Re: [ccp4bb] Problem with Rfree and Rfac
The R factor and Rfree will always diverge during refinement; the model is being modified to fit the working set, and not theFree set.. A largish difference at such a resolution is not surprising really. Your rebuilding has improved the FreeR but it isnt surprising thatit has not fallen below 38% Eleanor Sravanti Vaidya wrote: Hello CCP4i GURUs, I am working on a 3A resolution structure and I recently got a solution from Phaser. I have just started building and refining the structure. I started with Rfree-43 , R-41.7 and the first round of building and refining gave me Rfree- 40 and Rfac- 35.5. The Rfree and R do not decrease to the same extent during refinement (Refmac5). Further rounds of refinement increases the difference between R and Rfree (Rfree-38 Rfac- 30) I am worried that since this is just the beginning further rounds of building and refinement might drastically increase the gap between R and Rfree. On the positive note the FOM is increasing with refinement and the maps look better. I also see new density at several regions where I can build into. I tried to change the weighting factor and geometric parameters after I read the earlier posts here. The references noted earlier were extremely helpful. Here are some other details of my structure which might help you Space group- P1 21 1 Resolution- 3A Solvent content- 39% unit cell parameters- 63.3, 90.72 , 86.11, angles- 90, 91.5 , 90 Please enlighten me with your suggestions. Is it fine to go ahead with this refinement or is there a way I can reduce this difference between R and Rfree?? Your help will be greatly appreciated. As this is my first structure I am excited about it!!! Thank you Sravanti
Re: [ccp4bb] Re :Re: [ccp4bb] MAD phasing
You need to try both P61 22 and P6522 with the Se sites changed fron x,y,z to -x,-y,-z. The anom differences can be explained by either solution. But maybe these programs automatically check both hands? Eleanor Dodson Ethayathulla Abdulsamath wrote: hi BertThank you for your reply. Your are right about the values for your Pattersons and NatFourier, but the systematic absences and pointless suggested me to be P6122 and according to mathews coeff i get nbsp; 1 molecule with Vm=nbsp; 2.06 solvent=nbsp; 40.42. My protein has two SeMet sites so i searched for two sites. I have just pasted few reflections which showed me P61. then based on pointless i tried P6122nbsp;nbsp; 0nbsp;nbsp; 0nbsp;nbsp; 3nbsp;nbsp;nbsp;nbsp; 1.9nbsp;nbsp;nbsp;nbsp; 1.8nbsp;nbsp;nbsp;nbsp; 0.5nbsp;nbsp;nbsp;nbsp; 1.8nbsp;nbsp; 0nbsp;nbsp; 0nbsp;nbsp; 4nbsp;nbsp;nbsp;nbsp; 0.0nbsp;nbsp;nbsp;nbsp; 2.7nbsp;nbsp;nbsp;nbsp; 1.1nbsp;nbsp;nbsp;nbsp; 2.5nbsp;nbsp; 0nbsp;nbsp; 0nbsp;nbsp; 5nbsp;nbsp;nbsp;nbsp; 0.4nbsp;nbsp;nbsp;nbsp; 3.5nbsp;nbsp;nbsp; -0.5nbsp;nbsp;nbsp;nbsp; 3.3nbsp;nbsp; 0nbsp;nbsp; 0nbsp;nbsp; 6nbsp;nbsp; 427.5nbsp;nbsp;nbsp; 33.6nbsp;nbsp; 399.9nbsp;nbsp;nbsp; 30.3nbsp;nbsp; 0nbsp;nbsp; 0nbsp;nbsp; 7nbsp;nbsp;nbsp; 14.3nbsp;nbsp;nbsp;nbsp; 5.9nbsp;nbsp;nbsp;nbsp; 4.0nbsp;nbsp;nbsp;nbsp; 4.8nbsp;nbsp; 0nbsp;nbsp; 0nbsp;nbsp; 8nbsp;nbsp;nbsp;nbsp; 1.5nbsp;nbsp;nbsp;nbsp; 5.8nbsp;nbsp;nbsp; -4.4nbsp;nbsp;nbsp;nbsp; 6.3nbsp;nbsp; 0nbsp;nbsp; 0nbsp;nbsp; 9nbsp;nbsp;nbsp; 10.8nbsp;nbsp;nbsp;nbsp; 6.7nbsp;nbsp;nbsp; -2.1nbsp;nbsp;nbsp;nbsp; 7.5nbsp;nbsp; 0nbsp;nbsp; 0nbsp; 10nbsp;nbsp;nbsp;nbsp; 7.0nbsp;nbsp;nbsp;nbsp; 7.3nbsp;nbsp;nbsp;nbsp; 1.5nbsp;nbsp;nbsp;nbsp; 7.2nbsp;nbsp; 0nbsp;nbsp; 0nbsp; 11nbsp;nbsp;nbsp; -3.8nbsp;nbsp;nbsp;nbsp; 9.3nbsp;nbsp;nbsp; -2.7nbsp;nbsp;nbsp;nbsp; 8.4nbsp;nbsp; 0nbsp;nbsp; 0nbsp; 12nbsp;nbsp; 201.7nbsp;nbsp;nbsp; 19.1nbsp;nbsp; 198.6nbsp;nbsp;nbsp; 19.8nbsp;nbsp; 0nbsp;nbsp; 0nbsp; 13nbsp;nbsp;nbsp; 22.5nbsp;nbsp;nbsp;nbsp; 9.6nbsp;nbsp;nbsp; 12.5nbsp;nbsp;nbsp;nbsp; 9.9nbsp;nbsp; 0nbsp;nbsp; 0nbsp; 14nbsp;nbsp;nbsp;nbsp; 2.9nbsp;nbsp;nbsp; 10.7nbsp;nbsp;nbsp;nbsp; 