[ccp4bb] Crystal Optimization
Dear All, We got crystals for a 35 KDa protein with 323aa including His tag and linker. It was originally crystallized in 0.1M BisTris Propane pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350. Later we managed to obtain crystals with 0.1M BisTris pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350 as well. Crystals are 3 dimensional in shape and 0.2-0.3mm long. Maximum resolution obtained till now is 5.8Å. Tried various cryo conditions like Oil, glycerol, salt and sugars. However, the resolution hasn't improved. The crystal tends to break in the presence of glycerol. Kindly give your suggestions to improve the resolution. Thanks. Jobi
Re: [ccp4bb] Crystal Optimization
Dear Jobi, The usual suspects are: - add another purification step. In fact I'd do that first: * Ni-column * remove His-tag * Ni-column * gel filtration if you have done that: add another step to test for better crystals - additive screens - room temperature data set to check if freezing hampers the crystal - seeding - crystallisation at different temperatures - removal of the His-tag (or leave it on if it is cleaved) etc, etc, pp Cheers, Tim On Wed, Apr 13, 2011 at 05:44:12PM +0800, Jobichen Chacko wrote: Dear All, We got crystals for a 35 KDa protein with 323aa including His tag and linker. It was originally crystallized in 0.1M BisTris Propane pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350. Later we managed to obtain crystals with 0.1M BisTris pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350 as well. Crystals are 3 dimensional in shape and 0.2-0.3mm long. Maximum resolution obtained till now is 5.8Å. Tried various cryo conditions like Oil, glycerol, salt and sugars. However, the resolution hasn't improved. The crystal tends to break in the presence of glycerol. Kindly give your suggestions to improve the resolution. Thanks. Jobi -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] Crystal Optimization
Dear Jobi, For a crystal which is big in size(0.2-0.3mm is pretty big) while diffract poorly, dehydration (increase concentration of the precipitant slowly) is a good choice to improve diffraction, especially for those tends to crack during cryo. Also those regular optimization approaches: Additive screen etc. Sometimes cleave the tag or change it to another end will work. Cheers, Bingfa 发件人: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] 代表 Jobichen Chacko 发送时间: 2011年4月13日 17:44 收件人: CCP4BB@JISCMAIL.AC.UK 主题: [ccp4bb] Crystal Optimization Dear All, We got crystals for a 35 KDa protein with 323aa including His tag and linker. It was originally crystallized in 0.1M BisTris Propane pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350. Later we managed to obtain crystals with 0.1M BisTris pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350 as well. Crystals are 3 dimensional in shape and 0.2-0.3mm long. Maximum resolution obtained till now is 5.8Å. Tried various cryo conditions like Oil, glycerol, salt and sugars. However, the resolution hasn't improved. The crystal tends to break in the presence of glycerol. Kindly give your suggestions to improve the resolution. Thanks. Jobi
Re: [ccp4bb] Crystal Optimization
Dear Jobi, the paper of Heras and Martin 'Post-crystallization treatments for improving diffraction quality of protein crystals' is really helpful. Additionally, if you have lysines have you tried reductive methylation? Good luck, e On Wed, April 13, 2011 12:34, Bingfa Sun wrote: Dear Jobi, For a crystal which is big in size(0.2-0.3mm is pretty big) while diffract poorly, dehydration (increase concentration of the precipitant slowly) is a good choice to improve diffraction, especially for those tends to crack during cryo. Also those regular optimization approaches: Additive screen etc. Sometimes cleave the tag or change it to another end will work. Cheers, Bingfa 发件人: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] 代表 Jobichen Chacko 发送时间: 2011年4月13日 17:44 收件人: CCP4BB@JISCMAIL.AC.UK 主题: [ccp4bb] Crystal Optimization Dear All, We got crystals for a 35 KDa protein with 323aa including His tag and linker. It was originally crystallized in 0.1M BisTris Propane pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350. Later we managed to obtain crystals with 0.1M BisTris pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350 as well. Crystals are 3 dimensional in shape and 0.2-0.3mm long. Maximum resolution obtained till now is 5.8Å. Tried various cryo conditions like Oil, glycerol, salt and sugars. However, the resolution hasn't improved. The crystal tends to break in the presence of glycerol. Kindly give your suggestions to improve the resolution. Thanks. Jobi -- ** Eirini Gkougkoulia Department for Structural and Computational Biology Max F. Perutz Laboratories University of Vienna Campus Vienna Biocenter 5 A-1030 Vienna Austria
