Re: [ccp4bb] crystallization incubators
Dear Alexandra, I bought this one recently and it is very nice. Very low vibration and enough space for about 25 24-well plates plus maybe 50 96-well plates. The only problem I had is that in my case the temperature reading on the external LCD display is slightly off compared to the 'real' temperature inside. So its probably best to put one ore two thermometers inside if you really want to be at lets say 21 instead of 22 deg Celsius. best regards Michael Alexandra Deaconescu wrote: Hello everyone: I know this question has been posted before, but what models of crystal growth incubators do you recommend? I was thinking of a smaller benchtop incubator. I have found this from Torrey Pines Scientific, EchoTherm Chilling Incubator, Digital, Bench Top, Programmable, 115VAC, 50/60 Hz, 2 amp. 55 liter capacity. 4 racks provided. Positions for 6. UL, CSA and CE approved. *Price: $3380.00* I was wondering if anyone else knows of something better... Many thanks, Alex Alexandra M. Deaconescu Postdoctoral Fellow Grigorieff Laboratory Brandeis University Rosenstiel Center MS 029 415 South St. Waltham, MA 02454 USA For deliveries: Brandeis University Kalman Dock 415 South St. Waltham, MA 02454 USA
Re: [ccp4bb] How many reflections for Rfree?
Hi Sam As I said, if you have NCS (doesn't matter whether you restrained it or not) you need to be aware that the results may not be accurate, because there are well-documented ways in which NCS can affect Rfree completely unpredictably. With that in mind, if you refer to our Acta D paper http://journals.iucr.org/d/issues/1998/04/00/ad0030/ad0030.pdf and refer to equation 16: y = sqrt((1+ax)/(1-ax)) Here x is the ratio Natoms/Nrefls, y is the expected ratio Rfree/Rwork and a=2 for the case of restrained refinement with 4 variables per atom (x,y,z,B) (see text for explanation), so x = (6100+200)/32585 = 0.19334 and therefore y = sqrt((1+2*0.19334)/(1-2*0.19334)) = 1.5036, so since Rwork = 0.182 we get Rfree = 1.5036*0.182 = 0.274 with an error range at the 3 sigma level as I said of +/- 0.014. So I would say your actual Rfree = 0.267 is pretty well spot-on. Fig 1 in the paper shows the results for other PDB entries so you can compare. Note however that this by itself doesn't prove that your structure is optimal, all we've done is demonstrate that Rfree is (probably) optimal *assuming that the number of parameters that you used is optimal*, i.e. we took your estimate of 6300 atoms at face value. What happens is, assuming always that the refinement has converged (if it hasn't then it's not possible to draw any conclusions from Rfree), Rfree is sensitive (i.e. increases above its optimal minimum value) to both underfitting (either too few parameters, or the wrong choice of parameters, or refinement stuck in a false minimum) and overfitting (too many parameters) with the ideal at the minimum produced by these competing effects. The above is only a test for underfitting - getting the expected Rfree doesn't rule out overfitting (caused for example by being over-enthusiastic with water addition!) simply because it makes the assumption that the number of parameters you used is optimal, hence you must rule that out by verifying that you can't get a lower Rfree with a different parameterisation, say with fewer waters, then repeat the above test. Hope this helps. -- Ian -Original Message- From: U Sam [mailto:[EMAIL PROTECTED] Sent: 22 June 2008 04:47 To: Ian Tickle Subject: RE: [ccp4bb] How many reflections for Rfree? Hi Ian, I have nearly 6100 protein non-hydrogen atoms, 200 waters. I did not use NCS during the refinement although there are two molecule in the asymmetric unit. I use C2 space group. reflections for Rfree are selected randomly in CCP4. Now I believe you can suggest me something how to proceed. Thanks. Sam Date: Sat, 21 Jun 2008 14:42:49 +0100 From: [EMAIL PROTECTED] Subject: Re: [ccp4bb] How many reflections for Rfree? To: CCP4BB@JISCMAIL.AC.UK Hi U Well if you can tell me the total number of atoms in your PDB file (NOT counting any H atoms), I can estimate a range for the expected optimal Rfree assuming your value of Rwork and the no of reflns in your working set (~= 19x1715 = 32585 right?). If you have any NCS I can't promise my estimate will be very accurate, because it will also depend a lot on the nature of the NCS, how you selected your test set, how you restrained the NCS etc. I can already tell you that the range of optimal Rfree (+/- 3 sd's) will be ~ +/- 0.014 (i.e. +/- 3x0.267/sqrt(2x1715)) from whatever is the expected value. Cheers -- Ian -Original