0.0nbsp;nbsp;nbsp;nbsp; 8.5nbsp;nbsp; 0nbsp;nbsp; 0nbsp; 15nbsp;nbsp; -21.0nbsp;nbsp;nbsp; 12.6nbsp;nbsp;nbsp;nbsp; 1.4nbsp;nbsp;nbsp; 12.1nbsp;nbsp; 0nbsp;nbsp; 0nbsp; 16nbsp;nbsp;nbsp; -1.6nbsp;nbsp;nbsp; 12.6nbsp;nbsp;nbsp; 10.8nbsp;nbsp;nbsp; 12.3nbsp;nbsp; 0nbsp;nbsp; 0nbsp; 18nbsp; 6649.0nbsp;nbsp; 474.7nbsp; 6423.4nbsp;nbsp; 460.5nbsp;nbsp; 0nbsp;nbsp; 0nbsp; 19nbsp;nbsp;nbsp; -4.6nbsp;nbsp;nbsp; 16.5nbsp;nbsp;nbsp; -8.5nbsp;nbsp;nbsp; 15.1nbsp;nbsp; 0nbsp;nbsp; 0nbsp; 20nbsp;nbsp; -44.1nbsp;nbsp;nbsp; 17.0nbsp;nbsp; -16.3nbsp;nbsp;nbsp; 16.7nbsp;nbsp; 0nbsp;nbsp; 0nbsp; 21nbsp;nbsp;nbsp; 34.6nbsp;nbsp;nbsp; 20.7nbsp;nbsp;nbsp; 61.8nbsp;nbsp;nbsp; 18.5nbsp;nbsp; 0nbsp;nbsp; 0nbsp; 22nbsp;nbsp; -16.7nbsp;nbsp;nbsp; 12.5nbsp;nbsp; -15.4nbsp;nbsp;nbsp; 15.9nbsp;nbsp; 0nbsp;nbsp; 0nbsp; 23nbsp;nbsp; -46.2nbsp;nbsp;nbsp; 18.7nbsp;nbsp; -35.9nbsp;nbsp;nbsp; 18.2nbsp;pointless resultsBest Solutionnbsp;nbsp;nbsp;nbsp; space group P 61 2 2nbsp;nbsp; Reindex operator:nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp; [h,k,l]nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp; nbsp;nbsp; Laue group probability:nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp; 1.000nbsp;nbsp; Systematic absence probability:nbsp;nbsp;nbsp;nbsp; 0.963nbsp;nbsp; Total probability:nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp; 0.963nbsp;nbsp; Space group confidence:nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp; 0.949nbsp;nbsp; Laue group confidencenbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp; 1.000thank youEthayathullaOn Thu, 21 May 2009 19:44:27 -0400 Bert Van Den Berg wrote Re: [ccp4bb] MAD phasing Hi,My guess is that you don’t have the right space group. The values for your Pattersons and NatFourier are way too low, especially the NatFourier you would like to be at least 2-3 or so. In your assigned space group, do you have a reasonable value for nbsp;the matthews coefficient? Is there NCS? Are you finding the expected number of Se sites? Again, I would try to investigate alternative SGs.Also it would be a good idea to take the sites and refine them with SHARP.Good luck, Bert On 5/21/09 6:38 PM, Ethayathulla Abdulsamath wrote:helloI am have a MAD dataset collected upto 2.3A but anomalous signal is upto 3.5A. The dataset is in P6 and based on pointless and absences it is P6122. Overall Rlin/rsym is (0.07/ 0.06). the overall redundancy is 10. I have two Se peaks, i used solve/resolve to find those peaks. I used datasets from 3.5 to 20A to find peaks i got two good peaks of occ (1.4 amp; 0.92). I did analyze_solve and refined those peaks. Then i tried resolve to autobuild and phasing but it didnt work. The r-factor FC vs FP is 0.40 and FOM 0.6, although the
Re: [ccp4bb] Experimental design and response surface methods for crystal optimization
Hi Christian We often use a simple approach where people design their experiments using rational multivariate design, but then simply use the best well that is found as the centre of the next round of experiments. This means that you don't have to score all the wells - which is time-consuming! There's more info at http://www.douglas.co.uk/rat_des.htm Patrick -- patr...@douglas.co.uk Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Benda, Christian Sent: 15 May 2009 15:07 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Experimental design and response surface methods for crystal optimization Dear all I recently came across the experimental design method for systematic optimization of multiparametric problems like macromolecular crystal growth. (The response surface concept has been introduced to protein crystallization by Carter et al. (e.g. Meth. Mol. Biol, vol. 363) and should theoretically enable optimization of a multiparatmetric problem by simultaneous variation of several variables). I am wondering if anyone is actually making use of this method on a regular basis and what their experiences are. It would also be interesting to now if tools (web-based) are available to help in designing and evaluating experiments. Any comment appreciated! Thanks very much Christian
