[ccp4bb] Information on enzymes involved in TPP biosynthesis
Dear all, By some extraordinary chance, could someone provide me information about the temperature dependence of two E. coli enzymes: (1) Hydroxyethythiazole kinase: EC 2.7.1.50 (encoded by the gene thiM) (2) Enzyme participating in the synthesis of 4-amino-5-hydroxymethyl pyrimidine phosphate from 5-aminoimidazole ribotide (encoded by the gene thiC) Something like kCat(T), Km(T) would be magnificent. And if I am very, very lucky: more generally, any information/reference on the temperature dependence of other enzyme(s) involved in thimamine pyrophosphate biosynthesis would be much welcome. Many thanks in advance. Philippe Dumas Biophysics Structural biology team IBMC-CNRS, 15 rue René Descartes Strasbourg, France +33 (0)388 41 70 02
[ccp4bb] Off topic: Problem in reproducing crystal screen
I have obtained crystals in Hampton Crustal Screen 2, condition 26.The condition has the following components: 1. 0.2 M ammonium sulphate 2. 0.1 M MES buffer pH 6.5 3. 30% PEG mononmethyl ether 5,000. When I try to prepare the screen in-house, I use the Hampton stocks of 3.5 M ammonium sulphate and 50% PEG mononmethyl ether 5,000. I make the MES buffer using MES hydrate, SigmaUltra from Sigma and maintain the pH accurately. The water I use is filtered MilliQ. However, the PEG MME and the rest of the solution remains immiscible and thus, forms droplets of separated PEG when I set up crystallization. The original condition from Hampton has no such problems and gives good crystals. Can anyone let me know how to overcome the problem insolubility of PEG MME in the buffer mix? I have not faced such problems with other conditions which I prepare in house and have PEG in them. Further, it's not possible for me to use the original screen all the time. Thanks a lot! Regards -- Nazia Nasir PhD Scholar Protein Crystallography Lab National Institute of Immunology New Delhi
Re: [ccp4bb] Phaser question
Just to follow up on this: The enormous increase between the TFZ= and TFZ== numbers is surprising, so I focused on that in my original answer to Fred's question. However, if anyone wants to know the difference between these, it's documented on this web page: http://www.phaser.cimr.cam.ac.uk/index.php/Molecular_Replacement#Annotation. Best wishes, Randy Read On 5 Dec 2013, at 08:36, vellieux frederic.velli...@ibs.fr wrote: Hiyya all, I have a question about the latest Phaser output, concerning TFZ = and TFZ == . I do not know how to interpret outputs of the type TFZ = 5.2 TFZ == 54.1; TFZ = 5.8 TFZ == 63.0; TFZ = 6.4 TFZ == 19.7 (these are real TFZ figures coming from Phaser log files). I used to analyse the Phaser output using TFZ, when only a single number was given. Now with two figures to consider, I do not know what to think of it any more. Any ideas out there ? And best wishes for an enjoyable end-of-the-year season (be it in the cold or in the sun depending on which side of the planet you sit). Fred. -- Fred. Vellieux (B.Sc., Ph.D., hdr) IBS2 / ELMA Campus EPN 6 rue Jules Horowitz F-38042 Grenoble Tel: +33 457428605 (Fax: +33 438785494) -- 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] rotation peak number in new version phaser
Hi, I would only suggest using the brute rotation function if you want to use the rotate around option or do something other than a full rotation search, because the fast search followed by rescoring should give you essentially the same list of peaks (but faster). If you want to get a bigger list of orientations, you should be able to open the Expert parameters section of the ccp4i GUI for Phaser, then change the percent cutoff in the Purge rotation peaks line to something lower than the default of 75%. Alternatively, if you're running Phaser from a script, you can include commands like: PURGE ROT ENABLE ON PURGE ROT PERCENT 50.0 I've just tested and this works for me. Let me know if you're still having trouble with this. Best wishes, Randy Read On 5 Dec 2013, at 10:11, LISA science...@gmail.com wrote: hi all, I try to solve the crystal structures of a mult-domain protein with phaser. I cannot solve the structure by automatic search using domain structure as the models. I tried to do the rotation search using the fast or brute rotation function. I defined the rotation search peaks number is 1000. But I only get no more than 100 peaks. I tried the phaser in ccp4-6.3.0 and ccp4-6.4.0. But I can only get much less peaks than I required in the rotation. But the old version phaser can give about 1000 peaks if I use brute rotation function ask for 1000 peaks. How can I more rotation peaks in the new version phaser ? Thank you. Lisa -- 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] Off topic: Problem in reproducing crystal screen
Dear Nazia Nasir, did you warm up the solution a little? This makes working with PEG solutions a lot easier. You may also note the Hampton do not maintain the pH: they use the buffers as indicated but this does not mean that the final solution still has pH 6.5. With your ingredients it probably is, but e.g. with high concentrations of imidazole the final pH would differ a lot. Best, Tim On 12/06/2013 02:37 PM, Nazia Nasir Phd2009,ProteinCrystall.Lab wrote: I have obtained crystals in Hampton Crustal Screen 2, condition 26.The condition has the following components: 1. 0.2 M ammonium sulphate 2. 0.1 M MES buffer pH 6.5 3. 30% PEG mononmethyl ether 5,000. When I try to prepare the screen in-house, I use the Hampton stocks of 3.5 M ammonium sulphate and 50% PEG mononmethyl ether 5,000. I make the MES buffer using MES hydrate, SigmaUltra from Sigma and maintain the pH accurately. The water I use is filtered MilliQ. However, the PEG MME and the rest of the solution remains immiscible and thus, forms droplets of separated PEG when I set up crystallization. The original condition from Hampton has no such problems and gives good crystals. Can anyone let me know how to overcome the problem insolubility of PEG MME in the buffer mix? I have not faced such problems with other conditions which I prepare in house and have PEG in them. Further, it's not possible for me to use the original screen all the time. Thanks a lot! Regards -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
[ccp4bb] Insect cell SF9 expression
Dear all, Does any one has protocols for expression and making stable virus for SF-9 Cell, I was trying with Invitrogen manual but somehow it is not working, any alternative protocols which works quickly? I am new to thins insect cell expression. your suggestion and protocols are really helpful Thanking in advance Dhananjay.K Post Doctoral Research Associate Department of Biochemistry Indian Institute of Science Bangalore India
