Re: [ccp4bb] 100% Rmerge in high resolution shell
Usually this means that you have relatively high multiplicity, which give-or-take improves the I/sig(I) by sqrt(m) where m is the multiplicity, but also increases the Rmerge. For any given narrow shell of reflections, Rmerge ~ 0.8 / unmerged(I/sig(I)) merged(I/sig(I)) ~ sqrt(m) * unmerged(I/sig(I)) So it is perfectly possible to have unmerged I/sig(I) of 0.8 which will give you an Rmerge of around 1.0, and have I/sig(I) (merged) around 3, by having multiplciity 14 or so. I suggest that this is the case: if it is much lower than this there is something odd going on. For the merged I/sig(I) Rpim is much more instructive. I'd love it if people reported merged and unmerged I/sig(I), Rmerge, Rmeas, Rpim, CC1/2, ... as each of these tells something different. Best wishes, Graeme Possibly useful papers: http://www.nature.com/nsmb/journal/v4/n4/abs/nsb0497-269.html http://scripts.iucr.org/cgi-bin/paper?he0191 http://scripts.iucr.org/cgi-bin/paper?he0268 On 19 November 2013 06:43, Shanti Pal Gangwar gangwar...@gmail.com wrote: Dear All Can anyone explain the meaning and relevance of data when the Rmerge is 100% in high resolution shell and I/sig(I) is 3. Thanks -- regards Shanti Pal Gangwar School of Life Sciences Jawaharlal Nehru University New Delhi-110067 India Email:gangwar...@gmail.com
Re: [ccp4bb] Fix cell dimensions
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Niu, in XDS the respective keywords in XDS.INP are REFINE(IDXREF), REFINE(INTEGRATE) and REFINE(CORRECT). If you do not want the cell to be refined, you have to set all three keywords (i.e. ensure they are not commented out with a leading '!') in XDS.INP and you must not mention the word 'CELL' on the line. Best, Tim On 11/18/2013 10:48 PM, Niu Tou wrote: Dear All, Does any one know how to strictly fix the cell dimensions during data processing? In HKL2000 there is only a keyword to define the longest vector. In XDS there is a option to input cell parameters, but sometimes the program would not follow the input values and switch back to the one it thinks best. Any suggestions will be appreciated. Thanks! Best, Niu - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Icedove - http://www.enigmail.net/ iD8DBQFSiy4+UxlJ7aRr7hoRAqzjAKCd0E6SBGmgDro+eTVXkmr1nFdpmgCgnCgv FAVxVxvhKUSRkPTP63osrtQ= =d5uB -END PGP SIGNATURE-
Re: [ccp4bb] 100% Rmerge in high resolution shell
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Graeme, On 11/19/2013 09:02 AM, Graeme Winter wrote: [...] For the merged I/sig(I) Rpim is much more instructive. I'd love it if people reported merged and unmerged I/sig(I), Rmerge, Rmeas, Rpim, CC1/2, ... as each of these tells something different. Depending on where you publish the editor will ask you to use their standard layout for the table which was probably last updated in the 1990's given the presence of something as sophisticated as an Rfree... That's my recent experience, which undermined my preference for scientifically sound journals over tabloids. Unfortunately, it's the latter that funding agency like better ... Best, Tim Best wishes, Graeme Possibly useful papers: http://www.nature.com/nsmb/journal/v4/n4/abs/nsb0497-269.html http://scripts.iucr.org/cgi-bin/paper?he0191 http://scripts.iucr.org/cgi-bin/paper?he0268 On 19 November 2013 06:43, Shanti Pal Gangwar gangwar...@gmail.com wrote: Dear All Can anyone explain the meaning and relevance of data when the Rmerge is 100% in high resolution shell and I/sig(I) is 3. Thanks -- regards Shanti Pal Gangwar School of Life Sciences Jawaharlal Nehru University New Delhi-110067 India Email:gangwar...@gmail.com - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Icedove - http://www.enigmail.net/ iD8DBQFSizAgUxlJ7aRr7hoRAtW8AJ9faxDJ6Wz2F5frob8PlOOXne2ZMACfdGxv fA0SSd2GsXKQRqZwg6MHjOk= =fyIi -END PGP SIGNATURE-
Re: [ccp4bb] 100% Rmerge in high resolution shell