Re: [ccp4bb] Crystal Optimization
Hi Jobi, I also had some crystals that were highly sensitive to glycerol. In one case, I found that DMSO at 10-15% can cryo protect it, also the crystal could grow in the presence of 10% DMSO which essentially eliminated a soaking step. In another case, I grew the crystals in the presence of 5% glycerol(it won't take DMSO this time), then the crystal could survive 30% glycerol cryo soak. Also you may try using some smaller crystals as they are easier to freeze and may still diffract decently in synchrotron. Another thing to try is to crosslink the crystals with glutaraldehyde by vapor diffusion. The crystal will partially turn to a gel and will not break when soaked with cryos. In my hand this did produce tough, diffracting crystals, but didn't solve my high mosaicity problem. Of course, RT shooting should be tested first to make sure the poor diffraction is caused by the freezing. Zhijie From: Jobichen Chacko Sent: Wednesday, April 13, 2011 5:44 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystal Optimization Dear All, We got crystals for a 35 KDa protein with 323aa including His tag and linker. It was originally crystallized in 0.1M BisTris Propane pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350. Later we managed to obtain crystals with 0.1M BisTris pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350 as well. Crystals are 3 dimensional in shape and 0.2-0.3mm long. Maximum resolution obtained till now is 5.8Å. Tried various cryo conditions like Oil, glycerol, salt and sugars. However, the resolution hasn't improved. The crystal tends to break in the presence of glycerol. Kindly give your suggestions to improve the resolution. Thanks. Jobi
Re: [ccp4bb] methods to capture proteins from cell culture medium
Bei, How do you concentrate your media? We use tangential flow filtration. If you get a good filter (Millipore Spiral wound TFF is one example) it goes pretty quick, in ~2 hours you should be able to process 4L(concentrate+ dilute several time in buffer). We secrete proteins from insect cells, but something in the media will strip the Ni2+ from our IMAC column if we apply the sup directly. I have tried AS precipitation and peg precipitation from culture media. Both worked fine at the small scale, but when scaling up I ran into 2 issues: 1. You have to add huge amounts of AS (like kilograms), which increases your volume a good amount, 2. my 'pellets' would actually float on top of the media after spinning, which was tough to deal with. I have also tried loading the media onto a large Q column, but that didn't work well for me-fractions were too messy. I think you best option is to get a good TFF setup, do your concentration/buffer exchange, and go right to your IMAC column Nat Nat Clark Graduate Student Garman Lab Biochemistry and Molecular Biology Dept. UMass Amherst 2011/4/13 joybeiyang joybeiy...@gmail.com: Dear all, Thanks a lot for sharing, seems that either a HIC column or AS would work, and that's great, I should give both of them a try. I thought about HIC too, but do not know if it would work since the binding of protein to HIC need high salt conc. and I am not sure if the salt conc. in the sf900 or Hi5 medium is high enough (the formulation is secret, LOL), thus it is good to know that someone has succesful experience with HIC. Thank you very much again! Bei 2011-04-12 joybeiyang 发件人： mi...@chem.ucla.edu 发送时间： 2011-04-12 18:34:27 收件人： joybeiyang 抄送： CCP4BB@JISCMAIL.AC.UK 主题： Re: [ccp4bb] methods to capture proteins from cell culture medium Bei, I had a former labmate who had the same situation and would load somewhere between 6-8L of media directly onto a column. I don't remember what type of column it was, ion exchange may not be ideal if the ionic strength of your medium is high. I think it may have been a phenyl sepharose column. Good luck, Mike - Original Message - From: joybeiyang joybeiy...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, April 12, 2011 2:13:49 PM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] methods to capture proteins from cell culture medium Dear all, My protein of interest was expressed as secreted protein, so I have to collect the medium and change the buffer with sortorius Jet before I load the sample onto a IMAC, the buffer change step in my current protocol can last for 12hrs (I have to concentrate 4L to 200ml, then dilute it with lysis buffer and concentrate it again, then dilute and concentrate repeatedly) and is really boring and troublesome, besides I always observe protein loss during this step and the detergent in the medium usually concentrate as well in this step which would interfere with subsequent purification process. I am wondering if there are more convenient ways to capture the target protein from medium? How about the following: 1. directly load the medium onto a ion exchange column? 2. Amonium sulfate precipitation? 3. anyother thoughts? Thank you very much in advance! Best, Bei 2011-04-12 joybeiyang -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] program to calculate electron density at x,y,z (SUMMARY)
