[ccp4bb] How to know if ADP exists in the ATP-binding site of bacterial expressed proteins
Deer CCP4ers, How to know if ADP exists in the ATP-binding site of bacterial expressed proteins? Check the UV spectrum at 280 nm is one way, but there is no Trp in my protein. Is there any other conveniet way to find out? Thanks in advance Best wishes Xinghua Qin -- Xinghua Qin State Key Laboratory of Plant Physiology and biochemistry College of Biological Sciences China Agricultural University No.2, Yuan Ming Yuan West Road Haidian District, Beijing, China 100193 Tel: +86-10-62732672 E-mail: xinghua...@126.com
[ccp4bb] how to ignore spot overlap in imosflm?
Dear all, Thanks for all of the suggestions, It helps me a lot as I am a newcomer to the structural world. Increasing the "Profile Tolerance" parametersas Dr Harry Powell has pointed out can increase the completeness by ten percent (from 50% to 60%). I will try other people's advice soon. Although the completeness is quite low (just 50%), now I have determined the structure (Rfree=0.31, Rfactor=0.24, resolution=2.6). I will look into the map with COOT, and try to get the structure more beautiful. Thanks again Best wishes Xinghua Qin -- Xinghua Qin State Key Laboratory of Plant Physiology and biochemistry College of Biological Sciences China Agricultural University No.2, Yuan Ming Yuan West Road Haidian District, Beijing, China 100193 Tel: +86-10-62732672 E-mail: xinghua...@126.com At 2012-05-14 11:48:58,"Zhijie Li" wrote: Hi Xinghua, The total intensity of each reflection needs to be accurately quantitated in order to calculate the structure factors. Not only the dots need to be well separated in the 3D reciprocal space, but also a small area around the dots are often needed to calculate the background for subtraction. That is why when two dots are getting too close, the programs will reject both dots. The first thing you need to do is to inspect the images reported with large number of overlaps to see if the dots are really overlapping or just close to each other. If the dots are barely touching or just too close to each other, you can manipulate the SEPERATION parameter to force the program to take the closely spaced spots. But keep in mind that you may get less accurate integration by doing so. If many spots are really touching each other, normally we won't force the programs to use them. Then the proper remedy is to move the detector farther and collect the dataset again (also, try to optimize your f! reezing to get the mosaicity as low as possible). For how to play with the mosflm parameters, please read here: http://www.mrc-lmb.cam.ac.uk/harry/cgi-bin/keyword2.cgi?SEPARATION. What you need is probably CLOSE. The hazard of high percentage of overlaps: If the overlaps are only scattered in a whole dataset, it is OK, even if they make up 5-10% or even 20% of the whole dataset. It will only give you a lower completeness, which is not too detrimental to the structure solution. However, if large, continuous regions in the dataset are missing, that will cause you to have poorly defined regions in the calculated map, often seen as featureless stripes or layers in the map. Unfortunately, when you have closely spaced reflections, the latter is often the case. The proper solution is to collect the data at a greater detector distance to resolve the spots (after taking the test images, both imosflm and HKL2000 can simulate the collection run to help you to decide what distance you need). In cases that you have a long unit cell (>200A), the first thing you need to do is to align the long edge of the Unix cell with the rotational axis of the pin. In the difficult cases, you probably even need to shoot multiple crystals and combine the ! datasets to get enough completeness. Zhijie From:Xinghua Qin Sent: Sunday, May 13, 2012 10:22 PM To:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] how to ignore spot overlap in imosflm? Dear CCP4ers, We collected a diffraction dataset with high percentage of spot overlaps, It would be so kind to tell me how to ignore spot overlap in imosflm and explain the hazard of high percentage of spot overlaps. Thanks in advance. Best wishes Xinghua Qin -- Xinghua Qin State Key Laboratory of Plant Physiology and biochemistry College of Biological Sciences China Agricultural University No.2, Yuan Ming Yuan West Road Haidian District, Beijing, China 100193 Tel: +86-10-62732672 E-mail: xinghua...@126.com
[ccp4bb] how to ignore spot overlap in imosflm?
Dear CCP4ers, We collected a diffraction dataset with high percentage of spot overlaps, It would be so kind to tell me how to ignore spot overlap in imosflm and explain the hazard of high percentage of spot overlaps. Thanks in advance. Best wishes Xinghua Qin -- Xinghua Qin State Key Laboratory of Plant Physiology and biochemistry College of Biological Sciences China Agricultural University No.2, Yuan Ming Yuan West Road Haidian District, Beijing, China 100193 Tel: +86-10-62732672 E-mail: xinghua...@126.com
[ccp4bb] about the "working solution"
hi,everyone, Sorry for the off-topic question.I got two many crystals in the drop with long thin needle shape and crossed , after optimization, no big change appeared, and I got only a few needle clusters in the drop at most times. which one is better for next optimization step and suitale to be the "working solution"? In my opinion, the latter one has fewer nucleus, but the former one has better shape, did I got the point? Best wishes Xinghua Qin -- Xinghua Qin Room 4022, Center for Life Sciences, China Agricultural University, No.2, Yuan Ming Yuan West Road, Haidian District， Beijing，China，100193 Tel: +86-10-62732672
[ccp4bb] about partial plroteolysis
hello CCp4ers: Sorry about the off-topic question. My protein is a kinase which the predicted kinase domain is from 32aa to 319aa. But the protein sequence after partial proteolysis is from 52aa. what should I do? Are there any reasonable expanation? Thanks in advance! Xinghua Qin -- Xinghua Qin Room 4022, Center for Life Sciences, China Agricultural University, No.2, Yuan Ming Yuan West Road, Haidian District， Beijing，China，100193 Tel: +86-10-62732672
[ccp4bb] how to fix a single helice
Hi CCP4ers: Recently I am trying to fix a helice, but found that it is so hard for me because of the poor resolution(3.2A) and also a unvisible loop near it, so is there any way to just fix a helice ? can I get it out of the whole structure to refine it? Best regards -- 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 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 >> >> -- >> 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
[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
[ccp4bb] linear R factor in HKL2000
hi CCP4ers: I have a question about the linear R factor in HKL2000 scaling which is higher than 0.1, is this value must below 0.1? as the values are all above 0.1, even the deletion of the images can not work. so should I ignore it and move on? Best regards Xinghua Qin -- Xinghua Qin College of Biological Sciences No.2, Yuan Ming Yuan West Road Haidian District，Beijing，China，100193 Tel: +86-10-62732672