I've generally found that adding lines to the standard table works, and they are not removed by editors On 19 Nov 2013, at 09:32, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Graeme, On 11/19/2013 09:02 AM, Graeme Winter wrote: [...] For the merged I/sig(I) Rpim is much more instructive. I'd love it if people reported merged and unmerged I/sig(I), Rmerge, Rmeas, Rpim, CC1/2, ... as each of these tells something different. Depending on where you publish the editor will ask you to use their standard layout for the table which was probably last updated in the 1990's given the presence of something as sophisticated as an Rfree... That's my recent experience, which undermined my preference for scientifically sound journals over tabloids. Unfortunately, it's the latter that funding agency like better ... Best, Tim Best wishes, Graeme Possibly useful papers: http://www.nature.com/nsmb/journal/v4/n4/abs/nsb0497-269.html http://scripts.iucr.org/cgi-bin/paper?he0191 http://scripts.iucr.org/cgi-bin/paper?he0268 On 19 November 2013 06:43, Shanti Pal Gangwar gangwar...@gmail.com wrote: Dear All Can anyone explain the meaning and relevance of data when the Rmerge is 100% in high resolution shell and I/sig(I) is 3. Thanks -- regards Shanti Pal Gangwar School of Life Sciences Jawaharlal Nehru University New Delhi-110067 India Email:gangwar...@gmail.com - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Icedove - http://www.enigmail.net/ iD8DBQFSizAgUxlJ7aRr7hoRAtW8AJ9faxDJ6Wz2F5frob8PlOOXne2ZMACfdGxv fA0SSd2GsXKQRqZwg6MHjOk= =fyIi -END PGP SIGNATURE-
Re: [ccp4bb] translational pseudo symmetry
You would get a different MR solution in P41212 than in P43212 so you shouldnt test the SAME pdb in both SGS? Not sure I am understanding this though. Eleanor On 19 November 2013 05:02, #CHEN DAN# chen0...@e.ntu.edu.sg wrote: Hi Eleanor, I checked P43212 and P41212 by changing the header of mtz file and running refmac for the same PDB input. P43212 is a better match than P41212. Sincerely, Dan From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK on behalf of Eleanor Dodson eleanor.dod...@york.ac.uk Sent: Monday, November 18, 2013 8:47 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] translational pseudo symmetry I guess you have checked that P43212 is a better match than P41212? (And that you are running REFMAC against an mtz file with the same symmetry as the input PDB - you may need to change the SG in the mtz header by hand. mtzutils hklin P41212.mtz hklout P43212.mtz symm P43212 end Or vice versa.. Sorry - THIS IS CRAZY but there you are.. Re the pseudo translation -Randy summs up the situation very clearly. I would build my model by hand actually but I am sure PHASER does itwell too! Something I dont understand but maybe it is to do with your patterson sampling. Peak 3 is a consequence of Pk 1 and Pk2 - Pk 5 is the consequence of Pk 1 and Pk4 but the peak heights dont exactly fit.. Eleanor On 18 November 2013 10:19, Randy Read rj...@cam.ac.uk wrote: Dear Dan, First, you don't want to reprocess in the smaller cell. What xtriage is saying is that, if *and only if* the translation detected in the Patterson map were an exact crystallographic translation, then you would get the smaller cell. However, in order for that to be a plausible hypothesis, the Patterson peaks would have to be near to 100% of the origin peak. You actually seem to have a very interesting case, where the Patterson peaks are related by multiples of approximately the same translation. If you take a translation of 1/2,1/2,1/6 and multiply it by 1, 2 and 3, you get something close to the three biggest peaks in your Patterson (taking account of lattice translations), and these are related by the Patterson inversion centre to what you get if you multiply by 4 and 5. So the six molecules should be related to each other by something close to a repeated translation of 1/2,1/2,1/6. (You should check this in the solution that you already have.) If this were exact, you would have a smaller