[ccp4bb]
A back of the envellope calculation shows that your asymmetric unit contains most likely 4 molecules with Vm = 2.16 A3/Da, corresponding to 43% solvent. Searching for 16 molecules is thus nonsense. Remy Loris Vrije Universiteit Brussel On 17/08/14 08:54, Avisek Mondal wrote: Hello everyone, i am struggling with a problem.. My crystal was diffracted at 1.9A in P21 spacegoup with unit cell parameter a=87.7 b=93.9, c=111.78 ,beta=94.98 which contains 16 molecules per assymmetric unit (Molecular weight of the Protein+DNA =56 KDa. actually it is a complex of 40Kda tetramer and 23bpDNA ) .In solution, it shows tetrameric in nature The crystal structure of its homologous structure has been reported earlier (50%identical in amino acid seq.) and it was also a tetramer and its unit cell (also P21) was approximately 4 times less than mine. It showed 4 molecules per assymmetric unit. I didn't get any molecular replacement. All the programms are takings very long time to do it. Although the single crystal is untwinned i think it is a case of pseudosymmetry. Please help me if you have any good suggestion regarding molecular replacement other than experimental phasing.
Re: [ccp4bb] Structure Refinement
A very likely possibility (but there may be others) is merohedral twinning, which can and often does occur in this space group, and these are typical R-values you would get stuck to in case of partial merohedral twinning. Checking the log file of truncate should be informative in this respect. Also: there cannot be three molecules in the unit cell as this space group has six asymmetric units. You probably mean three molecules in the asymmetric unit. Since crystallography is an exact science, one needs to be correct. Remy Loris Vrije Universiteit Brussel On 15/02/13 21:35, Muhammed bashir Khan wrote: Dear All I have a data at 2.75A. I process it in Space group P3121, using HKL3000. Run a molrep,find three molecule in a unit cell. I am trying to refine it with phenix, the R and R-free stuck at 34 and 41 respectively. Crystal: The crystal seems multiple thin plates and I tried to freeze the possible single crystal (plate). Any suggestion would be highly appreciated!! Bashir Muhammad Bashir Khan ** Structural Genome Consortium (SGC) University of Toronto Canada
Re: [ccp4bb] side chain density
Dear Faisal, You definitely do not mutate to alanine as that would imply for the future user of your pdb file that it is a mutant. Some people feel they have to keep the side chain but put the occupancies at zero. I think this is a bad practice and strongly oppose to it as for the future user of your deposited pdb file, who often is not a crystallographer, you suggest a specific conformation for your side chain that may be interpreted in terms of biology, while in reality it addopts a huge, disordered ensemble of conformations. Personally I am of the opinion that you should simply remove the side chain atoms (but keep the residue name). And that is the same as what you do with a whole loop that is disordered. I think it is lso the most common practice in deposited structures. Remy Loris Vrije Universiteit Brussel On 09/11/12 20:22, Faisal Tarique wrote: Dear all i have solved a structure ( at 2A resolution) whose Rwork and Rfree is 22 and 25 respectively..the Ramachandran plot shows 90% of the residues in the most favorable region and with 6 residues in generously allowed and no residues in disallowed region. But in some areas i can see density missing for side chains ( in loop regions )..i have question do i need to mutate them to alanine or leave them as such..The density fit analysis in COOT ( traffic light) showing those regions with side chain as red.. thanx in advance Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] MR solution
Dear Rui, If your search model is itself a homodimer, you expect to find 2 equivalent solution And indeed in your case: 50.3 + 128.6 = 178.3 (= aboput 180) 0.2 + 179.8 = 180 219.8 - 38.7 = 181.1 (= about 180) indicating that both solutions are crystallographically equivalent. What does worry me is the apparent special position that you obtain for the translation solution. Are you sure you do not have a higher symmetry or cetered space group? Remy Loris Vrije Universiteit Brussel and VIB On 07/11/12 17:39, rui wrote: Hi, Dear group, I recently collected a dataset about 2.5 A and integrated with P4. When I tried phaser I got a sol file looks like this, is this real solution? Is LLG high enough? SOLU SET RFZ=5.2 TFZ=10.3 PAK=0 LLG=239 LLG=366 TFZ==20.9 SOLU SPAC P 41 SOLU 6DIM ENSE ensemble1 EULER 50.265 0.217 219.800 FRAC 0.50029 0.49916 -0.00408 BFAC -0.04000 SOLU ENSE ensemble1 VRMS 0.639 SOLU SET RFZ=5.2 TFZ=10.0 PAK=0 LLG=239 TFZ==11.2 LLG=365 SOLU SPAC P 41 SOLU 6DIM ENSE ensemble1 EULER 128.607 179.813 38.677 FRAC 0.49991 0.49905 -0.00208 BFAC -0.06264 SOLU ENSE ensemble1 VRMS 0.642 Looks like there are two solutions,but in the output pdb file, I only see one solution (homo-tetramer). So I took this output pdb and run first round of refinement with Refmac, somehow, the structure fall apart if I use restraint refinement( lots of atoms are not connected any more ). If I use rigid body refinement, it's ok and Rfree is 52%. Does that mean the solution might not be correct? What's the acceptable Rfree for the initial refinement? Thanks a lot. Rui
Re: [ccp4bb] Problem with making PDB from Coot
Seconday structure assignement by Pymol is poor and unreliable to say the least. This behavior is very common. Best is to assign secondary structure using other software and then tell Pymol explicitely from where to where the starnds and helices run Remy Loris Vriej universiteit Brussel On 23/07/12 23:46, meisam nosrati wrote: Dear CCP4ers It seems like the text has not showed up, but I have a problem with making pdbs from coot. As I refine my structure ( with MR solution ) the beta sheets become loops while H-bondings are still there. I am not sure, if the problem is originating from making PDBs from coot. I will appriciate your help Meisam On Mon, Jul 23, 2012 at 5:31 PM, Meisam Nosrati meisam.nosr...@gmail.com mailto:meisam.nosr...@gmail.com wrote:
Re: [ccp4bb] do you think it is interesting?