Re: [ccp4bb] How to improve crystal which is twinning?
If you haven't already used it, I would certainly try the approach of microseeding into screens since you already have crystals http://scripts.iucr.org/cgi-bin/paper?S0907444907007652 -- patr...@douglas.co.uk mailto:patr...@douglas.co.uk Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of HanJie_HCT Tai Sent: 17 May 2009 13:27 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] How to improve crystal which is twinning? Hi, I have a 22kDa protein that the floopy N C terminus have been deleted. It was crystallized in 35%MPD/0.1m Tris (pH 8.5)/0.2M/(NH4)2SO4 in 2 - 3 days. Previously, a few small twining crystals were grown in this condition. I tried Hampton Research 96-additive screen . Additives such as glycerol, dioxane, etc didn't work well to improve/reduce twining issue. The 0.3% DMSO is the best additive I found that can grow a single crystal in the 1(pro)+0.8(buff)+0.2(add) drop. However, If looking careful under microscope it may be some other crystals growing inside that single crystal(hardly see under the microscope, but I can see it in other drops). I conducted the x-ray diffraction experiment for this 0.1x 0.1 mm crystal for which no cryoprotectant is required. The highest resolution is 2A but there are a lot of smear on the diffraction pattern. Thus, the index procedures failed due to the crystal quality. Do you have any brilliant ideas to improve the crystal growing condition in my case in order to get a truly single crystal? regards, Heng Windows Live(tm): Keep your life in sync. Check it out. http://windowslive.com/explore?ocid=TXT_TAGLM_BR_life_in_synch_052009
[ccp4bb] Mr Bump
Dear all, I am testing Mr BUMP on vista and run into the following error message, after it has performed fasta and clustal alignements, it downloads several pdb files such as: PDB Download log: Chain:1qyc_B Download Log: Attempting to download the PDB file for 1qyc from WWPDB site: URL: ftp://ftp.wwpdb.org/pub/pdb/data/structures/all/pdb/pdb1qyc.ent.gz Download Log: This will be saved to: C:/Users/Thierry/Desktop/LAR-NAP\search_7\PDB_files\1qyc.pdb But the files which are saved in this directory are in fact: C:/Users/Thierry/Desktop/LAR-NAP\search_7\PDB_files\1qyc.pdb.gz and C:/Users/Thierry/Desktop/LAR-NAP\search_7\PDB_files\pdb1qyc.ent and afterwards, when executing PDBclip, I get the following message: Processing PDB files (PDBclip): --- PDB Processing log: Model 1qyc_B - Could not find the unprocessed PDB file for this model. and afterwards: - Process PDBs - Processing file: 1qyc.pdb, chain: A --- modifying with coord_format. Coord_format command line: C:\CCP4-Packages\ccp4-6.1.1\bin\coord_format.exe xyzin C:/Users/Thierry/Desktop/LAR-NAP\search_7\PDB_files\1qyc.pdb xyzout C:/Users/Thierry/Desktop/LAR-NAP\search_7\data\1qyc_A\pdbclip\cf_1qyc_A.pdb PDB download log: coord_format failed for 1qyc_A PDB download log: log file can be found at: C:/Users/Thierry/Desktop/LAR-NAP\search_7\data\1qyc_A\pdbclip\coord_format_1qyc_A.log In this coord_format_1qyc_A.log file, I have the following: File: C:/Users/Thierry/Desktop/LAR-NAP\search_7\PDB_files\1qyc.pdb Cannot be opened for reading CCP4 library signal ccp4_general:Cannot find input file (Error) raised in ccp4setenv It seems that the downloaded and uncompressed files are not the apropriate ones. How can I get around this problem? thank you very much for your help -- Thierry GRANIER CNRS UMR 5248 CBMN Bâtiment B8 Avenue des Facultés Université Bordeaux 1 33405 Talence Cedex FRANCE Tél: 33 5 40 00 24 52 Fax: 33 5 40 00 22 00 email: t.gran...@cbmn.u-bordeaux.fr
[ccp4bb] MALLS equipment: SUMMARY
