Re: [ccp4bb] effective iodide conc. for SAD data
Jacob, The main reason why it is not common practice to saturate every crystal with every heavy metal under the sun is radiation damage. X-ray absorption increases very rapidly with atomic number (third power), so on the order of 100 mM of heavy atom is usually enough to cut your crystal's useful life in half. It doesn't matter if the heavy atom is in the lattice or not. At 12 keV the photoelectron cascade crashes through about 3 microns worth of organic matter before finally coming to rest (Cole, 1969). The program RADDOSE (Murray et al. 2004, 2005, Paithankar et al 2009, 2010) was written to calculate expected crystal lifetime given your buffer concentrations, etc. For example, with a 50% solvent crystal, 250 mM iodide in the solvent channels is dose doubling (life halving). That is, the number of photons you will get scattered into spots before the crystal is dead will be half that which you would get with no iodine. This is because the iodine-soaked crystal is absorbing twice as much energy per incident photon, but scattering at pretty much the same rate with or without the iodine. 500 mM iodine will cut the useful life to 1/3 of what it would be with no iodine, and 1 M will cut it to 1/5th. (250/([iodine]+250)). For Cs, 200 mM is the dose-doubling buffer concentration. For Rb it is 1 M. For Cl, it is 2.4 M, and for fluoride there is no concentration that doubles the dose because water absorbs more than fouoride. This is why I recommend using low-Z buffers in crystallography whenever possible. I have listed dose-doubling concentrations of a few common elements on the last page of this document: http://bl831.als.lbl.gov/damage_rates.pdf and a fairly comprehensive look up table is here: http://bl831.als.lbl.gov/~jamesh/pickup/dose_doubler_solc50.txt The dose doubling concentration does depend on the wavelength. The worst one at 1 A is tungsten (57 mM), but this is not all that different from tantalum (62 mM) or mercury (74 mM), or gold (76 mM), or uranium (105 mM). So, by and large ~100 mM concentration of anything heavy will double the dose per scattered photon. Note that this is the concentration of the atoms, not molecules. The dose-doubling concentration of Ta6Br12 clusters is 9.5 mM at 1 A. As for the original poster's question, 20 mM iodide disrupting your crystallization is actually a good sign that it's binding, but the bad news is that it appears your crystals don't like that. Nevertheless it could be worth a shot, you can estimate your expected Bijvoet ratio assuming one site per molecule using this little web app I wrote: http://bl831.als.lbl.gov/xtalsize.html In general, a Bijvoet ratio of 3% or so is needed to solve a structure (the current world record is 0.5% and lots of multiplicity). The above web page will also tell you how many crystals you need if you type in their size in all three dimensions. but this estimate assumes that you don't have high concentrations of heavy metals in your solution! So, if it says you can get away with one crystal but you know your have a dose-doubling concentration of something, then you're going to need to average data from two crystals, etc. Good luck! -James Holton MAD Scientist On 5/3/2012 8:29 AM, Jacob Keller wrote: I have wondered for a long time now why it is not standard practice for all crystallization protein stocks to contain either Br- or I- ions instead of Cl-, even for cationic buffers like TRIS, which could be titrated with HBr or HI to get in the 10+ mM range. Also, one could use Cs or Rb for the cations (and titrate anionic buffers with the respective hydroxides). What's there to lose? The gain is obviously the possible anomalous signal (always helpful), and one might pick up additional interesting and possibly physiologically-relevant halide or alkali metal sites. Seems that structural genomics people might standardize this into the pipeline as well, and thereby potentially cut out SeMet protein production in many if not most cases. JPK On Thu, May 3, 2012 at 9:05 AM, Jan Abendroth jan.abendr...@gmail.com mailto:jan.abendr...@gmail.com wrote: Hi Rajesh, it can be a bit all over the place: For quick soaks, we typically use 500mM-1000mM. A good starting point might be to simply replace the NaCl concentration in your protein buffer. By some serendipity we also managed to solve a structure by I/S SAD after a 1mM NaI soak. One iodide had found its way into a nice binding pocket. For co-crystallization, mostly 200mM should be fine. Another approach could be to supplement your cryo buffer with iodide, replacing NaCl. NaI is highly soluble in ethylene glycol. Also see here: http://www.ncbi.nlm.nih.gov/pubmed/21359836 Good luck! Cheers, Jan -- Jan Abendroth Emerald Bio Seattle / Bainbridge Island WA, USA home: Jan.Abendroth_at_gmail.com http://Jan.Abendroth_at_gmail.com work:
Re: [ccp4bb] effective iodide conc. for SAD data
James Holton wrote: In general, a Bijvoet ratio of 3% or so is needed to solve a structure (the current world record is 0.5% and lots of multiplicity). The above web page will also tell you how many crystals you need if you type in their size in all three dimensions. but this estimate assumes that you don't have high concentrations of heavy metals in your solution! So, if it says you can get away with one crystal but you know your have a dose-doubling concentration of something, then you're going to need to average data from two crystals, etc. I don't know of any systematic studies, but in my experience if you've got weak anomalous signal you're better off with multi-crystal phasing than multi-crystal merging. Pete
[ccp4bb] effective iodide conc. for SAD data
Dear All, I have very thin crystals but diffracting. I was not able to handle them easily for iodide soak. I always lost the crystals during manipulation and other big crystals obtained after seeding doesn't even give any diffraction. I tried for co-crystallizing with NaI. The crystals appear only up to 20 mM in 1:2 (3ul drop of 1 ul protein and 2ul reservoir).Is this concentration of iodide is enough for SAD data ( if it had good incorporation) ?I appreciate your help. ThanksRajesh
Re: [ccp4bb] effective iodide conc. for SAD data
Hi Rajesh, it can be a bit all over the place: For quick soaks, we typically use 500mM-1000mM. A good starting point might be to simply replace the NaCl concentration in your protein buffer. By some serendipity we also managed to solve a structure by I/S SAD after a 1mM NaI soak. One iodide had found its way into a nice binding pocket. For co-crystallization, mostly 200mM should be fine. Another approach could be to supplement your cryo buffer with iodide, replacing NaCl. NaI is highly soluble in ethylene glycol. Also see here: http://www.ncbi.nlm.nih.gov/pubmed/21359836 Good luck! Cheers, Jan -- Jan Abendroth Emerald Bio Seattle / Bainbridge Island WA, USA home: Jan.Abendroth_at_gmail.com work: JAbendroth_at_embios.com http://www.emeraldbiostructures.com On May 3, 2012, at 6:51 AM, Rajesh Kumar wrote: Dear All, I have very thin crystals but diffracting. I was not able to handle them easily for iodide soak. I always lost the crystals during manipulation and other big crystals obtained after seeding doesn't even give any diffraction. I tried for co-crystallizing with NaI. The crystals appear only up to 20 mM in 1:2 (3ul drop of 1 ul protein and 2ul reservoir). Is this concentration of iodide is enough for SAD data ( if it had good incorporation) ? I appreciate your help. Thanks Rajesh
Re: [ccp4bb] effective iodide conc. for SAD data
Iodide is a fantastic derivative. One does not need a lot with modern X-ray equipment, careful data collection, and great software. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Rajesh Kumar [ccp4...@hotmail.com] Sent: Thursday, May 03, 2012 8:51 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] effective iodide conc. for SAD data Dear All, I have very thin crystals but diffracting. I was not able to handle them easily for iodide soak. I always lost the crystals during manipulation and other big crystals obtained after seeding doesn't even give any diffraction. I tried for co-crystallizing with NaI. The crystals appear only up to 20 mM in 1:2 (3ul drop of 1 ul protein and 2ul reservoir). Is this concentration of iodide is enough for SAD data ( if it had good incorporation) ? I appreciate your help. Thanks Rajesh
Re: [ccp4bb] effective iodide conc. for SAD data
Rajesh, Why not try a soak-in-place by adding iodide and/or cryoprotectant directly to the crystallization drop? Then you only have to fish out the crystal once, minimizing handling. I usually do this by preparing an artificial mother liquor solution that has 125% of the final desired concentration of cryoprotectant and/or ligand. Add 4 volumes of this solution to your crystallization drop all at once or in stages. (I usually add 0.25, 0.25, 0.5, 1.0, and 2.0 drop volumes a few minutes apart). The drop can be incubated over its reservoir well during this procedure between additions to minimize evaporation. This often works well for fragile crystals or crystals that are sensitive to evaporation of the drop. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline 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 On 5/3/2012 9:51 AM, Rajesh Kumar wrote: Dear All, I have very thin crystals but diffracting. I was not able to handle them easily for iodide soak. I always lost the crystals during manipulation and other big crystals obtained after seeding doesn't even give any diffraction. I tried for co-crystallizing with NaI. The crystals appear only up to 20 mM in 1:2 (3ul drop of 1 ul protein and 2ul reservoir). Is this concentration of iodide is enough for SAD data ( if it had good incorporation) ? I appreciate your help. Thanks Rajesh