Message- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of U Sam Sent: 20 June 2008 21:23 To: Mark J. van Raaij; ccp4bb@jiscmail.ac.uk Subject: RE: [ccp4bb] How many reflections for Rfree? I use CCP4i, refmac5 for the refinement using data of 2.45 angstrom. My R and Rfree is 0.182 and 0.267 respectively. For calculating Rfree ,5% of random data (1715 reflections) was used . So I see there is a difference of about 8.5% between R and Rfree. Is this difference reasonable ? Any idea how can I improve Rfree and difference between R and Rfree gets less than 5%. Thanks, _ Need to know now? Get instant answers with Windows Live Messenger. http://www.windowslive.com/messenger/connect_your_way.html?oci d=TXT_TAGLM_WL_Refresh_messenger_062008 _ The i'm Talkathon starts 6/24/08. For now, give amongst yourselves. http://www.imtalkathon.com?source=TXT_EML_WLH_LearnMore_GiveAmongst Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this
Re: [ccp4bb] query on DNA-protein complex preparation for crystallization
Hi Artem Evdokimov Thank you for the mail. I have synthesized DMT-on oligos in our laboratory. Deprotection was performed treating with ammonium hydroxide for 15 hours at 55C. Then, DMT-on oligo was separated from off using RPHPLC. DMT was cleaved by treating with 20% glacial acetic acid for one hour. Then, DMT-off DNA was separated from DMT, again using RPHPLC. Lyophilized DMT-off oligos were dissolved in 3 mL of Milli Q water and dialysed against 2 L of milli Q water for 4hrs by changing water 2 times. Then complemntary oligos are concentrated around 1.0 mM and mixed them and concentrated further to 1.5 mM (duplex). 2 mM (final concentration) of Magensium chloride was added to oligos and concentrated to half of the volume. While concentrating oligos become viscos and white precipitate. however, annealing did not help to dissolve the white precipitate. I kept oligos in distilled water, without adusting pH. Please can you mail if I iginite DNA on metal spatual, eiether burns or not, what it indicates? Thanking you Rajakumara [EMAIL PROTECTED] wrote: Hi, How did you synthesize the DNA? I assume external vendor (so few people make their own these days)? How was the DNA purified? Sometimes if only a 'desalting' step is used there may be 'other chemicals' in the mix. Also, what pH was your DNA at, and in what buffer (if any)? If your DNA degraded you may have Pi in solution, which forms insoluble precipitates with many counterions. So, first of all I would check your white precipitate - does it dissolve in anything at all? If it does dissolve, what pH does it have? Does it run on an agarose gel? When you ignite a speck of it on a clean metal spatula - does it burn or does it just sit there (and what color does it become). Normally you can prepare DNA-protein complexes in a variety of ways, including direct addition, concentration, counterdialysis, etc. Regards, Artem -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of E rajakumar Sent: Saturday, June 21, 2008 5:48 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] query on DNA-protein complex preparation for crystallization Dear All Sorry for non-crystallography question. I have synthesized two complementary strands of 16 bases in length for making duplex DNA and co-crystallization with DNA binding protein. I have mixed two complementary strands of 1:1 molar ratio (0.5 mM) in water and concentrated to 1.5 mM (Duplex), while concentrating solution becomes viscous and turned to white precipitate. However, adding 2 mM Magnesium chloride followed by annealing (heating at 90C for 10 minutes and followed by cooling to room temperature) did not help to dissolve the white precipitate. Please can you give me suggestions on following queries? 1.How do I dissolve white precipitate? Is increasing divalent cation or keeping duplex in particular pH could help in dissolving the precipitate? 2.How do I prepare DNA-protein complex? I mean, can I mix diluted DNA and protein in 1:1 molar ratio and concentrate further? Any guidance in this regard will be appreciated. Sorry, foregot to mention that any references in this regards will be great help. Thank you in Advance Rajakumara E. Rajakumara Postdoctoral Fellow Strcutural Biology Program Memorial Sloan-Kettering Cancer Center New York-10021 NY 001 212 639 7986 (Lab) 001 917 674 6266 (Mobile) Send instant messages to your online friends http://uk.messenger.yahoo.com E. Rajakumara Postdoctoral Fellow Strcutural Biology Program Memorial Sloan-Kettering Cancer Center New York-10021 NY 001 212 639 7986 (Lab) 001 917 674 6266 (Mobile) Send instant messages to your online friends http://uk.messenger.yahoo.com
