Re: [ccp4bb] how to optimize small rod-shaped crystals
Hi, thank you for you reply. Could you tell me if you try to dye membrane protein crystal with detergent? Thanks. Y.B. 2010-11-18 yybbll 发件人： Matthew Bratkowski 发送时间： 2010-11-17 02:58:40 收件人： CCP4BB 抄送： 主题： Re: [ccp4bb] how to optimize small rod-shaped crystals I like using Izit dye from Hampton (http://hamptonresearch.com/product_detail.aspx?cid=4&sid=41&pid=33) to check if crystals are protein or salt. If the crystals are protein, the dye should absorb rather readily into the crystals and turn them blue, while the rest of the drop will eventually turn clear. Quite likely, excess dye will also crystallize out as well. Salt crystals will not soak in the dye, and the rest of the drop may remain blue for several days. Using Izit is easy and saves a lot of time. In my experience, I have gotten a lot of false positives from phosphate crystallization conditions, so you want to be sure that the crystals are not salt before you waste any time on optimizing them. Matt On Tue, Nov 16, 2010 at 3:23 PM, Ulli Hain wrote: If you have a polarizer on your microscope you could see if they are extremely dichroic, in which case they may be salt. You could also open the well up and see if they are heavy - do they sink immediately when you lift them to the top of the drop? This could also indicate salt. But I agree with other posts that they do not seem to small to mount, especially with the new mesh loops they make. -Ulli Adelaide-Ulricke P. Hain Johns Hopkins University Bloomberg School of Public Health Biochemistry & Molecular Biology Quoting yybbll : > 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 > > > > Laurie Betts > ? 2010-11-16 17:13:32 > CCP4BB > ??? > ??? [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 >
Re: [ccp4bb] how to optimize small rod-shaped crystals
Agree. two comments for your reference: 1. When you have glycerol in your protein buffer, always add same % glycerol in your reservoir solution.You have 10% glycerol in your protein buffer, but not in your reservoir solution, the glycerol will overcome all the other facts and "grasp" water from reservoir to your drop, make your protein more diluted and hard to growing big crystal, try to add 10% glycerol in your reservoir solution to balance the glycerol force, and the vapor will go from drop to reservoir as normal, you may have an other lucky direction. 2. Ni--phosphate crystal will be colored and very hard to form crystal in normal condition, if you worry about Ni treat your sample with EDTA than dialysis against out your protein buffer. Deqian --- On Tue, 11/16/10, Clement Angkawidjaja wrote: From: Clement Angkawidjaja Subject: Re: [ccp4bb] how to optimize small rod-shaped crystals To: CCP4BB@JISCMAIL.AC.UK Date: Tuesday, November 16, 2010, 8:19 PM I strongly agree with Eric Larson’s suggestion on trying to see the diffraction of your crystal. The most straightforward solution. Other suggestions may work too, but there are chances they will still give you false positives. If you need bigger crystals, try to slow down the nucleation (use lower temperature, different ratio of protein:crystallant, etc). Clement From: yybbll Sent: Wednesday, November 17, 2010 2:42 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] how to optimize small rod-shaped crystals 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
Re: [ccp4bb] how to optimize small rod-shaped crystals
There was a thread about this topic--dyes--a little while back. It seemed that many many dyes work, not just methylene blue. I believe there was even an over-the-counter mercury-bromine compound (merbromin?) which was suggested by Artem, as it would provide a nice derivative as well. JPK On Tue, Nov 16, 2010 at 9:03 PM, Filip Van Petegem wrote: > One problem with a dye like methylene blue is that it tends to crystallize > in certain conditions commonly found in crystallization screens (e.g. some > that are high in PEG) making them less useful in such conditions. Has > anybody systematically tested alternative dyes and found one that is more > soluble? > Cheers > Filip Van Petegem > > > > On Tue, Nov 16, 2010 at 6:15 PM, Jim Pflugrath > wrote: >> >> With Izit or other dyes, you might wish to do a positive control with bona >> fide protein crystals and a negative control with bona fide salt crystals. >> >> >> From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of >> Matthew Bratkowski >> Sent: Tuesday, November 16, 2010 7:58 PM >> To: CCP4BB@JISCMAIL.AC.UK >> Subject: Re: [ccp4bb] how to optimize small rod-shaped crystals >> >> I like using Izit dye from Hampton >> (http://hamptonresearch.com/product_detail.aspx?cid=4&sid=41&pid=33) to >> check if crystals are protein or salt. If the crystals are protein, the dye >> should absorb rather readily into the crystals and turn them blue, while the >> rest of the drop will eventually turn clear. Quite likely, excess dye will >> also crystallize out as well. Salt crystals will not soak in the dye, and >> the rest of the drop may remain blue for several days. >> Using Izit is easy and saves a lot of time. In my experience, I have >> gotten a lot of false positives from phosphate crystallization conditions, >> so you want to be sure that the crystals are not salt before you waste any >> time on optimizing them. >> Matt >> > > > -- > Filip Van Petegem, PhD > Assistant Professor > The University of British Columbia > Dept. of Biochemistry and Molecular Biology > 2350 Health Sciences Mall - Rm 2.356 > Vancouver, V6T 1Z3 > > phone: +1 604 827 4267 > email: filip.vanpete...@gmail.com > http://crg.ubc.ca/VanPetegem/ >
Re: [ccp4bb] how to optimize small rod-shaped crystals
