[ccp4bb] To Trim or Not to To Trim
Hi All, I'm trying to crowdsource an opinion on how people deal with modelling side chains with poorly resolved electron or cryoEM density. My preference is to model the sidechain and allow the B-factors to go high in refinement to represent that the side chain is flexible. However, I'm aware that some people truncate sidechains if density is not present to justify modelling. I've also seen models where the sidechain is modelled but with zero occupancy if density isn't present. Is there a consensus and justifying arguments for why one approach is better? Cheers, Rhys To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Questionable Ligand Density - Part 2
Correction! I mean't 6BV0 et. al. Many apologies!!! Thanks Paul Brear for pointing this out. Rhys On Wed, 24 Jul 2019 at 21:36, Rhys Grinter wrote: > Hi All, > > Thanks for all the helpful comments and discussion surrounding my last > post. I've been doing a little more investigation into this issue and > wanted to see if people were able to provide me with some additional > opinions/insights. > > I investigated the PDB entries for the lead deposition author of the > 6MO0-1-2 series: Lin, Y.-L. ( > http://www.rcsb.org/pdb/results/results.do?tabtoshow=Current=50E454B). > With some slightly alarming results. > > A number of additional entries appear to have ligands associated with > questionable density.: > > 6VB0,1,2,3,4 and 6BUY – Density does not support amino acids modelled and > there are questionable interactions with protein and solvent (Published > Scientific Reports 2017) > > 4NZ8, 4NAQ – Questionable density for peptide modelled, bad/unrealistic > interactions with protein (Published PNAS 2012) > > 4HOM – Questionable density for peptide modelled. Fo-Fc map density on > refinement seems more interconnected than would be indicated by water, > however density does not fit peptide modelled. Bad/unrealistic interactions > with protein observed, specifically Phe7 of peptide (chain B) (Published > PNAS 2012, same paper as 5NZ8 and 4NAQ) > > 4KXD et. al. – Questionable density for amino acids in both EDS maps and > re-refined with SF and model minus ligand. Poor R-factors obtained for 4KXD > on refinement (W 0.27,F 0.33) (Published JBC 2013) > > These constitute many of entries associated with this author (some other > not mentioned appear are okay) and it makes it seem much less likely to me > that modelling of the structures from my previous post are a result of lack > of experience or poor supervision. > > It is in my opinion a huge problem for the field and science in general, > if structures like this are making it into publications in well respected > journals. > > Best Wishes, > > Rhys > > -- > Dr Rhys Grinter > NHMRC Postdoctoral Researcher > Monash University > +61 (0)3 9902 9213 > +61 (0)403 896 767 > -- Dr Rhys Grinter NHMRC Postdoctoral Researcher Monash University +61 (0)3 9902 9213 +61 (0)403 896 767 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] Questionable Ligand Density - Part 2
Hi All, Thanks for all the helpful comments and discussion surrounding my last post. I've been doing a little more investigation into this issue and wanted to see if people were able to provide me with some additional opinions/insights. I investigated the PDB entries for the lead deposition author of the 6MO0-1-2 series: Lin, Y.-L. ( http://www.rcsb.org/pdb/results/results.do?tabtoshow=Current=50E454B). With some slightly alarming results. A number of additional entries appear to have ligands associated with questionable density.: 6VB0,1,2,3,4 and 6BUY – Density does not support amino acids modelled and there are questionable interactions with protein and solvent (Published Scientific Reports 2017) 4NZ8, 4NAQ – Questionable density for peptide modelled, bad/unrealistic interactions with protein (Published PNAS 2012) 4HOM – Questionable density for peptide modelled. Fo-Fc map density on refinement seems more interconnected than would be indicated by water, however density does not fit peptide modelled. Bad/unrealistic interactions with protein observed, specifically Phe7 of peptide (chain B) (Published PNAS 2012, same paper as 5NZ8 and 4NAQ) 4KXD et. al. – Questionable density for amino acids in both EDS maps and re-refined with SF and model minus ligand. Poor R-factors obtained for 4KXD on refinement (W 0.27,F 0.33) (Published JBC 2013) These constitute many of entries associated with this author (some other not mentioned appear are okay) and it makes it seem much less likely to me that modelling of the structures from my previous post are a result of lack of experience or poor supervision. It is in my opinion a huge problem for the field and science in general, if structures like this are making it into publications in well respected journals. Best Wishes, Rhys -- Dr Rhys Grinter NHMRC Postdoctoral Researcher Monash University +61 (0)3 9902 9213 +61 (0)403 896 767 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] Questionable Ligand Density: 6MO0, 6MO1, 6MO2
Hi All, I was chatting with a colleague during a recent synchrotron visit and they'd recently come across some ligand/drug bound structures associated with a paper recently published in a high impact factor journal. They had pulled the associated SFs from the PDB and found that the electron density associated with these ligands didn't match that reported in the paper and certainly wasn't sufficient to model the alleged ligand. I also pulled the structure factors and after refinement in the presence/absence of the alleged ligand I also feel that the density present does not warrant modelling of the ligand. I was hoping that the community might be able to give me an outside opinion on these datasets (PDB IDs: 6MO0, 6MO1, 6MO2) and if the problem associated with the data is verified, provide some advice on how to proceed. This isn't the first occasion I've seen ligand bound structures with questionable density deposited in association with papers in well respected journals. Despite improvements to validation I feel that this problem is widespread. Best Regards, Rhys -- Dr Rhys Grinter NHMRC Postdoctoral Researcher Monash University +61 (0)3 9902 9213 +61 (0)403 896 767 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] PhD opportunity on Mycobacterial structural biology at Monash University, Melbourne
