Re: [ccp4bb] Refinement with refmac05 and methionine density
Hi Jeorge, The simplest answer is that you have multiple positions for the Methionine. So you can try adding in an alternate position for it. However, you didn’t mention the sigma cutoff or e-/A^3 for either of your density maps. Its quite possible that your fofc map is just scaled way down and what you are seeing is an exaggeration of what’s barely there. Hope this helps. Cheers! - Greg --- Greg Costakes, Ph.D. Department of Biochemistry University of Cambridge 80 Tennis Court Road Cambridge, CB2 1GA United Kingdom From: jeorgemarley thomas Sent: Wednesday, November 12, 2014 8:16 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Refinement with refmac05 and methionine density Dear All, I am refining the structure with refmac05, and after each refinement the density around this methionine is flipping. and it is difficult to say where this methionine actually have the density. please suggest what I should I do. I am attaching the snap here. Thanks in Advance for your kind suggestions Jeorge --- This email is free from viruses and malware because avast! Antivirus protection is active. http://www.avast.com
Re: [ccp4bb] few questions about resolving new structure through MR
The fact that you have a 10% split between R/Rfree means your solution is heavily model biased (rule of thumb is a split of 5%). An Rfree of 0.55 would imply randomness. So unfortunately in this case, I dont think that you have an actual solution. You could try MR with a poly-A form of the homology model to see if you get a better phaser solution. Then proceed with the refinement while being careful to keep the R/Rfree within 5% and slowly build in the residues of the rest of your protein based on adequate electron density. Hope this helps. - Greg --- Greg Costakes, Ph.D. Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 - Original Message - From: Zhihong Yu nkyuz...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, November 7, 2013 11:36:51 AM Subject: [ccp4bb] few questions about resolving new structure through MR Hi, all I'm a rookie in resolving a brand new structure. I have some questions for my current case and look forward to some suggestions. Now I’m working on a protein like this: N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a diffraction data just to 3.5Å, and there is no complete homology structure in pdb bank, but only a homology structure (named as structureX later) for domainB with ~30% sequence identity, so I have some questions as following: 1. Is it possible to find a resolution through MR approach using structureX as a search model? Especially considering that the resolution is only 3.5Å. Currently I just tried once using phaser and refine the structure, I can get a R/Rfree of 0.45/0.55, and it looks like most of backbone in the structureX, especially those within helix or sheet, can be well described by 2Fo-Fc density. Is this primary result promising or not? 2. If it’s possible, what’s the general optimal procedure I should follow? Really thanks for any advice and suggestions! Zhihong
Re: [ccp4bb] balbes solution
Hi Faisal, The solutions from BALBES have already gone through multiple rounds of refinement which minimize the R/Rfree. If you make a number of drastic changes to side chains or add waters, the R/Rfree should go down. However if the structure fits the density well (with no side chains needing major adjustment) and you have not yet added waters... you will not notice the R/Rfree go down. What are you R/Rfree values? --- Greg Costakes PhD Candidate Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 ** Hard work often pays of in time, but Procrastination always pays off now ** - Original Message - From: Faisal Tarique faisaltari...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, August 7, 2012 4:40:38 PM Subject: [ccp4bb] balbes solution Dear all i submitted one job in BALBES at YSBL server. The final outcome are showing the result to be definite solution by stating it to be a 99%solution. but when i am refining with refmac, Rwork and Rfree is not coming down despite my several tries. In COOT i can see tye missing density for loop region. the data is of 2.5 angstrom resolution. -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] Summary for scale out data
Hi Uma, The scale.log file contains all of those values. They're all near the bottom of the file. Also, once you refine your structure, refmac will print out most of those numbers in the .pdb file. --- Greg Costakes PhD Candidate Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 ** Hard work often pays of in time, but Procrastination always pays off now ** - Original Message - From: Uma Ratu rosiso2...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, July 31, 2012 9:27:37 AM Subject: [ccp4bb] Summary for scale out data Dear All: I process my data using HKL2000. After scale, the program generates several files, including .out, .log and .sca. Is there a way that I can generate a summary report file from the .out file, such as Rmerge, total number of observation, and completeness so on. XDS give a nice report with such information. With HKL, one has to dig into the tables to find out. Does CCP4 have a program to run such task? Thank you for advice Uma
Re: [ccp4bb] Crystal Optimization