Re: [ccp4bb] effective iodide conc. for SAD data
I have wondered for a long time now why it is not standard practice for all crystallization protein stocks to contain either Br- or I- ions instead of Cl-, even for cationic buffers like TRIS, which could be titrated with HBr or HI to get in the 10+ mM range. Also, one could use Cs or Rb for the cations (and titrate anionic buffers with the respective hydroxides). What's there to lose? The gain is obviously the possible anomalous signal (always helpful), and one might pick up additional interesting and possibly physiologically-relevant halide or alkali metal sites. Seems that structural genomics people might standardize this into the pipeline as well, and thereby potentially cut out SeMet protein production in many if not most cases. JPK On Thu, May 3, 2012 at 9:05 AM, Jan Abendroth jan.abendr...@gmail.comwrote: Hi Rajesh, it can be a bit all over the place: For quick soaks, we typically use 500mM-1000mM. A good starting point might be to simply replace the NaCl concentration in your protein buffer. By some serendipity we also managed to solve a structure by I/S SAD after a 1mM NaI soak. One iodide had found its way into a nice binding pocket. For co-crystallization, mostly 200mM should be fine. Another approach could be to supplement your cryo buffer with iodide, replacing NaCl. NaI is highly soluble in ethylene glycol. Also see here: http://www.ncbi.nlm.nih.gov/pubmed/21359836 Good luck! Cheers, Jan -- Jan Abendroth Emerald Bio Seattle / Bainbridge Island WA, USA home: Jan.Abendroth_at_gmail.com work: JAbendroth_at_embios.com http://www.emeraldbiostructures.com On May 3, 2012, at 6:51 AM, Rajesh Kumar wrote: Dear All, I have very thin crystals but diffracting. I was not able to handle them easily for iodide soak. I always lost the crystals during manipulation and other big crystals obtained after seeding doesn't even give any diffraction. I tried for co-crystallizing with NaI. The crystals appear only up to 20 mM in 1:2 (3ul drop of 1 ul protein and 2ul reservoir). Is this concentration of iodide is enough for SAD data ( if it had good incorporation) ? I appreciate your help. Thanks Rajesh -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] effective iodide conc. for SAD data
Interesting idea. The only caveat that springs to mind is that the more useful anions (e.g., iodide and bromide) are on the chaotropic end of the Hofmeister series and may potentially destabilize protein structure or protein-protein interactions, which might complicate co-crystallization starting from known conditions, especially at higher concentrations of anion. Alternate cations may be less problematic. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline 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 On 5/3/2012 11:29 AM, Jacob Keller wrote: I have wondered for a long time now why it is not standard practice for all crystallization protein stocks to contain either Br- or I- ions instead of Cl-, even for cationic buffers like TRIS, which could be titrated with HBr or HI to get in the 10+ mM range. Also, one could use Cs or Rb for the cations (and titrate anionic buffers with the respective hydroxides). What's there to lose? The gain is obviously the possible anomalous signal (always helpful), and one might pick up additional interesting and possibly physiologically-relevant halide or alkali metal sites. Seems that structural genomics people might standardize this into the pipeline as well, and thereby potentially cut out SeMet protein production in many if not most cases. JPK On Thu, May 3, 2012 at 9:05 AM, Jan Abendroth jan.abendr...@gmail.com mailto:jan.abendr...