Re: [ccp4bb] combining AutoMR and brute
No, we haven't made that possible, largely because we can't see circumstances where this would be desirable. However, you can get as close as you want to a brute force search by changing the criteria for rescoring the results of the fast search. If you include the command FINAL TRA SELECT PERCENT 65 (changing from the default of PERCENT 75), then all fast translation peaks greater than 0.9*65=58.5% of the maximum difference from the mean will be rescored, and, after rescoring, everything above 65% of the maximum will be kept. You can do this from ccp4i on the line labelled "Final selection criterion for TRANSLATION search peaks". If you only want to adjust which fast translation peaks are rescored but not which are kept (which is closer to running the brute translation search), then you can include the command FINAL TRA STEP 1 SELECT PERCENT 50 to rescore everything above 50% of the maximum from the fast translation search. To do this from ccp4i, you have to open the "Additional parameters" pane, then under "Criteria for the translation search" change "Rescore criterion". Instead of PERCENT, you can also specify the criteria for which peaks to keep by SIGMA (i.e. Z-score), NUMBER or even ALL. So if you said FINAL TRA STEP 1 SELECT ALL this would essentially be like using the brute translation search, except that the sampling of points is one a fractional grid instead of the more efficient hexagonal-close-packed grid used for the brute translation search. My feeling is that this would just be a waste of computer time. I don't think we've seen a case where rescoring anything below about 50% of the maximum has made a difference -- but would appreciate hearing otherwise if someone has had a different experience! Best wishes, Randy Read On 22 Dec 2009, at 00:25, Francois Berenger wrote: > Hello, > > Is there any way to search in AutoMR mode but have the > translation only being brute forced? > > Regards, > F. -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
Re: [ccp4bb] where I have been going wrong in crystallization?
Thanks to those who replied. The humour comes of course from a slight feeling that there is some truth in the 'respectful' version. Proteins are wonderful subtle machines so perhaps we should be careful and thoughtful when we expose them to the horrors of most crystallization conditions! Which takes me to the original point of my search - which was for a dialysis method for lysozyme. I was trying to set up a micro dialysis format for my protein but wanted to test it first. I am using spectrapore 8K dialysis membrane and wondered if that would retain lysozyme enough to get crystals (I'd like to stick with this cut-off as the physical and sealing properties of other membranes might be different). Or if someone has a higher MW cheap alternative protein/condition? Best wishes - agus 'Bliadhna Mhath Ur Dhuibh Uile' as we say in gaelic. Martyn - Martyn Symmons Cambridge - Original Message From: David Briggs To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, 21 December, 2009 17:26:15 Subject: Re: [ccp4bb] where I have been going wrong in crystallization? Hi Martyn, this recipe tends to work for me... Lysozyme: 50 mg/ml in 0.1 M Sodium Acetate pH 4.8 Reagent: 8% w/v Sodium Chloride, 0.1 M Sodium Acetate pH 4.8 Additional Reagents: Index Reagent 8, 22, 28, 31, 34, 40, 58, 59, 69, 86, 88 Mix equal amounts of lysozyme with reagent, incubate at 4 or 22 degrees Celsius. Batch or vapor diffusion works fine. ==> Direct copy/paste from http://hamptonresearch.com/experiments.aspx <== HTH and Happy Christmas. Cheers, Dave David C. Briggs PhD University of Manchester E-mail: david.c.bri...@manchester.ac.uk Twitter: @xtaldave Skype: DocDCB 2009/12/21 MARTYN SYMMONS : > Dear All > checking out the Lysozyme crystallization methods on the web I liked the > Rigaku Instructions that I found: > (http://www.rigaku.com/protein/crystallization.html) > > "...create a drop of 3ul lysozyme solution, and 3 ul of well solution, > respectfully, for a total drop size of 6ul..." > > So perhaps sometimes I am just not respectful enough to deserve crystals ? > >good wishes to all > regards, > Martyn > --- > Martyn Symmons > MRC-MBU Cambridge UK > 'Chan fhiosrach mur feòraich.' > Gaelic proverb - > Nothing asked, nothing learned. >
Re: [ccp4bb] where I have been going wrong in crystallization?