Dear all, here is a summary of the few responses that I have got from my posting on MALLS equipment suggestions. 2 labs have WYATT miniDawn system. One is connected to an Agilent HPLC. Seems to be quite good. 1 lab has a Waters equipment connected to an Akta FPLC, which seems to be more appropriate than Akta purifier because on this latter, the pumps have a very short oscillation time, creating some serious noise. More advices are still welcome. Yours Marc -- Marc Graille, PhD Yeast Structural Genomics, IBBMC CNRS UMR8619 Bat 430 Universite Paris Sud 91405 Orsay Cedex NEW PHONE NUMBER Tel: (33) 1 69 15 31 57 Fax: (33) 1 69 85 37 15
[ccp4bb] create cif for Zn atom
Dear Friends, I have Zn atoms in my pdb file. So, I think I need to run elbow to create the cif otherwise refinement stops. I did the following and got the error as follows: phenix.elbow generate_easy_r4.pdb --do-all -- electronic Ligand Builder Optimisation Workbench (eLBOW) 1.4 3 - Nigel W. Moriarty (nwmoria...@lbl.gov) -- Random number seed: 664322001 0:00 Parsing Parsing Parsing Parsing Parsing Parsing Parsing Parsing Parsing P No molecule read Use --all-residues to view residues if this is a PDB file Can anyone suggests at this point, what I should do? Thanking you in advance... Raja Explore and discover exciting holidays and getaways with Yahoo! India Travel http://in.travel.yahoo.com/
[ccp4bb] Subject: off topic, purification from insect media and TFF/CFF
Hey, I have a GST fusion protein secreted into express5 (with hint of serum), it does not bind beads. Changing pH with 4xPBS limits ppt and enables binding but in a scale up I think it makes sense to use a TFF/CFF setup for concentration and diafiltration. Does anybody have advice about purifying proteins from insect media? Is TFF the right choice? And any recommendations for TFF system/brand will be appreciated. Cheers, Mark
Re: [ccp4bb] Tips on fitting poorly defined loop regions
Hi Drew, Sorry for the slightly late reply. Something that 'might' be able to help (though I fully agree with Dale's reply) is the procedure for local density improvement described here: Acta Cryst. (1997). D53, 540-543 Local Improvement of Electron-Density Maps. We are currently in the process of implementing this in phenix. We have a preliminary command line tool (phenix.grow_density) ,and though it's not anywhere near ready for general use yet, if you want we could have a look at your data, as we are currently looking for test cases. Anybody else who feels they may benefit from this procedure and is willing to let us test it on their data is also welcome to contact me offline. Lester Carter PSI Technology Portal: http://technology.lbl.gov/portal/ LBNL , Berkeley X-RAL-MFrom: aw...@med.nyu.edu X-RAL-Connect: ictmailer1.itd.rl.ac.uk [130.246.192.56] Date: Wed, 20 May 2009 15:45:43 +0100 Reply-to: Drew Waight aw...@med.nyu.edu Sender: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK Hello all, I'm just finishing up my first structure, a membrane protein with 5 fold NCS. The native dataset is good to about 2.1A. The R factors are good, around .22/.26 without waters. The only problem is that I'm having a difficult time fitting what looks to be a very important (potential binding site) loop region of about 20 AA or so because the density is poor. I have relied heavily on the NCS to solve the rest of the structure, but releasing the constraints doesn't particularily help. I can see green blobs here and there in the difference map, and there is some continuous density at .8 sigma but its much poorer than the rest of the protein. (which is also mostly alpha helical) I would imagine that this is a common problem and am wondering if there are any tips from the experts seeing as I am extremely new to this. Thank you all, I have already learned a great deal from this board. Drew P.S. I have been using CCP4, Coot, Refmac, DM, Resolve (for dm), and Phenix refine
