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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 data<https://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 attachment<https://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 <[log in to unmask]>: > Transmit Report: > > [log in to unmask]에게 메일 발송을 5번 시도했지만 실패하였습니다. > (실패 이유 : 554 Transaction failed. 402 Local User Inbox Full ( > [log in to unmask]) 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;[log in to unmask] > Diagnostic-Code: smtp; 554 error - Transaction failed. 402 Local User > Inbox Full ([log in to unmask]) 10,61440,61437(163.152.6.98) > Action: failed > Status: 4.0.0 > > > ---------- Forwarded message ---------- > From: Amit Kumar <[log in to unmask]> > To: [log in to unmask] > 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 <[log in to unmask]>wrote: > >> 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 <[log in to unmask]> 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. >> ================================= >> >> > > >