Re: [ccp4bb] [Err] Re: [ccp4bb] minimum acceptable sigma level for very small ligand and more
Thanks all for your replies. I am attaching links of the active site view of both Native data https://www.dropbox.com/s/1n7a0nf59rc5bna/NativeNoSoak.png?m=(without any ligand soaked, at 1.7 angs.) and the soaked datahttps://www.dropbox.com/s/5s5ao486fg0op8p/Soaked.png?m=(1.9 angs, but the ligand has not been modelled). *Native crystal and soaked crystal were grown in identical conditions* and the map quality is good for both data, except for 7 residues in a loop and the N and C terminus. *The 2Fo-Fc level for the attached maps is 0.7 and the Fo-Fc for both maps is 3.0*. The sigma level I am talking about is of coot. I also checked the difference map for the soaked data one by one and found no other place where the ligand could fit. My ligand fits, even though not perfectly, in the density shown in the attachmenthttps://www.dropbox.com/s/5s5ao486fg0op8p/Soaked.png?m=. Looks like something is there but to see that clearly I have to go below 0.7 sigma. I was not sure if this is something acceptable and hence this mail. I will be trying Buster and the Rapid map (of the PNAS paper mentioned earlier in the thread) and let you know if the map quality improves. Amit 2014-03-19 22:13 GMT+05:30 spam_mas...@korea.ac.kr: Transmit Report: wc_2...@korea.ac.kr에게 메일 발송을 5번 시도했지만 실패하였습니다. (실패 이유 : 554 Transaction failed. 402 Local User Inbox Full ( wc_2...@korea.ac.kr) 10,61440,61437(163.152.6.98)) 참고 실패 이유에 대한 설명 User unknown :메일을 수신할 사용자가 존재하지 않음 Socket connect fail:수신 메일 서버와 연결 실패 DATA write fail:수신 메일 서버로 메세지 송신 실패 DATA reponse fail :수신 메일 서버로부터 메세지 수신 실패 Final-Recipient: rfc822;wc_2...@korea.ac.kr Diagnostic-Code: smtp; 554 error - Transaction failed. 402 Local User Inbox Full (wc_2...@korea.ac.kr) 10,61440,61437(163.152.6.98) Action: failed Status: 4.0.0 -- Forwarded message -- From: Amit Kumar amit7urmcc...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Cc: Date: Wed, 19 Mar 2014 21:46:13 +0530 Subject: Re: [ccp4bb] minimum acceptable sigma level for very small ligand and more Before putting in the ligand, there is a clear density for three extra atoms at more than 3 sigma in the Fo-Fc map but for other atoms the density appears around those 3 peaks in the 2Fo-Fc map only and not in the Fo-Fc. Thanks for your reply. Amit On Wed, Mar 19, 2014 at 8:57 PM, Sampson, Jared jared.samp...@nyumc.orgwrote: Hi Amit - I don’t know of a universally accepted minimum sigma level, but I tend not to attempt to fit anything that doesn’t have clearly interpretable density at 1.0 sigma (in a 2Fo-Fc map). A map level of 0.5-0.6 sigma is very low, and to me, it’s very likely that you’re seeing what you want to see and fitting your ligand into noise. Is there any positive Fo-Fc density (greater than about 2-3 sigma) at that location if you remove the ligand? Do you see an appreciable decrease in Rfree when you add the ligand? That would be an indicator to me that you have placed it correctly. Especially if identification of the ligand binding site is the primary purpose of this structure, I think you need stronger evidence of its presence. You may also want to try co-crystallization, which may reduce the effect a full soak has on the diffraction of the crystal. Cheers, Jared -- Jared Sampson Xiangpeng Kong Lab NYU Langone Medical Center 550 First Avenue New York, NY 10016 212-263-7898 http://kong.med.nyu.edu/ On Mar 19, 2014, at 10:39 AM, Amit Kumar amit7urmcc...@gmail.com wrote: Hello, My protein is 26 kDa and the resolution of the data is 1.90 angs. My ligand is 174 Daltons. and it was soaked into the crystal. Ligand is colored and the crystal after soaking takes up intense color. However if we soak more than optimum, the color deepens in intensity but the crystal diffracts no more. So perhaps the ligand's occupancy can not be the 1.00. After model building I see ligand density, starting to appear at 0.7 sigma and clear at 0.5-0.6 sigma, close to the protein residue where it should bind. Occupancy is ~0.6 after the refinement and B factors for the atoms of the ligand range from 30-80. Questions I have (1) What is the acceptable sigma level for very small ligands for peer review/publication? (2) I did refinement by Refmac and by Phenix refine, separately. The map quality for the ligand is better after the refmac refinement than after the Phenix refinement. Why is such a difference and which one should I trust? I used mostly default parameters for both (Phenix and Refmac) before the refinement. Thanks for your time. Amit This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error
[ccp4bb] minimum acceptable sigma level for very small ligand and more
Hello, My protein is 26 kDa and the resolution of the data is 1.90 angs. My ligand is 174 Daltons. and it was soaked into the crystal. Ligand is colored and the crystal after soaking takes up intense color. However if we soak more than optimum, the color deepens in intensity but the crystal diffracts no more. So perhaps the ligand's occupancy can not be the 1.00. After model building I see ligand density, starting to appear at 0.7 sigma and clear at 0.5-0.6 sigma, close to the protein residue where it should bind. Occupancy is ~0.6 after the refinement and B factors for the atoms of the ligand range from 30-80. Questions I have (1) What is the acceptable sigma level for very small ligands for peer review/publication? (2) I did refinement by Refmac and by Phenix refine, separately. The map quality for the ligand is better after the refmac refinement than after the Phenix refinement. Why is such a difference and which one should I trust? I used mostly default parameters for both (Phenix and Refmac) before the refinement. Thanks for your time. Amit
Re: [ccp4bb] minimum acceptable sigma level for very small ligand and more
