Re: [ccp4bb] Heavy Atom Phasing
Rhys, If crystals grow reproducibly and within a reasonable timeframe, I would always do co-crystallization, particularly if the HA is a good anomalous scatterer. We have had good success with this method, including a recent membrane protein structure. Even if you get crystals that are not isomorphous with the native, SIRAS is easy to do these days. Also broaden your soaking screens of HAs (if you haven't already), to include mercury compounds, which don't always need a Cys to bind well and love hydrophobic nooks and crannies, and metal clusters (like tantalum bromide). As this is a beta-barrel membrane protein, iodine might also be a way to go. Good luck, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Jul 27, 2014, at 5:48 PM, RHYS GRINTER wrote: > Hi All, > > I thought I might put a question to the community, with the hope of getting > some tips of the best way to proceed with my heavy atom phasing problem. > I'm working on solving the structure of an integral beta-barrel membrane > protein of approximately 100 kDa. I've crystallised protein, growing some > very flimsy needle like crystals, and collected datasets to around 3.1 A. > I then produced selenomet derivative protein and repeated crystallisation > trials in the same conditions and also repeated broad screens, however the > derivative protein failed to produce crystals that diffracted beyond 10 A (in > fact it barely crystallises at all). > So I've moved on to heavy atom soaks and have had some success with > tetrachloroplatinate and tetranitroplatinate compounds, in that the crystals > didn't dissolve (as they did with gold and samarium compounds) and diffracted > to some degree. I collected SAD data to around 6.5 A from these crystals and > there seems to be anomolous signal. However, while I get a good CC of 0.4 > from HYSS in phenix with this dataset and the phaser EP FOM is 0.56, the maps > before and after DM are uninterpretable. I'm guessing the quality and > resolution of the data I collected just aren't good enough (the data is > reasonably anisotropic). > I performed the metal soaking, by taking a small amount of the platinate salt > and adding it to the crystallisation drop as the crystals are extremely > fragile and don't stand up well to handling through a soaking or cryo > solution. Leaving the crystals to soak for 48 hours and then, freezing them > directly. The solution is on the border of cryoprotection (the conditions has > PEG2000MME and PVP and the precipitants), but with native crystals this > doesn't seem to be a parameter which affects diffraction. The crystals are > very variable in performance, so while I feel that the heavy atom soaking has > compromised their diffractability to a degree, inherent variation may play a > part. > > What I was wondering is if some one with more experience than me found > themselves in this position, how would they proceed? Questions which spring > to mind are, how much heavy atom compound do people add and how long do they > soak for? Is there anyway I can squeeze something out of the anomalous data I > have, given I have 'reasonable' native data, or will poor quality data give > spuriously positive statistics for heavy atom phasing? And are there any > tricks people have experienced to improve performance of crystals like these > (aside from the usual seeding, additives, different detergents etc which I > have spend a fair bit of time on optimization already). > > Thanks in advance, > > Rhys
Re: [ccp4bb] Heavy Atom Phasing
Rhys, since you can obtain good native crystals, I would try Xenon phasing. I have recently heard several success stories at the Australian Synchrotron, and it just seems to be a straightforward and rational way. Molecular Replacement should also be an option. I did this with beta-barrel proteins quite a bit, and even wrote a program for elliptic distortion of the model, to arrive at models that may be closer to the target. good luck, Kay
Re: [ccp4bb] Heavy Atom Phasing
Dear Rhys, the humidity control device at Diamond and ESRF (HC1) is apparently especially recommended for crystals that show large variability. You might want to give this a try, collect a few wedges at RT off many crystals and then Blend them. This approach might increase resolution and, a la W. Hendrickson as mentioned already, signal. Andreas On 27/07/2014 10:48, RHYS GRINTER wrote: Hi All, I thought I might put a question to the community, with the hope of getting some tips of the best way to proceed with my heavy atom phasing problem. I'm working on solving the structure of an integral beta-barrel membrane protein of approximately 100 kDa. I've crystallised protein, growing some very flimsy needle like crystals, and collected datasets to around 3.1 A. I then produced selenomet derivative protein and repeated crystallisation trials in the same