[ccp4bb] vacancy for Java Web Application Developer at Diamond
Dear all, this new vacancy at Diamond may be of interest for some readers of this BB which is focused on further development of ISPyB at Diamond. Full details of the post can be found here: http://www.diamond.ac.uk/Home/Jobs/Current/DIA0737_TH.html Informal inquiries can be made to myself (martin.wa...@diamond.ac.uk ) or Alun Ashton (alun.ash...@diamond.ac.ukmailto:alun.ash...@diamond.ac.uk) Thanks Martin
Re: [ccp4bb] Fun Question - Is multiple isomorphous replacement an obsolete technique?
Why would anyone ignore the anomalous data they had collected? It will always help the phasing, and decide the hand for you.. Eleanor On 6 Jun 2012, at 03:55, Stefan Gajewski wrote: Hey! I was just wondering, do you know of any recent (~10y) publication that presented a structure solution solely based on MIR? Without the use of any anomalous signal of some sort? When was the last time you saw a structure that was solved without the use of anomalous signal or homology model? Is there a way to look up the answer (e.g. filter settings in the RCSB) I am not aware of? Thanks, S. (Disclaimer: I am aware that isomorpous data is a valuable source of information)
[ccp4bb] how to interpret DALI search results
Dear ALL; After we solved our structure by anomalous scattering, we did a DALI search. Here are the results but it is not easy to draw meaningful conclusions whether our structure represents a novel fold or is homologous to others. Basically the Z-score is between 2 and 6.4 since our structure only contains 130 residues. Sequence identity is between 5 to 15%. The RMSD of structural alignment is between 2.5 to 6 angstrom. Any suggests to interpret the DALI results? Many thanks, Jerry McCully
Re: [ccp4bb] how to interpret DALI search results
if this is the first (or second, or third) time you do a DALI search, take the list output from DALI, start from the top and superpose each structure with yours and look at the superpositions with your favourite superposition software. This is very educational and the only way you get a feeling for what numbers mean. Quoting Jerry McCully: Dear ALL; After we solved our structure by anomalous scattering, we did a DALI search. Here are the results but it is not easy to draw meaningful conclusions whether our structure represents a novel fold or is homologous to others. Basically the Z-score is between 2 and 6.4 since our structure only contains 130 residues. Sequence identity is between 5 to 15%. The RMSD of structural alignment is between 2.5 to 6 angstrom. Any suggests to interpret the DALI results? Many thanks, Jerry McCully Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] how to interpret DALI search results
...I meant visualisation software of course... Quoting VAN RAAIJ , MARK JOHAN: if this is the first (or second, or third) time you do a DALI search, take the list output from DALI, start from the top and superpose each structure with yours and look at the superpositions with your favourite superposition software. This is very educational and the only way you get a feeling for what numbers mean. Quoting Jerry McCully: Dear ALL; After we solved our structure by anomalous scattering, we did a DALI search. Here are the results but it is not easy to draw meaningful conclusions whether our structure represents a novel fold or is homologous to others. Basically the Z-score is between 2 and 6.4 since our structure only contains 130 residues. Sequence identity is between 5 to 15%. The RMSD of structural alignment is between 2.5 to 6 angstrom. Any suggests to interpret the DALI results? Many thanks, Jerry McCully Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] how to interpret DALI search results
On Tuesday, June 12, 2012 02:29:13 pm Jerry McCully wrote: Dear ALL; After we solved our structure by anomalous scattering, we did a DALI search. Here are the results but it is not easy to draw meaningful conclusions whether our structure represents a novel fold or is homologous to others. I don't think the question is it homologous can ever be answered by a DALI score, whether it is high or low. For that you need to think about sequence families, inferred evolutionary history, shared function, etc. If your structure has a biological function related to that of your DALI hit, I'd be inclined to consider seriously whether they could be distant homologs. If not, I doubt you will convince anyone based only on a vaguely similar fold. A sequence identity of 15% is really low, but that is presumeably only a pairwise comparison. You should try PSIBLAST or similar to see if both your protein and the DALI hit are recognizably members of the same sequence family or superfamily. Ethan Basically the Z-score is between 2 and 6.4 since our structure only contains 130 residues. Sequence identity is between 5 to 15%. The RMSD of structural alignment is between 2.5 to 6 angstrom. Any suggests to interpret the DALI results? Many thanks, Jerry McCully -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
[ccp4bb] how to get phase of huge complex