Hi Martyn, When it comes to micro-dialysis, you could check the following paper: http://www.jbc.org/content/260/25/13580.full.pdf+html (freely available, check in particular the appendix for the micro-dialysis setup that was used, that was for heat-labile enterotoxin) Fred. PS I know this is not exactly answering your question but still you might find this setup useful MARTYN SYMMONS wrote: Thanks to those who replied. The humour comes of course from a slight feeling that there is some truth in the 'respectful' version. Proteins are wonderful subtle machines so perhaps we should be careful and thoughtful when we expose them to the horrors of most crystallization conditions! Which takes me to the original point of my search - which was for a dialysis method for lysozyme. I was trying to set up a micro dialysis format for my protein but wanted to test it first. I am using spectrapore 8K dialysis membrane and wondered if that would retain lysozyme enough to get crystals (I'd like to stick with this cut-off as the physical and sealing properties of other membranes might be different). Or if someone has a higher MW cheap alternative protein/condition? Best wishes - agus 'Bliadhna Mhath Ur Dhuibh Uile' as we say in gaelic. Martyn - Martyn Symmons Cambridge
Re: [ccp4bb] Coot pudding? (a.k.a N-linked carbohydrate addition)
If I can chip into this somewhat sacrilegiously-named thread 1) I *would* use real-space refinement :), specifically Sphere Refinement. You can dial down the density weight if needed, of course. 2) the documentation on refining carbohydrates in Coot has recently been updated http://www.biop.ox.ac.uk/coot/doc/coot/Refining-Carbohydrates.html 3) Coot does not (yet) correct chiral centre inversions in glycosidic linkages on refinement 4) or delete the O1s :) Paul. Robbie Joosten wrote: Dear Steve, I would also use Damian's approach, but the sequence of the core should be NAG-NAG-BMA-(MAN)2. This is improtant because the correct stereochemistry restraints for beta-mannose can only be applied when you call the residue BMA. Building carbohydrates also comes with special validation requirements. PDB-care and CARP are both very usefull. Unfortunately, the service is currently down (http://www.dkfz.de/spec/glycosciences.de). Just make sure the links between your carbs are correct and, please, remove the O1 atoms when needed ;) Cheers, Robbie Joosten Date: Mon, 21 Dec 2009 17:48:31 -0800 From: dceki...@scripps.edu Subject: Re: [ccp4bb] Coot pudding? (a.k.a N-linked carbohydrate addition) To: CCP4BB@JISCMAIL.AC.UK Steve, My general strategy is to start with an "ideal" glycan (an Asn linked to NAG-NAG-(MAN)3 ) and superimpose the Asn on the residue from my protein. Then you can move the whole glycan as a rigid body until the Asn and first NAG are roughly positioned. Then you can tweak any sugars further out on the chain to get them to fit. Unless you have really great density, usually it is best to avoid real pace refine zone. Better to fit the sugars using the manual rigid body fitting tools, do the best you can, then REFMAC usually brings them in OK. I have some models that I could send you if you need them. Best, Damian Ekiert Soisson, Stephen M wrote: Hi everyone- I was searching for some information on what might be the best way to add N-linked sugars in coot, and Google has let me down. Searching "adding sugars in coot" returns a very nice recipe for Coot Pudding. ***_Recipe for_/ Coot//__/_ Pudding - American_/ Coots/* It has plenty of fat,/ sugar/, and starch, and probably some calcium from the milk.* ...* The/ coots/ will not tolerate/ adding/ eggs in any form, so this is an egg* ...* ///_www.beaky_//_*coot*.com/pudding.html_/// / -/ _Similar_ // I did not know that coots had such an aversion to eggs. :) Anyway, would anyone have any top tips on adding N-linked sugars using coot? I can import the NAG monomers, but linking them up to the protein seems non-trivial Many thanks in advance, Steve Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] HKL2000 and Fedora 12
Thanks! The 75 dpi fonts fixed it!
