drop within 4 days. And one week a lot of crystals appeared in the
| drops.
|
| Our question is all of these crystals are too small to check them by X-ray diffraction and SDS-PAGE. We are not sure they are protein crystals or salt
| crystals. Our condition seems difficult to produce salt crystal. But I am a little warry because we use reloaded our sample to small Ni-resin column to
| reduce the concentration of detergent. Maybe some nickel ion dropped off, and then our protein sample contained some this ion. And nickel ion may react
| with phosphate, and then produced nickel phosphate crystal. Could somebody tell me if it is possible?
|
| I attach some photos of our crystals. Could somebody give me some suggestions about how to optimize this type crystal to get bigger crystal?
|
| Thanks a lot!
|
| Yibin
|
|
| 2010-11-16
|
| _________________________________________________________________________________________________________________________________________________________
| yybbll
|
| _________________________________________________________________________________________________________________________________________________________
| 发件人: Laurie Betts
| 发送时间: 2010-11-16 17:13:32
| 收件人: CCP4BB
| 抄送:
| 主题: [ccp4bb] expression of Cys-rich small protein
| All -
|
| We are trying to express for structural studies a 257 AA eukaryotic intracellular (also possibly nuclear) protein (predicted to be single domain
| all-helical) that has 12 Cysteines. No known metal-binding function not that it couldn't happen. So far (E. coli) it expressed solubly as MBP fusion
| (with an N-terminal region deleted predicted disordered) until cleavage of MBP, then it's not soluble, including detergents added. THe MBP fusion is
| usually soluble aggregate so we assume that our part is not folded right. We have so far assumed it needs a lot of reducing agent (5 mM DTT or TCEP).
| Thinking of trying chaperones and insect cells next.
|
| Any experience out there that might help? Mostly I wonder about all the cysteines. Don't really know if that is the problem.
|
| Laurie Betts
|
|
|