Dear All, There is an exciting opportunity to do a PhD co-supervised by Dr. Chris Greening and myself at Monash University, Melbourne. The project will investigate the structural basis for persistence in Mycobacterium tuberculosis, through the structural characterisation of the F420 biosynthesis pathway. This multidisciplinary project will combine structural techniques (crystallography, cryo-EM), with biophysics, biochemistry and microbiology. https://www.findaphd.com/search/ProjectDetails.aspx?PJID=74941 As well as being situated in Melbourne the worlds most liveable city, Monash University is within walking distance of the Australian Synchrotron and is home to a cutting edge Cryo-EM microscope facility. Making it a great place to do structural biology. Interested candidates should send their CV, academic transcript, and a brief outline of research interests and motivation to chris.green...@monash.edu. Please share with your colleagues and anyone interesting in starting a PhD in this exciting area. Best Wishes, Rhys -- Dr Rhys Grinter Sir Henry Wellcome Fellow Monash University +61 (0)3 9902 9213 +61 (0)403 896 767 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] Stable Refinement as Low(ish) resolution - Follow up
Hi All, Thanks for all the useful comments. Buster gave me quite stable refinement and nice maps, so I've been working with that so far. I'll work through the other suggestions however, and see what gives the best results. Cheers, Rhys -- Dr Rhys Grinter Sir Henry Wellcome Fellow Monash University +61 (0)3 9902 9213 +61 (0)403 896 767
[ccp4bb] Stable Refinement as Low(ish) resolution
Dear All, I'm currently in the process of refining a low(ish) resolution structure at 3.2 Ang, with a fair level of anisotropy. I processed the data through the anisotropy server (https://services.mbi.ucla.edu/anisoscale/), which elliptically truncated the data to 4.0, 3.8 and 3.2 Ang. This really improved the maps and allowed me to trace the majority of the chain and build most side chains. The R-factors are reasonable (0.29 work and 0.35 free respectively). but I'm having trouble with over fitting in refinement as I continue to refine. What parameters/restraints would the community generally use when refining this kind of structure? Additionally Refmac doesn't seem to read the structure factors from the anisotropy server output file properly, giving vastly inflated R values and strange looking maps. Cheers, Rhys -- Dr Rhys Grinter Sir Henry Wellcome Fellow Monash University +61 (0)3 9902 9213 +61 (0)403 896 767
[ccp4bb] Twinning/Spacegroup Woes
Dear All, As I have approached my crystallography from a biological perspective, sometimes so of the more mathematical/geometrical aspects sometimes perplex me. I was wondering if anyone would be able to clarify what is going on with some problematic crystals I'm working on. I've grown crystals of a protein which forms a concentration dependent oligomer. This is almost certainly a physiological oligomer and probably is a hexamer at maximum oligomerisation (although maybe a trimer). These crystals diffract poorly, however after some optimisation I managed to collect data to around 3.6 A, with a predicted space group of P6322 with unit cell dimensions of 177, 177, 150 . In order to improve diffraction I performed dehydration on these crystals. This seemed to improve diffraction to around 3A (although as the crystals are quite variable attribution of effect is a little difficult), however the best space group I can find for indexing is C2221 with a unit cell of 177, 310, 151, XDS doesn't process the data when I force the previous P6322 SG. It seems also that the C2221 space group isn't the correct choice as the merging stats are worse than I would expect from looking at the diffraction pattern. Additionally, the intensity statistics from both space groups suggests twinning. Although for the P6322 space group it says twinning is not possible. If this is the case what is causing these abnormal intensities and is this related to my SG ambiguity? Also what is the best what to proceed with processing in this case? Cheers, Rhys -- Dr Rhys Grinter Sir Henry Wellcome Fellow Monash University +61 (0)3 9902 9213 +61 (0)403 896 767
[ccp4bb] Improving MR map contrast
Hi All, I recently obtained a molecular replacement solution for a 100kDa protein I'm working on, using an ensemble of low sequence identity models for around 1/4 of the protein into 2.6A data. I got initial Tfz scores of around 8.2 (LLG of 100 or so), which I improved to 14 (LLG 350) which manual rebuilding of the placed fragment. There were enough local features in the map to rebuild the sequence on my protein for this fragment. However, I now have the problem that there is very little contrast in the rest of the map to build into, and the flexibility of the rest of the protein makes it uncertain where the rest of the domains will go. Refinement of my model so far doesn't seem stable and so far attempts at autobuilding have unsurprisingly failed. I was wondering if someone had an idea of how to improve the contrast of my map so I can place the other domains and build the rest of my protein? The solvent content of the crystal is around 55%. Cheers, -- Dr Rhys Grinter Sir Henry Wellcome Fellow Monash University +61 (0)3 9902 9213 +61 (0)403 896 767 -- Dr Rhys Grinter Sir Henry Wellcome Fellow Monash University +61 (0)3 9902 9213 +61 (0)403 896 767