cell, but it's not exact, and one way in which it is not exact is that the translations along z are not exactly multiples of 1/6. This is reminiscent of a structure that we recently collaborated with Mariusz Jaskolski and Zbyszek Dauter to solve (paper accepted for publication in Acta D). In that case, there are seven translations of approximately 0,0,1/7. The difficulty with cases like this is figuring out how to break the exact symmetry. Any solution that has approximately the right translations will basically fit the data, but you need to find the right combination of deviations from the exact symmetry to get an optimal answer. If you get the wrong deviations from exact symmetry, the refinement will stall, and this may be the problem that you're facing. You can deal with problems like this in Phaser by using the TNCS NMOL 6 command (to say that there are 6 copies related by repeated applications of the same translation). You should tell Phaser to use the 1/2,1/2,0.174 vector (TNCS TRA VECTOR 0.5 0.5 0.174), and hopefully this will break the symmetry in a way that subsequent rigid-body refinement can deal with. I'm happy to give you more advice on this, off-line, because this kind of case isn't something that we've figured out how to deal with automatically yet. The optimal approach probably involves getting a deeper understanding of commensurate modulation, which is another way of thinking about pseudo-translations. Best wishes, Randy Read On 18 Nov 2013, at 09:19, #CHEN DAN# chen0...@e.ntu.edu.sg wrote: Dear experts, I am working on one dataset (2.5A) which was processed using space group P43212 ( 107.9, 107.9, 313.7; 90, 90, 90). After running MR with 6 molecules in ASU and one round of refmac, the R factors are high (38%/45%). I ran phenix.xtriage and found that translational pseudo symmetry is likely present. It suggested that the space group is I4122 with the unit cell about 1/3 smaller (I paste the patterson analyses below). I tried to reprocess the data to get the suggested space group and unit cell using HKL2000. But the index always gives a long c axis about 313A. Could you provide any suggestions on how to proceed? Patterson analyses -- Largest Patterson peak with length larger than 15 Angstrom Frac. coord.:0.5000.5000.174 Distance to origin : 93.757 Height (origin=100) : 55.763 p_value(height) :3.018e-05 The reported
Re: [ccp4bb] Fix cell dimensions
Dear Niu,
concerning XDS: there is no way, using just XDS.INP, to force the IDXREF step
to use a cell that is not compatible with the reflections that COLSPOT found.
But I can think of two ways to reach your goal (even if I believe that
crystallographically it makes no sense to ignore every second existing
reflection).
a) stop after the IDXREF step that uses all reflections, and gives you the 40A
axis that you want to halve (suppose it is the a axis). Next, edit SPOT.XDS
and remove
all lines which have (in this example) odd h. This can be accomplished with
mv SPOT.XDS SPOT.XDS.all
awk '{if ($5%2==0) print $0}' SPOT.XDS.all SPOT.XDS
Then, run IDXREF and subsequent steps - they will use the short a axis.
b) stop after the IDXREF step that uses all reflections, and gives you the 40A
axis that you want to halve (suppose it is the a axis). Next, edit two lines
in XPARM.XDS: line 4, which has the cell parameters (halve a in this line), and
line 5, which has the direction of the a axis in space (halve all 3 values).
Then, run DEFPIX and subsequent steps - they will use the short a axis.
Hope this helps,
Kay
On Mon, 18 Nov 2013 17:03:06 -0500, Niu Tou niutou2...@gmail.com wrote:
Dear Andrew,
As previously I posted a MR case which has a significant 95% off origin
peak, some experts suggested to reprocess the data with cutting one axis to
half, from 40A to 20A. I tried HKL2000 and XDS, none of them is willing to
give a solution with 20A, even I specify it in XDS script. So I wonder is
there any way to force this work to be done. Thanks!