Re: [ccp4bb] query on DNA-protein complex preparation for crystallization
Check the purity of the DNA in solution: A(260 nm)/A(280) = 1.8 for fully deprotected DNA, and you should see a nice clean simple curve with a peak very close to 260 nm. Check it on a denaturing gel. Smearing indicates incomplete deprotection. This is usually the cause of solubility problems. Sometimes resuspending in a strong cationic buffer (say 100 mM Tris pH 8.5) might be required. For crystallization it is probably best to have Na+ or K+ as a counterion, rather than Mg++. So you need to dialize against a high concentration of monovalent salt first, not just deionized water. On Jun 22, 2008, at 9:10 AM, E rajakumar wrote: Hi Artem Evdokimov Thank you for the mail. I have synthesized DMT-on oligos in our laboratory. Deprotection was performed treating with ammonium hydroxide for 15 hours at 55C. Then, DMT-on oligo was separated from off using RPHPLC. DMT was cleaved by treating with 20% glacial acetic acid for one hour. Then, DMT-off DNA was separated from DMT, again using RPHPLC. Lyophilized DMT-off oligos were dissolved in 3 mL of Milli Q water and dialysed against 2 L of milli Q water for 4hrs by changing water 2 times. Then complemntary oligos are concentrated around 1.0 mM and mixed them and concentrated further to 1.5 mM (duplex). 2 mM (final concentration) of Magensium chloride was added to oligos and concentrated to half of the volume. While concentrating oligos become viscos and white precipitate. however, annealing did not help to dissolve the white precipitate. I kept oligos in distilled water, without adusting pH. Please can you mail if I iginite DNA on metal spatual, eiether burns or not, what it indicates? Thanking you Rajakumara [EMAIL PROTECTED] wrote: Hi, How did you synthesize the DNA? I assume external vendor (so few people make their own these days)? How was the DNA purified? Sometimes if only a 'desalting' step is used there may be 'other chemicals' in the mix. Also, what pH was your DNA at, and in what buffer (if any)? If your DNA degraded you may have Pi in solution, which forms insoluble precipitates with many counterions. So, first of all I would check your white precipitate - does it dissolve in anything at all? If it does dissolve, what pH does it have? Does it run on an agarose gel? When you ignite a speck of it on a clean metal spatula - does it burn or does it just sit there (and what color does it become). Normally you can prepare DNA-protein complexes in a variety of ways, including direct addition, concentration, counterdialysis, etc. Regards, Artem -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of E rajakumar Sent: Saturday, June 21, 2008 5:48 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] query on DNA-protein complex preparation for crystallization Dear All Sorry for non-crystallography question. I have synthesized two complementary strands of 16 bases in length for making duplex DNA and co-crystallization with DNA binding protein. I have mixed two complementary strands of 1:1 molar ratio (0.5 mM) in water and concentrated to 1.5 mM (Duplex), while concentrating solution becomes viscous and turned to white precipitate. However, adding 2 mM Magnesium chloride followed by annealing (heating at 90C for 10 minutes and followed by cooling to room temperature) did not help to dissolve the white precipitate. Please can you give me suggestions on following queries? 1.How do I dissolve white precipitate? Is increasing divalent cation or keeping duplex in particular pH could help in dissolving the precipitate? 2.How do I prepare DNA-protein complex? I mean, can I mix diluted DNA and protein in 1:1 molar ratio and concentrate further? Any guidance in this regard will be appreciated. Sorry, foregot to mention that any references in this regards will be great help. Thank you in Advance Rajakumara E. Rajakumara Postdoctoral Fellow Strcutural Biology Program Memorial Sloan-Kettering Cancer Center New York-10021 NY 001 212 639 7986 (Lab) 001 917 674 6266 (Mobile) Send instant messages to your online friends http://uk.messenger.yahoo.com E. Rajakumara Postdoctoral Fellow Strcutural Biology Program Memorial Sloan-Kettering Cancer Center New York-10021 NY 001 212 639 7986 (Lab) 001 917 674 6266 (Mobile) Send instant messages to your online friends http://uk.messenger.yahoo.com
Re: [ccp4bb] query on DNA-protein complex preparation for crystallization
