Re: [ccp4bb] Matthews coefficient
To reply to the original question, can the calculated Matthews coefficient be wrong?: have you read the paper by Matthews? There is a distribution of solvent content in macromolecular (protein) crystals. It has a peak and tails on both sides. Meaning that there are outliers in the distribution, i.e. crystals with very little solvent, and crystals with plenty of solvent. They are not really outliers since, as in any physical phenomenon, some data points fall outside 1 sigma or 2 sigmas from the mean. Exceptions do exist. What you cannot have however, is an asymmetric unit that contains too many molecules/subunits than what is physically possible. Molecular replacement calculations can be performed searching for 1, 2, 3,... N copies in the asymmetric unit. This is what I personally do because at first I do not know the content of the asymmetric unit in my crystal. Fred. Message du 25/06/10 07:54 De : xinghua qin A : CCP4BB@JISCMAIL.AC.UK Copie à : Objet : Re: [ccp4bb] Matthews coefficient Hi everyone: Thanks for all the responses. The Matthaws-cell content analysis programe in CCP4 package gives the results: 47% solvent content and 3 molecules in asu with 87% confidence. the space group is P3121. how to carry out self rotation function? can phaser do that work? If there areTwo moleculars in asu ,no clashes and the TF values are above 10.but if three, the clashes are too much and TF valur is about five. And how to calculate the solvent content? Is the calculated solvent content with the Matthews-cell content analysis programe not always right? Best regards Xinghua Qin On Fri, Jun 25, 2010 at 10:42 AM, Vineet Gaur wrote: Hi It would b good if u can mention the solvent content and space group along with the no. of molecules in asu/ u can carry out self rotation function to check the no of mol in asu best vineet On Fri, Jun 25, 2010 at 6:42 AM, xinghua qin wrote: hi CCPeers The Matthews coefficient of my protein is 3 calculated with matthews-cell content analysis CCP4 programe with 87% confidence , but when doing the refinement the third molecular couldn't get into the unit cell because of too many clashes.Deletion of the clashed AA did not work well, Then I used two molecules in the unit cell, After refining with Refmac, I found that the R factor is 0.29, R free is 0.40.I believe the value can be better with the real space refinement.But the question is that can the calculated Matthews coefficient be wrong? Best regards Xinghua Qin
Re: [ccp4bb] Matthews coefficient
Dear Xinghua, the solvent content provided by yhe Matthews-program is certainly correct. Whether or not it applies to you protein is a different issue. With only 2 molecules in the asymmetric unit the solvent content rises to 65%, and this is stll perfectly fine for proteins. So carry out molecular replacement with only two molecules and start model building and refinement. Your previous message sounds as though to started refinement directly after molecular replacement. I would strongly discourage you from this practice (which somehow seems to be stuck in the heads of many people). After molecular replacement you should check the result with the model building program of your choice and correct as many errors as possible before running a refinement program. Tim On Fri, Jun 25, 2010 at 01:54:05PM +0800, xinghua qin wrote: Hi everyone: Thanks for all the responses. The Matthaws-cell content analysis programe in CCP4 package gives the results: 47% solvent content and 3 molecules in asu with 87% confidence. the space group is P3121. how to carry out self rotation function? can phaser do that work? If there areTwo moleculars in asu ,no clashes and the TF values are above 10.but if three, the clashes are too much and TF valur is about five. And how to calculate the solvent content? Is the calculated solvent content with the Matthews-cell content analysis programe not always right? Best regards Xinghua Qin On Fri, Jun 25, 2010 at 10:42 AM, Vineet Gaur vineetgaur1...@gmail.comwrote: Hi It would b good if u can mention the solvent content and space group along with the no. of molecules in asu/ u can carry out self rotation function to check the no of mol in asu best vineet On Fri, Jun 25, 2010 at 6:42 AM, xinghua qin xtal...@gmail.com wrote: hi CCPeers The Matthews coefficient of my protein is 3 calculated with matthews-cell content analysis CCP4 programe with 87% confidence , but when doing the refinement the third molecular couldn't get into the unit cell because of too many clashes.Deletion of the clashed AA did not work well, Then I used two molecules in the unit cell, After refining with Refmac, I found that the R factor is 0.29, R free is 0.40.I believe the value can be better with the real space refinement.But the question is that can the calculated Matthews coefficient be wrong? Best regards Xinghua Qin -- Xinghua Qin College of Biological Sciences No.2, Yuan Ming Yuan West Road Haidian District，Beijing，China，100094 Tel: +86-10-62732672 -- Xinghua Qin College of Biological Sciences No.2, Yuan Ming Yuan West Road Haidian District，Beijing，China，100094 Tel: +86-10-62732672 -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] Matthews coefficient