Best,
Niu
On Mon, Nov 18, 2013 at 4:56 PM, Andrew Leslie and...@mrc-lmb.cam.ac.ukwrote:
Dear Niu,
It depends on which part of processing you are referring
to, i.e. the indexing step or the integration step. In MOSFLM there is no
way to enforce cell dimensions during indexing, but providing there is an
indexing solution that has cell dimensions close to the ones you want, you
can enforce a (slightly) different set of cell dimensions during the
integration step. Normally other refined parameters will ensure that you
still get a good prediction of spot positions.
I suspect that this can be done in other programs too.
Without knowing why you want to do this, I cannot comment on whether this
is the best procedure to follow.
Best wishes,
Andrew
On 18 Nov 2013, at 21:48, Niu Tou niutou2...@gmail.com wrote:
Dear All,
Does any one know how to strictly fix the cell dimensions during data
processing? In HKL2000 there is only a keyword to define the longest
vector. In XDS there is a option to input cell parameters, but sometimes
the program would not follow the input values
and switch back to the one it thinks best. Any suggestions will be
appreciated. Thanks!
Best,
Niu
Re: [ccp4bb] 100% Rmerge in high resolution shell
Graeme wrote: ... Rpim is much more instructive. ... as each of these tells something different. I have to ask: Why is Rpim much more instructive? I'm trying to figure this out still. Can one please summarize what are best practices with all these numbers and how each of these tells something different? Another problem that I see is that folks can adjust their sigmas many different ways without knowing they have adjusted their sigmas. And they can be adjusted incorrectly when they are adjusted. BTW, Graeme is correct about lots of multiple low I/sigI observations for each Bragg reflection in a resolution shell will lead to 100% (or higher) Rmerge with I/sigI of 3. This assumes no systematic errors and only randomly distributed random errors (a rare if not impossible situation, I would think). I will defer to others about what the relevance of that is. Thanks for any insights, Jim From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Graeme Winter [graeme.win...@gmail.com] Sent: Tuesday, November 19, 2013 2:02 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] 100% Rmerge in high resolution shell Usually this means that you have relatively high multiplicity, which give-or-take improves the I/sig(I) by sqrt(m) where m is the multiplicity, but also increases the Rmerge. For any given narrow shell of reflections, Rmerge ~ 0.8 / unmerged(I/sig(I)) merged(I/sig(I)) ~ sqrt(m) * unmerged(I/sig(I)) So it is perfectly possible to have unmerged I/sig(I) of 0.8 which will give you an Rmerge of around 1.0, and have I/sig(I) (merged) around 3, by having multiplciity 14 or so. I suggest that this is the case: if it is much lower than this there is something odd going on. For the merged I/sig(I) Rpim is much more instructive. I'd love it if people reported merged and unmerged I/sig(I), Rmerge, Rmeas, Rpim, CC1/2, ... as each of these tells something different. Best wishes, Graeme Possibly useful papers: http://www.nature.com/nsmb/journal/v4/n4/abs/nsb0497-269.html http://scripts.iucr.org/cgi-bin/paper?he0191 http://scripts.iucr.org/cgi-bin/paper?he0268 On 19 November 2013 06:43, Shanti Pal Gangwar gangwar...@gmail.commailto:gangwar...@gmail.com wrote: Dear All Can anyone explain the meaning and relevance of data when the Rmerge is 100% in high resolution shell and I/sig(I) is 3. Thanks -- regards Shanti Pal Gangwar School of Life Sciences Jawaharlal Nehru University New Delhi-110067 India Email:gangwar...@gmail.commailto:email%3agangwar...@gmail.com
Re: [ccp4bb] 100% Rmerge in high resolution shell
Dear Ed, when it comes to deciding about the high-resolution cutoff, I agree that the paired-refinement technique should be used - even more so as it does not require any of the data quality indicators! But my posting was meant in a more general sense (i.e. not only talking about high-resol cutoff): crystallography is complicated enough that we need the right means and tools, which should enable us to make the right decisions. For instance, we need to decide which detector distance to choose, which oscillation range to use, at which frame to cut in case of radiation damage, what to do in case of anisotropy, which data to merge, which datasets to choose for phasing, MR, refinement ... ... . The tools, means and decisions need to be based on the right concepts. I find that the historical concepts (centered on Rmerge), when it comes to data quality indicators, are quite awkward and do not enable understanding of the problems, rather, they obscure the solutions. I hope that you find the concepts that I mention logical and helpful. best, Kay On Tue, 19 Nov 2013 10:27:16 -0500, Ed Pozharski epozh...