Re: [ccp4bb] Coot pudding? (a.k.a N-linked carbohydrate addition)
For an "ideal" glycan, you could used a model from a high resolution structure, or something that has been energy minimized, etc. Mostly I find this helps in getting the sugars in about the right place (keeping bond lengths and angles reasonable). I perhaps could stand to fiddle more and maybe I'll look through the updated documentation. Last I tried, the Asn-NAG1 linkage wasn't enforced, with would allow the whole glycan to slide down into the protein, or off into space. I am typically building relatively small glycans (2-5 residues) into ~3A data, so the density itself doesn't keep things in place very well. Regarding BMA vs MAN: When I have tried to used BMA in REFMAC, it doesn't seem to recognize it and requires a library file. But if you use MAN, it adds a MODRES record to the header, enforcing beta-mannose geometry. Not sure if this is just a REFMAC version issue or what. Best, Damian Paul Emsley wrote: If I can chip into this somewhat sacrilegiously-named thread 1) I *would* use real-space refinement :), specifically Sphere Refinement. You can dial down the density weight if needed, of course. 2) the documentation on refining carbohydrates in Coot has recently been updated http://www.biop.ox.ac.uk/coot/doc/coot/Refining-Carbohydrates.html 3) Coot does not (yet) correct chiral centre inversions in glycosidic linkages on refinement 4) or delete the O1s :) Paul. Robbie Joosten wrote: Dear Steve, I would also use Damian's approach, but the sequence of the core should be NAG-NAG-BMA-(MAN)2. This is improtant because the correct stereochemistry restraints for beta-mannose can only be applied when you call the residue BMA. Building carbohydrates also comes with special validation requirements. PDB-care and CARP are both very usefull. Unfortunately, the service is currently down (http://www.dkfz.de/spec/glycosciences.de). Just make sure the links between your carbs are correct and, please, remove the O1 atoms when needed ;) Cheers, Robbie Joosten Date: Mon, 21 Dec 2009 17:48:31 -0800 From: dceki...@scripps.edu Subject: Re: [ccp4bb] Coot pudding? (a.k.a N-linked carbohydrate addition) To: CCP4BB@JISCMAIL.AC.UK Steve, My general strategy is to start with an "ideal" glycan (an Asn linked to NAG-NAG-(MAN)3 ) and superimpose the Asn on the residue from my protein. Then you can move the whole glycan as a rigid body until the Asn and first NAG are roughly positioned. Then you can tweak any sugars further out on the chain to get them to fit. Unless you have really great density, usually it is best to avoid real pace refine zone. Better to fit the sugars using the manual rigid body fitting tools, do the best you can, then REFMAC usually brings them in OK. I have some models that I could send you if you need them. Best, Damian Ekiert Soisson, Stephen M wrote: Hi everyone- I was searching for some information on what might be the best way to add N-linked sugars in coot, and Google has let me down. Searching "adding sugars in coot" returns a very nice recipe for Coot Pudding. ***_Recipe for_/ Coot//__/_ Pudding - American_/ Coots/* It has plenty of fat,/ sugar/, and starch, and probably some calcium from the milk.* ...* The/ coots/ will not tolerate/ adding/ eggs in any form, so this is an egg* ...* ///_www.beaky_//_*coot*.com/pudding.html_/// / -/ _Similar_ // I did not know that coots had such an aversion to eggs. :) Anyway, would anyone have any top tips on adding N-linked sugars using coot? I can import the NAG monomers, but linking them up to the protein seems non-trivial Many thanks in advance, Steve Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
[ccp4bb] PhD position at the ESRF in Grenoble, France
