Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
Hi Thierry, We have worked on a number of proteins where the oligomerisation state differs from that found in solution. A recent example is a project we worked on involving the HC fragment of tetanus toxin (see Qazi et al., J Mol Biol. 2007 Jan 5;365(1):123-34) which crystallises as a monomer. The discovery of it multimerising was somewhat serendipitous in that the protein (like so many these days) was purified using a his-tag and then crystallised. We planned to use SAXS to study proposed conformational changes and looked at the protein on a native gel as part of our standard preparation for these experiments. The native gel showed the protein multimerised and the combination of the SAXS and excellent crystal structure from the Isaacs group led to new ideas about the function of the toxin. A few points: 1. Crystallisation conditions can be very selective for different oligomerisation states of protein and other parameters ( e.g., pH, ionic strength, oxidation state, exogenous ligands,...) besides concentration can affect the equilibria controlling whether a protein appears to be a monomer or not as the case may be. 2. It is always worth looking carefully at native gels or other sizing data for any protein which is crystallised. With the great expression systems out there using affinity tags this sometimes gets forgotten. 3. Be aware that truncated forms of proteins (even small deletions) may affect oligomerisation states (we have also seen this with a membrane bound receptor we are working with now and I think there are other similar examples in the literature) best wishes, Kate
Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
hi thierry we had a case where Spo0A in the phospho form was a dimer in solution, and in the non-phospho form was a monomer. in the crystal, we had monomers of the phospho form, and (domain-swapped) dimers of the non-phospho form. turns out that the crystallisation conditions affected the behaviour of the protein, converting a M->D Kd from nanomolar to micromolar in the first instance, and the acid pH of the crystallisation solution in the latter promoted domain- swapping. the biochemistry where we sorted all this out is: http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WK7-457CYHW-HN&_user=224739&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C14659&_version=1&_urlVersion=0&_userid=224739&md5=5d3b66d5d8d662753ec43bc94a173880 rick Fischmann, Thierry wrote: Dear fellow crystallographers, This is a question which is not CCP4-related. Is anybody aware of a protein which is known to be a dimer in solution (say by SEC), and yet crystallizes as a monomer? Wouldn’t the high concentration in the crystallization drop further favor dimerization? In other words, if a protein crystallizes as a monomer, can I conclude that it does not form biologically relevant dimers in solution? Thank you in advance for your replies. Thierry * This message and any attachments are solely for the intended recipient. If you are not the intended recipient, disclosure, copying, use or distribution of the information included in this message is prohibited -- Please immediately and permanently delete. -- R. J. Lewis Professor of Structural Biology Institute for Cell and Molecular Biosciences Faculty of Medical Sciences Tel: +44 (0)191 222 5482 University of Newcastle Fax: +44 (0)191 222 7424 Newcastle upon Tyne, NE2 4HH, UKEmail: [EMAIL PROTECTED]
[ccp4bb] Offtopic: FAD enzymatic assay: a little bit more about my enzyme
Dear all, Thank you for all your kind replies. Here is a little bit more about the enzyme and how I carry out the assay at the first place. My enzyme is a lipid desaturase, originally from plant but overexpressed in bacteria. FAD serves as a co-factor for this enzyme, in which FAD is reduced to FADH2. My goal is set up an assay that would allows me to continuously monitor the progress of the reaction. And I didn't want to use HPLC to analyze the final product since that would take a lot time and we don't have an instrument readily available to us. I wish FAD could be an alternative way since FAD will have different Abs in reduced or oxidized forms. I set up assay is a regular lab setting (not anaerobic), add FAD, substrate, ions and incubate. I finally add enzyme to initialize the reaction. I expect to see some decrease of the Ab at 450 nM. But I didn't. I have several concerns, one is the autooxidisability of FAD, how fast FADH2 would be reoxidized by O2 in the air or by the O2 dissolved in solution. The second cocern is how fast the FAD reaction will go. Please advise. Thank you! Mike
Re: [ccp4bb] Offtopic: FAD enzymatic assay: a little bit more about my enzyme