This may be somethng similar Domain swapping of a llama VHH domain builds a crystal-wide beta-sheet structure. Spinelli S, Desmyter A, Frenken L, Verrips T, Tegoni M, Cambillau C. FEBS Lett. 2004 Apr 23;564(1-2):35-40. Remy Loris Vrije Universiteit Brussel On 18/06/12 15:49, anna anna wrote: Hi all! I'd like your opinion about a structure I solved. Apart from protein structure itself, I think that my protein xtallized in an odd way! The biological unit is a dimer while the asymmetric unit is a tetramer (red cartoon in the figure) resulting from domain swapping between two dimers. The strange thing is that swapping connects infinite monomers and, rather than a xtal, my diffracting object seems a multilayer of endless linear polymers, a kind of papyrus with greek fret-like fibers. The figure shows the orientation of the polymers in each layer. I'd like to know if some of you have already seen a similar pattern or it is weird as I think! I'm further racking my brain to figure out a biological implication of this behaviour, I thought something like plaque formation but I can't find support in literature. All suggestions are welcome!! Cheers, Anna
Re: [ccp4bb] Off-topic: Supplying PDB file to reviewers
Dear Marc, The only way a reviewer can really judge the quality ofa structure and verify the claims made in a structural biology manuscript is by having access to the pdb files and x-ray data. I have myself as a reviewer requested co-ordinates and data for this purpose, and the results can be quite revealing, both in positive and negative sense. I have no problem myself to provide data when requested and even to release structures before publication. The latter has helped me for a paper that was accepted last week where one of the referees wrote in hes/her report that he/she could only judge the paper correctly because the data were available for downloading. Thus I am a strong advocate for all structural data to be made available to referees upon submission of a manuscript. Science and scientific publishing requires a great deal of mutual trust. This is not only for the reader or referee who has to trust that the work is correct and not fraudulent. It also goes in the opposite way where the author has to trust the referees and editors to be honest. Only in this way can the system work. Otherwise, just keep everything for yourself and don't publish. In the 23 years that I am active in science I had only a single case where I genuinely believe a referee misused his position. This to say that 99.99% of the referee reports are honest, although not necessarily in agreement with your own vision. Remy Loris Vrije Universiteit Brussel and VIB On 19/04/12 00:34, Marc Kvansakul wrote: Dear CCP4BBlers, I was wondering how common it is that reviewers request to have a copy of the PDB coordinate file for the review purpose. I have just been asked to supply this by an editor after several weeks of review, after one of the reviewers requested a copy. Not having ever been asked to do this before I feel just a tad uncomfortable about handing this over… Your opinions would be greatly appreciated. Best wishes Marc Dr. Marc Kvansakul Laboratory Head, NHMRC CDA Fellow Dept. of Biochemistry| La Trobe University | Bundoora Rm 218, Phys Sci Bld 4, Kingsbury Drive, Melbourne, 3086, Australia T: 03 9479 2263 | F: 03 9479 2467 | E: m.kvansa...@latrobe.edu.au |
Re: [ccp4bb] MR problem
Dear Ping, First thing to ask: Do you know with 100% certainty if your crystals contain a complex? If your crystals are large enough (and you have more than one) you can check this on SDS-PAGE or with mass-spec as long as you are sure to remove all surrounding mother liquid that may contaminate your result. Both proteins should be observed very clearly (80%-20% would be a contamination rather than a co-crystal!). Experience tells us that even with high affinity (nanomolar or better) and preformed complexes, still often only one of the two partners will crystallize. Second: does your second protein contain more than one domain? If so, there may be domain movements that obscure the desnity or even lead to a completely wrong MR solution. Try rigid body refinement with individual domains first. If this does not work, try MR with the individual domains. The MR solution of your second protein may also be wrong for any other reason. Check if you can trust the solution based on the statistics provided in the log file. If the LLG is only marginally better after adding the second protein, it will probably be wrong. Check also if there is no real solution hanging around but rejected by the program because (for example) it has just one clash too many. Remy Loris Vrije Universiteit Brussel and VIB On 04/12/11 05:18, Ping Wang wrote: Dear all, Recently I have a dataset with a protein complex including two proteins. Each structure of the single protein is available. When I use phaser to solve the phase, one protein got good density while the other was not so ideal. In order to get a better phase, I try to cut the flexible region of the bad protein. But the result shows no improvement. Does anyone have some suggestion for me? Thanks! Ping