Hello, I am not a crystallographer, so I will ask a maybe stupid question. When you say After molecular replacement you should check the result with the model building program of your choice and correct as many errors as possible before running a refinement program. Can this step be automated in some way (without any human intervention)? Are there programs to do this? I am interested even if some program does only one part of the job. Thanks a lot, Francois. Tim Gruene wrote: Dear Xinghua, the solvent content provided by yhe Matthews-program is certainly correct. Whether or not it applies to you protein is a different issue. With only 2 molecules in the asymmetric unit the solvent content rises to 65%, and this is stll perfectly fine for proteins. So carry out molecular replacement with only two molecules and start model building and refinement. Your previous message sounds as though to started refinement directly after molecular replacement. I would strongly discourage you from this practice (which somehow seems to be stuck in the heads of many people). After molecular replacement you should check the result with the model building program of your choice and correct as many errors as possible before running a refinement program. Tim On Fri, Jun 25, 2010 at 01:54:05PM +0800, xinghua qin wrote: Hi everyone: Thanks for all the responses. The Matthaws-cell content analysis programe in CCP4 package gives the results: 47% solvent content and 3 molecules in asu with 87% confidence. the space group is P3121. how to carry out self rotation function? can phaser do that work? If there areTwo moleculars in asu ,no clashes and the TF values are above 10.but if three, the clashes are too much and TF valur is about five. And how to calculate the solvent content? Is the calculated solvent content with the Matthews-cell content analysis programe not always right? Best regards Xinghua Qin On Fri, Jun 25, 2010 at 10:42 AM, Vineet Gaur vineetgaur1...@gmail.comwrote: Hi It would b good if u can mention the solvent content and space group along with the no. of molecules in asu/ u can carry out self rotation function to check the no of mol in asu best vineet On Fri, Jun 25, 2010 at 6:42 AM, xinghua qin xtal...@gmail.com wrote: hi CCPeers The Matthews coefficient of my protein is 3 calculated with matthews-cell content analysis CCP4 programe with 87% confidence , but when doing the refinement the third molecular couldn't get into the unit cell because of too many clashes.Deletion of the clashed AA did not work well, Then I used two molecules in the unit cell, After refining with Refmac, I found that the R factor is 0.29, R free is 0.40.I believe the value can be better with the real space refinement.But the question is that can the calculated Matthews coefficient be wrong? Best regards Xinghua Qin -- Xinghua Qin College of Biological Sciences No.2, Yuan Ming Yuan West Road Haidian District,Beijing,China,100094 Tel: +86-10-62732672 -- Xinghua Qin College of Biological Sciences No.2, Yuan Ming Yuan West Road Haidian District,Beijing,China,100094 Tel: +86-10-62732672
Re: [ccp4bb] Matthews coefficient
Hi, No such thing as a stupid question. If the resolution is sufficient, arp_warp does a good job. But it is a reconstruction and REFINEMENT program. But checking the proper packing (that there are indeed contacts in the 3 dimensions of space to form the crystal): takes only 10 seconds using a graphics program, so there is no need to automate that. And trying to do everything completely automatically (without any human intervention and checks) can lead to big problems (for example if the space group has been wrongly assigned). You may end up publishing a wrong structure if you rely entirely on totally automated crystallographic software without checking anything. Fred. Francois Berenger wrote: Hello, I am not a crystallographer, so I will ask a maybe stupid question. When you say After molecular replacement you should check the result with the model building program of your choice and correct as many errors as possible before running a refinement program. Can this step be automated in some way (without any human intervention)? Are there programs to do this? I am interested even if some program does only one part of the job. Thanks a lot, Francois.