Re: [ccp4bb] Post-translational modification of cystine
The large atom of the end of the modification looks wrong to me for BME. If it were BME it would just be an oxygen right? From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of RHYS GRINTER [r.grinte...@research.gla.ac.uk] Sent: 14 January 2015 00:48 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Post-translational modification of cystine Hi All, I think everyone enjoys this game, so to save me a trawl through the literature can anyone help me interpret this density? The density on the end extents to 7.4 sigma, so something reasonably large. I guess it's some kind of PTM of the cystine residues, but nothing specific springs to mind. the crystallization conditions were 0.1 M CITRIC ACID, PH 5.0, 2.0 M NACL. Cheers, Rhys
[ccp4bb] P31 or P32
Hi All, This will no doubt show something of my ignorance with experimental phasing, however I'm currently working on solving a 3.8A SAD dataset with Pt anomalous signal. Both Shelx and Xtriage see reasonable anomalous signal to about 7A and seem to get statistics suggesting a solution when I run SAD phasing using Autosol in Phenix. After density modification I get pretty nice looking maps with clear solvent channels and interconnected density for protein, however there is very little difference in map R-factor between the hands and the maps from both the P31 and P32 solution appear comparable in structure. Obviously DM is struggling to break the hand ambiguity, however can some one tell me if what I'm seeing represents a definite solution? And what is the best way to proceed from here? Cheers, Rhys
Re: [ccp4bb] P31 or P32
Hi Eleanor, The data is perfectly twinned, with the original auto processing assigning a point group of P312, but I reprocessed it in P3 and it seems to be behaving okay. Are there any additional precautions I should take at the phasing stage with twinned data? BW Rhys From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Eleanor Dodson [eleanor.dod...@york.ac.uk] Sent: 16 December 2014 15:13 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] P31 or P32 Sorry - of course it can.. Sorry. Only if the twinning is perfect then you apparently get a higher symmetry.. Eleanor On 16 December 2014 at 14:26, Tim Gruene t...@shelx.uni-ac.gwdg.demailto:t...@shelx.uni-ac.gwdg.de wrote: Dear Rhys, I would try to place idealised secondary structure elements with coot into the density - at this resolution they probably fit both hands, but you may see a difference when you do e.g. rigid body refinement. Best, Tim On 12/16/2014 10:39 AM, RHYS GRINTER wrote: Hi All, This will no doubt show something of my ignorance with experimental phasing, however I'm currently working on solving a 3.8A SAD dataset with Pt anomalous signal. Both Shelx and Xtriage see reasonable anomalous signal to about 7A and seem to get statistics suggesting a solution when I run SAD phasing using Autosol in Phenix. After density modification I get pretty nice looking maps with clear solvent channels and interconnected density for protein, however there is very little difference in map R-factor between the hands and the maps from both the P31 and P32 solution appear comparable in structure. Obviously DM is struggling to break the hand ambiguity, however can some one tell me if what I'm seeing represents a definite solution? And what is the best way to proceed from here? Cheers, Rhys -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
[ccp4bb] Tetrachloroplatinate coordination
Hi All, I was wondering if anyone knew off the top of their heads if there are any specific protein side chain residues which coordinate or react with Tetrachloroplatinate ions. I'm working on a low-res structure and was wondering if I could use the sites of platinum coordination as a starting point for assigning sequence. My crystal conditions were at pH 7.5. Cheers, Rhys
[ccp4bb] Heavy Atom Phasing
Hi All, I thought I might put a question to the community, with the hope of getting some tips of the best way to proceed with my heavy atom phasing problem. I'm working on solving the structure of an integral beta-barrel membrane protein of approximately 100 kDa. I've crystallised protein, growing some very flimsy needle like crystals, and collected datasets to around 3.1 A. I then produced selenomet derivative protein and repeated crystallisation trials in the same conditions and also repeated broad screens, however the derivative protein failed to produce crystals that diffracted beyond 10 A (in fact it barely crystallises at all). So I've moved on to heavy atom soaks and have had some success with tetrachloroplatinate and tetranitroplatinate compounds, in that the crystals didn't dissolve (as they did with gold and samarium compounds) and diffracted to some degree. I collected SAD data to around 6.5 A from these crystals and there seems to be anomolous signal. However, while I get a good CC of 0.4 from HYSS in phenix with this dataset and the phaser EP FOM is 0.56, the maps before and after DM are uninterpretable. I'm guessing the quality and resolution of the data I collected just aren't good enough (the data is reasonably anisotropic). I performed the metal soaking, by taking a small amount of the platinate salt and adding it to the crystallisation drop as the crystals are extremely fragile and don't stand up well to handling through a soaking or cryo solution. Leaving the crystals to soak for 48 hours and then, freezing them directly. The solution is on the border of cryoprotection (the conditions has PEG2000MME and PVP and the precipitants), but with native crystals