On Tue, 2011-04-12 at 22:54 -0400, Edward A. Berry wrote: What about doing the Fourier summation at the precise location requested, in order to not calculate the map or interpolate at all? Input would be the mtz file rather than map file. eab One advantage of map interpolation is speed - my code (admittedly not optimized for speed) is about 6x slower than sftools/mapman combination (the advantage of fast fourier transform and explicit use of symmetry). But these days it is only 20ms per atom on a 40,000 reflection dataset (on a modest 2.33GHz Core2 Q8200 CPU). -- Hurry up before we all come back to our senses! Julian, King of Lemurs
[ccp4bb] Methods to calculate the importance of mutated residue on the stability of protein
Dear all, Do anyone know the way to estimate the importance of mutation contributing to the stability of protein? Thanks for the help. Heng-Keat
Re: [ccp4bb] methods to capture proteins from cell culture medium
Hi Bei, For the extracellular protein I worked on in graduate school, I typically purified it from 4 L preps in LB media. The standard protocol was to do a crude low cut with ammonium sulfate cut followed by precipitation of the protein with a high cut. The pellet was then resuspended, dialyzed and loaded on an S-sepharose column. I experimented with taking the media (after spinning out the cells) and diluting 1:1 with a low ionic strength buffer and loading directly onto the S-sepharose column. This worked, but the loading time for 8 L was so long it was not worth it. Another option might have been to use bulk media to bind the protein in a batch step. I never tested this. A final option that we explored was Expanded-Bed Adsorption Chromatography. This would allow us to get rid of the initial centrifugation step to remove the cells from the media and would allow fast loading with a high speed pump. We priced out the media, column and pump from Pharmacia at the time, but never ended up purchasing the system. The technology looked promising and should have worked well for our system, but we decided that we did not need to do too many more preps for the project and just used the standard protocol. Regards, Mitch -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of joybeiyang Sent: Tuesday, April 12, 2011 2:14 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] methods to capture proteins from cell culture medium Dear all, My protein of interest was expressed as secreted protein, so I have to collect the medium and change the buffer with sortorius Jet before I load the sample onto a IMAC, the buffer change step in my current protocol can last for 12hrs (I have to concentrate 4L to 200ml, then dilute it with lysis buffer and concentrate it again, then dilute and concentrate repeatedly) and is really boring and troublesome, besides I always observe protein loss during this step and the detergent in the medium usually concentrate as well in this step which would interfere with subsequent purification process. I am wondering if there are more convenient ways to capture the target protein from medium? How about the following: 1. directly load the medium onto a ion exchange column? 2. Amonium sulfate precipitation? 3. anyother thoughts? Thank you very much in advance! Best, Bei 2011-04-12 joybeiyang
Re: [ccp4bb] Methods to calculate the importance of mutated residue on the stability of protein
Hi Keat, Check this http://cupsat.tu-bs.de/ (CUPSAT: Cologne University Protein Stability Analysis Tool) Hope it serves your purpose. Gauri On Wed, Apr 13, 2011 at 9:50 AM, Heng Keat Tam t...@bio.chemie.uni-freiburg.de wrote: Dear all, Do anyone know the way to estimate the importance of mutation contributing to the stability of protein? Thanks for the help. Heng-Keat
Re: [ccp4bb] Crystal Optimization
Hi Jobi- You might want to try using drop ratios ---we have had great success with this many times. My favorite additive screen is using the Hampton Research Crystal Screen HT as an additive screen. I usually start by adding 5% to the well---this has often yielded good crystals where the traditional 96 reagent additive screens and the detergent screens do not. Good Luck! Annie From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jobichen Chacko Sent: Wednesday, April 13, 2011 5:44 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystal Optimization Dear All, We got crystals for a 35 KDa protein with 323aa including His tag and linker. It was originally crystallized in 0.1M BisTris Propane pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350. Later we managed to obtain crystals with 0.1M BisTris pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350 as well. Crystals are 3 dimensional in shape and 0.2-0.3mm long. Maximum resolution obtained till now is 5.8Å. Tried various cryo conditions like Oil, glycerol, salt and sugars. However, the resolution hasn't improved. The crystal tends to break in the presence of glycerol. Kindly give your suggestions to improve the resolution. Thanks. Jobi