Re: [ccp4bb] Matthews coefficient
Vellieux Frederic wrote: Hi, No such thing as a stupid question. If the resolution is sufficient, arp_warp does a good job. But it is a reconstruction and REFINEMENT program. But checking the proper packing (that there are indeed contacts in the 3 dimensions of space to form the crystal): takes only 10 seconds using a graphics program, so there is no need to automate that. Well, if my colleague sends you his 300,000 computer-generated models to use in MR, I am sure you will like to automate the job. ;) And trying to do everything completely automatically (without any human intervention and checks) can lead to big problems (for example if the space group has been wrongly assigned). You may end up publishing a wrong structure if you rely entirely on totally automated crystallographic software without checking anything. Sure, an expert is always useful to check things at some moment. And, I also agree that things made only in the computer have to be checked using some experimental data in order to be relied on. Francois. Fred. Francois Berenger wrote: Hello, I am not a crystallographer, so I will ask a maybe stupid question. When you say After molecular replacement you should check the result with the model building program of your choice and correct as many errors as possible before running a refinement program. Can this step be automated in some way (without any human intervention)? Are there programs to do this? I am interested even if some program does only one part of the job. Thanks a lot, Francois.
Re: [ccp4bb] Matthews coefficient
On Fri, Jun 25, 2010 at 12:41 AM, Francois Berenger beren...@riken.jpwrote: When you say After molecular replacement you should check the result with the model building program of your choice and correct as many errors as possible before running a refinement program. Can this step be automated in some way (without any human intervention)? Are there programs to do this? I am interested even if some program does only one part of the job. Both ARP/wARP and phenix.autobuild will do this (again, given data of sufficiently high quality). But I disagree with the original statement. Molecular replacement is typically done at reduced resolution, and particularly where the search model is different in conformation or sequence from the protein in the crystal, there will be many errors, even if the solution is generally excellent. Attempting to correct each of these manually is a waste of time, since the refinement or rebuilding programs will do it faster and better. If there are severe problems like overlapping molecules, this will become obvious very quickly whether or not automation is used. Is it even possible to solve, rebuild, and refine a structure from MR in the wrong space group? I would expect the R-factors to be unreasonably high and the maps uninterpretable. I do agree that inspecting the lattice visually after MR is a good idea (I like PyMOL for this). I don't know whether there are programs that will do this automatically - it would be a useful tool to have. As always, there will still be corner cases, where the search model is too remote for the automated steps to work and manual fixing is required, or you get packing clashes before all copies are found and need to prune the search model, or the crystal resolution is too poor, and so on. It is also possible to waste time trying to improve a nonsensical solution - until your R-free is well below 0.5, you can't be certain that you've solved the structure, and if the automated methods don't push it below 0.4, it's probably worth checking your MR solution to make sure it's already as good as possible. The more general point, that no one should publish or deposit a structure without ever thoroughly checking the maps, validation, and lattice contacts, I of course agree with - but I haven't heard anyone propose doing away with human intervention altogether, nor are any of the automated pipelines anywhere close to making this feasible. -Nat
Re: [ccp4bb] Matthews coefficient
Dear Xinghua, Of course, you can have 2 molecules in the asu. However, if things don't it is worth to double-check your space group or check for twinning. Cheers christian xinghua qin wrote: hi CCPeers The Matthews coefficient of my protein is 3 calculated with matthews-cell content analysis CCP4 programe with 87% confidence , but when doing the refinement the third molecular couldn't get into the unit cell because of too many clashes.Deletion of the clashed AA did not work well, Then I used two molecules in the unit cell, After refining with Refmac, I found that the R factor is 0.29, R free is 0.40.I believe the value can be better with the real space refinement.But the question is that can the calculated Matthews coefficient be wrong? Best regards Xinghua Qin -- Xinghua Qin College of Biological Sciences No.2, Yuan Ming Yuan West Road Haidian District，Beijing，China，100094 Tel: +86-10-62732672 -- ___ Dr. Christian Biertümpfel Laboratory of Molecular Biology NIDDK/National Institutes of Health phone: +1 301 402 4647 9000 Rockville Pike, Bldg. 5, Rm. B1-03 fax: +1 301 496 0201 Bethesda, MD 20892-0580 USA ___