Have you tried multiple rounds of micro-seeding? --- Greg Costakes PhD Candidate Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 ** Hard work often pays of in time, but Procrastination always pays off now ** - Original Message - From: Muhammed bashir Khan muhammad.bashir.k...@univie.ac.at To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, July 10, 2012 1:22:28 PM Subject: [ccp4bb] Crystal Optimization Dear All; Could somebody give a nice suggestion how the following type crystal could be optimized, I almost tried everything. Crystal Image is attached Crystal condition: 20% w/v PEG3350 and 200mM NaCl. Thanks in advance Bashir -- Muhammad Bashir Khan ** Structural Genome Consortium (SGC). University of Toronto Toronto, Canada
[ccp4bb] Bond Length Outliers (correction)
Ahh yes, I looked at the wrong line. My Rmsd bond angle is 2.55 degrees (not bond length). MolProbity states that my only abnormal angle is 124.23 degrees between O--C--N of an Arg. Real Space Refinement does not change anything and Regularizing the zone completely distorts the backbone. Any suggestions on how to fix this? --- Greg Costakes PhD Candidate Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 - Original Message - From: Bernard D. Santarsiero b...@uic.edu To: Greg Costakes gcost...@purdue.edu Sent: Thursday, February 16, 2012 11:42:55 AM Subject: Re: [ccp4bb] Bond Length Outliers Greg, Your RMSD on bond lengths should be around 0.01A (your structure vs. idealized library), and the RMSD on bond angles should be around 1.5deg. You must be using an incorrect value of the weight factor between structure factors and geometric factors, and relying too heavily on structure factors. Bernie On Thu, February 16, 2012 10:31 am, Greg Costakes wrote: I am currently in the final steps of refining a 1.3A structure and am coming across a slight problem. According the the pdb file, I have an Rmsd bond length of 2.55. MolProbity identifies three outliers which correspond to the bond lengths of: Asp: C--O , bond length = 1.2A Arg: C--O , bond length = 1.15A Ala: N--Ca , bond length = 1.43A Real space refinement in Coot does not help and if I Regularize the zone it completely distorts the backbone. So my question is, how do I fix these bond length outliers? Do I need to be concerned with them? Any advice will be much appreciated. Thank you! --- Greg Costakes PhD Candidate Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907
Re: [ccp4bb] Problem with getting Rfree and Rf down
It would seem that you have a large model bias. Rule of thumb is to keep R/Rfree within 5% of each other. If you find that the numbers are separating during refinement you need to reduce the weighting factor (dont use automatic) during refinement. What is your overall redundancy? Higher redundancies (7 or so) do tend to increase overall R/Rfree. Dont worry so much about getting the R-factor down as supposed to keeping it close to the Rfree. --- Greg Costakes PhD Candidate Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 ** Hard work often pays of in time, but Procrastination always pays off now ** - Original Message - From: Sam Arnosti meisam.nosr...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, January 23, 2012 4:48:50 PM Subject: [ccp4bb] Problem with getting Rfree and Rf down Hi every one I have some crystals in the space group P3121. I collect 180 frames of data. My crystals do not diffract better than at most 2.0 angstrom, but the Rf barely goes below 23%, and Rfree also remains somewhere between 28-33%. I have tried to refine my data as much as I can. I do not know whether the problem is because of the bad diffraction or collecting extra frames. The structure factors are also high but they get better as the crystals diffract better. Thanks Sam
Re: [ccp4bb] Problem with getting Rfree and Rf down
Whoops, I misspoke... I meant Rsym and Rmerge increase with higher redundancies. --- Greg Costakes PhD Candidate Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 ** Hard work often pays of in time, but Procrastination always pays off now ** - Original Message - From: Dale Tronrud det...@uoxray.uoregon.edu To: Greg Costakes gcost...@purdue.edu Cc: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, January 24, 2012 12:43:43 PM Subject: Re: [ccp4bb] Problem with getting Rfree and Rf down Is this observation about redundancies a general rule that I missed? It seems rather surprising to me. What have results have others seen? Dale Tronrud On 01/24/12 07:23, Greg Costakes wrote: snip... Higher redundancies (7 or so) do tend to increase overall R/Rfree. snip... --- Greg Costakes PhD Candidate Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 ** Hard work often pays of in time, but Procrastination always pays off now ** *From: *Sam Arnosti meisam.nosr...@gmail.com *To: *CCP4BB@JISCMAIL.AC.UK *Sent: *Monday, January 23, 2012 4:48:50 PM *Subject: *[ccp4bb] Problem with getting Rfree and Rf down Hi every one I have some crystals in the space group P3121. I collect 180 frames of data. My crystals do not diffract better than at most 2.0 angstrom, but the Rf barely goes below 23%, and Rfree also remains somewhere between 28-33%. I have tried to refine my data as much as I can. I do not know whether the problem is because of the bad diffraction or collecting extra frames. The structure factors are also high but they get better as the crystals diffract better. Thanks Sam
Re: [ccp4bb] extra density ??