@umaryland.edu wrote: Dear Kay, I wonder what is your opinion of the following proposition. None of the data quality indicators derived from data alone matter too much. Let me explain what I mean by this. Ultimately, I truly don't care what value of Rmerge, Rpim, or even CC1/2 data processing produces from the set of frames I toss at it. Surely it is important to keep an eye on them to verify that dataset is kosher and to obtain an *initial* estimate of the resolution limit of useful data. But the actual values never deter from trying to solve the structure. If it cannot be solved - well, then none of the aforementioned indicators matter at all. If it is solved, the only remaining question is how far the useful data goes. And that should be determined using Karplus-Diederichs (KD) test. I do use CC1/2~0.5 as initial resolution cutoff these days, but before finalizing any model I run the KD test. I do look at where the Rpim, I/sigma and CC1/2 end up at the resolution edge, but only out of curiosity and to adjust my perception of how they correlate with true resolution. And I think that efforts should be targeted at optimizing KD test as a tool rather than being distracted by outdated approaches that were proposed in the computationally-handicapped times. It is entirely possible that all this is exactly what you said, just with different wording. But I guess more wording is still needed given that people keep asking about Rmerge. Cheers, Ed. On Tue, 2013-11-19 at 14:22 +, Kay Diederichs wrote: Hi Jim, of course the issue of crystallographic data quality indicators deserves a somewhat more appropriate (or at least more permanent, and peer-reviewed) means of dissemination than CCP4BB. Nevertheless I'll sum up some of the most important points I can think of: A) all data quality indicators measure precision, not accuracy B) there are those data quality indicators that measure the precision of unmerged data: (Rsym=)Rmerge, Rmeas, (I/sigma)_unmerged and those that measure the precision of merged data: Rpim, Rsplit (the FEL community uses this; same as R_mrgd_I - see DiederichsKarplus 1997), CC1/2, (I/sigma)_merged The merged indicators usually differ by a factor of sqrt(m) from their unmerged counterparts, where m is multiplicity. Rsplit (~R_mrgd_I) and CC1/2 compare random half-datasets which may be more robust than just hoping that the explicit sqrt(m) law holds (it only holds for unrelated errors). There is no unmerged counterpart of CC1/2. C) Since downstream steps use intensities, it is preferable to use a data quality indicator that does not require sigma to be estimated, because the authors of the different data processing programs/algorithms have different ideas how this should be done. This rules out I/sigma as a useful quality indicator - at least as soon as different programs look at the same data. D) Merged data quality indicators are more useful because we are using merged data for downstream steps (phasing, molecular replacement, refinement), so we need to know _their_ precision, not that of the unmerged data. E) Rpim and Rsplit are calculated from intensities and have a different asymptotic behaviour than model R-values (Rwork, Rfree), so they cannot be meaningfully be compared with model R-values (i.e. their numerical value tells you nothing about the Rwork/Rfree your model can be refined to). This is very different from CC1/2 - it can be used to calculate CC*, a quantity that is the upper limit of what the CC of the model intensities against the experimental intensities can reach. I'll stop here. Most of this may be at variance with what we were all brought up with, but it's time for a change! best, Kay On Tue, 19 Nov 2013 13:18:19 +, Jim Pflugrath jim.pflugr...@rigaku.com wrote: Graeme wrote:
Re: [ccp4bb] distinguish ligand binding sites within a protein