Hi Thank you for the mail. It seems your correct. A(260 nm)/A(280) of one oligo is around 1.0 and peak is around 272. Other oligo's(260 nm)/A(280) is around 1.5. Can I know what is the absorbance peak of base protecting N-benzoyl group. Is it possible to do deprotection of base after mixing complementary strands? Can you suggest me what is the volume of ammonium hydroxide will be used for 1uM oligo of 16 bases in length and how much time heating shoul be done? thanking you rajakumara --- William Scott [EMAIL PROTECTED] wrote: Check the purity of the DNA in solution: A(260 nm)/A(280) = 1.8 for fully deprotected DNA, and you should see a nice clean simple curve with a peak very close to 260 nm. Check it on a denaturing gel. Smearing indicates incomplete deprotection. This is usually the cause of solubility problems. Sometimes resuspending in a strong cationic buffer (say 100 mM Tris pH 8.5) might be required. For crystallization it is probably best to have Na+ or K+ as a counterion, rather than Mg++. So you need to dialize against a high concentration of monovalent salt first, not just deionized water. On Jun 22, 2008, at 9:10 AM, E rajakumar wrote: Hi Artem Evdokimov Thank you for the mail. I have synthesized DMT-on oligos in our laboratory. Deprotection was performed treating with ammonium hydroxide for 15 hours at 55C. Then, DMT-on oligo was separated from off using RPHPLC. DMT was cleaved by treating with 20% glacial acetic acid for one hour. Then, DMT-off DNA was separated from DMT, again using RPHPLC. Lyophilized DMT-off oligos were dissolved in 3 mL of Milli Q water and dialysed against 2 L of milli Q water for 4hrs by changing water 2 times. Then complemntary oligos are concentrated around 1.0 mM and mixed them and concentrated further to 1.5 mM (duplex). 2 mM (final concentration) of Magensium chloride was added to oligos and concentrated to half of the volume. While concentrating oligos become viscos and white precipitate. however, annealing did not help to dissolve the white precipitate. I kept oligos in distilled water, without adusting pH. Please can you mail if I iginite DNA on metal spatual, eiether burns or not, what it indicates? Thanking you Rajakumara [EMAIL PROTECTED] wrote: Hi, How did you synthesize the DNA? I assume external vendor (so few people make their own these days)? How was the DNA purified? Sometimes if only a 'desalting' step is used there may be 'other chemicals' in the mix. Also, what pH was your DNA at, and in what buffer (if any)? If your DNA degraded you may have Pi in solution, which forms insoluble precipitates with many counterions. So, first of all I would check your white precipitate - does it dissolve in anything at all? If it does dissolve, what pH does it have? Does it run on an agarose gel? When you ignite a speck of it on a clean metal spatula - does it burn or does it just sit there (and what color does it become). Normally you can prepare DNA-protein complexes in a variety of ways, including direct addition, concentration, counterdialysis, etc. Regards, Artem -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of E rajakumar Sent: Saturday, June 21, 2008 5:48 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] query on DNA-protein complex preparation for crystallization Dear All Sorry for non-crystallography question. I have synthesized two complementary strands of 16 bases in length for making duplex DNA and co-crystallization with DNA binding protein. I have mixed two complementary strands of 1:1 molar ratio (0.5 mM) in water and concentrated to 1.5 mM (Duplex), while concentrating solution becomes viscous and turned to white precipitate. However, adding 2 mM Magnesium chloride followed by annealing (heating at 90C for 10 minutes and followed by cooling to room temperature) did not help to dissolve the white precipitate. Please can you give me suggestions on following queries? 1.How do I dissolve white precipitate? Is increasing divalent cation or keeping duplex in particular pH could help in dissolving the precipitate? 2.How do I prepare DNA-protein complex? I mean, can I mix diluted DNA and protein in 1:1 molar ratio and concentrate further? Any guidance in this regard will be appreciated. Sorry, foregot to mention that any references in this regards will be great help. Thank you in Advance Rajakumara E. Rajakumara Postdoctoral Fellow Strcutural Biology Program Memorial Sloan-Kettering Cancer Center New York-10021 NY 001 212 639 7986 (Lab) 001 917 674 6266 (Mobile) Send instant messages to your online friends http://uk.messenger.yahoo.com E. Rajakumara Postdoctoral Fellow
Re: [ccp4bb] query on DNA-protein complex preparation for crystallization