conditions and also repeated broad screens, however the derivative protein failed to produce crystals that diffracted beyond 10 A (in fact it barely crystallises at all). So I've moved on to heavy atom soaks and have had some success with tetrachloroplatinate and tetranitroplatinate compounds, in that the crystals didn't dissolve (as they did with gold and samarium compounds) and diffracted to some degree. I collected SAD data to around 6.5 A from these crystals and there seems to be anomolous signal. However, while I get a good CC of 0.4 from HYSS in phenix with this dataset and the phaser EP FOM is 0.56, the maps before and after DM are uninterpretable. I'm guessing the quality and resolution of the data I collected just aren't good enough (the data is reasonably anisotropic). I performed the metal soaking, by taking a small amount of the platinate salt and adding it to the crystallisation drop as the crystals are extremely fragile and don't stand up well to handling through a soaking or cryo solution. Leaving the crystals to soak for 48 hours and then, freezing them directly. The solution is on the border of cryoprotection (the conditions has PEG2000MME and PVP and the precipitants), but with native crystals this doesn't seem to be a parameter which affects diffraction. The crystals are very variable in performance, so while I feel that the heavy atom soaking has compromised their diffractability to a degree, inherent variation may play a part. What I was wondering is if some one with more experience than me found themselves in this position, how would they proceed? Questions which spring to mind are, how much heavy atom compound do people add and how long do they soak for? Is there anyway I can squeeze something out of the anomalous data I have, given I have 'reasonable' native data, or will poor quality data give spuriously positive statistics for heavy atom phasing? And are there any tricks people have experienced to improve performance of crystals like these (aside from the usual seeding, additives, different detergents etc which I have spend a fair bit of time on optimization already). Thanks in advance, Rhys -- Andreas Förster Crystallization and X-ray Facility Manager Centre for Structural Biology Imperial College London
Re: [ccp4bb] Heavy Atom Phasing
Hi Rhys, Have you tried quick back-soaks into solutions lacking the heavy atoms? This can reduce radiation decay caused by absorption of X-rays by overabundant heavy atoms. Best, Chris > On Jul 27, 2014, at 4:48 PM, RHYS GRINTER > wrote: > > Hi All, > > I thought I might put a question to the community, with the hope of getting > some tips of the best way to proceed with my heavy atom phasing problem. > I'm working on solving the structure of an integral beta-barrel membrane > protein of approximately 100 kDa. I've crystallised protein, growing some > very flimsy needle like crystals, and collected datasets to around 3.1 A. > I then produced selenomet derivative protein and repeated crystallisation > trials in the same conditions and also repeated broad screens, however the > derivative protein failed to produce crystals that diffracted beyond 10 A (in > fact it barely crystallises at all). > So I've moved on to heavy atom soaks and have had some success with > tetrachloroplatinate and tetranitroplatinate compounds, in that the crystals > didn't dissolve (as they did with gold and samarium compounds) and diffracted > to some degree. I collected SAD data to around 6.5 A from these crystals and > there seems to be anomolous signal. However, while I get a good CC of 0.4 > from HYSS in phenix with this dataset and the phaser EP FOM is 0.56, the maps > before and after DM are uninterpretable. I'm guessing the quality and > resolution of the data I collected just aren't good enough (the data is > reasonably anisotropic). > I performed the metal soaking, by taking a small amount of the platinate salt > and adding it to the crystallisation drop as the crystals are extremely > fragile and don't stand up well to handling through a soaking or cryo > solution. Leaving the crystals to soak for 48 hours and then, freezing them > directly. The solution is on the border of cryoprotection (the conditions has > PEG2000MME and PVP and the precipitants), but with native crystals this > doesn't seem to be a parameter which affects diffraction. The crystals are > very variable in performance, so while I feel that the heavy atom soaking has > compromised their diffractability to a degree, inherent variation may play a > part. > > What I was wondering is if some one with more experience than me found > themselves in this position, how would they proceed? Questions which spring > to mind are, how much heavy atom compound do people add and how long do they > soak for? Is there anyway I can squeeze something out of the anomalous data I > have, given I have 'reasonable' native data, or will poor quality data give > spuriously positive statistics for heavy atom phasing? And are there any > tricks people have experienced to improve performance of crystals like these > (aside from the usual seeding, additives, different detergents etc which I > have spend a fair bit of time on optimization already). > > Thanks in advance, > > Rhys