One problem with a dye like methylene blue is that it tends to crystallize in certain conditions commonly found in crystallization screens (e.g. some that are high in PEG) making them less useful in such conditions. Has anybody systematically tested alternative dyes and found one that is more soluble? Cheers Filip Van Petegem On Tue, Nov 16, 2010 at 6:15 PM, Jim Pflugrath wrote: > With Izit or other dyes, you might wish to do a positive control with > bona fide protein crystals and a negative control with bona fide salt > crystals. > > -- > *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of > *Matthew > Bratkowski > *Sent:* Tuesday, November 16, 2010 7:58 PM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* Re: [ccp4bb] how to optimize small rod-shaped crystals > > I like using Izit dye from Hampton ( > http://hamptonresearch.com/product_detail.aspx?cid=4&sid=41&pid=33) to > check if crystals are protein or salt. If the crystals are protein, the dye > should absorb rather readily into the crystals and turn them blue, while the > rest of the drop will eventually turn clear. Quite likely, excess dye will > also crystallize out as well. Salt crystals will not soak in the dye, and > the rest of the drop may remain blue for several days. > > Using Izit is easy and saves a lot of time. In my experience, I have > gotten a lot of false positives from phosphate crystallization conditions, > so you want to be sure that the crystals are not salt before you waste any > time on optimizing them. > > Matt > > > -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] how to optimize small rod-shaped crystals
I strongly agree with Eric Larson’s suggestion on trying to see the diffraction of your crystal. The most straightforward solution. Other suggestions may work too, but there are chances they will still give you false positives. If you need bigger crystals, try to slow down the nucleation (use lower temperature, different ratio of protein:crystallant, etc). Clement From: yybbll Sent: Wednesday, November 17, 2010 2:42 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] how to optimize small rod-shaped crystals 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
Re: [ccp4bb] how to optimize small rod-shaped crystals
With Izit or other dyes, you might wish to do a positive control with bona fide protein crystals and a negative control with bona fide salt crystals. _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Matthew Bratkowski Sent: Tuesday, November 16, 2010 7:58 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] how to optimize small rod-shaped crystals I like using Izit dye from Hampton (http://hamptonresearch.com/product_detail.aspx?cid=4 <http://hamptonresearch.com/product_detail.aspx?cid=4&sid=41&pid=33> &sid=41&pid=33) to check if crystals are protein or salt. If the crystals are protein, the dye should absorb rather readily into the crystals and turn them blue, while the rest of the drop will eventually turn clear. Quite likely, excess dye will also crystallize out as well. Salt crystals will not soak in the dye, and the rest of the drop may remain blue for several days. Using Izit is easy and saves a lot of time. In my experience, I have gotten a lot of false positives from phosphate crystallization conditions, so you want to be sure that the crystals are not salt before you waste any time on optimizing them. Matt
Re: [ccp4bb] how to optimize small rod-shaped crystals
I like using Izit dye from Hampton ( http://hamptonresearch.com/product_detail.aspx?cid=4&sid=41&pid=33) to check if crystals are protein or salt. If the crystals are protein, the dye should absorb rather readily into the crystals and turn them blue, while the rest of the drop will eventually turn clear. Quite likely, excess dye will also crystallize out as well. Salt crystals will not soak in the dye, and the rest of the drop may remain blue for several days. Using Izit is easy and saves a lot of time. In my experience, I have gotten a lot of false positives from phosphate crystallization conditions, so you want to be sure that the crystals are not salt before you waste any time on optimizing them. Matt On Tue, Nov 16, 2010 at 3:23 PM, Ulli Hain wrote: > If you have a polarizer on your microscope you could see if they are > extremely dichroic, in which case they may be salt. You could also open the > well up and see if they are heavy - do they sink immediately when you lift > them to the top of the drop? This could also indicate salt. But I agree with > other posts that they do not seem to small to mount, especially with the new > mesh loops they make. > -Ulli > > Adelaide-Ulricke P. Hain > Johns Hopkins University > Bloomberg School of Public Health > Biochemistry & Molecular Biology > > > Quoting yybbll : > > > 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 > > > > > > > > Laurie Betts > > ? 2010-11-16 17:13:32 > > CCP4BB > > ??? > > ??? [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 > > >
Re: [ccp4bb] how to optimize small rod-shaped crystals
If you have a polarizer on your microscope you could see if they are extremely dichroic, in which case they may be salt. You could also open the well up and see if they are heavy - do they sink immediately when you lift them to the top of the drop? This could also indicate salt. But I agree with other posts that they do not seem to small to mount, especially with the new mesh loops they make. -Ulli Adelaide-Ulricke P. Hain Johns Hopkins University Bloomberg School of Public Health Biochemistry & Molecular Biology Quoting yybbll : > 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 > > > > Laurie Betts > ? 2010-11-16 17:13:32 > CCP4BB > ??? > ??? [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 >
Re: [ccp4bb] how to optimize small rod-shaped crystals
Hi Yibin, MiTeGen offers different loops that make it easier to pick really tiny crystals which can be diffracted at the synchrotron. If you would like to optimize small needles or rod shaped crystals, you may try an additive screen. You may also try to lower the rate of crystal growth. Perhaps increasing the amount of protein may help. Or you may also try macro-seeding,,,where you pick out one tiny crystal and seed it into the drop with same conditions. Good luck! --Subhangi
Re: [ccp4bb] how to optimize small rod-shaped crystals
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 | |
