[ccp4bb] cracking crystal
Dear All, Could you please help me solve this problem? I have a sensitive crystal, the mother liquor is 10% PEG4k+ 100 mM Tris-buffer pH 8.5, crystal is big and good but very sensitive, when i put it in cryoprotectants, cracking happened. First i used artificial mother liquor + 25% v/v glycerol, slowly decrease to + 12.5% v/v glycerol, all crystals cracked after immersed, finally i tried 50% mineral oil + 50% paratone, it still cracked (4- 5 cracks, but not broken) . even this cracked crystal can give me 3.2 angstrom diffraction, so no cracked crystal might give me 2.0+ angstrom diffraction. Now i don't have many crystals to try so many cryoprotectants, so anybody has experience on this? any suggestion would be greatly appreciated. Thank you. Fengxiale
Re: [ccp4bb] cracking crystal
Dear Fengxiale, I would suggest two thing you can try: 1) shock-freeze the crystal: pick it up from the crystallisation plate with the mounting loop and only briefly (=1s) dip it into the cryo-condition, then freeze it in a bath of liquid nitrogen. I would try both glycerol and PEG400. - try to find the minimum required amount of glycerol and PEG400 by testing various conditions withouth the crystal: freeze the cryo-solution in a large loop and take a picture. Go high enough with the cryoprotectant to avoid any ice-rings at all. 2) transfer the crystals _really_ slowly! Make solutions in steps of 5% cryo-protectant in the mother liquor and transfer the crystal with a pipette. If the crystal is too big to go through the pipette tip, cut off a small piece with a scalpel. After each step observe the crystal, check whether you observe any cracks. Wait for 5-10min before going to the next concentration. This way you can check various cryo-protectants like PEG400, MPD, glycerol, sugars (fructose, sucrose)... Since you don't have to many crystal, you can use the cracked parts for this experiment, so you only have to sacrifice one or two crystals. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Tue, 1 Sep 2009, Fengxia Liu wrote: Dear All, Could you please help me solve this problem? I have a sensitive crystal, the mother liquor is 10% PEG4k+ 100 mM Tris-buffer pH 8.5, crystal is big and good but very sensitive, when i put it in cryoprotectants, cracking happened. First i used artificial mother liquor + 25% v/v glycerol, slowly decrease to + 12.5% v/v glycerol, all crystals cracked after immersed, finally i tried 50% mineral oil + 50% paratone, it still cracked (4- 5 cracks, but not broken) . even this cracked crystal can give me 3.2 angstrom diffraction, so no cracked crystal might give me 2.0+ angstrom diffraction. Now i don't have many crystals to try so many cryoprotectants, so anybody has experience on this? any suggestion would be greatly appreciated. Thank you. Fengxiale
Re: [ccp4bb] cracking crystal
We have had very good success with fragile, crack-prone crystals by doing gradual soaks in increasing glucose. Besides the osmotic pressure/solvent composition changes induced by cryopreservation, another reason crystals crack is drop dehydration. You can minimize dehydration of exposed drops by carrying out transfers in the cold room (I hate that, but it sometimes works), or by replacing coverslips over the well to maintain humidity. The most gentle method we use is our "no-fail" cryoprotection procotol, which you can find at: http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Mounting+Protein+Crystals#No_fail_cryoprotection We have successfuly cryopreserved crystals that could not be frozen any other way. Fengxia Liu wrote: Dear All, Could you please help me solve this problem? I have a sensitive crystal, the mother liquor is 10% PEG4k+ 100 mM Tris-buffer pH 8.5, crystal is big and good but very sensitive, when i put it in cryoprotectants, cracking happened. First i used artificial mother liquor + 25%v/v glycerol, slowly decrease to + 12.5%v/v glycerol, all crystals cracked after immersed, finally i tried 50% mineral oil + 50% paratone, it still cracked (4- 5 cracks, but not broken). even this cracked crystal can give me 3.2 angstrom diffraction, so no cracked crystal might give me 2.0+ angstrom diffraction. Now i don't have many crystals to tryso many cryoprotectants, so anybody has experience on this? any suggestion would be greatly appreciated. Thank you. Fengxiale -- Roger S. Rowlett Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu
Re: [ccp4bb] cracking crystal
One thing that I have had work in the past is just increase the percentage of PEG. I think something around 30-35% may work for PEG4K, maybe a little higher. Most PEGS will work as a cryo protectant if the concentration is high enough. Also as mentioned before you could try stepping up the glycerol concentration very gradually. I had a colleague who said the protein he worked on in grad school had to be stepped up 1% increments or it would crack and fall apart. IIRC he had better then 2.0 ang. data for the final structure. Leonard Thomas Ph.D. Macromolecular Crystallography Laboratory Manager University of Oklahoma Department of Chemistry and Biochemistry 620 Parrington Oval Norman, OK 73019 lmtho...@ou.edu http://barlywine.chem.ou.edu Office: (405)325-1126 Lab: (405)325-7571 On Sep 1, 2009, at 5:25 AM, Fengxia Liu wrote: Dear All, Could you please help me solve this problem? I have a sensitive crystal, the mother liquor is 10% PEG4k+ 100 mM Tris-buffer pH 8.5, crystal is big and good but very sensitive, when i put it in cryoprotectants, cracking happened. First i used artificial mother liquor + 25% v/v glycerol, slowly decrease to + 12.5% v/v glycerol, all crystals cracked after immersed, finally i tried 50% mineral oil + 50% paratone, it still cracked (4- 5 cracks, but not broken) . even this cracked crystal can give me 3.2 angstrom diffraction, so no cracked crystal might give me 2.0+ angstrom diffraction. Now i don't have many crystals to try so many cryoprotectants, so anybody has experience on this? any suggestion would be greatly appreciated. Thank you. Fengxiale
Re: [ccp4bb] cracking crystal
Have you tried to grow the crystals in the presence of small amounts of cryoprotectants? For example, use sucrose, glycerol, ethylene glycol, trehalose, glucose, sorbitol, various salts in the crystal growth. One can use concentrations of the additives in the 1% to 4% (w/v or v/v) range. The hypothesis you are testing is this: Does the additive in the tested concentration have any effect on crystal growth? If yes, don't use that additive. If no, go onto the next test: At what higher concentration does the additive have any effect on crystal growth? You can imagine where this leads. Perhaps once grown in a little bit of cryoprotectant, then swishing through a higher concentration will not cause cracking. Also, do you suspect that the cryoprotectants that you have used bind to your protein? Do they by chance mimic substrate, product, or effector? And then there is always cross-linking with glutaraldehyde. Good luck! Jim _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Fengxia Liu Sent: Tuesday, September 01, 2009 5:25 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] cracking crystal Dear All, Could you please help me solve this problem? I have a sensitive crystal, the mother liquor is 10% PEG4k+ 100 mM Tris-buffer pH 8.5, crystal is big and good but very sensitive, when i put it in cryoprotectants, cracking happened. First i used artificial mother liquor + 25% v/v glycerol, slowly decrease to + 12.5% v/v glycerol, all crystals cracked after immersed, finally i tried 50% mineral oil + 50% paratone, it still cracked (4- 5 cracks, but not broken) . even this cracked crystal can give me 3.2 angstrom diffraction, so no cracked crystal might give me 2.0+ angstrom diffraction. Now i don't have many crystals to try so many cryoprotectants, so anybody has experience on this? any suggestion would be greatly appreciated. Thank you. Fengxiale
Re: [ccp4bb] cracking crystal