Mike: It seems FAD is readily dissociable for your protein.Then, trust me, you got to do it anaerobically in order to see the 450nm decrease upon reduction of the enzyme by the substrate(better use excess substrate). Sincerely, Hongnan Cao UCR Date: Tue, 2 Dec 2008 11:11:53 -0800From: [EMAIL PROTECTED]: [ccp4bb] Offtopic: FAD enzymatic assay: a little bit more about my enzymeTo: CCP4BB@JISCMAIL.AC.UK Dear all,Thank you for all your kind replies.Here is a little bit more about the enzyme and how I carry out the assay at the first place.My enzyme is a lipid desaturase, originally from plant but overexpressed in bacteria. FAD serves as a co-factor for this enzyme, in which FAD is reduced to FADH2. My goal is set up an assay that would allows me to continuously monitor the progress of the reaction. And I didn't want to use HPLC to analyze the final product since that would take a lot time and we don't have an instrument readily available to us. I wish FAD could be an alternative way since FAD will have different Abs in reduced or oxidized forms. I set up assay is a regular lab setting (not anaerobic), add FAD, substrate, ions and incubate. I finally add enzyme to initialize the reaction. I expect to see some decrease of the Ab at 450 nM. But I didn't.I have several concerns, one is the autooxidisability of FAD, how fast FADH2 would be reoxidized by O2 in the air or by the O2 dissolved in solution. The second cocern is how fast the FAD reaction will go.Please advise.Thank you!Mike _ 新版手机MSN，新功能，新体验！满足您的多彩需求！ http://mobile.msn.com.cn
Re: [ccp4bb] Offtopic: FAD enzymatic assay: a little bit more about my enzyme
Hi Michael, Fraser et al writes that in case of Synechococcus phytoene desaturase 'NAD+ and NADP+ were observed to be involved, whilst FAD was an ineffective electron acceptor ' Biochem J. 1993 May 1;291 ( Pt 3):687-92 HTH Guenter PS The enzyme itself has no flavin bound? michael nelson wrote: Dear all, Thank you for all your kind replies. Here is a little bit more about the enzyme and how I carry out the assay at the first place. My enzyme is a lipid desaturase, originally from plant but overexpressed in bacteria. FAD serves as a co-factor for this enzyme, in which FAD is reduced to FADH2. My goal is set up an assay that would allows me to continuously monitor the progress of the reaction. And I didn't want to use HPLC to analyze the final product since that would take a lot time and we don't have an instrument readily available to us. I wish FAD could be an alternative way since FAD will have different Abs in reduced or oxidized forms. I set up assay is a regular lab setting (not anaerobic), add FAD, substrate, ions and incubate. I finally add enzyme to initialize the reaction. I expect to see some decrease of the Ab at 450 nM. But I didn't. I have several concerns, one is the autooxidisability of FAD, how fast FADH2 would be reoxidized by O2 in the air or by the O2 dissolved in solution. The second cocern is how fast the FAD reaction will go. Please advise. Thank you! Mike -- *** Priv.Doz.Dr. Guenter Fritz Fachbereich Biologie Sektion Naturwissenschaften Universitaet Konstanz http://www.biologie.uni-konstanz.de/fritz Universitaetsstrasse 10 Postfach M665 D-78457 Konstanz e-mail: [EMAIL PROTECTED] Tel. Office: +49-(0)7531 88 3205 Tel. Lab : +49-(0)7531 88 3687 Fax: +49-(0)7531 88 2966
Re: [ccp4bb] Offtopic: FAD enzymatic assay: a little bit more about my enzyme
Does the FAD actually dissociate on each turnover, or does it remain bound and transfer electrons to an acceptor? Succinate dehydrogenase is an example of the latter, and it can be readily assayed using a mediater phenazine methosulfate to accept the electrons and transfer them to a redox dye dichlorophenol-indophenol. The reaction is monitored in real time by watching the disappearance of the intense blue color of DCIP, monitored at 600 nm I think. Google for SDH DCIP PMS will probably give a recipe. michael nelson wrote: Dear all, Thank you for all your kind replies. Here is a little bit more about the enzyme and how I carry out the assay at the first place. My enzyme is a lipid desaturase, originally from plant but overexpressed in bacteria. FAD serves as a co-factor for this enzyme, in which FAD is reduced to FADH2. My goal is set up an assay that would allows me to continuously monitor the progress of the reaction. And I didn't want to use HPLC to analyze the final product since that would take a lot time and we don't have an instrument readily available to us. I wish FAD could be an alternative way since FAD will have different Abs in reduced or oxidized forms. I set up assay is a regular lab setting (not anaerobic), add FAD, substrate, ions and incubate. I finally add enzyme to initialize the reaction. I expect to see some decrease of the Ab at 450 nM. But I didn't. I have several concerns, one is the autooxidisability of FAD, how fast FADH2 would be reoxidized by O2 in the air or by the O2 dissolved in solution. The second cocern is how fast the FAD reaction will go. Please advise. Thank you! Mike
[ccp4bb] site mutation evaluation
Dear All, I am trying to mutate a single amino acid in a PDB to see if the mutant disturbs ligand binding. Does anyone know any software that can do such work? Thanks a lot!