I think that at this point you're better off looking at a professional literature. For example: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=102833 Artem -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of E rajakumar Sent: Sunday, June 22, 2008 1:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] query on DNA-protein complex preparation for crystallization Hi Thank you for the mail. It seems your correct. A(260 nm)/A(280) of one oligo is around 1.0 and peak is around 272. Other oligo's(260 nm)/A(280) is around 1.5. Can I know what is the absorbance peak of base protecting N-benzoyl group. Is it possible to do deprotection of base after mixing complementary strands? Can you suggest me what is the volume of ammonium hydroxide will be used for 1uM oligo of 16 bases in length and how much time heating shoul be done? thanking you rajakumara --- William Scott [EMAIL PROTECTED] wrote: Check the purity of the DNA in solution: A(260 nm)/A(280) = 1.8 for fully deprotected DNA, and you should see a nice clean simple curve with a peak very close to 260 nm. Check it on a denaturing gel. Smearing indicates incomplete deprotection. This is usually the cause of solubility problems. Sometimes resuspending in a strong cationic buffer (say 100 mM Tris pH 8.5) might be required. For crystallization it is probably best to have Na+ or K+ as a counterion, rather than Mg++. So you need to dialize against a high concentration of monovalent salt first, not just deionized water. On Jun 22, 2008, at 9:10 AM, E rajakumar wrote: Hi Artem Evdokimov Thank you for the mail. I have synthesized DMT-on oligos in our laboratory. Deprotection was performed treating with ammonium hydroxide for 15 hours at 55C. Then, DMT-on oligo was separated from off using RPHPLC. DMT was cleaved by treating with 20% glacial acetic acid for one hour. Then, DMT-off DNA was separated from DMT, again using RPHPLC. Lyophilized DMT-off oligos were dissolved in 3 mL of Milli Q water and dialysed against 2 L of milli Q water for 4hrs by changing water 2 times. Then complemntary oligos are concentrated around 1.0 mM and mixed them and concentrated further to 1.5 mM (duplex). 2 mM (final concentration) of Magensium chloride was added to oligos and concentrated to half of the volume. While concentrating oligos become viscos and white precipitate. however, annealing did not help to dissolve the white precipitate. I kept oligos in distilled water, without adusting pH. Please can you mail if I iginite DNA on metal spatual, eiether burns or not, what it indicates? Thanking you Rajakumara [EMAIL PROTECTED] wrote: Hi, How did you synthesize the DNA? I assume external vendor (so few people make their own these days)? How was the DNA purified? Sometimes if only a 'desalting' step is used there may be 'other chemicals' in the mix. Also, what pH was your DNA at, and in what buffer (if any)? If your DNA degraded you may have Pi in solution, which forms insoluble precipitates with many counterions. So, first of all I would check your white precipitate - does it dissolve in anything at all? If it does dissolve, what pH does it have? Does it run on an agarose gel? When you ignite a speck of it on a clean metal spatula - does it burn or does it just sit there (and what color does it become). Normally you can prepare DNA-protein complexes in a variety of ways, including direct addition, concentration, counterdialysis, etc. Regards, Artem -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of E rajakumar Sent: Saturday, June 21, 2008 5:48 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] query on DNA-protein complex preparation for crystallization Dear All Sorry for non-crystallography question. I have synthesized two complementary strands of 16 bases in length for making duplex DNA and co-crystallization with DNA binding protein. I have mixed two complementary strands of 1:1 molar ratio (0.5 mM) in water and concentrated to 1.5 mM (Duplex), while concentrating solution becomes viscous and turned to white precipitate. However, adding 2 mM Magnesium chloride followed by annealing (heating at 90C for 10 minutes and followed by cooling to room temperature) did not help to dissolve the white precipitate. Please can you give me suggestions on following queries? 1.How do I dissolve white precipitate? Is increasing divalent cation or keeping duplex in particular pH could help in dissolving the precipitate? 2.How do I prepare DNA-protein complex? I mean, can I mix diluted DNA and protein in 1:1 molar ratio and concentrate further? Any guidance in this regard will be appreciated. Sorry, foregot to mention that any