this doesn't seem to be a parameter which affects diffraction. The crystals are very variable in performance, so while I feel that the heavy atom soaking has compromised their diffractability to a degree, inherent variation may play a part. What I was wondering is if some one with more experience than me found themselves in this position, how would they proceed? Questions which spring to mind are, how much heavy atom compound do people add and how long do they soak for? Is there anyway I can squeeze something out of the anomalous data I have, given I have 'reasonable' native data, or will poor quality data give spuriously positive statistics for heavy atom phasing? And are there any tricks people have experienced to improve performance of crystals like these (aside from the usual seeding, additives, different detergents etc which I have spend a fair bit of time on optimization already). Thanks in advance, Rhys
Re: [ccp4bb] Best compounds for heavy atom soaks
Hi All, A truly herculean response! Thanks everyone, I will process all of the information and come up with a strategy. Rhys
[ccp4bb] Best compounds for heavy atom soaks
Hello message board, My group has some crystals of an interesting protein to take to the synchrotron in a couple of weeks. We won't be able to prepare and crystallise a SelMet derivative during that time period, but we have loads of crystals sitting around. The diffraction isn't great, we see maybe 3.5 at home but might be enough to get over the line. It will be a very difficult MR target, so we were thinking of soaking so crystals with heavy atomic compounds that we have lying around. I was wondering if people had any suggestions of compounds that people have used successfully for experimental phasing and maybe concentrations to use and soaking time. Cheers, Rhys
[ccp4bb] tricky mr problem
Hi all, I have been attempting to find a MR solution for a low resolution data set (3.9A), with pretty poor merging stats of a 22 strand membrane beta barrel I'm working on. I've created a trimmed poly-alanine from a structure of 17% identity, that gives a solution with a Tfz of 14.3 with two molecules per asu (llg is around 900). I'm guessing this in a genuine solution, but the map is too poor to build into. Does anyone have any advice as to proceed from here? It may be just a case of needing better resolution data to work with, but would this indicate that Selenomet derivative crystals won't be needed for this structure? Cheers, Rhys
Re: [ccp4bb] Concentrating purified membrane protein
Hi Raji, DDM has quite a large micelle so you might be alright with a 100kDa cut off concentrator, I've had the same experience with a 30kDa protein in LDAO being comfortably contained by a 100kDa concentrator. I would try with a small amount of you sample and see if significant amounts of protein are found in the flow through. Is your protein tagged? if so you could bind it to a small volume (1ml) affinity column, exchange it into the lowest DDM conc. it is stable in (0.01% ?) and elute with a very steep gradient. If this didn't give you the desired concentration at least it would minimize the volume to be concentrated (and thus the final detergent concentration). You could also try and intermediate cut off size concentrators you have available (20,30,50kDa etc) until you find biggest one that retain the sample. Good luck Rhys Grinter From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Raji Edayathumangalam [r...@brandeis.edu] Sent: 14 July 2013 01:47 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Concentrating purified membrane protein Hi Folks, Sorry for the non-ccp4 post. I have purified an 18kDa membrane protein and want to concentrate the protein from gel filtration fractions, which are in buffer containing 0.05% DDM (well above the CMC for DDM). My colleague was able to concentrate a 30kDa membrane protein using a 100kDa MWCO concentrator but I am not sure if I can do the same without losing protein in the flowthrough. On the other hand, if use too low a MWCO for the concentrator, then I'm concerned that I may end up concentrating the DDM and end up with too much detergent in the final sample. Any tips about how to concentrate my low MW protein without concentrating the DDM? Many thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Split Crystal Dataprocessing
Hi All, Thanks for all your responses and help. I'll be trying all of these things over the coming couple of weeks. Rhys From: pshawstew...@gmail.com [pshawstew...@gmail.com] On Behalf Of Patrick Shaw Stewart [patr...@douglas.co.uk] Sent: 04 July 2013 16:09 To: RHYS GRINTER Subject: Re: [ccp4bb] Split Crystal Dataprocessing Dear Rhys I'm not sure if your group up there in Gla.ac.ukhttp://Gla.ac.uk uses random microseeding on a routine basis as soon as you get your first hits, but if you don't I would strongly suggest that you try it. The chances are that you can avoid the sort of problems you are facing. I've posted lots of messages to this bb on this topic, and you can find info by searching in google for MMS or rMMS crystallization. It's very important not to get sucked down one particular path by focussing too much on one hit from a screen. That's why it so helpful to run an rMMS screen as soon as you can. The method also gives you better hits right out of the screen, and also allows you to control the number of crystals per drop. All this is explained at eg http://www.douglas.co.uk/mms.htm Best wishes Patrick On 2 July 2013 15:43, RHYS GRINTER r.grinte...@research.gla.ac.ukmailto:r.grinte...