Re: [ccp4bb] Crystal Optimization
Dear Jobi, See if you can slowly increase the PEG concentration to 28 - 30% and that will be a good cryo. Since BIS Tris Propane is the buffer, I think, 28% 3350 should work fine. If the crystals crack by going straight to 28% PEG 3350, 0.1M Bis Tris Propane pH:6.5, 0.2M Potassium thiocyanate solution, put the crystal in a 24% solution and slowly increase the concentration by adding the higher concentration well solution. It seems to be a good idea to keep the crystals for 2 or 3 mts in a solution before increasing the PEG concentration (well, this seems to vary with protein). Also remember to include any salts in the cryo (at least half of the concentration) that may be already in the protein solution. Kind regards, Mathews -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jobichen Chacko Sent: Wednesday, April 13, 2011 2:44 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystal Optimization Dear All, We got crystals for a 35 KDa protein with 323aa including His tag and linker. It was originally crystallized in 0.1M Bis Tris Propane pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350. Later we managed to obtain crystals with 0.1M BisTris pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350 as well. Crystals are 3 dimensional in shape and 0.2-0.3mm long. Maximum resolution obtained till now is 5.8Å. Tried various cryo conditions like Oil, glycerol, salt and sugars. However, the resolution hasn't improved. The crystal tends to break in the presence of glycerol. Kindly give your suggestions to improve the resolution. Thanks. Jobi
Re: [ccp4bb] Methods to calculate the importance of mutated residue on the stability of protein
Hi Heng-Keat, you can try our new server (beta version) at: http://babel.ucmp.umu.se/prosms/ Cheers, /Uwe On 2011-04-13 15:50, Heng Keat Tam wrote: Dear all, Do anyone know the way to estimate the importance of mutation contributing to the stability of protein? Thanks for the help. Heng-Keat -- Dr. Uwe H. Sauer, Associate Professor BioCrystallography BioInformatics Group Computational Life Science Cluster, CLiC Umea Centre for Molecular Research, UCMR Centre for Chemical Biology, KBC Deptartment of Chemistry Umea University, Linnaeus vag 6, SE-901 87 Umea, Sweden Tel: +46-(0)90-786-5930 FAX: +46-(0)90-786-7655 e-mail: uwe.sa...@chem.umu.se URL: http://soul.ucmp.umu.se/~ucmp/uhs/
Re: [ccp4bb] Promoting oligomer dissociation
Many thanks Jacob and Mark for your questions/suggestions. In response: So what happened with the non-reducing gel? (If the DTT was fresh, there should be no problem, but if not...) The gel is very clear, it shows the same exact pattern as the reducing gels. Both high and low MW fractions run at the same MW. I used -80C stocks of the native protein for which the DTT was buffer exchanged out (3, ~15-fold exchanges via concentration/dilution). Technically, there is a tiny amount in there. But I don't think it's disulfides based on this experiment. Concerning the freshness of added DTT, it's pretty much as fresh as our solid DTT at -20C is fresh. I make all buffers containing DTT just before use and I do it by first adding all the components to ddH2O except the DTT; then I put the buffer at -20C until it gets cold ~4C. Then I pH it and add the DTT and move it to the column. you're right, I read too quickly over the 2 orders, and understood 2-fold. Is it possible the oligomer peak contains higher aggregates that inhibit crystallisation and do not separate well on your gel filtration column? In that case, perhaps very high-g centrifugation could remove them, or a gel filtration column that separates better in that MW-range. Or perhaps the tail fractions of the oligomer peak on your current column might be already conformationally pure enough. Concerning this point made by Mark, there might be something to this. On gels these fractions do tend to appear slightly dirty in that there are a couple very faint bands at high MW. Also, When I rerun individual peak fractions back through gel filtration, the high MW samples sometime result in additional light-absorbing matter at even higher MW closer to the void fractions. Crystallization drops for the high MW fractions appear less soluble even at lower concentrations than the lower MW fractions. However, the unclipped, lower MW samples also are less soluble than their clipped counterparts (which raises the issue of the importance of the tag). I'm still playing with optimization of these samples as well because who knows really.