http://www.esrf.eu/Jobs/Research/CFR355 Subject: Kinetic Crystallography to Probe for Catalytic Mechanism and Protein Loop Motions in Glycosyltransferases General Framework: Glycosyltransferases are an enormous class of enzymes responsible for the biosynthesis of oligosaccharides, polysaccharides and glycoconjugates. They catalyse the transfer of a sugar from a donor substrate, usually a nucleotide sugar, to an acceptor. Glycosyltransferase reactions can occur with either retention or inversion of the anomeric configuration of the transferred sugar. While the catalytic mechanisms of glycoside hydrolyses are well characterized, many uncertainties remain concerning those of glycosyltransferases. The proposed project aims at investigating the catalytic mechanisms of an inverting glycosyltransferase and of a retaining one, by kinetic crystallography with the use of caged compounds based on the nucleotide-sugar substrate. Description of the thesis work: The successful candidate will be in charge of the production of recombinant glycosyltransferases. He/she will set up the crystallization assays of the enzymes and of the complexes with the caged compounds provided by our chemist collaborators. If needed, site-directed mutagenesis will be performed in order to alter the kinetic characteristic of the reaction for facilitating the kinetic crystallography study. The student will optimize by microspectrophotometry the conditions for efficient photocleavage of the caged compounds, first in frozen protein solutions, then in crystals. Using the Temperature Derivative Fluorescence Microspectrophotometry method, he will find a temperature range in which solvent rearrangements will allow the enzymatic reaction to proceed. He/she will then collect diffraction data on protein crystals to solve: (i) the structure of the protein/caged compound complex; (ii) the structure of the protein/end product complex after complete cleavage by strong light irradiation; (iii) the structure of putative intermediate states of the enzymatic reaction. Overall, these data should provide snapshots of the catalytic mechanism and protein loop motions. Place of Work: ESRF in Grenoble. Supervisors: Dr. Antoine Royant ((+33) (0)4 76 88 17 46; antoine.royant at esrf.fr) & Dr. Serge Pérez (+33) (0)4 76 88 21 81; serge.perez at esrf.fr). General Conditions: You should hold a degree in either Physical Chemistry, Chemistry, Biochemistry or Structural Biology allowing enrolment for a PhD, such as an MSc, Master 2 de Recherche, Laurea or equivalent. Contract of two years renewable (subject to satisfactory progress) for one year. Gross salary around 2268 €/month. (The applicant will be responsible for arranging his/her academic registration and for paying the fees (if any)). The ESRF is an equal opportunity employer and encourages applications from disabled persons. If you are interested, please send us an e-mail (recruitment at esrf.fr) with your address, and we will provide you with an application form. Or print out an application form on the World Wide Web http://www.esrf.fr/Jobs/Applying. In addition to the application form, you should provide us with a detailed CV and the names of two referees. Deadline: 15-02-2010 Contract type: Non-permanent contract (CDD) Please send your application (form, covering letter and CV) to: peritore at esrf.fr with Subject: 'PhD Thesis Student (f/m), Kinetic Crystallography to Probe for Catalytic Mechanism and Protein Loop Motions in Glycosyltransferases'
Re: [ccp4bb] Coot pudding? (a.k.a N-linked carbohydrate addition)
There is no excuse for using MAN to mean both alpha and beta mannose. It is easy to take a MAN-b-D.cif file and modify it to a BMA.cif. BMA is already in the monomer library that comes with ccp4 (but I think it does not have geometry descriptions), and the last time I checked, there was not a BMA.cif in the default library that ships with phenix.refine, which makes using the standard beta-D-mannose monomer name one tiny step more difficult then using MAN for both alpha and beta mannose. No surprise then that nearly 30% of all sugar entries have incorrect names and geometries in the PDB (according to the paper that describes PDB-care). Engin On 12/22/09 9:29 AM, Damian Ekiert wrote: For an "ideal" glycan, you could used a model from a high resolution structure, or something that has been energy minimized, etc. Mostly I find this helps in getting the sugars in about the right