@research.gla.ac.uk wrote: Hi All, I collected some data on the weekend on forked crystal, I collected data on this crystal at the base before the crystal split into two. The crystal didn't stand up well to the radiation damage so I shot a number of places along the crystal and got maybe 45 degrees of good data per position. Auto-processing failed on all but one data set, this dataset processed to 3.99 A, but only with around 80% completeness. However looking at the diffraction images I see spots in the first 45 degrees to at least 3.2 angstroms. I tried quickly to manually process in mosflm, but I noticed that many of the spots appear to be in fact made up to two very closely located spots. This data was collected at a micro-focus station so it was impossible to tell this without careful analysis of the spots. I guess these spots are an indication that the lattice was splitting even at this point. As a relative novice at data processing, I'm wondering if this kind of data is processable and if so what is the best strategy (or if I should just get back to the bench and grow some more crystals) and program to use? Cheers, Rhys -- patr...@douglas.co.ukmailto:patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart 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
[ccp4bb] Split Crystal Dataprocessing
Hi All, I collected some data on the weekend on forked crystal, I collected data on this crystal at the base before the crystal split into two. The crystal didn't stand up well to the radiation damage so I shot a number of places along the crystal and got maybe 45 degrees of good data per position. Auto-processing failed on all but one data set, this dataset processed to 3.99 A, but only with around 80% completeness. However looking at the diffraction images I see spots in the first 45 degrees to at least 3.2 angstroms. I tried quickly to manually process in mosflm, but I noticed that many of the spots appear to be in fact made up to two very closely located spots. This data was collected at a micro-focus station so it was impossible to tell this without careful analysis of the spots. I guess these spots are an indication that the lattice was splitting even at this point. As a relative novice at data processing, I'm wondering if this kind of data is processable and if so what is the best strategy (or if I should just get back to the bench and grow some more crystals) and program to use? Cheers, Rhys
Re: [ccp4bb] Puzzling observation about size exclusion chromatography
Hi Zhen, I'm not sure that binding to a monoclonal antibody is good evidence that the protein is in a natively folded state. I would be suspicious of such a result as the protein could be improperly, which is causing it to interact with the column matrix. It could be useful to use some other techniques (Activity Assay, Circular Dichroism, DSC, Native Page etc. to validate the refolding). Best, Rhys From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Patrick Loll [pat.l...@drexel.edu] Sent: 20 June 2013 20:39 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Puzzling observation about size exclusion chromatography If your protein elutes very late, that means it's binding to the column matrix (so all estimates of size go into the trash). Check to see that the ionic strength of buffer is reasonable (equivalent to, say, 150 mM NaCl). If so, then the only solution is to go to a different matrix type. Pat On 20 Jun 2013, at 3:09 PM, Zhang, Zhen wrote: Dear all, I just observed a puzzling phenomenon when purifying a refolded protein with size exclusion chromatography. The protein was solubilized by 8M Urea and refolded by dialysis against 500mM Arginine in PBS. The protein is 40KDal and is expected to be a trimer. The puzzling part is the protein after refolding always eluted at 18ml from the superdex S200 column (10/300), which is calculated to be 5KDal by standard. However, the fractions appear to be at 40KDal with SDS PAGE and the protein is functional in term of in vitro binding to the protein-specific monoclonal antibody. I could not explain the observation and I am wondering if anyone has the similar experience or has an opinion on this. Any comments are welcome. Thanks. Zhen The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail.
Re: [ccp4bb] Calcium ions in enzymes
My work with colicin M class bacteriocins shows that they require Ca2+ (or Mg or Mn) for catalysis: 1 Grinter, R., Roszak, A. W., Cogdell, R. J., Milner, J. J. and Walker, D. (2012) The Crystal Structure of the Lipid II-degrading Bacteriocin Syringacin M Suggests Unexpected Evolutionary Relationships between Colicin M-like Bacteriocins. J. Biol. Chem. 287, 38876-3 Best Regards, Rhys From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Wei Liu [we...@me.com] Sent: 31 May 2013 11:25 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Calcium ions in enzymes Dear all, As we all know, many proteins contain calcium ions. Does anyone know if there are reported cases where calcium ions play a catalytic role rather than a structural role in enzymes? Best Wei Liu
Re: [ccp4bb] Calcium ions in enzymes
In our case, the Ca ion is essential for activity but not correct folding. The enzyme requires Ca2+ (Mg or Mn) for activity. The crystal structure shows a single Ca2+ ion coordinated by a key catalytic aspartate and two backbone carbonyls. Mutagenesis of the key Asparate abolishes enzyme activity, and the presence of the Ca2+ ion in the structure. From: Eleanor Dodson [eleanor.dod...@york.ac.uk] Sent: 31 May 2013 12:49 To: RHYS GRINTER Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] Calcium ions in enzymes I would think a Google search would make some suggestions for you. There are lots of cases of proteins which require Calcium to function, but it is a bit chicken-and-egg-y - can the protein only function after it folds correctly, and is the Ca essential for that folding? On 31 May 2013 11:54, RHYS GRINTER r.grinte...@research.gla.ac.ukmailto:r.grinte...@research.gla.ac.uk wrote: My work with colicin M class bacteriocins shows that they require Ca2+ (or Mg or Mn) for catalysis: 1 Grinter, R., Roszak, A. W., Cogdell, R. J., Milner, J. J. and Walker, D. (2012) The Crystal Structure of the Lipid II-degrading Bacteriocin Syringacin M Suggests Unexpected Evolutionary Relationships between Colicin M-like Bacteriocins. J. Biol. Chem. 287, 38876-3 Best Regards, Rhys From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Wei Liu [we...@me.commailto:we...@me.com] Sent: 31 May 2013 11:25 To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Calcium ions in enzymes Dear all, As we all know, many proteins contain calcium ions. Does anyone know if there are reported cases where calcium ions play a catalytic role rather than a structural role in enzymes? Best Wei Liu