Re: [ccp4bb] Matthews coefficient
It is out of principle misleading to think of the Matthews coefficient as one hard number. Xinghua mentions it actually: 'with 87 % confidence' (although 'confidence as in confidence interval' is a statistically too hard term here imho). The Matthews probability calculator gives a more complete picture of the probable values and their distribution. http://www.ruppweb.org/mattprob/default.html BR -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Christian Biertuempfel Sent: Friday, June 25, 2010 9:34 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Matthews coefficient Dear Xinghua, Of course, you can have 2 molecules in the asu. However, if things don't it is worth to double-check your space group or check for twinning. Cheers christian xinghua qin wrote: hi CCPeers The Matthews coefficient of my protein is 3 calculated with matthews-cell content analysis CCP4 programe with 87% confidence , but when doing the refinement the third molecular couldn't get into the unit cell because of too many clashes.Deletion of the clashed AA did not work well, Then I used two molecules in the unit cell, After refining with Refmac, I found that the R factor is 0.29, R free is 0.40.I believe the value can be better with the real space refinement.But the question is that can the calculated Matthews coefficient be wrong? Best regards Xinghua Qin -- Xinghua Qin College of Biological Sciences No.2, Yuan Ming Yuan West Road Haidian District,Beijing,China,100094 Tel: +86-10-62732672 -- ___ Dr. Christian Biert�mpfel Laboratory of Molecular Biology NIDDK/National Institutes of Health phone: +1 301 402 4647 9000 Rockville Pike, Bldg. 5, Rm. B1-03 fax: +1 301 496 0201 Bethesda, MD 20892-0580 USA ___
[ccp4bb] Matthews coefficient
hi CCPeers The Matthews coefficient of my protein is 3 calculated with matthews-cell content analysis CCP4 programe with 87% confidence , but when doing the refinement the third molecular couldn't get into the unit cell because of too many clashes.Deletion of the clashed AA did not work well, Then I used two molecules in the unit cell, After refining with Refmac, I found that the R factor is 0.29, R free is 0.40.I believe the value can be better with the real space refinement.But the question is that can the calculated Matthews coefficient be wrong? Best regards Xinghua Qin -- Xinghua Qin College of Biological Sciences No.2, Yuan Ming Yuan West Road Haidian District，Beijing，China，100094 Tel: +86-10-62732672
Re: [ccp4bb] Matthews coefficient
Hi everyone: Thanks for all the responses. The Matthaws-cell content analysis programe in CCP4 package gives the results: 47% solvent content and 3 molecules in asu with 87% confidence. the space group is P3121. how to carry out self rotation function? can phaser do that work? If there areTwo moleculars in asu ,no clashes and the TF values are above 10.but if three, the clashes are too much and TF valur is about five. And how to calculate the solvent content? Is the calculated solvent content with the Matthews-cell content analysis programe not always right? Best regards Xinghua Qin On Fri, Jun 25, 2010 at 10:42 AM, Vineet Gaur vineetgaur1...@gmail.comwrote: Hi It would b good if u can mention the solvent content and space group along with the no. of molecules in asu/ u can carry out self rotation function to check the no of mol in asu best vineet On Fri, Jun 25, 2010 at 6:42 AM, xinghua qin xtal...@gmail.com wrote: hi CCPeers The Matthews coefficient of my protein is 3 calculated with matthews-cell content analysis CCP4 programe with 87% confidence , but when doing the refinement the third molecular couldn't get into the unit cell because of too many clashes.Deletion of the clashed AA did not work well, Then I used two molecules in the unit cell, After refining with Refmac, I found that the R factor is 0.29, R free is 0.40.I believe the value can be better with the real space refinement.But the question is that can the calculated Matthews coefficient be wrong? Best regards Xinghua Qin -- Xinghua Qin College of Biological Sciences No.2, Yuan Ming Yuan West Road Haidian District，Beijing，China，100094 Tel: +86-10-62732672 -- Xinghua Qin College of Biological Sciences No.2, Yuan Ming Yuan West Road Haidian District，Beijing，China，100094 Tel: +86-10-62732672