Re: [ccp4bb] Heavy Atom Phasing
How about merging multiple crystals together a la Wayne Hendrickson's most recent papers? JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Boaz Shaanan Sent: Sunday, July 27, 2014 6:34 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Heavy Atom Phasing Hi, While some useful phase information could perhaps be extracted from the data you currently have, I'd suggest to try the method of quick soak described here: Acta Cryst. (2002). D58, 1092-1098 rather than the long soak that you tried. I guess that you also tried complete new screens for the SeMet crystals and just didn't get well diffracting crystals, right? Cheers, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of RHYS GRINTER [r.grinte...@research.gla.ac.uk] Sent: Monday, July 28, 2014 12:48 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Heavy Atom Phasing Hi All, I thought I might put a question to the community, with the hope of getting some tips of the best way to proceed with my heavy atom phasing problem. I'm working on solving the structure of an integral beta-barrel membrane protein of approximately 100 kDa. I've crystallised protein, growing some very flimsy needle like crystals, and collected datasets to around 3.1 A. I then produced selenomet derivative protein and repeated crystallisation trials in the same conditions and also repeated broad screens, however the derivative protein failed to produce crystals that diffracted beyond 10 A (in fact it barely crystallises at all). So I've moved on to heavy atom soaks and have had some success with tetrachloroplatinate and tetranitroplatinate compounds, in that the crystals didn't dissolve (as they did with gold and samarium compounds) and diffracted to some degree. I collected SAD data to around 6.5 A from these crystals and there seems to be anomolous signal. However, while I get a good CC of 0.4 from HYSS in phenix with this dataset and the phaser EP FOM is 0.56, the maps before and after DM are uninterpretable. I'm guessing the quality and resolution of the data I collected just aren't good enough (the data is reasonably anisotropic). I performed the metal soaking, by taking a small amount of the platinate salt and adding it to the crystallisation drop as the crystals are extremely fragile and don't stand up well to handling through a soaking or cryo solution. Leaving the crystals to soak for 48 hours and then, freezing them directly. The solution is on the border of cryoprotection (the conditions has PEG2000MME and PVP and the precipitants), but with native crystals this doesn't seem to be a parameter which affects diffraction. The crystals are very variable in performance, so while I feel that the heavy atom soaking has compromised their diffractability to a degree, inherent variation may play a part. What I was wondering is if some one with more experience than me found themselves in this position, how would they proceed? Questions which spring to mind are, how much heavy atom compound do people add and how long do they soak for? Is there anyway I can squeeze something out of the anomalous data I have, given I have 'reasonable' native data, or will poor quality data give spuriously positive statistics for heavy atom phasing? And are there any tricks people have experienced to improve performance of crystals like these (aside from the usual seeding, additives, different detergents etc which I have spend a fair bit of time on optimization already). Thanks in advance, Rhys
Re: [ccp4bb] Heavy Atom Phasing
Hi, While some useful phase information could perhaps be extracted from the data you currently have, I'd suggest to try the method of quick soak described here: Acta Cryst. (2002). D58, 1092-1098 rather than the long soak that you tried. I guess that you also tried complete new screens for the SeMet crystals and just didn't get well diffracting crystals, right? Cheers, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of RHYS GRINTER [r.grinte...