Re: [ccp4bb] MR problem
Dear Ping, First thing to ask: Do you know with 100% certainty if your crystals contain a complex? If your crystals are large enough (and you have more than one) you can check this on SDS-PAGE or with mass-spec as long as you are sure to remove all surrounding mother liquid that may contaminate your result. Both proteins should be observed very clearly (80%-20% would be a contamination rather than a co-crystal!). Experience tells us that even with high affinity (nanomolar or better) and preformed complexes, still often only one of the two partners will crystallize. Second: does your second protein contain more than one domain? If so, there may be domain movements that obscure the desnity or even lead to a completely wrong MR solution. Try rigid body refinement with individual domains first. If this does not work, try MR with the individual domains. The MR solution of your second protein may also be wrong for any other reason. Check if you can trust the solution based on the statistics provided in the log file. If the LLG is only marginally better after adding the second protein, it will probably be wrong. Check also if there is no real solution hanging around but rejected by the program because (for example) it has just one clash too many. Remy Loris Vrije Universiteit Brussel and VIB On 04/12/11 05:18, Ping Wang wrote: Dear all, Recently I have a dataset with a protein complex including two proteins. Each structure of the single protein is available. When I use phaser to solve the phase, one protein got good density while the other was not so ideal. In order to get a better phase, I try to cut the flexible region of the bad protein. But the result shows no improvement. Does anyone have some suggestion for me? Thanks! Ping
Re: [ccp4bb] how to Collecting Data from Long Unit Cell Axes ?
Ideally you should rotate your crystal around the long axis during data collection. This is however easier said than done since often such crystals grow a plates with the long axis perpendicular to the plane of the plate. As long as the long axis is more or less oriented perpendicular to the x-ray beam (+/-45 deg but depending on the severity of the case) you will get away with it and you might even consider large oscillation ranges. For orientations with the long axis in a direction close to the X-ray beam, you will have to reduce the oscillation range, perhaps even to 0.1deg to avoid excessive spot overlap. Here things depend heavily on mosaicity, which should be low (preferably 0.2 deg). Otherwise spots may always overlap, even on still images. Very often (in my experience at least) crystals with one particularly long axis have high symmetry, and collecting a large wedge covering the good orientations can be sufficient. Of course I don't know if you are that lucky. If you are not sure which part to collect, collect at least 180 deg to be sure you covered the whole unique part of reciprocal space. BTW, you didn't say what is long. My post extreme case until now was P3121 with a=b= 50, c = 360 and this resulted in very good 2.4 A data from a rystal with mosaicity 0.1-0.15. Crystals with mosiacity around 1.0 deg were useless because of too much overlap. Remy Op 5/04/2011 7:05, dengzq1987 schreef: hello all, does anyone have the experience of Collecting Data from Long Unit Cell Axes ? I have a crystal that diffracts to about 4 A. in some /direction/ the spots overlap. we can't use the data to index .we think it is because that there is a long unit cell axes. so is there any method to solve this problem? best wishes. 2011-04-05 dengzq1987
[ccp4bb] Off-topic: chemical modification on thiol groups
I am looking for a reagent (and vendor) that will irreversible put a raher bulky substituent on a free SH group and that does not react with free amines (or other potential reactive groups present on a protein surface). The connection with crystallography is that it is required for an experiment asked by a referee necessary to confirms or reject a hypothesis that results from a crystal structure. For this experiment it is essential that the reaction is not reversible (so no S-S bond formation). Remy Loris Vrije Universiteit Brussel
Re: [ccp4bb] peptide for co-crystallization