Hi all, My work is to solve huge complex containing 4 different proteins and total molecular weight is about 300 KD. I can purify the complex by co-expression them in E.coli. This complex contains 8 protein A, 2 protein B and 1 protein C and D. protein B and protein C have homology structures deposited in PDB database. No homology structure available for protein A and D, which contribute 60% of the whole molecular weight for the complex. Now I am trying to find a way to solve the phase of this complex. I am thinking of use sad or mad with se-Met. There total 111 Met residues in this complex. Is it possible to solve this complex by se-Met? Does someone have experience to solve huge complex structure with se-met? It is also very welcome for all the suggestion. Thank you. All the best, Lisa
Re: [ccp4bb] how to get phase of huge complex
Do you have crystals? Do they diffract? If so, to what resolution? What resolution do you require to answer your biological question? F On Jun 12, 2012, at 7:46 PM, LISA science...@gmail.com wrote: Hi all, My work is to solve huge complex containing 4 different proteins and total molecular weight is about 300 KD. I can purify the complex by co-expression them in E.coli. This complex contains 8 protein A, 2 protein B and 1 protein C and D. protein B and protein C have homology structures deposited in PDB database. No homology structure available for protein A and D, which contribute 60% of the whole molecular weight for the complex. Now I am trying to find a way to solve the phase of this complex. I am thinking of use sad or mad with se-Met. There total 111 Met residues in this complex. Is it possible to solve this complex by se-Met? Does someone have experience to solve huge complex structure with se-met? It is also very welcome for all the suggestion. Thank you. All the best, Lisa
[ccp4bb] Where to get a thermo-cycling controlled chamber
Hi everybody, My experiment need me to try a thermo-cycling method for protein crystallization. So I need a kind of temperature chamber, which can changes temperature quickly, precisely and steadily. Furthermore, it should be relatively quiet when running, because I'm afraid vibration will affect the crystallization of my protein. I just need a small one, which can control temperature from 0 C to 50 C. I'm looking for this for some time, and still don't know where to get it. If anyone can provide information about where to purchase, or how to solve the thermo-cycling control if there's no commercial merchandise, I will appreciate that very much. Any other suggestions are welcome. Jun Ma 2012-06-12
Re: [ccp4bb] how to get phase of huge complex
If you want to use se-Met, you might want to start by labeling only one protein at a time. For example, if you have A,B,C,D, grow crystals like this: se-A, B, C, D A, se-B, C, D, etc. Then try combinations of 2, then 3, then if you haven't got the phases you need, try all 4. And remember, if 2 different combinations diffract and they are isomorphous, then you can try MIR too. Also, if your complex can stay intact on a gel shift, look at this paper: http://www.ncbi.nlm.nih.gov/pubmed/10903954 James On Jun 12, 2012, at 8:46 PM, LISA wrote: Hi all, My work is to solve huge complex containing 4 different proteins and total molecular weight is about 300 KD. I can purify the complex by co-expression them in E.coli. This complex contains 8 protein A, 2 protein B and 1 protein C and D. protein B and protein C have homology structures deposited in PDB database. No homology structure available for protein A and D, which contribute 60% of the whole molecular weight for the complex. Now I am trying to find a way to solve the phase of this complex. I am thinking of use sad or mad with se-Met. There total 111 Met residues in this complex. Is it possible to solve this complex by se-Met? Does someone have experience to solve huge complex structure with se-met? It is also very welcome for all the suggestion. Thank you. All the best, Lisa
Re: [ccp4bb] Where to get a thermo-cycling controlled chamber
Why wouldn't a peltier-cooled/heated (PCR) thermal cycler be adaptable? Roger Rowlett On Jun 12, 2012 11:15 PM, Jun Ma mrmar2...@gmail.com wrote: Hi everybody, My experiment need me to try a thermo-cycling method for protein crystallization. So I need a kind of temperature chamber, which can changes temperature quickly, precisely and steadily. Furthermore, it should be relatively quiet when running, because I'm afraid vibration will affect the crystallization of my protein. I just need a small one, which can control temperature from 0 C to 50 C. I'm looking for this for some time, and still don't know where to get it. If anyone can provide information about where to purchase, or how to solve the thermo-cycling control if there's no commercial merchandise, I will appreciate that very much. Any other suggestions are welcome. Jun Ma 2012-06-12
Re: [ccp4bb] how to get phase of huge complex