place (keeping bond lengths and angles reasonable). I perhaps could stand to fiddle more and maybe I'll look through the updated documentation. Last I tried, the Asn-NAG1 linkage wasn't enforced, with would allow the whole glycan to slide down into the protein, or off into space. I am typically building relatively small glycans (2-5 residues) into ~3A data, so the density itself doesn't keep things in place very well. Regarding BMA vs MAN: When I have tried to used BMA in REFMAC, it doesn't seem to recognize it and requires a library file. But if you use MAN, it adds a MODRES record to the header, enforcing beta-mannose geometry. Not sure if this is just a REFMAC version issue or what. Best, Damian Paul Emsley wrote: If I can chip into this somewhat sacrilegiously-named thread 1) I *would* use real-space refinement :), specifically Sphere Refinement. You can dial down the density weight if needed, of course. 2) the documentation on refining carbohydrates in Coot has recently been updated http://www.biop.ox.ac.uk/coot/doc/coot/Refining-Carbohydrates.html 3) Coot does not (yet) correct chiral centre inversions in glycosidic linkages on refinement 4) or delete the O1s :) Paul. Robbie Joosten wrote: Dear Steve, I would also use Damian's approach, but the sequence of the core should be NAG-NAG-BMA-(MAN)2. This is improtant because the correct stereochemistry restraints for beta-mannose can only be applied when you call the residue BMA. Building carbohydrates also comes with special validation requirements. PDB-care and CARP are both very usefull. Unfortunately, the service is currently down (http://www.dkfz.de/spec/glycosciences.de). Just make sure the links between your carbs are correct and, please, remove the O1 atoms when needed ;) Cheers, Robbie Joosten Date: Mon, 21 Dec 2009 17:48:31 -0800 From: dceki...@scripps.edu Subject: Re: [ccp4bb] Coot pudding? (a.k.a N-linked carbohydrate addition) To: CCP4BB@JISCMAIL.AC.UK Steve, My general strategy is to start with an "ideal" glycan (an Asn linked to NAG-NAG-(MAN)3 ) and superimpose the Asn on the residue from my protein. Then you can move the whole glycan as a rigid body until the Asn and first NAG are roughly positioned. Then you can tweak any sugars further out on the chain to get them to fit. Unless you have really great density, usually it is best to avoid real pace refine zone. Better to fit the sugars using the manual rigid body fitting tools, do the best you can, then REFMAC usually brings them in OK. I have some models that I could send you if you need them. Best, Damian Ekiert Soisson, Stephen M wrote: Hi everyone- I was searching for some information on what might be the best way to add N-linked sugars in coot, and Google has let me down. Searching "adding sugars in coot" returns a very nice recipe for Coot Pudding. ***_Recipe for_/ Coot//__/_ Pudding - American_/ Coots/* It has plenty of fat,/ sugar/, and starch, and probably some calcium from the milk.* ...* The/ coots/ will not tolerate/ adding/ eggs in any form, so this is an egg* ...* ///_www.beaky_//_*coot*.com/pudding.html_/// / -/ _Similar_ // I did not know that coots had such an aversion to eggs. :) Anyway, would anyone have any top tips on adding N-linked sugars using coot? I can import the NAG monomers, but linking them up to the protein seems non-trivial Many thanks in advance, Steve Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then dele
Re: [ccp4bb] Coot pudding? (a.k.a N-linked carbohydrate addition)
I am also having problems with the branching. The fucose linked to the first NAG being linked to the second NAG of the carbohydrate chain. So one has a linear chain NAG-FUC-NAG-MAN-MAN-MAN instead of the FUC being a branch out from the first NAG. i.e. : NAG-NAG-BMA-BMA || FUC MAN Has anybody solved such problem in coot. Enrico. -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449 Lab LTMB, SIMOPRO, IBiTec-S, CEA Saclay, 91191 Gif-sur-Yvette Cedex FRANCE http://www-dsv.cea.fr/en/ibitecs/82 http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
[ccp4bb] Electrophysiology Homolog to CCP4BB?