It would help if we knew the crystallization conditions and the protein solution components. Also, what are the map sigmas and e/A^3? It seems odd that there isnt positive density on the Fo-Fc map across the entire blob seen on the 2Fo-Fc map, considering you do not have anything modeled in. My guess is that your Fo-Fc map is at a much lower e/A^3 compared to your 2Fo-Fc map and what you are seeing is basically just greatly amplified noise. Try placing 3 waters in the blobs you see on the 2Fo-Fc map and see if that helps. --- Greg Costakes PhD Candidate Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 ** Hard work often pays of in time, but Procrastination always pays off now ** - Original Message - From: stacy William stacy.biot...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, January 19, 2012 1:44:30 AM Subject: [ccp4bb] extra density ?? Dear All, I am working on plant proteins and solved a structure, there is an extra density which i cannot fix . I am attaching the coot image , can anybody suggest me what it could be THANKS :)
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
The only thing you can do is to move the detector in and collect higher resolution. Then you can determine what the I/sigI ratio is for each of your resolution shells and manually set the resolution limit once you run HKL2000. As far as the R/Rfree values go, you will need to manually set the weighting factor if you are using refmac. I have found that during the initial rigid body refinement and the first few rounds of restrained refinement, it works well if you keep the weighting factor low (0.01-0.9), then as you go through a few rounds, gradually increase the weighting factor. But keep in mind that as you increase the weighting factor, the difference between R/Rfree will also increase. -- Greg Costakes PhD Candidate Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 -- -- From: Mittal, Seema seema.mit...@umassmed.edu Sent: Friday, May 20, 2011 5:28 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] how to remove part of data with bad signal to noise ratio Hi All, I am currently working on a 3A resolution dataset. The scaled file shows the following statistics (scroll down to the end of this email). It is P212121 space group with R merge of 8.8%. My question is : Is there a way to selectively use only the data with I/Sigma value of 2 and more for refinement? And how do i achieve this using refmac? I am aware that this would come at the cost of compromising data completeness. Any suggestions/help would be greatly appreciated. Thanks much, Seema Mittal Department of Biochemistry Molecular Pharmacology 970L Lazare Research Building University of Massachusetts Medical School 364 Plantation Street Worcester, MA 01605 Shell I/Sigma in resolution shells: LowerUpper % of of reflections with I / Sigma less than limit limit 0 1 2 3 5 10 20 20 total 50.00 6.46 2.0 3.8 5.3 6.27.6 12.5 34.3 65.0 99.3 6.46 5.13 0.7 2.2 3.9 5.38.2 15.7 36.6 63.4 100.0 5.13 4.48 1.3 2.8 4.0 5.89.3 13.8 27.3 72.7 100.0 4.48 4.07 0.7 1.7 4.0 5.47.9 13.9 35.4 64.1 99.5 4.07 3.78 1.8 3.6 5.1 6.9 11.8 20.8 49.6 47.3 96.9 3.78 3.56 1.5 3.8 6.7 8.7 13.3 26.7 65.4 30.8 96.2 3.56 3.38 0.8 3.2 7.1 8.9 12.9 31.1 76.6 20.0 96.6 3.38 3.23 2.0 4.8 8.1 14.8 23.4 44.8 84.7 12.7 97.5 3.23 3.11 4.1 9.2 13.8 18.4 29.6 51.0 86.0 11.0 96.9 3.11 3.00 2.4 8.6 13.9 18.8 30.6 53.9 92.44.596.9 All hkl1.7 4.3 7.19.8 15.3 28.0 58.1 39.9 98.0 Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2 R-fac R-fac 50.00 6.46 511.720.0 8.8 1.098 0.065 0.073 6.46 5.13 284.610.1 6.3 1.047 0.062 0.064 5.13 4.48 500.917.0 8.8 1.007 0.062 0.069 4.48 4.07 446.117.4 9.2 1.032 0.069 0.070 4.07 3.78 307.114.5 8.4 1.065 0.089 0.092 3.78 3.56 243.413.8 7.9 1.033 0.108 0.112 3.56 3.38 182.312.0 8.3 1.083 0.132 0.134 3.38 3.23 136.510.4 7.7 1.048 0.155 0.151 3.23 3.11 107.4 9.2 7.3 1.096 0.184 0.163 3.11 3.0091.0 8.7 7.3 1.0440.215 0.201 All reflections287.713.5 8.0 1.055 0.088 0.082