[ccp4bb] Post-doctoral positions available at Emory University, Atlanta
Macromolecular Machines involved in Translation and mRNA Transport Post-doctoral positions are available immediately in the group of Christine Dunham at Emory University School of Medicine. We are interested in two separate but related projects: 1) the structure and function of RNA and RNA-protein complexes involved in translation regulation on the ribosome and 2) structure and function of RNA- protein complexes required during messenger RNA recognition for processing and transport. Our group primarily uses the structural biology technique of X-ray crystallography in addition to complementary biochemical and biophysical techniques to address function in vitro. The laboratory is located in the Department of Biochemistry at Emory University School of Medicine where we have state-of-the-art in-house crystallographic facilities and crystallization robots. We also have access to dedicated synchrotron beamtime at the Advanced Photon Source (APS) at Argonne National Laboratory in Chicago. Funding is provided by the NASA Astrobiology Institute as a collaborative project (http://astrobiology.nasa.gov/nai/teams/can5/gatech ). Interested applicants should have a Ph.D. degree in biochemistry, molecular biology or structural biology. X-ray crystallographic experience is preferred but not essential. The ideal candidate must be highly motivated, possess excellent communication skills and the ability to work in a collaborative and team-oriented environment. To apply for this position, please e-mail a CV including a list of publications, a brief statement describing your scientific interests and the names and contact information of three references to Christine Dunham: [EMAIL PROTECTED] Laboratory website: http://www.biochem.emory.edu/labs/cmdunha Christine M. Dunham, Ph.D. Department of Biochemistry Emory University School of Medicine 1510 Clifton Road, NE, G223 Atlanta, Georgia 30322 1-404-712-1756 (office) 1-404-727-4928 (lab) -2738 (fax)
Re: [ccp4bb] site mutation evaluation
Pymol the question is, into how much trouble do you want to get ? MD simulations ? Energy minimisation ? Then you will need to do more than just mutate on the sreen one residue with Pymol. Jürgen On 2 Dec 2008, at 17:29, Hongmin Zhang wrote: Dear All, I am trying to mutate a single amino acid in a PDB to see if the mutant disturbs ligand binding. Does anyone know any software that can do such work? Thanks a lot! - Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch
Re: [ccp4bb] site mutation evaluation
Yes, it is better to have MD or energy minimization. Otherwise, with only view on the screen, we can't tell if the mutated residue would disturb ligand binding because of the side chain flexibility. Best! Hongmin On Wed, Dec 3, 2008 at 12:50 PM, Juergen Bosch <[EMAIL PROTECTED]>wrote: > Pymol > the question is, into how much trouble do you want to get ? > MD simulations ? Energy minimisation ? Then you will need to do more than > just mutate on the sreen one residue with Pymol. > > Jürgen > > On 2 Dec 2008, at 17:29, Hongmin Zhang wrote: > > Dear All, >> I am trying to mutate a single amino acid in a PDB to see if the mutant >> disturbs ligand binding. Does anyone know any software that can do such >> work? >> Thanks a lot! >> > > - > Jürgen Bosch > University of Washington > Dept. of Biochemistry, K-426 > 1705 NE Pacific Street > Seattle, WA 98195 > Box 357742 > Phone: +1-206-616-4510 > FAX: +1-206-685-7002 > Web: http://faculty.washington.edu/jbosch > > The information in this e-mail is intended only for the person to whom it > is > addressed. If you believe this e-mail was sent to you in error and the > e-mail > contains patient information, please contact the Partners Compliance > HelpLine at > http://www.partners.org/complianceline . If the e-mail was sent to you in > error > but does not contain patient information, please contact the sender and > properly > dispose of the e-mail. > >
Re: [ccp4bb] site mutation evaluation