Since there are some crystallographers who also do electrophysiology / patch clamping, I thought it might be possible that there is a homologous BB for those techniques, and that a posting here would be a good means to find such, i.e., Is anybody aware of a BB similar (or greater) in vivacity and enthusiasm to CCP4BB, but which deals with electrophysiology techniques? (Or at least an Artem homolog?) Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Coot pudding? (a.k.a N-linked carbohydrate addition)
Hi Damian, I have used both Coot and Refmac for glycan modelling/refinement in the past, last time was 2 or 3 years ago. They both behaved correctly for me. To get the correct behaviour, you must have the correct LINK cards in the header. At the time, the Chemical ID BMA did not exist in the PDB. There was only MAN. Presumably, the PDB had originally decided that the saccahride linkage is a bit like the peptide bond, where it can be one sort or another, so you have to specify it explicitly. But convenience, or avoiding confusion, must have been the reason for the recent creation of BMA. If Coot/Refmac do not have it in their library, well you can add it easily into the correct folder on your disk, by editing the MAN-b-D.cif entry and calling it BMA.cif. Garib told me, when I was grappling with this problem, that Refmac works out from the coordinates which form of the saccahride linkage it is and imposes the correct restraints. Admittedly, there must be room for confusion if modelling is not accurate enough. So BMA is better all round. The branching of the glycan tree does have a good template in PDB entry 1LGB, first deposited in 1994. It has been reviewed to include BMA earlier this year. The branching pattern is correct, so care must be taken to enter the correct numbers in the LINK cards if you are to reproduce a similar branching pattern in your glycan. Incidentally, if you search the PDB for chemical id BMA, there are apparently 36 entries. 1LGB was not among them, and I don't know why. I hope this is useful. Good Luck. Pierre Rizkallah Dr. Pierre Rizkallah, Senior Lecturer in Structural Biology, WHRI, School of Medicine, Cardiff University, Heath Park, CF14 4XN email: rizkall...@cf.ac.uk phone + 44 29 2074 2248 >>> Damian Ekiert 22/12/09 5:46 PM >>> For an "ideal" glycan, you could used a model from a high resolution structure, or something that has been energy minimized, etc. Mostly I find this helps in getting the sugars in about the right place (keeping bond lengths and angles reasonable). I perhaps could stand to fiddle more and maybe I'll look through the updated documentation. Last I tried, the Asn-NAG1 linkage wasn't enforced, with would allow the whole glycan to slide down into the protein, or off into space. I am typically building relatively small glycans (2-5 residues) into ~3A data, so the density itself doesn't keep things in place very well. Regarding BMA vs MAN: When I have tried to used BMA in REFMAC, it doesn't seem to recognize it and requires a library file. But if you use MAN, it adds a MODRES record to the header, enforcing beta-mannose geometry. Not sure if this is just a REFMAC version issue or what. Best, Damian Paul Emsley wrote: > If I can chip into this somewhat sacrilegiously-named thread > > 1) I *would* use real-space refinement :), specifically Sphere > Refinement. You can dial down the > density weight if needed, of course. > > 2) the documentation on refining carbohydrates in Coot has recently been > updated > > http://www.biop.ox.ac.uk/coot/doc/coot/Refining-Carbohydrates.html > > 3) Coot does not (yet) correct chiral centre inversions in glycosidic > linkages on refinement > > 4) or delete the O1s :) > > Paul. > > > > Robbie Joosten wrote: > >> Dear Steve, >> >> I would also use Damian's approach, but the sequence of the core should be >> NAG-NAG-BMA-(MAN)2. This is improtant because the correct stereochemistry >> restraints for beta-mannose can only be applied when you call the residue >> BMA. >> Building carbohydrates also comes with special validation requirements. >> PDB-care and CARP are both very usefull. Unfortunately, the service is >> currently down (http://www.dkfz.de/spec/glycosciences.de). Just make sure >> the links between your carbs are correct and, please, remove the O1 atoms >> when needed ;) >> >> Cheers, >> Robbie Joosten >> >> >> >> >>> Date: Mon, 21 Dec 2009 17:48:31 -0800 >>> From: dceki...@scripps.edu >>> Subject: Re: [ccp4bb] Coot pudding? (a.k.a N-linked carbohydrate addition) >>> To: CCP4BB@JISCMAIL.AC.UK >>> >>> Steve, >>> >>> My general strategy is to start with an "ideal" glycan (an Asn linked to >>> NAG-NAG-(MAN)3 ) and superimpose the Asn on the residue from my protein. >>> Then you can move the whole glycan as a rigid body until the Asn and >>> first NAG are roughly positioned. Then you can tweak any sugars further >>> out on the chain to get them to fit. Unless you have really great >>> density, usually it is best to avoid real pace refine zone. Better to >>> fit the sugars using the manual rigid body fitting tools, do the best >>> you can, then REFMAC usually brings them in OK. >>> >>> I have some models that I could send you if you need them. >>> >>> Best, >>> >>> Damian Ekiert >>> >>> >>> >>> Soisson, Stephen M wrote: >>> >>> Hi everyone- >>>