[ccp4bb] Membrane Protein Optimisation
Hi All, A quick question if you've ever worked on membrane proteins, I'm trying to optimize crystals for bacterial integral outer membrane protein I'm working on. I'm getting some fairly modest rod like crystals in a 0.1M Tris pH 7.5, 30% PEG 600, 0.03M MgCl2 condition. However I get lots and lots of birefringent gel forming in the same condition, I get the feeling that this is Detergent/Protein complex and is robbing my crystals of material to grow bigger. These crystals diffract to 5 A so I'd quite like to make them bigger and better, Cheers, Rhys
Re: [ccp4bb] Membrane Protein Optimisation
Thanks for the suggestions so far, I should have given a little more information in my initial post. The protein I'm working on is an Gram-Neg Beta-Barrel about 100kDa, likely with 22 strands. I'm currently crystallising in bOG, although my next optimisation step is to try a range of detergents. I'm using a refolding protocol which relies heavily in LDAO (one of the only cost effective detergents for the volumes I'm using), I refold, nickel purify (N-terminal tag, signal peptide removed) , do a size exclusion step (S200), then detergent exchange into bOG using a nickel column. The BOG concentration is obviously high 0.8-1%, which might be causing the gels? The gels don't look like classic phase separation to me (from soluble proteins that is, this is my first membrane protein), they are not spherical, the tend to float or sit on the bottom and are semi-solid, around 30-100uM diameter. These screens I've set up are classic screens for membrane proteins (mem-plus, mem-start, PGA etc.) and the drops aren't dried up. The gels are strongly birefringent, but the drop is not. Additionally the conditions in my initial screen which yielded crystals seemed biased for the formation of the gels. Thanks again for the help, I feel I might have a long raod to optimisation ahead of me. Rhys From: Jim Fairman [fairman@gmail.com] Sent: 09 May 2013 20:58 To: RHYS GRINTER Cc: ccp4bb Subject: Re: [ccp4bb] Membrane Protein Optimisation With information you've provided I have multiple suggestions for you, some of which you may have already tried: 1. OMPs can be fairly particular about which detergents they will crystallize in. Try exchanging the protein into a different detergent/detergent mixture and then set up new trays in your favorite broad matrix screens. DDM, DM, LDAO, OG, and C8E4 are a few of my favorites for OMPs, but there are many others. This can be done using a size exclusion column as the final purification step for your protein where the column is equilibrated with the appropriate detergent. This step will also let you know how well behaved the protein is in that detergent via the shape/height of the peak. This won't help you optimize your current condition, but it may lead to different/new conditions with even better crystals. As Pascal suggested, try to carefully choose which MW cut-off concentrator you end up using. Minimizing the amount of detergent concentration that occurs during your concentration step(s) is optimal. 2. Attempt crystallization using DHCP/CHAPSO bicelles. You can buy them pre-made from MemX Biosciences or make them yourself using a published protocol from David Bowie's lab. These have been used to crystallize the mitochondrial beta barrel protein VDAC and I had success crystallizing intimin in them. David Bowie also has a JoVE article on the bicelle crystallization method. 3. Attempt crystallization using Lipidic Cubic Phases. This was the saving grace for my post-doc project. Neither detergent nor bicelle crystallization produced crystals that were of sufficient quality, but the crystals from LCP all diffracted to the 2.0 angstrom resolution range. If you're unfamiliar with the technique, there are several nice videos on JoVE describing it by Vadim Cherezov and Martin Caffrey. 4. Alter your construct. Small changes in the construct (ie: deletion or addition of 1-2 residues on the N- or C-terminus) often led to drastically different crystallization behavior of several OMPs in my hands. Cheers, Jim On Thu, May 9, 2013 at 8:34 AM, RHYS GRINTER r.grinte...@research.gla.ac.ukmailto:r.grinte...@research.gla.ac.uk wrote: Hi All, A quick question if you've ever worked on membrane proteins, I'm trying to optimize crystals for bacterial integral outer membrane protein I'm working on. I'm getting some fairly modest rod like crystals in a 0.1M Tris pH 7.5, 30% PEG 600, 0.03M MgCl2 condition. However I get lots and lots of birefringent gel forming in the same condition, I get the feeling that this is Detergent/Protein complex and is robbing my crystals of material to grow bigger. These crystals diffract to 5 A so I'd quite like to make them bigger and better, Cheers, Rhys -- Jim Fairman, Ph D. Crystal Core Leader I Emerald BioStructureshttp://www.emeraldbiostructures.com/ Tel: 206-780-8914 Cell: 240-479-6575 E-mail: fairman@gmail.commailto:fairman@gmail.com jfair...@embios.commailto:jfair...@embios.com
Re: [ccp4bb] salt or not?