[ccp4bb] Postdoctoral position available
This notice is posted on behalf of Wim Hol. Please send inquiries to him at the email address at the bottom of the page. Postdoctoral Position Available Laboratory of Wim Hol Department of Biochemistry, School of Medicine University of Washington, Seattle, USA Structural Biology of the type II secretion system from pathogenic bacteria The projects in Wim Hol’s protein crystallography group at the University of Washington are all focused on providing a basis for development of new therapeutics for tropical diseases. This particular postdoctoral position is within a collaborative project which aims to unravel the architecture, mechanism of action and biogenesis of the” type II secretion system” (T2SS). The sophisticated T2SS occurs in many pathogenic bacteria. This machinery is responsible for translocating a wide variety of proteins in a folded state from the periplasm across the outer membrane into the extracellular milieu. One of these proteins is cholera toxin which has been studied intensively in the Hol lab. The large T2SS consists of ~14 different proteins that span the inner and the outer membrane, and is associated with a secretion ATPase in the cytoplasm which provides the energy for the secretion process. Another remarkable feature of the T2SS is a helical sub-assembly in the periplasm ! which is likely serving as a piston pushing cholera toxin and other exoproteins through a pore in the outer membrane. The successful candidate will have the opportunity to: (i) carry out protein expression and protein chemistry studies to obtain insight into protein-protein interactions involving the T2SS from pathogenic bacteria like Vibrio cholera, enterotoxigenic E. coli, and other bacteria; (ii) purify and characterize multi-membrane protein complexes; (iii) determine high resolution crystal structures of multi-protein sub-complexes of the machinery; (iv) apply electron microscopy techniques in the laboratory of Dr. Tamir Gonen to enhance our insight into the architecture and functioning of the T2SS; (v) combine the results of electron microscopy and crystallography. For more information regarding the laboratory of Wim Hol see the following websites: http://www.bmsc.washington.edu/WimHol/ http://depts.washington.edu/biowww/faculty/hol.html and regarding the laboratory of Tamir Gonen: http://depts.washington.edu/biowww/faculty/gonen.html START DATE: Immediately INSTITUTION: Department of Biochemistry Biomolecular Structure Center School of Medicine Box 357742 University of Washington Seattle, WA, 98195 USA Requirements Experience with membrane protein preparation, characterization and crystallography. Application If you are interested, please send your CV, including a description of your experience and technical know-how, a list of publications and presentations, and names and email addresses of three references able to assess your scientific experience and capabilities to: wg...@u.washington.edu
[ccp4bb] Inside Diamond
This is much better than Coronation Steet! http://blog.the-scientist.com/2011/04/11/multipole-wigglers/ Rex Palmer Birkbeck College
[ccp4bb] Comparing two proteins
Dear All What is the best program to use for comparing two protein structures which are very similar both structurally and wrt aa sequence? ie to get the rms deviations both generally and in selected regions. Rex Palmer Birkbeck College
Re: [ccp4bb] Comparing two proteins
Hi Rex, (not claiming it is the best) may be you can use structure comparison tool that allows you to load a bunch of similar structures, superpose them and corresponding maps, and it will highlight various differences (Ramachandran, rotamers, secondary structure, etc..). Have a look at page #17 here: http://cci.lbl.gov/~afonine/pavel_phenix_general.pdf Pavel. On Wed, Apr 13, 2011 at 1:18 PM, REX PALMER rex.pal...@btinternet.comwrote: Dear All What is the best program to use for comparing two protein structures which are very similar both structurally and wrt aa sequence? ie to get the rms deviations both generally and in selected regions. Rex Palmer Birkbeck College
[ccp4bb] about RMSD bond lengths and angles
Dear All, I am wondering about the ranges of RMSD bond lengths and angles. What are the acceptable ranges for these two values? Is there some statistics for them? Thanks and best wishes.