...and I prefer to use PFPE (perfluoropolyether, trade name FOMBLIN) as an oil since it is less viscous, has no reactivity and is a low temp lubricant that freezes easily. Small molecule crystallographers have used it for years. Kris - Kris F. Tesh, Ph D Director, Macromolecular Products Rigaku Americas Corporation 9009 New Trails Drive The Woodlands, TX 77381 USA 001 281 362 2300 x 144 From: Leonard Thomas lmtho...@ou.edu To: CCP4BB@JISCMAIL.AC.UK Date: 09/01/2009 10:09 AM Subject: Re: [ccp4bb] cracking crystal One thing that I have had work in the past is just increase the percentage of PEG. I think something around 30-35% may work for PEG4K, maybe a little higher. Most PEGS will work as a cryo protectant if the concentration is high enough. Also as mentioned before you could try stepping up the glycerol concentration very gradually. I had a colleague who said the protein he worked on in grad school had to be stepped up 1% increments or it would crack and fall apart. IIRC he had better then 2.0 ang. data for the final structure. Leonard Thomas Ph.D. Macromolecular Crystallography Laboratory Manager University of Oklahoma Department of Chemistry and Biochemistry 620 Parrington Oval Norman, OK 73019 lmtho...@ou.edu http://barlywine.chem.ou.edu Office: (405)325-1126 Lab: (405)325-7571 On Sep 1, 2009, at 5:25 AM, Fengxia Liu wrote: Dear All, Could you please help me solve this problem? I have a sensitive crystal, the mother liquor is 10% PEG4k+ 100 mM Tris-buffer pH 8.5, crystal is big and good but very sensitive, when i put it in cryoprotectants, cracking happened. First i used artificial mother liquor + 25% v/v glycerol, slowly decrease to + 12.5% v/v glycerol, all crystals cracked after immersed, finally i tried 50% mineral oil + 50% paratone, it still cracked (4- 5 cracks, but not broken) . even this cracked crystal can give me 3.2 angstrom diffraction, so no cracked crystal might give me 2.0+ angstrom diffraction. Now i don't have many crystals to try so many cryoprotectants, so anybody has experience on this? any suggestion would be greatly appreciated. Thank you. Fengxiale
Re: [ccp4bb] cracking crystal
You can always give this one a try: http://www.xtals.org/crystal_cryo.pdf Artem Nothing is built on stone; all is built on sand, but we must build as if the sand were stone Jorge Luis Borges _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Fengxia Liu Sent: Tuesday, September 01, 2009 5:25 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] cracking crystal Dear All, Could you please help me solve this problem? I have a sensitive crystal, the mother liquor is 10% PEG4k+ 100 mM Tris-buffer pH 8.5, crystal is big and good but very sensitive, when i put it in cryoprotectants, cracking happened. First i used artificial mother liquor + 25% v/v glycerol, slowly decrease to + 12.5% v/v glycerol, all crystals cracked after immersed, finally i tried 50% mineral oil + 50% paratone, it still cracked (4- 5 cracks, but not broken) . even this cracked crystal can give me 3.2 angstrom diffraction, so no cracked crystal might give me 2.0+ angstrom diffraction. Now i don't have many crystals to try so many cryoprotectants, so anybody has experience on this? any suggestion would be greatly appreciated. Thank you. Fengxiale
Re: [ccp4bb] cracking crystal
Hi Fengxiale, Have you tried no cryoprotection? 10% PEG4K is probably too low but you may get lucky. Since your crystal is already in PEG, you may try increasing the concentration of the PEG or try just plain ethylene glycol, which may be more gentle on your crystal than sticking it in glycerol. You say you started with 25% glycerol and slowly decreased to 12.5%. Are you doing this by dialysis or by just sequentially transferring your crystal from one solution to the next? Dialysis is much more gentle and you should be going the other direction - i.e. from low conc. of cryoprotectant to high conc (not high to low like you did). How long are you soaking your crystal in the cryoprotectant solution? Do crystals crack immediately or does it take some time? Oftentimes just a quick dip is sufficient. How well does your crystal diffract at room temperature? Perhaps 3.2 A is the limit even in the absence of freezing. Good luck, Eric __ Eric Larson, PhD MSGPP Consortium Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 On Tue, 1 Sep 2009, Fengxia Liu wrote: Dear All, Could you please help me solve