Re: [ccp4bb] Coot pudding? (a.k.a N-linked carbohydrate addition)
Pierre, For what its worth, I just tried using "Real Space Refine Zone" on something I am working on now (2.8A resolution, in coot 0.5.2, build 1691). With the "LINKR" record generated by REFMAC5, my glycan slithers down into the protein density. Similarly, using "Regularize Zone" does not enforce the Asn-NAG linkage. I also tried replacing the "R" from LINKR with a space, but it doesn't make a difference. My records read: 1 2 3 4 5 6 7 8 12345678901234567890123456789012345678901234567890123456789012345678901234567890 LINKRC1 NAG A 401 ND2 ASN A 21NAG-ASN LINK C1 NAG A 401 ND2 ASN A 21NAG-ASN However, I checked the page that Paul mentioned in an earlier email, and it states: "LINK and LNKR cards are not used to determine the geometry of the restraints." If other people don't have this problem, I would love to know how to get this to work because, admittedly, placing everything using other tools is a pain. Best, Damian Pierre Rizkallah wrote: Hi Damian, I have used both Coot and Refmac for glycan modelling/refinement in the past, last time was 2 or 3 years ago. They both behaved correctly for me. To get the correct behaviour, you must have the correct LINK cards in the header. At the time, the Chemical ID BMA did not exist in the PDB. There was only MAN. Presumably, the PDB had originally decided that the saccahride linkage is a bit like the peptide bond, where it can be one sort or another, so you have to specify it explicitly. But convenience, or avoiding confusion, must have been the reason for the recent creation of BMA. If Coot/Refmac do not have it in their library, well you can add it easily into the correct folder on your disk, by editing the MAN-b-D.cif entry and calling it BMA.cif. Garib told me, when I was grappling with this problem, that Refmac works out from the coordinates which form of the saccahride linkage it is and imposes the correct restraints. Admittedly, there must be room for confusion if modelling is not accurate enough. So BMA is better all round. The branching of the glycan tree does have a good template in PDB entry 1LGB, first deposited in 1994. It has been reviewed to include BMA earlier this year. The branching pattern is correct, so care must be taken to enter the correct numbers in the LINK cards if you are to reproduce a similar branching pattern in your glycan. Incidentally, if you search the PDB for chemical id BMA, there are apparently 36 entries. 1LGB was not among them, and I don't know why. I hope this is useful. Good Luck. Pierre Rizkallah Dr. Pierre Rizkallah, Senior Lecturer in Structural Biology, WHRI, School of Medicine, Cardiff University, Heath Park, CF14 4XN email: rizkall...@cf.ac.uk phone + 44 29 2074 2248 Damian Ekiert 22/12/09 5:46 PM >>> For an "ideal" glycan, you could used a model from a high resolution structure, or something that has been energy minimized, etc. Mostly I find this helps in getting the sugars in about the right place (keeping bond lengths and angles reasonable). I perhaps could stand to fiddle more and maybe I'll look through the updated documentation. Last I tried, the Asn-NAG1 linkage wasn't enforced, with would allow the whole glycan to slide down into the protein, or off into space. I am typically building relatively small glycans (2-5 residues) into ~3A data, so the density itself doesn't keep things in place very well. Regarding BMA vs MAN: When I have tried to used BMA in REFMAC, it doesn't seem to recognize it and requires a library file. But if you use MAN, it adds a MODRES record to the header, enforcing beta-mannose geometry. Not sure if this is just a REFMAC version issue or what. Best, Damian Paul Emsley wrote: If I can chip into this somewhat sacrilegiously-named thread 1) I *would* use real-space refinement :), specifically Sphere Refinement. You can dial down the density weight if needed, of course. 2) the documentation on refining carbohydrates in Coot has recently been updated http://www.biop.ox.ac.uk/coot/doc/coot/Refining-Carbohydrates.html 3) Coot does not (yet) correct chiral centre inversions in glycosidic linkages on refinement 4) or delete the O1s :) Paul. Robbie Joosten wrote: Dear Steve, I would also use Damian's approach, but the sequence of the core should be NAG-NAG-BMA-(MAN)2. This is improtant because the correct stereochemistry restraints for beta-mannose can only be applied when you call the residue BMA. Building carbohydrates also comes with special validation requirements. PDB-care and CARP are both very usefull. Unfortunately, the service is currently down (http://www.dkfz.de/spec/glycosciences.de). Just make sure the links between your carbs are correct and, please, remove the O1 atoms w