@gmail.com wrote: Hi Rajesh, it can be a bit all over the place: For quick soaks, we typically use 500mM-1000mM. A good starting point might be to simply replace the NaCl concentration in your protein buffer. By some serendipity we also managed to solve a structure by I/S SAD after a 1mM NaI soak. One iodide had found its way into a nice binding pocket. For co-crystallization, mostly 200mM should be fine. Another approach could be to supplement your cryo buffer with iodide, replacing NaCl. NaI is highly soluble in ethylene glycol. Also see here: http://www.ncbi.nlm.nih.gov/pubmed/21359836 Good luck! Cheers, Jan -- Jan Abendroth Emerald Bio Seattle / Bainbridge Island WA, USA home: Jan.Abendroth_at_gmail.com http://Jan.Abendroth_at_gmail.com work: JAbendroth_at_embios.com http://www.emeraldbiostructures.com On May 3, 2012, at 6:51 AM, Rajesh Kumar wrote: Dear All, I have very thin crystals but diffracting. I was not able to handle them easily for iodide soak. I always lost the crystals during manipulation and other big crystals obtained after seeding doesn't even give any diffraction. I tried for co-crystallizing with NaI. The crystals appear only up to 20 mM in 1:2 (3ul drop of 1 ul protein and 2ul reservoir). Is this concentration of iodide is enough for SAD data ( if it had good incorporation) ? I appreciate your help. Thanks Rajesh -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu mailto:j-kell...@northwestern.edu ***
Re: [ccp4bb] effective iodide conc. for SAD data
Please see my poster at the ACA 2012 meeting. See also: (1) Dauter, Z., Dauter, M. Rajashankar, K.R. (2000) Acta Cryst. D56, 232-237. (2) Nagem, R.A.P, Dauter, Z. Polikarpov, I. (2001) Acta Cryst. D57, 996-1002. :) From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Roger Rowlett [rrowl...@colgate.edu] Sent: Thursday, May 03, 2012 11:00 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] effective iodide conc. for SAD data Interesting idea. The only caveat that springs to mind is that the more useful anions (e.g., iodide and bromide) are on the chaotropic end of the Hofmeister series and may potentially destabilize protein structure or protein-protein interactions, which might complicate co-crystallization starting from known conditions, especially at higher concentrations of anion. Alternate cations may be less problematic. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline 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.edumailto:rrowl...@colgate.edu On 5/3/2012 11:29 AM, Jacob Keller wrote: I have wondered for a long time now why it is not standard practice for all crystallization protein stocks to contain either Br- or I- ions instead of Cl-, even for cationic buffers like TRIS, which could be titrated with HBr or HI to get in the 10+ mM range. Also, one could use Cs or Rb for the cations (and titrate anionic buffers with the respective hydroxides). What's there to lose? The gain is obviously the possible anomalous signal (always helpful), and one might pick up additional interesting and possibly physiologically-relevant halide or alkali metal sites. Seems that structural genomics people might standardize this into the pipeline as well, and thereby potentially cut out SeMet protein production in many if not most cases. JPK On Thu, May 3, 2012 at 9:05 AM, Jan Abendroth jan.abendr...@gmail.commailto:jan.abendr...@gmail.com wrote: Hi Rajesh, it can be a bit all over the place: For quick soaks, we typically use 500mM-1000mM. A good starting point might be to simply replace the NaCl concentration in your protein buffer. By some serendipity we also managed to solve a structure by I/S SAD after a 1mM NaI soak. One iodide had found its way into a nice binding pocket. For co-crystallization, mostly 200mM should be fine. Another approach could be to supplement your cryo buffer with iodide, replacing NaCl. NaI is highly soluble in ethylene glycol. Also see here: http://www.ncbi.nlm.nih.gov/pubmed/21359836 Good luck! Cheers, Jan -- Jan Abendroth Emerald Bio Seattle / Bainbridge Island WA, USA home: Jan.Abendroth_at_gmail.comhttp://Jan.Abendroth_at_gmail.com work: JAbendroth_at_embios.com http://www.emeraldbiostructures.com On May 3, 2012, at 6:51 AM, Rajesh Kumar wrote: Dear All, I have very thin crystals but diffracting. I was not able to handle them easily for iodide soak. I always lost the crystals during manipulation and other big crystals obtained after seeding doesn't even give any diffraction. I tried for co-crystallizing with NaI. The crystals appear only up to 20 mM in 1:2 (3ul drop of 1 ul protein and 2ul reservoir). Is this concentration of iodide is enough for SAD data ( if it had good incorporation) ? I appreciate your help. Thanks Rajesh -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu ***
Re: [ccp4bb] effective iodide conc. for SAD data
Dear Rajesh, another method to incorporate Iodine into your crystal is by simply placing a drop of KI/I2 solution next to the crystallization drop. Have a look here: Acta Cryst. (2006). D62, 280-289 New methods to prepare iodinated derivatives by vaporizing iodine labelling (VIL) and hydrogen peroxide VIL (HYPER-VIL) Worked for me with salt concentration sensitive crystal and a derivatization time 10 min. Good luck, Florian Am 03.05.12 15:51, schrieb Rajesh Kumar: Dear All, I have very thin crystals but diffracting. I was not able to handle them easily for iodide soak. I always lost the crystals during manipulation and other big crystals obtained after seeding doesn't even give any diffraction. I tried for co-crystallizing with NaI. The crystals appear only up to 20 mM in 1:2 (3ul drop of 1 ul protein and 2ul reservoir). Is this concentration of iodide is enough for SAD data ( if it had good incorporation) ? I appreciate your help. Thanks Rajesh