Hello, we work with proteins that have typically several chemically identical binding sites (viral capsid proteins fully assembled or as multimeric assembly-intermediates). Depending on how long at which concentrations they are soaked the chemically identical ligand pockets within one asymmetric unit are typically occupied to different levels purely because of individual crystal contacts and accessibility. I therefore think that neither soaking with different concentrations nor B-factor analysis are solid methods to determine some sort of relative affinities. I'd suggest to design mutants for either binding site and ITC measurements with the mutant proteins. This might also tell you if some sort of co-op exists between both sites. Baerbel Quoting Ed Pozharski epozh...@umaryland.edu: IMHO, while explaining binding affinity from a structure is fun, it does not prove anything. Assuming that I understand your situation correctly, you can (relatively) easily find out from experiment which pocket has higher affinity. Just do soaks with different ligand concentrations - the expectation is that the weaker binding site will become partially occupied first. On Tue, 2013-11-19 at 04:58 +, Xiaodi Yu wrote: Hi Wei: Based on the structure, you can calculate the binding surface between the protein and the ligand. Maybe the two binding pockets will give you two different numbers. And the larger one usually can have the higher binding affinity. You also can analyse how the ligand interacts with the protein though hydrophobic or electrostatic interaction , etc? the last, you may also compare the b factors of the ligand or the protein binding pocket regions after you refining the structure. These things may give you some hints about which binding site is more strong. Dee __ Date: Mon, 18 Nov 2013 22:45:58 -0500 From: wei.shi...@gmail.com Subject: Re: [ccp4bb] distinguish ligand binding sites within a protein To: CCP4BB@JISCMAIL.AC.UK Thank you so much for the suggestions, Tomas! Yes, my ligand is a small molecule. I have the crystal structure of the ligands bound to the protein, do I still need to computationally dock the ligand to the two pockets, can I calculate the parameters of binding directly using the crystal structure? Best, Wei On Mon, Nov 18, 2013 at 9:03 PM, Tomas Malinauskas tomas.malinaus...@gmail.com wrote: Dear Wei Shi, is your ligand a small molecule? If it is a small molecule, I would try to computationally dock the small molecule to two pockets separately using AutoDock, and look at the estimated free energies of binding. Best wishes, Tomas On Mon, Nov 18, 2013 at 8:55 PM, Wei Shi wei.shi...@gmail.com wrote: Hi all, I got the crystal structure of a transcription factor, and every monomer binds two molecules of the same ligand in different binding pockets. And I also did the ITC experiment, titrating the ligand into the protein, and got a U-shaped curve. The binding affinity for the first binding site is higher than the second binding site. I am wondering whether I could computationally determine from the protein-ligand complex structure that which binding site has higher affinity for the ligand and correlate the binding sites with the parameters I got from ITC experiment. Thank you so much! Best, Wei -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse / -- Bärbel Blaum, Ph.D. Interfakultäres Institut für Biochemie (IFIB) Hoppe-Seyler-Strasse 4 D-72076 Tübingen Germany +49 70 71 29 73 375
[ccp4bb] PostDoc - Structural Biology - Cancer
Dear CCP4ers, a post-doctoral position is available immediately in the laboratory of Dr. Alessandro Vannini, within the Division of Structural Biology at The Institute of Cancer Research in Chelsea, London, UK. We are looking for highly motivated individuals with a strong interest in structural characterization of multi-subunit macromolecular complexes. We currently employ single particle electron microscopy analysis and X-ray crystallography, as well as biochemical and biophysical analysis, to elucidate the role of the RNA Polymerase III and associated factors in cancer development. The Division of Structural Biology has managed facilities for protein crystallography (Bruker Microstar and CCD detector and crystallisation robots), cryo-electron microscopy (FEI Tecnai F20 and T12), and protein production with expertise in multi-subunit expression (insect cell, yeast and bacterial expression, including a 60 L fermentor). The Division is also well equipped with equipment for biophysical analysis (e.g. ITC, fluorescence, multi-angle light scattering). Applicants should possess a PhD (or equivalent) in biochemistry or molecular biology and a sound knowledge in production and purification of macromolecular complexes for structural biology analysis. Previous experience with purification of multi-subunit complexes and/or biochemistry of protein-nucleic acid complexes would be beneficial. Previous experience in protein crystallography and/or single particle electron microscopy would be desirable. Appointment will be on Fixed Term Contract for 3 years in the first instance, with a starting salary in the range of £28,425 to £34,944 p.a. inclusive (based on previous post-doctoral experience). CV and covering letter must be submitted online at http://www.icr.ac.uk/jobsearch Job Ref. No. 1378320 Closing Date: 29th November 2014 For informal inquiries you can contact me directly at alessandro.vann...