this problem? I have a sensitive crystal, the mother liquor is 10% PEG4k+ 100 mM Tris-buffer pH 8.5, crystal is big and good but very sensitive, when i put it in cryoprotectants, cracking happened. First i used artificial mother liquor + 25% v/v glycerol, slowly decrease to + 12.5% v/v glycerol, all crystals cracked after immersed, finally i tried 50% mineral oil + 50% paratone, it still cracked (4- 5 cracks, but not broken) . even this cracked crystal can give me 3.2 angstrom diffraction, so no cracked crystal might give me 2.0+ angstrom diffraction. Now i don't have many crystals to try so many cryoprotectants, so anybody has experience on this? any suggestion would be greatly appreciated. Thank you. Fengxiale
[ccp4bb] Fw: [ccp4bb] cracking crystal
I have been asked about a supplier. I know of at least 4: 1. Aldrichwww.sigmaalldrich.com Fomblin Y LVAC 14/6Cat # 317934-100G100g 2. Lancaster/Alfa Aesar www.alfa.com/CGI-BIN/LANSAWEB?WEBEVENT+L0178567F6B740600C68505B+ALF+ENG Fomblin YR-1800 Item # L17548 5 or 25g 3. Specialty Fluids www.vacuumoil.com/fomblin.htm Lots of Fomblins 1kg 4. Hampton Research www.hamptonresearch.com/product_detail.aspx?cid=4sid=138pid=551 PFO-X175/08 Cat # HR2-814 1ml The price per unit is very different for each supplier, so check it out for yourself. And, if you get into this sort of stuff, the following link has the properties and specifications of the different oil types: http://www.vacuumoil.com/msdspdfs/productinfo/fom_vac.pdf One additional tidbit: if you are using this to retard evaporation of a volatile organic, consider saturating the oil before placing your crystal in the oil or the oil on the drop! It takes very little to saturate and is unlikely to add solvent to the crystal or drop. Enjoy, Kris - Kris F. Tesh, Ph D Director, Macromolecular Products Rigaku Americas Corporation 9009 New Trails Drive The Woodlands, TX 77381 USA 001 281 362 2300 x 144 - Forwarded by Kris Tesh/Rigaku on 09/01/2009 03:25 PM - From: Kris Tesh/Rigaku To: CCP4BB@JISCMAIL.AC.UK Date: 09/01/2009 10:33 AM Subject: Re: [ccp4bb] cracking crystal ...and I prefer to use PFPE (perfluoropolyether, trade name FOMBLIN) as an oil since it is less viscous, has no reactivity and is a low temp lubricant that freezes easily. Small molecule crystallographers have used it for years. Kris - Kris F. Tesh, Ph D Director, Macromolecular Products Rigaku Americas Corporation 9009 New Trails Drive The Woodlands, TX 77381 USA 001 281 362 2300 x 144 From: Leonard Thomas lmtho...@ou.edu To: CCP4BB@JISCMAIL.AC.UK Date: 09/01/2009 10:09 AM Subject: Re: [ccp4bb] cracking crystal One thing that I have had work in the past is just increase the percentage of PEG. I think something around 30-35% may work for PEG4K, maybe a little higher. Most PEGS will work as a cryo protectant if the concentration is high enough. Also as mentioned before you could try stepping up the glycerol concentration very gradually. I had a colleague who said the protein he worked on in grad school had to be stepped up 1% increments or it would crack and fall apart. IIRC he had better then 2.0 ang. data for the final structure. Leonard Thomas Ph.D. Macromolecular Crystallography Laboratory Manager University of Oklahoma Department of Chemistry and Biochemistry 620 Parrington Oval Norman, OK 73019 lmtho...@ou.edu http://barlywine.chem.ou.edu Office: (405)325-1126 Lab: (405)325-7571 On Sep 1, 2009, at 5:25 AM, Fengxia Liu wrote: Dear All, Could you please help me solve this problem? I have a sensitive crystal, the mother liquor is 10% PEG4k+ 100 mM Tris-buffer pH 8.5, crystal is big and good but very sensitive, when i put it in cryoprotectants, cracking happened. First i used artificial mother liquor + 25% v/v glycerol, slowly decrease to + 12.5% v/v glycerol, all crystals cracked after immersed, finally i tried 50% mineral oil + 50% paratone, it still cracked (4- 5 cracks, but not broken) . even this cracked crystal can give me 3.2 angstrom diffraction, so no cracked crystal might give me 2.0+ angstrom diffraction. Now i don't have many crystals to try so many cryoprotectants, so anybody has experience on this? any suggestion would be greatly appreciated. Thank you. Fengxiale