Re: [ccp4bb] effective iodide conc. for SAD data
Did you locate the Iodine(s)? Did you have iodotyrosine, or I^- bound at anion binding sites? There are two distinct methods based on I2/I-. Florian Sauer wrote: Dear Rajesh, another method to incorporate Iodine into your crystal is by simply placing a drop of KI/I2 solution next to the crystallization drop. Have a look here: Acta Cryst. (2006). D62, 280-289 New methods to prepare iodinated derivatives by vaporizing iodine labelling (VIL) and hydrogen peroxide VIL (HYPER-VIL) Worked for me with salt concentration sensitive crystal and a derivatization time 10 min. Good luck, Florian Am 03.05.12 15:51, schrieb Rajesh Kumar: Dear All, I have very thin crystals but diffracting. I was not able to handle them easily for iodide soak. I always lost the crystals during manipulation and other big crystals obtained after seeding doesn't even give any diffraction. I tried for co-crystallizing with NaI. The crystals appear only up to 20 mM in 1:2 (3ul drop of 1 ul protein and 2ul reservoir). Is this concentration of iodide is enough for SAD data ( if it had good incorporation) ? I appreciate your help. Thanks Rajesh
Re: [ccp4bb] effective iodide conc. for SAD data
Yes I did and could solve the structure by SAD. In total 8 I atoms found. 6 bound at anion binding sites. 2 as I2. None covalently bound to tyrosine. Florian Am 03.05.12 19:42, schrieb Edward A. Berry: Did you locate the Iodine(s)? Did you have iodotyrosine, or I^- bound at anion binding sites? There are two distinct methods based on I2/I-. Florian Sauer wrote: Dear Rajesh, another method to incorporate Iodine into your crystal is by simply placing a drop of KI/I2 solution next to the crystallization drop. Have a look here: Acta Cryst. (2006). D62, 280-289 New methods to prepare iodinated derivatives by vaporizing iodine labelling (VIL) and hydrogen peroxide VIL (HYPER-VIL) Worked for me with salt concentration sensitive crystal and a derivatization time 10 min. Good luck, Florian Am 03.05.12 15:51, schrieb Rajesh Kumar: Dear All, I have very thin crystals but diffracting. I was not able to handle them easily for iodide soak. I always lost the crystals during manipulation and other big crystals obtained after seeding doesn't even give any diffraction. I tried for co-crystallizing with NaI. The crystals appear only up to 20 mM in 1:2 (3ul drop of 1 ul protein and 2ul reservoir). Is this concentration of iodide is enough for SAD data ( if it had good incorporation) ? I appreciate your help. Thanks Rajesh
Re: [ccp4bb] effective iodide conc. for SAD data
Dear All, I really thank everyone who gave me excellent suggestions. I will try them to see which one works better for me and my crystals. Again thanks a lot for your help. Regards, Rajesh Date: Thu, 3 May 2012 20:56:21 +0200 From: florian.sa...@embl-hamburg.de Subject: Re: [ccp4bb] effective iodide conc. for SAD data To: CCP4BB@JISCMAIL.AC.UK Yes I did and could solve the structure by SAD. In total 8 I atoms found. 6 bound at anion binding sites. 2 as I2. None covalently bound to tyrosine. Florian Am 03.05.12 19:42, schrieb Edward A. Berry: Did you locate the Iodine(s)? Did you have iodotyrosine, or I^- bound at anion binding sites? There are two distinct methods based on I2/I-. Florian Sauer wrote: Dear Rajesh, another method to incorporate Iodine into your crystal is by simply placing a drop of KI/I2 solution next to the crystallization drop. Have a look here: Acta Cryst. (2006). D62, 280-289 New methods to prepare iodinated derivatives by vaporizing iodine labelling (VIL) and hydrogen peroxide VIL (HYPER-VIL) Worked for me with salt concentration sensitive crystal and a derivatization time 10 min. Good luck, Florian Am 03.05.12 15:51, schrieb Rajesh Kumar: Dear All, I have very thin crystals but diffracting. I was not able to handle them easily for iodide soak. I always lost the crystals during manipulation and other big crystals obtained after seeding doesn't even give any diffraction. I tried for co-crystallizing with NaI. The crystals appear only up to 20 mM in 1:2 (3ul drop of 1 ul protein and 2ul reservoir). Is this concentration of iodide is enough for SAD data ( if it had good incorporation) ? I appreciate your help. Thanks Rajesh
