Re: [ccp4bb] how to get phase of huge complex
Lisa, As others have said, using careful data collection and the modern program suites available (SHARP, Phenix, etc.), a 300 KD complex with 111 Se-Met residues should be quite solvable. But you didn't state is what is in the asymmetric unit (the important figure): one complex with 111 Se-Met residues or 111 Se-Met residues and 300KD in the ASU. We recently solved a structure by SAD with 750 KD of protein and 114 Se-Met residues in the ASU. The anomalous signal at 3.1 A was strong enough to find 109 Se-Met residues and trace about 70% of the chain after the first round of phasing. While we had the potential of NCS to work with, conformational changes between the different monomers meant that NCS methods could not be used in the initial phasing. Trying also James' suggestion of differential labeling and/or including MIRAS methods (Hg, Pt, Sm, etc.) with Se-Met protein should increase your chances. With modern beamlines, you can tune to the most optimal wavelengths for data collection. Good hunting, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Jun 12, 2012, at 10:46 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
[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
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] 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] 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