If you don't have crystals yet, you'll find getting it to crystallize is your main problem. Finding 111 sites should be feasible without other tricks than very careful data collection (see below); if you have two or more copies in the ASU, you may find you need to do what the ribosome guys did, namely use other derivatives (e.g TaBr clusters) to locate your seleniums, and then phase. If your resolution is low, you definitely want to do 2- or 3-wavelength MAD. So yes, it's definitely feasible. If you have crystals... von Delft, Inoue, et al. 'Structure of E. coli ketopantoate hydroxymethyl transferase complexed with ketopantoate and Mg2+, solved by locating 160 selenomethionine sites'. /Structure /11, no. 8 (2003): 985--996. http://www.cell.com/structure/retrieve/pii/S0969212603001588 On 13/06/2012 03:55, Francis E Reyes wrote: Do you have crystals? Do they diffract? If so, to what resolution? What resolution do you require to answer your biological question? F On Jun 12, 2012, at 7:46 PM, LISAscience...@gmail.com wrote: Hi all, My work is to solve huge complex containing 4 different proteins and total molecular weight is about 300 KD. I can purify the complex by co-expression them in E.coli. This complex contains 8 protein A, 2 protein B and 1 protein C and D. protein B and protein C have homology structures deposited in PDB database. No homology structure available for protein A and D, which contribute 60% of the whole molecular weight for the complex. Now I am trying to find a way to solve the phase of this complex. I am thinking of use sad or mad with se-Met. There total 111 Met residues in this complex. Is it possible to solve this complex by se-Met? Does someone have experience to solve huge complex structure with se-met? It is also very welcome for all the suggestion. Thank you. All the best, Lisa
Re: [ccp4bb] Where to get a thermo-cycling controlled chamber
Hi Jun, In this following article an metallic adapter was used to mount a 192 well sitting drop plate on a conventional thermocycler. http://journals.iucr.org/d/issues/2006/05/00/av5047/av5047bdy.html. However I guess there would be significant evaporation and condensation if you use vapor diffusion setup and cycle the temperatures. On the other hand, if you do microbatch under oil, then you should be able to easily setup the experiment on a 96-tube PCR plate and mount it on a PCR machine. I guess the temperature can be more precisely controlled this way with no special equipment needed. Especially, the evaporation and condensation during thermo cycles would probably be less problematic. Zhijie From: Jun Ma Sent: Tuesday, June 12, 2012 11:15 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Where to get a thermo-cycling controlled chamber Hi everybody, My experiment need me to try a thermo-cycling method for protein crystallization. So I need a kind of temperature chamber, which can changes temperature quickly, precisely and steadily. Furthermore, it should be relatively quiet when running, because I'm afraid vibration will affect the crystallization of my protein. I just need a small one, which can control temperature from 0 C to 50 C. I'm looking for this for some time, and still don't know where to get it. If anyone can provide information about where to purchase, or how to solve the thermo-cycling control if there's no commercial merchandise, I will appreciate that very much. Any other suggestions are welcome. Jun Ma 2012-06-12
Re: [ccp4bb] how to get phase of huge complex
On Tue, Jun 12, 2012 at 8:53 PM, Frank von Delft frank.vonde...@sgc.ox.ac.uk wrote: Finding 111 sites should be feasible without other tricks than very careful data collection (see below); if you have two or more copies in the ASU, you may find you need to do what the ribosome guys did, namely use other derivatives (e.g TaBr clusters) to locate your seleniums, and then phase. With 40% of the complex having homologues in the PDB, you may be able to place those subunits by MR, then use the phases from the incomplete model to locate the seleniums. -Nat
Re: [ccp4bb] how to get phase of huge complex
Hi Lisa, hi all, Please do not discard the alternative method(s) of conventional heavy atoms. Co-crystallisation or heavy-atom containing mother liquor soaks. You may remember that monster complexes have been solved in the past by such methods, and sometimes there are difficulties in crystallising the Se-Met version of a protein (the native protein gives crystals, the Se-Met version does not). And there is still some work going on regarding the development of novel (lanthanide-based) heavy-atom compounds that may provide both isomorphous and anomalous differences, the anomalous signal being extremely useful in the case of lanthanides and can be used on its own to solve 3D structures (one can then go back to the structure of the native macromolecule or complex if need be). See e.g. Talon, R. et al. (2011), J. Sync. Rad. 18, 74-78 (PMID: 21169697) and http://www.natx-ray.com/products/catalogue_consum_CSM002.html HTH, Fred. F.M.D. Vellieux (B.Sc., Ph.D., hdr) Institut de Biologie Structurale J.-P. Ebel CEA CNRS UJF LBM/ELMA 41 rue Jules Horowitz 38027 Grenoble Cedex 01 France Tel: +33 (0) 438789605 (direct line), +33 (0) 663482891 (mobile phone) Fax: +33 (0) 438785494 e-mail: frederic.velli...@ibs.fr LISA wrote: Hi all, My work is to solve huge complex containing 4 different proteins and total molecular weight is about 300 KD. I can purify the complex by co-expression them in E.coli. This complex contains 8 protein A, 2 protein B and 1 protein C and D. protein B and protein C have homology structures deposited in PDB database. No homology structure available for protein A and D, which contribute 60% of the whole molecular weight for the complex. Now I am trying to find a way to solve the phase of this complex. I am thinking of use sad or mad with se-Met. There total 111 Met residues in this complex. Is it possible to solve this complex by se-Met? Does someone have experience to solve huge complex structure with se-met? It is also very welcome for all the suggestion. Thank you. All the best, Lisa