Before putting in the ligand, there is a clear density for three extra atoms at more than 3 sigma in the Fo-Fc map but for other atoms the density appears around those 3 peaks in the 2Fo-Fc map only and not in the Fo-Fc. Thanks for your reply. Amit On Wed, Mar 19, 2014 at 8:57 PM, Sampson, Jared jared.samp...@nyumc.orgwrote: Hi Amit - I don't know of a universally accepted minimum sigma level, but I tend not to attempt to fit anything that doesn't have clearly interpretable density at 1.0 sigma (in a 2Fo-Fc map). A map level of 0.5-0.6 sigma is very low, and to me, it's very likely that you're seeing what you want to see and fitting your ligand into noise. Is there any positive Fo-Fc density (greater than about 2-3 sigma) at that location if you remove the ligand? Do you see an appreciable decrease in Rfree when you add the ligand? That would be an indicator to me that you have placed it correctly. Especially if identification of the ligand binding site is the primary purpose of this structure, I think you need stronger evidence of its presence. You may also want to try co-crystallization, which may reduce the effect a full soak has on the diffraction of the crystal. Cheers, Jared -- Jared Sampson Xiangpeng Kong Lab NYU Langone Medical Center 550 First Avenue New York, NY 10016 212-263-7898 http://kong.med.nyu.edu/ On Mar 19, 2014, at 10:39 AM, Amit Kumar amit7urmcc...@gmail.com wrote: Hello, My protein is 26 kDa and the resolution of the data is 1.90 angs. My ligand is 174 Daltons. and it was soaked into the crystal. Ligand is colored and the crystal after soaking takes up intense color. However if we soak more than optimum, the color deepens in intensity but the crystal diffracts no more. So perhaps the ligand's occupancy can not be the 1.00. After model building I see ligand density, starting to appear at 0.7 sigma and clear at 0.5-0.6 sigma, close to the protein residue where it should bind. Occupancy is ~0.6 after the refinement and B factors for the atoms of the ligand range from 30-80. Questions I have (1) What is the acceptable sigma level for very small ligands for peer review/publication? (2) I did refinement by Refmac and by Phenix refine, separately. The map quality for the ligand is better after the refmac refinement than after the Phenix refinement. Why is such a difference and which one should I trust? I used mostly default parameters for both (Phenix and Refmac) before the refinement. Thanks for your time. Amit This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. =
Re: [ccp4bb] minimum acceptable sigma level for very small ligand and more
Hi, First, there is nothing magical about contouring a map at 1 rms. The standard deviation of the electron density values really has no relationship to the rms of those values, and appears to generally be much smaller. This is discussed quite brilliantly in the recent paper http://www.ncbi.nlm.nih.gov/pubmed/24363322 If you have a ligand with low occupancy you expect you will have to dial down the contour level to see it. The question isn't how low can you go but does your model fit ALL the available data and is there any other model that will also fit those data. Even if a ligand has low occupancy it still must have good bond lengths and angles and must make reasonable interactions with the rest of the molecule. One of your observations is that full occupancy cracks the crystal. It would be good if your model explains this observation as well. If your ligand is present 60% of the time, what is there the other 40%? Usually when there is a partially occupied ligand there is water present the rest of the time. The apparent superposition of the ligand and the water will result in some density that is strong. Those strong bits will give clues about the minor conformation. The low occupancy water model must also make sense in terms of hydrogen bonds and bad contacts. Remember, if you are looking at lower than usual rms contours in the 2Fo-Fc style map you must evaluate your refined model by looking at lower contour levels in your Fo-Fc style map. You can't give your model a free ride by excusing a weak density map but then blowing off weak difference peaks. You must be very careful to consider alternative models and to accepts that sometimes you just can't figure these things out. Just because the density is weak does not mean that you can give it a pass for not fitting the model. The model has to fit the density, and fit it better than any other model. You must also make clear to your readers what the occupancy of your ligand is and the quality of the maps that lead you to this conclusion. Dale Tronrud P.S. I have had great experiences with the maps produced by Buster for looking at weak ligand density. I have also published a model with a 0.35 occupancy ligand although the resolution there was 1.3 A. On 03/19/2014 07:39 AM, Amit Kumar wrote: Hello, My protein is 26 kDa and the resolution of the data is 1.90 angs. My ligand is 174 Daltons. and it was soaked into the crystal. Ligand is colored and the crystal after soaking takes up intense color. However if we soak more than optimum, the color deepens in intensity but the crystal diffracts no more. So perhaps the ligand's occupancy can not be the 1.00. After model building I see ligand density, starting to appear at 0.7 sigma and clear at 0.5-0.6 sigma, close to the protein residue where it should bind. Occupancy is ~0.6 after the refinement and B factors for the atoms of the ligand range from 30-80. Questions I have (1) What is the acceptable sigma level for very small ligands for peer review/publication? (2) I did refinement by Refmac and by Phenix refine, separately. The map quality for the ligand is better after the refmac refinement than after the Phenix refinement. Why is such a difference and which one should I trust? I used mostly default parameters for both (Phenix and Refmac) before the refinement. Thanks for your time. Amit