@icr.ac.uk
Re: [ccp4bb] distinguish ligand binding sites within a protein
If I understand the original post correctly, the binding sites in question are not chemically identical. While it's possible that lattice may invert the order in which sites are occupied, it is not very likely given that affinity gap is sufficient to be observable by ITC. Mutagenesis is a good option too. On Tue, 2013-11-19 at 17:12 +0100, Bärbel Blaum wrote: Hello, we work with proteins that have typically several chemically identical binding sites (viral capsid proteins fully assembled or as multimeric assembly-intermediates). Depending on how long at which concentrations they are soaked the chemically identical ligand pockets within one asymmetric unit are typically occupied to different levels purely because of individual crystal contacts and accessibility. I therefore think that neither soaking with different concentrations nor B-factor analysis are solid methods to determine some sort of relative affinities. I'd suggest to design mutants for either binding site and ITC measurements with the mutant proteins. This might also tell you if some sort of co-op exists between both sites. Baerbel Quoting Ed Pozharski epozh...@umaryland.edu: IMHO, while explaining binding affinity from a structure is fun, it does not prove anything. Assuming that I understand your situation correctly, you can (relatively) easily find out from experiment which pocket has higher affinity. Just do soaks with different ligand concentrations - the expectation is that the weaker binding site will become partially occupied first. On Tue, 2013-11-19 at 04:58 +, Xiaodi Yu wrote: Hi Wei: Based on the structure, you can calculate the binding surface between the protein and the ligand. Maybe the two binding pockets will give you two different numbers. And the larger one usually can have the higher binding affinity. You also can analyse how the ligand interacts with the protein though hydrophobic or electrostatic interaction , etc? the last, you may also compare the b factors of the ligand or the protein binding pocket regions after you refining the structure. These things may give you some hints about which binding site is more strong. Dee __ Date: Mon, 18 Nov 2013 22:45:58 -0500 From: wei.shi...@gmail.com Subject: Re: [ccp4bb] distinguish ligand binding sites within a protein To: CCP4BB@JISCMAIL.AC.UK Thank you so much for the suggestions, Tomas! Yes, my ligand is a small molecule. I have the crystal structure of the ligands bound to the protein, do I still need to computationally dock the ligand to the two pockets, can I calculate the parameters of binding directly using the crystal structure? Best, Wei On Mon, Nov 18, 2013 at 9:03 PM, Tomas Malinauskas tomas.malinaus...@gmail.com wrote: Dear Wei Shi, is your ligand a small molecule? If it is a small molecule, I would try to computationally dock the small molecule to two pockets separately using AutoDock, and look at the estimated free energies of binding. Best wishes, Tomas On Mon, Nov 18, 2013 at 8:55 PM, Wei Shi wei.shi...@gmail.com wrote: Hi all, I got the crystal structure of a transcription factor, and every monomer binds two molecules of the same ligand in different binding pockets. And I also did the ITC experiment, titrating the ligand into the protein, and got a U-shaped curve. The binding affinity for the first binding site is higher than the second binding site. I am wondering whether I could computationally determine from the protein-ligand complex structure that which binding site has higher affinity for the ligand and correlate the binding sites with the parameters I got from ITC experiment. Thank you so much! Best, Wei -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse / -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born.