In that case you might want to use CNS with model_anneal.inp or model_minimize.inp, or the equivalents in phenix Jürgen On 2 Dec 2008, at 21:29, Hongmin Zhang wrote: Yes, it is better to have MD or energy minimization. Otherwise, with only view on the screen, we can't tell if the mutated residue would disturb ligand binding because of the side chain flexibility. Best! Hongmin On Wed, Dec 3, 2008 at 12:50 PM, Juergen Bosch <[EMAIL PROTECTED] > wrote: Pymol the question is, into how much trouble do you want to get ? MD simulations ? Energy minimisation ? Then you will need to do more than just mutate on the sreen one residue with Pymol. Jürgen On 2 Dec 2008, at 17:29, Hongmin Zhang wrote: Dear All, I am trying to mutate a single amino acid in a PDB to see if the mutant disturbs ligand binding. Does anyone know any software that can do such work? Thanks a lot! - Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail. - Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch
Re: [ccp4bb] site mutation evaluation
Thanks! I think we still can't tell if the mutant would disturb ligand binding or not. Best! Hongmin On Wed, Dec 3, 2008 at 2:15 PM, Juergen Bosch <[EMAIL PROTECTED]>wrote: > In that case you might want to use CNS with model_anneal.inp or > model_minimize.inp, or the equivalents in phenix > Jürgen > > On 2 Dec 2008, at 21:29, Hongmin Zhang wrote: > > Yes, it is better to have MD or energy minimization. Otherwise, with only > view on the screen, we can't tell if the mutated residue would disturb > ligand binding because of the side chain flexibility. > Best! > Hongmin > > On Wed, Dec 3, 2008 at 12:50 PM, Juergen Bosch <[EMAIL PROTECTED]>wrote: > >> Pymol >> the question is, into how much trouble do you want to get ? >> MD simulations ? Energy minimisation ? Then you will need to do more than >> just mutate on the sreen one residue with Pymol. >> >> Jürgen >> >> On 2 Dec 2008, at 17:29, Hongmin Zhang wrote: >> >> Dear All, >>> I am trying to mutate a single amino acid in a PDB to see if the mutant >>> disturbs ligand binding. Does anyone know any software that can do such >>> work? >>> Thanks a lot! >>> >> >> - >> Jürgen Bosch >> University of Washington >> Dept. of Biochemistry, K-426 >> 1705 NE Pacific Street >> Seattle, WA 98195 >> Box 357742 >> Phone: +1-206-616-4510 >> FAX: +1-206-685-7002 >> Web: http://faculty.washington.edu/jbosch >> >> The information in this e-mail is intended only for the person to whom it >> is >> addressed. If you believe this e-mail was sent to you in error and the >> e-mail >> contains patient information, please contact the Partners Compliance >> HelpLine at >> http://www.partners.org/complianceline . If the e-mail was sent to you in >> error >> but does not contain patient information, please contact the sender and >> properly >> dispose of the e-mail. >> >> > > - > Jürgen Bosch > University of Washington > Dept. of Biochemistry, K-426 > 1705 NE Pacific Street > Seattle, WA 98195 > Box 357742 > Phone: +1-206-616-4510 > FAX: +1-206-685-7002 > Web: http://faculty.washington.edu/jbosch > >
[ccp4bb] Temperature factor discrepancy
Dear all, I have a 3 A structure refined with REFMAC which gives consistently average atomic B-factors of 40 A2, whereas the B factor from a Wilson plot is about 60 A2. Is there any explanation for such a discrepancy? There are no obvious problems: No twinning, spacegroup P21 with two molecules in the asu, no proper ncs symmetry. No pathologic Wilson plot, complete and redundant dataset (although collected on several crystals with serious problems due to radiation damage). Interestingly, the Wilson plot of the Fcalc values is about 60 A2 as for Fobs in the output dataset. Yours Wim -- *** Wim Burmeister Professeur, Membre de l'Institut Universitaire de France Unit of Virus Host Cell Interactions (UVHCI) UMR5233 UJF-EMBL-CNRS 6 rue Jules Horowitz B.P. 181, F-38042 Grenoble Cedex 9 FRANCE E-mail: [EMAIL PROTECTED] Tel:+33 (0) 476 20 72 82 Fax: +33 (0) 476 20 94 00 http://www.uvhci.fr ***