[ccp4bb] NCSMASK question
Hi,
I want to generate a mask using NCSMASK; however, whenever I tried to add
the SYMMETRY keyword, and output mask cannot be opened in coot. The
following is my script and I was testing on PDB# 2VZ8. Thanks in advance
for any suggestions:
ncsmask xyzin ${EOMDATA}/2VZ8.pdb mskout 2VZ8-ncs.msk eof
SYMMETRY 4
END
eof
Hailiang
Re: [ccp4bb] about RMSD bond lengths and angles
This has been discussed multiple times on bb, also BMC p640. The restraint target standard uncertainty provides an upper limit54 for a reasonable bond or angle r.m.s.d. from targets within the model (approximately 0.02 Å and 1.9deg, respectively), but makes no further assumption where these values de facto should be in an optimal refi nement of a protein structure.73 Assume at one extreme a low-resolution torsion angle refi nement with fixed bond lengths, where the bond length deviation from targets will be zero. At the other extreme, an unrestrained refi nement at ultra-high resolution will refl ect the bond length variation as observed in small molecules, around ~0.02 Å. In between the values will vary, depending on the properly selected overall restraint weight that minimizes R-free (or preferably LL-free).73 The notion that the r.m.s.d. from targets should be the same for all structures and that it should be close to small molecule values is not correct. br From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jiamu Du Sent: Wednesday, April 13, 2011 2:06 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] about RMSD bond lengths and angles Dear All, I am wondering about the ranges of RMSD bond lengths and angles. What are the acceptable ranges for these two values? Is there some statistics for them? Thanks and best wishes.
Re: [ccp4bb] about RMSD bond lengths and angles
Thank you. On Wed, Apr 13, 2011 at 6:03 PM, Bernhard Rupp (Hofkristallrat a.D.) hofkristall...@gmail.com wrote: This has been discussed multiple times on bb, also BMC p640. The restraint target standard uncertainty provides an upper limit54 for a reasonable bond or angle r.m.s.d. from targets within the model (approximately 0.02 Å and 1.9deg, respectively), but makes no further assumption where these values *de facto *should be in an optimal refi nement of a protein structure.73 Assume at one extreme a low-resolution torsion angle refi nement with fixed bond lengths, where the bond length deviation from targets will be zero. At the other extreme, an unrestrained refi nement at ultra-high resolution will refl ect the bond length variation as observed in small molecules, around ~0.02 Å. In between the values will vary, depending on the properly selected overall restraint weight that minimizes *R*-free (or preferably –*LL*-free).73 The notion that the r.m.s.d. from targets should be the same for all structures and that it should be close to small molecule values is not correct. br *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Jiamu Du *Sent:* Wednesday, April 13, 2011 2:06 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] about RMSD bond lengths and angles Dear All, I am wondering about the ranges of RMSD bond lengths and angles. What are the acceptable ranges for these two values? Is there some statistics for them? Thanks and best wishes.
Re: [ccp4bb] Comparing two proteins
On Apr 13, 2011, at 4:00 PM, Rex Palmer wrote: What is the best program to use for comparing two protein structures which are very similar both structurally and wrt aa sequence? ie to get the rms deviations both generally and in selected regions. Best is kind of subjective, but you can use the MatchMaker tool in Chimera to superimpose the structures and show the superposition. MatchMaker also has an option to show the corresponding sequence alignment. The alignment will have a histogram of the column RMSD values running across the top. You can drag the mouse on the alignment to select/highlight the corresponding parts of the structure and to show the RMSD of the dragged region in the alignment's region- browser window. MatchMaker described here: http://www.cgl.ucsf.edu/chimera/current/docs/ContributedSoftware/matchmaker/matchmaker.html Chimera obtainable here: http://www.cgl.ucsf.edu/chimera --Eric Eric Pettersen UCSF Computer Graphics Lab http://www.cgl.ucsf.edu