Re: [ccp4bb] distinguish ligand binding sites within a protein
Hi Ed, you are right about the original question, but what I mean is this: if the occupancies (and B-factors) differ so much in crystals with IDENTICAL binding sites, i.e. identical affinities, does this not show that occupancies (and B-factors) do not reflect affinities alone, but equally local packing? There might be individual cases in which such effects can be neglected, but generally I think trying to determine affinities from crystal soaks is, hmm, not very good pratice, simply because there are other dedicated methods to do it that suffer less from side effects. Including the docking approach. Kind regards, Baerbel Quoting Ed Pozharski epozh...@umaryland.edu: If I understand the original post correctly, the binding sites in question are not chemically identical. While it's possible that lattice may invert the order in which sites are occupied, it is not very likely given that affinity gap is sufficient to be observable by ITC. Mutagenesis is a good option too. On Tue, 2013-11-19 at 17:12 +0100, Bärbel Blaum wrote: Hello, we work with proteins that have typically several chemically identical binding sites (viral capsid proteins fully assembled or as multimeric assembly-intermediates). Depending on how long at which concentrations they are soaked the chemically identical ligand pockets within one asymmetric unit are typically occupied to different levels purely because of individual crystal contacts and accessibility. I therefore think that neither soaking with different concentrations nor B-factor analysis are solid methods to determine some sort of relative affinities. I'd suggest to design mutants for either binding site and ITC measurements with the mutant proteins. This might also tell you if some sort of co-op exists between both sites. Baerbel Quoting Ed Pozharski epozh...@umaryland.edu: IMHO, while explaining binding affinity from a structure is fun, it does not prove anything. Assuming that I understand your situation correctly, you can (relatively) easily find out from experiment which pocket has higher affinity. Just do soaks with different ligand concentrations - the expectation is that the weaker binding site will become partially occupied first. On Tue, 2013-11-19 at 04:58 +, Xiaodi Yu wrote: Hi Wei: Based on the structure, you can calculate the binding surface between the protein and the ligand. Maybe the two binding pockets will give you two different numbers. And the larger one usually can have the higher binding affinity. You also can analyse how the ligand interacts with the protein though hydrophobic or electrostatic interaction , etc? the last, you may also compare the b factors of the ligand or the protein binding pocket regions after you refining the structure. These things may give you some hints about which binding site is more strong. Dee __ Date: Mon, 18 Nov 2013 22:45:58 -0500 From: wei.shi...@gmail.com Subject: Re: [ccp4bb] distinguish ligand binding sites within a protein To: CCP4BB@JISCMAIL.AC.UK Thank you so much for the suggestions, Tomas! Yes, my ligand is a small molecule. I have the crystal structure of the ligands bound to the protein, do I still need to computationally dock the ligand to the two pockets, can I calculate the parameters of binding directly using the crystal structure? Best, Wei On Mon, Nov 18, 2013 at 9:03 PM, Tomas Malinauskas tomas.malinaus...@gmail.com wrote: Dear Wei Shi, is your ligand a small molecule? If it is a small molecule, I would try to computationally dock the small molecule to two pockets separately using AutoDock, and look at the estimated free energies of binding. Best wishes, Tomas On Mon, Nov 18, 2013 at 8:55 PM, Wei Shi wei.shi...@gmail.com wrote: Hi all, I got the crystal structure of a transcription factor, and every monomer binds two molecules of the same ligand in different binding pockets. And I also did the ITC experiment, titrating the ligand into the protein, and got a U-shaped curve. The binding affinity for the first binding site is higher than the second binding site. I am wondering whether I could computationally determine from the protein-ligand complex structure that which binding site has higher affinity for the ligand and correlate the binding sites with the parameters I got from ITC experiment. Thank you so much! Best, Wei -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy.