Dear Daniel, We used peptides (up to 40 aa long) from BioSynthesis, Lewisville, Texas (www.biosyn.com) at 90% purity with great success. The material is guaranteed to be 90% (or whatever you ask) pure but in reality it is a lot better. (I have no commerical interest here myself). In one case we even crystallized a complex with peptide that was only supposed to be 80% pure (and very nice electron density, crystals diffracting to 1.7 A). Remy Loris Vrije Universiteit Brussel Daniel Jin schreef: Hi, We would like to order two peptides for co-crystallizaiton. I notice that there is a big price jump for peptide over 95% pure and over 98% pure. Do you think 95% purity is good enough? Another choice is to order crude peptides and purify by ourselves. Crude peptide is much cheaper. But we will then have to put in time to do multiple HPLC runs, reagent, cost for Mass Spec verification... Does anyone have suggestion about which one will be more efficient? Thanks. Best, Chen No virus found in this incoming message. Checked by AVG - http://www.avg.com Version: 8.0.138 / Virus Database: 270.6.9/1637 - Release Date: 8/27/2008 7:01 AM
[ccp4bb] Phd studentship in structural biology and biophysics
A Phd studentship is available in the group of Prof. Remy Loris to work on the structural biology and biophysics of the MazEF toxin-antitoxin module from Staphylococcus aureus. The project centres around crystal structure determination of the MazEF module and its complexes, interaction studies using a variety of biophysical techniques and, in collaboration with Prof. Ambrose Cheung (Dartmouth Medical School, USA), the development of small molecule drugs that interfere with the interaction between MazE and MazF. Further information concerning bacterial toxin-antitoxin modules can be found in Buts et al. (2005) Toxin-antitoxin modules as bacterial metabolic stress managers. Trends Brioche. Sci. 30(12), 672-679. Enquiries can be send to Prof. Remy Loris ([EMAIL PROTECTED]). Please attach a recent CV and the names of at least two referents. Remy Loris Vrije Universiteit Brussel http://www.vib.be/ http://www.structuralbiology.be/
Re: [ccp4bb] is it Ok to freeze
Typically crystals of small organic compounds do not require freezing as there are no solvent channels. They do in general not suffer from radiation damage at room temperature the way protein crystals do. Occasionally they are mounted in a capillary instead of simply glueing them to a goniometer if they are air sensitive. In principle freezing should not damage the crystals, but one still may have to be carefull if the crystals are large. I think you risk increasing mosiacity, and any manipulation that is not needed will on average only reduce the quality of the specimen rather than improve it Remy Loris Vrije Univesiteit Brussel Jayashankar wrote: Dear Scientists and Friends, I am not sure, whether organic crystals need to be in cryo stream necessarily during data collection from an in house xray machine . How most of the organic crystals have been solved mostly? -- S.Jayashankar (A bit confused new generation researcher). Research Student Institute for Biophysical Chemistry Hannover Medical School Germany
[ccp4bb] Opening for a X-ray facility/computing manager
The VIB Department of Molecular and Cellular Interactions at the Vrije Universiteit Brussel is looking for a highly motivated X-ray facility/computing manager. The successful candidate will provide expert support to our in house structural biology community and will conduct research in the area of structural biology through internal and/or external collaborations. The department currently contains four groups involved in structural biology totalling around 40 people. A Bio-NMR group is to be established beginning next year. Details on the research conducted in the department can be found on our websites (http://www.vib.be/ and http://ultr.vub.ac.be/). Experience with UNIX/LINUX system administration is a must and experience with the computational side of protein crystallography is highly desirable. The main service duties will include: - Maintaining and upgrading the crystallographic and NMR computing facilities and reassuring of data back-up - Maintenance of the in-house X-ray data collection facilities - Organizing and streamlining synchrotron trips Interested candidates should send their cv to Remy Loris ([EMAIL PROTECTED]) together with the names and contact details of at least two reference persons. Salary will be discussed and will depend on the level of experience and qualifications of the successful candidate. Remy Loris Vrije Universiteit Brussel Pleinlaan 2 1050 Brussel Belgium
Re: [ccp4bb] glycosylation sites]
I think you need to look at this paper, although already quite old: Imberty A, Pérez S. Stereochemistry of the N-glycosylation sites in glycoproteins. Protein Eng. 1995 Jul;8(7):699-709. Remy Loris Vrije Universiteit Brussel Ronnie WEi wrote: I was asked this question by a colleague. Has anyone looked into where glycosylation occurs most frequently on a protein- loops, alpha-helices or beta-strands? Thanks for your input! Best, Ronnie Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now. http://us.rd.yahoo.com/evt=51733/*http://mobile.yahoo.com/;_ylt=Ahu06i62sR8HDtDypao8Wcj9tAcJ