@research.gla.ac.uk] Sent: Monday, July 28, 2014 12:48 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Heavy Atom Phasing Hi All, I thought I might put a question to the community, with the hope of getting some tips of the best way to proceed with my heavy atom phasing problem. I'm working on solving the structure of an integral beta-barrel membrane protein of approximately 100 kDa. I've crystallised protein, growing some very flimsy needle like crystals, and collected datasets to around 3.1 A. I then produced selenomet derivative protein and repeated crystallisation trials in the same conditions and also repeated broad screens, however the derivative protein failed to produce crystals that diffracted beyond 10 A (in fact it barely crystallises at all). So I've moved on to heavy atom soaks and have had some success with tetrachloroplatinate and tetranitroplatinate compounds, in that the crystals didn't dissolve (as they did with gold and samarium compounds) and diffracted to some degree. I collected SAD data to around 6.5 A from these crystals and there seems to be anomolous signal. However, while I get a good CC of 0.4 from HYSS in phenix with this dataset and the phaser EP FOM is 0.56, the maps before and after DM are uninterpretable. I'm guessing the quality and resolution of the data I collected just aren't good enough (the data is reasonably anisotropic). I performed the metal soaking, by taking a small amount of the platinate salt and adding it to the crystallisation drop as the crystals are extremely fragile and don't stand up well to handling through a soaking or cryo solution. Leaving the crystals to soak for 48 hours and then, freezing them directly. The solution is on the border of cryoprotection (the conditions has PEG2000MME and PVP and the precipitants), but with native crystals this doesn't seem to be a parameter which affects diffraction. The crystals are very variable in performance, so while I feel that the heavy atom soaking has compromised their diffractability to a degree, inherent variation may play a part. What I was wondering is if some one with more experience than me found themselves in this position, how would they proceed? Questions which spring to mind are, how much heavy atom compound do people add and how long do they soak for? Is there anyway I can squeeze something out of the anomalous data I have, given I have 'reasonable' native data, or will poor quality data give spuriously positive statistics for heavy atom phasing? And are there any tricks people have experienced to improve performance of crystals like these (aside from the usual seeding, additives, different detergents etc which I have spend a fair bit of time on optimization already). Thanks in advance, Rhys
[ccp4bb] Heavy Atom Phasing
Hi All, I thought I might put a question to the community, with the hope of getting some tips of the best way to proceed with my heavy atom phasing problem. I'm working on solving the structure of an integral beta-barrel membrane protein of approximately 100 kDa. I've crystallised protein, growing some very flimsy needle like crystals, and collected datasets to around 3.1 A. I then produced selenomet derivative protein and repeated crystallisation trials in the same conditions and also repeated broad screens, however the derivative protein failed to produce crystals that diffracted beyond 10 A (in fact it barely crystallises at all). So I've moved on to heavy atom soaks and have had some success with tetrachloroplatinate and tetranitroplatinate compounds, in that the crystals didn't dissolve (as they did with gold and samarium compounds) and diffracted to some degree. I collected SAD data to around 6.5 A from these crystals and there seems to be anomolous signal. However, while I get a good CC of 0.4 from HYSS in phenix with this dataset and the phaser EP FOM is 0.56, the maps before and after DM are uninterpretable. I'm guessing the quality and resolution of the data I collected just aren't good enough (the data is reasonably anisotropic). I performed the metal soaking, by taking a small amount of the platinate salt and adding it to the crystallisation drop as the crystals are extremely fragile and don't stand up well to handling through a soaking or cryo solution. Leaving the crystals to soak for 48 hours and then, freezing them directly. The solution is on the border of cryoprotection (the conditions has PEG2000MME and PVP and the precipitants), but with native crystals this doesn't seem to be a parameter which affects diffraction. The crystals are very variable in performance, so while I feel that the heavy atom soaking has compromised their diffractability to a degree, inherent variation may play a part. What I was wondering is if some one with more experience than me found themselves in this position, how would they proceed? Questions which spring to mind are, how much heavy atom compound do people add and how long do they soak for? Is there anyway I can squeeze something out of the anomalous data I have, given I have 'reasonable' native data, or will poor quality data give spuriously positive statistics for heavy atom phasing? And are there any tricks people have experienced to improve performance of crystals like these (aside from the usual seeding, additives, different detergents etc which I have spend a fair bit of time on optimization already). Thanks in advance, Rhys