What else in in the conditions? Calcium Sulphate/Phosphate is poorly soluble, so if there is any sulphate or phosphate in your condition I would be suspicious. The age of the plate is also a bad sign, as evaporation over an extended time can lead to salt crystals. Check the well solution for crystals, if there are any then it's almost certainly salt. Softness and lack of birefringence are cautiously good signs, however the only way to know for sure is to stick them in an x-ray beam, which is always worth while for a crystal. Good luck Rhys From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Careina Edgooms [careinaedgo...@yahoo.com] Sent: 15 April 2013 11:18 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] salt or not? Dear ccp4 I have been performing trials on a protein DNA complex for a while now and have not seen any crystals form. Today I checked an old plate (over a month old) and I see 4 large crystals. *excitement* Three of them look tetragonal in shape (like a pyramid) and one of them looks hexagonal. I do not know if they are salt or protein. There is calcium chloride in the buffer. They feel quite soft to touch. They do not cause much birefringence. One of them does not seem to absorb much izit. It did go a bit blue but not entirely. How can I tell if this crystal is protein or not? Do you think its worth trying to see how it diffracts? Also, does Izit affect diffraction/ protein structures at all? Could I use a crystal with Izit in a diffraction experiment and ultimately to get the structure? Best Careina
Re: [ccp4bb] protein crystals or salt crystals
They look like other phosphate crystals that I've seen, but have to shoot them to tell. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nat Echols [nathaniel.ech...@gmail.com] Sent: 07 February 2013 22:30 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] protein crystals or salt crystals If SPG buffer is what I think it is, that means you have a significant concentration of inorganic phosphate, which forms salt crystals when mixed with divalent metal ions. -Nat On Thu, Feb 7, 2013 at 2:24 PM, amro selem amro_selem2...@yahoo.com wrote: Hallo my colleagues. i hope every one doing ok . i did screening since two weeks . i noticed today this crystals. i don`t know either it salt or protein crystal . my protein has zero tryptophan so i could distinguish by UV camera. the condition was conditions: 0.1M SPG buffer pH 8 and 25%PEG 1500. in addition to Nickle chlorid 1mM. best regards Amr
Re: [ccp4bb] unidentified density
Is this density on an 2 fold axis? From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Sudhir Kumar [sudhir.1...@gmail.com] Sent: 17 October 2012 12:07 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] unidentified density Dear all, I have been working on the crystal structure of an enzyme in which I found the unidentified density (see images in attachments). Crystallization condition has Peg 1000, Peg 1500, Ethylene glycol, Tris and MPD. Does any one has any idea what it could be? Thanks -- best regards Sudhir Kumar Research Scholar C/O Dr. S. Gourinath Structural Biology Laboratory SLS, JNU, New Delhi-110067
[ccp4bb] Modelling Flexible Regions
Hi All, This is a fairly basic question coming from a novice crystallographer, but I was wonder at what point it's reasonable to say 'I can't model that loop!'. I'm currently working on a couple of structures and there's density for where the a flexible loop on both structures goes, but there isn't good density for side chains (even aromatic ones) and even if the loop can be closed it doesn't seem like the Ramachandran plot will ever be happy. Both data sets are around 2A so it's not a question of poor density overall I was wondering if there was a consensus for when density is unmodelable, or if there are any tips for fitting residues into poor density. Thanks in advance, Rhys Grinter PhD Candidate University of Glasgow
[ccp4bb] Tricky MR problem
Thanks for your help everyone! It seems that the Balbes pipeline, followed by density modification in Phenix has done the trick Rhys
[ccp4bb] Tricky MR problem
Hi All, I'm currently working on solving the structure of a protein by molecular replacement. The protein is around 30kDa and likely has a two beta-prism domains, linked by a long curved two stranded sheet based on the structure of an analogue. There are also a number of other structures which represent a single homologous beta-prism domain. I've tried to find MR solution using the analogue and various truncation/AA substitution models based on it with no success. I've also tried single domain ensembles of the other homologous structures, also with no success. I think the problem is the overall sequence homology is quite low between my protein and the available structures (35% for the analogue and around 20% for the other models. I was curious as to how someone with more experience would tackle this problem. Just for background, the datasets I have are 2 to 2.7 angstroms with pretty nice stats. The space group is most likely C2221 with two molecules per ASU (giving around 58% solvent). Thanks, Rhys Grinter PhD Candidate University of Glasgow
[ccp4bb] Iron induced reduction of crystal
Hi All, I'm currently working of a protein with a ferredoxin protein with anIron-Sulphur cluster. I was harvesting some crystals the other day and a piece of my scalpel blade broke off and ended up in the well solution. I Sealed the well without noticing, the shard of iron oxidised and the crystals lost most of their red colour: Ordinary crystals: http://s1058.photobucket.com/albums/t401/__Rhys__/?action=viewcurrent=MBPR_Rodcluster2edit.png Crystals from Blade containing well: http://s1058.photobucket.com/albums/t401/__Rhys__/?action=viewcurrent=MBPR_Bleached.png My explanation for this (If someone has a different one that'd be great too) Is that the oxidation of the metallic iron the well, created reducing conditions in the chamber and reduced the iron-sulphur cluster (reduced ferredoxin is much less strongly coloured). Which got me to thinking...Could this be applied as a technique to create reducing conditions in protein crystallography, as the use of reducing agents isn't always practical. Cheers, Rhys Grinter PhD Candidate University of Glasgow
[ccp4bb] Molecular replacement of chimeric protein
Hi All, Thanks for the all the advice on my previous question regarding the likely photo-reduction of my crystals. I've now collected a data set on these crystals. The protein is a chimera of two domains of known structure (Ferredoxin domain and Colicin domain), what's the best way to use all of this information in molecular replacement i.e. can you use two search models simultaneously in Phaser? Thanks again Rhys Grinter
[ccp4bb] Ferredoxin Containing Crystal Bleaching
Hi All, I was collecting some data at home on a crystal from a protein containing a ferredoxin domain. This crystal was originally red-brown in colour, but after 16h of data collection, a large portion of it appears not be colourless. The crystal still seems to diffract fine (beyond 2A). I assume this it due in some way to the radiation exposure but was wondering out curiosity whether someone had experienced this before and had a specific explanation for it. And if this will effect the integrity of the data. Cheers Rhys Grinter PhD Candidate University of Glasgow
Re: [ccp4bb] Expressed protein hinders cell lysis?