Re: [ccp4bb] Fw: [ccp4bb] cracking crystal
The internet is incredible...within about 2 minutes, a few people had read my post and found that Aldrich was a broken link...there is only one l in Aldrich. www.sigmaaldrich.com or www.sigmaaldrich.com/sigma-aldrich/home.html Please forgive me for the error and the multiple post. Kris - Kris F. Tesh, Ph D Director, Macromolecular Products Rigaku Americas Corporation 9009 New Trails Drive The Woodlands, TX 77381 USA 001 281 362 2300 x 144 From: Kris Tesh kris.t...@rigaku.com To: CCP4BB@JISCMAIL.AC.UK Date: 09/01/2009 04:20 PM Subject: [ccp4bb] Fw: [ccp4bb] cracking crystal I have been asked about a supplier. I know of at least 4: 1. Aldrichwww.sigmaalldrich.com Fomblin Y LVAC 14/6Cat # 317934-100G100g 2. Lancaster/Alfa Aesar www.alfa.com/CGI-BIN/LANSAWEB?WEBEVENT+L0178567F6B740600C68505B+ALF+ENG Fomblin YR-1800 Item # L17548 5 or 25g 3. Specialty Fluids www.vacuumoil.com/fomblin.htm Lots of Fomblins 1kg 4. Hampton Research www.hamptonresearch.com/product_detail.aspx?cid=4sid=138pid=551 PFO-X175/08 Cat # HR2-814 1ml The price per unit is very different for each supplier, so check it out for yourself. And, if you get into this sort of stuff, the following link has the properties and specifications of the different oil types: http://www.vacuumoil.com/msdspdfs/productinfo/fom_vac.pdf One additional tidbit: if you are using this to retard evaporation of a volatile organic, consider saturating the oil before placing your crystal in the oil or the oil on the drop! It takes very little to saturate and is unlikely to add solvent to the crystal or drop. Enjoy, Kris - Kris F. Tesh, Ph D Director, Macromolecular Products Rigaku Americas Corporation 9009 New Trails Drive The Woodlands, TX 77381 USA 001 281 362 2300 x 144 - Forwarded by Kris Tesh/Rigaku on 09/01/2009 03:25 PM - From: Kris Tesh/Rigaku To: CCP4BB@JISCMAIL.AC.UK Date: 09/01/2009 10:33 AM Subject: Re: [ccp4bb] cracking crystal ...and I prefer to use PFPE (perfluoropolyether, trade name FOMBLIN) as an oil since it is less viscous, has no reactivity and is a low temp lubricant that freezes easily. Small molecule crystallographers have used it for years. Kris - Kris F. Tesh, Ph D Director, Macromolecular Products Rigaku Americas Corporation 9009 New Trails Drive The Woodlands, TX 77381 USA 001 281 362 2300 x 144 From: Leonard Thomas lmtho...@ou.edu To: CCP4BB@JISCMAIL.AC.UK Date: 09/01/2009 10:09 AM Subject: Re: [ccp4bb] cracking crystal One thing that I have had work in the past is just increase the percentage of PEG. I think something around 30-35% may work for PEG4K, maybe a little higher. Most PEGS will work as a cryo protectant if the concentration is high enough. Also as mentioned before you could try stepping up the glycerol concentration very gradually. I had a colleague who said the protein he worked on in grad school had to be stepped up 1% increments or it would crack and fall apart. IIRC he had better then 2.0 ang. data for the final structure. Leonard Thomas Ph.D. Macromolecular Crystallography Laboratory Manager University of Oklahoma Department of Chemistry and Biochemistry 620 Parrington Oval Norman, OK 73019 lmtho...@ou.edu http://barlywine.chem.ou.edu Office: (405)325-1126 Lab: (405)325-7571 On Sep 1, 2009, at 5:25 AM, Fengxia Liu wrote: Dear All, Could you please help me solve this problem? I have a sensitive crystal, the mother liquor is 10% PEG4k+ 100 mM Tris-buffer pH 8.5, crystal is big and good but very sensitive, when i put it in cryoprotectants, cracking happened. First i used artificial mother liquor + 25% v/v glycerol, slowly decrease to + 12.5% v/v glycerol, all crystals cracked after immersed, finally i tried 50% mineral oil + 50% paratone, it still cracked (4- 5 cracks, but not broken) . even this cracked crystal can give me 3.2 angstrom diffraction, so no cracked crystal might give me 2.0+ angstrom diffraction. Now i don't have many crystals to try so many cryoprotectants, so anybody has experience on this? any suggestion would be greatly appreciated. Thank you. Fengxiale