Hi, I often see no real change in change in solution appearance after sonication mediated lysis, with proteins which yield low amounts or no soluble protein in E. coli. I've had a look at the solution post lysis under the microscope and the cells are infact lysed, it's just the presence of high levels of inclusion bodies means the solution remains turbid. Check your pre and post lysis solution under the microscope to see if you see the same thing. Cheers, Rhys From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of J. Valencia S. [valen...@gene.nagoya-u.ac.jp] Sent: 21 June 2012 13:44 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Expressed protein hinders cell lysis? Greetings, everyone. We need to ask your advice on an issue with one of our proteins expressed in E. coli Rosetta cells. This yeast-derived protein has a very low yield compared to others we work with, and we think it is because the cells are hard to lyse: even after 3 cycles in a cell cracker the solution barely changes colour. We have no problems lysing Rosetta cells expressing other yeast-derived soluble proteins, and we usually obtain enough for our crystallisation screens. For the aforementioned protein we have already tried using STAR cells, varying the contents of the lysis buffer, sonicating, or adding FeSO4 to the solution (we think the protein binds Fe or Mn because it is yellow), but to no avail. Searching the ccp4bb archive and other resources did not help, so we would like to ask 2 questions to the community in order to focus our efforts better: 1. How can a recombinant protein make a cell harder to lyse? 2. Do you have any suggestions to avoid this effect? We appreciate any input, and will be sure to post a summary for future reference once this issue is solved. Sincerely, -- J. Valencia S. PhD student CGR-NU
[ccp4bb] Model submission
Dear All, I'm working out the finer details on a structural paper for submission to JBC. I'm having a slight problem with how to present my data. I've got a high resolution (1.46 A) truncated structure of the protein with the N-terminal 38aa removed. I've also got data from lower resolution (2.68 A) crystals of the full length protein. There's no significant difference between the high res and low res proteins in the shared region (amino acid 38+) (r.m.s.d 0.46 A), and the while there is broken density for the first 38aa from the full length data it's too poor to model into. I want to present a figure which shows the density corresponding to the first 38aa and where that fits with the rest if protein molecule. What I'm unsure of it whether I will be required by the journal to submit a model from the lower resolution data to the PDB in order to present this figure. Bearing in mind the density doesn't allow any additional residues to be modelled compared to the high res. structure. Your opinions or advice on how best to present this data would be welcomed. Cheers, Rhys
[ccp4bb] Strange Density
Dear Community, As I'm a relatively new to protein crystallography this might turn out to be an obvious question, however. I'm working on the structure of a enzyme requiring Ca2+ for activity and with calcium coordinated in the active site by Asp and 2x backbone carbonyl groups, in a crystal structure with Ca in the crystallisation conditions (http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_TD_15A.jpg). When Ca is omitted from the crystallizing conditions and a divalent chelator (EGTA) is added the crystals are of significantly lower resolution (3.13A). Refinement of this data reveals density for a molecule coordinated by the Ca coordinating Asp and backbone, however this density is significantly further away (3.4-3.8A) too far away for water or a strongly coordinated divalent cation(http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_EGTA_315.jpg). The density is also much weaker than for Ca in the previous model disappearing at 3.5 sigma. The crystallisation conditions for the Ca free condition is: 0.1M Tris/Bicine buffer [pH 8.5] 8% PEG 8000 30% Ethylene Glycol 1mM EGTA The protein was purified by nickel affinity/SEC and dialysed into: 20mM NaCl 20mM Tris [pH 8.0] A colleague suggested that sulphate or phosphate could fit at these distances, but these ions have not been added at any stage of the crystallisation process. Could anyone give me some insight into what this density might represent? Thanks in advance, Rhys Grinter PhD Candidate University of Glasgow
