Re: [ccp4bb] Protein models with Cofactors, metal clusters and heme
Hi Joel, Have you tried Alphafill: AlphaFold Filled (alphafill.eu)<https://alphafill.eu/> https://alphafill.eu/ AlphaFill: enriching AlphaFold models with ligands and cofactors Maarten L. Hekkelman, Ida de Vries, Robbie P. Joosten & Anastassis Perrakis Nature Methods volume 20, pages205–213 (2023) https://www.nature.com/articles/s41592-022-01685-y Good luck, Joao Joao M. Dias, Ph.D. Principal Scientist Pfizer Structural and Molecular Sciences Building 220/ room 3263, MS-8220-3224 445 Eastern Point Rd. Groton, CT 06340 From: CCP4 bulletin board On Behalf Of Joel Tyndall Sent: Wednesday, March 1, 2023 4:10 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [EXTERNAL] [ccp4bb] Protein models with Cofactors, metal clusters and heme Hi, I have a slightly off topic question around structure models, e.g. Homology modelling. It may appear that Alpha fold etc could make homology modelling somewhat redundant. However I have noticed that in at least two structures, either a cofactor or heme are not present on the Alphafold models (that I am aware of). Is anyone aware of any webserver/package that will model these cofactors reliably (outside of modeller)? Many thanks J Joel Tyndall | BSc(Hons) PhD Professor in Medicinal Chemistry School of Pharmacy | He Rau Kawakawa University of Otago | Te Whare Wānanga o Otāgo PO Box 56 9054 Dunedin | Ōtepoti New Zealand | Aotearoa Ph: 64 3 479 7293 Website | pharmacy.otago.ac.nz<https://urldefense.com/v3/__http:/pharmacy.otago.ac.nz__;!!H9nueQsQ!-8ToqYWLFzEomDSOyXoLqbjfu9cvBGCMHVQQL7xOvV9gs9bpoPV1GEZgbVvGdJiS5UoZz1DgybeZrnr_RfC3-xPE$> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1<https://urldefense.com/v3/__https:/www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1__;!!H9nueQsQ!-8ToqYWLFzEomDSOyXoLqbjfu9cvBGCMHVQQL7xOvV9gs9bpoPV1GEZgbVvGdJiS5UoZz1DgybeZrnr_RQYnWTSP$> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Off topic - 3D models demonstrating drug binding to students
Hi Ed, The future is already here... and virtual reality is your friend. This is a field that will likely be revolutionized in the near future, and your students would get an advantage by being exposed to these new technologies. You could check the following: https://www.labcompare.com/10-Featured-Articles/577506-VR-for-Science-Drug-Discovery-and-More-in-the-Virtual-World/ https://nanome.ai/ I wonder how they will respond to an academic collaboration, but you might be very surprised. Let me know if you need more info or if you want me to make the introduction. Good luck, Joao Joao M. Dias, Ph.D. Principal Scientist Pfizer Structural and Molecular Sciences Building 220/ room 3263, MS-8220-3224 445 Eastern Point Rd. Groton, CT 06340 From: CCP4 bulletin board On Behalf Of Edward Snell Sent: Wednesday, February 15, 2023 3:13 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [EXTERNAL] [ccp4bb] Off topic - 3D models demonstrating drug binding to students Dear CCP4, I apologize if this is off topic but I thought this may be a good community to ask. Before we re-invent the wheel this end, we are looking for any commercial source of three-dimensional macromolecular models that can be used to teach how drugs are designed to fit to protein targets. Our audience is mid to advanced high-school students and we are looking for materials that can withstand being passed around the students and can teach basic concepts of how structure can inform ligand binding. There are commercial sources of these models for macromolecules that we are aware of, but few if any that allow us to manipulate a drug and show how it fits to the model. We would love to find ones where a student may have name recognition of the drug concerned and we can show how it would fit to the target of that drug. Any experience in this area and a source of such models would be greatly appreciated. We enjoy our interactions with these students and have the advantage of a professional high-school science teacher/science curriculum developer on staff with Nicole Terranova (copied on the email). Our program related to this is being developed and if anyone has any experiences with this nature of teaching, any offline feedback would be appreciated. Thank you, Eddie Edward Snell Ph.D. President and CEO | Hauptman-Woodward Medical Research Institute Director | NSF BioXFEL Science and Technology Center Professor, Materials Design and Innovation | University at Buffalo, SUNY Fellow of the American Crystallographic Association - The Structural Science Society p: +1 716 898 8631 | f: +1 716 898 8660 e: esn...@hwi.buffalo.edu<mailto:esn...@hwi.buffalo.edu> skype: eddie.snell Hauptman-Woodward Medical Research Institute 700 Ellicott Street | Buffalo, NY 14203-1102 hwi.buffalo.edu<https://urldefense.com/v3/__https:/hwi.buffalo.edu/__;!!H9nueQsQ!9I3n8FaljxVxUlya3-fNm9prByPB0RNfVyoMOonG6xlREiNN0LgYqtvzrdCbgMScGtWFMkx8uIVATPGcZswt$> [hwi-logo-primary-horizontal] To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1<https://urldefense.com/v3/__https:/www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1__;!!H9nueQsQ!9I3n8FaljxVxUlya3-fNm9prByPB0RNfVyoMOonG6xlREiNN0LgYqtvzrdCbgMScGtWFMkx8uIVATM2LkF3n$> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] [EXTERNAL] [ccp4bb] outliers
Hi James, My initial thought is that a 3-sigma bond length deviation is significant. When we make an experiment and observe something that doesn’t correspond to what we expect, it could be an error, or it could be that we found the thread for a new discovery. If the experimental data supports that deviation, I would look at the experimental data carefully because that can show you a surprise. Maybe it is a different bond than you were expecting? Maybe a different geometry? I assume you are talking about a protein crystal structure that was collected at the synchrotron, but you should analyze the data and consider what is the resolution of your data? What is the completeness? What is the B-factor? How did you refine those bonds? If you have low resolution you might have more variability, but we typically impose more restrictions during refinement and might not observe those variations. When you represent your crystal structure by a model, you have to consider that in a crystal you have an average of structures in the crystal lattice, and an average in time, where radiation damage is also occurring . If the experiment is well done, the data might point you to something relevant. Maybe the atoms you are considering C-C bond are not what you thought and therefore the bond is different. One example can be observed in interactions of proteins with metals, where initially we refine the model thinking we have one metal and it shows a mixture of metals, or a completely different one. I know you will do a fluorescence scan in your MAD beamline to sort it out Best wishes, Joao -Original Message- From: CCP4 bulletin board On Behalf Of James Holton Sent: Tuesday, November 8, 2022 5:22 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [EXTERNAL] [ccp4bb] outliers OK, so lets suppose there is this bond in your structure that is stretched a bit. Is that for real? Or just a random fluke? Let's say for example its a CA-CB bond that is supposed to be 1.529 A long, but in your model its 1.579 A. This is 0.05 A too long. Doesn't seem like much, right? But the "sigma" given to such a bond in our geometry libraries is 0.016 A. These sigmas are typically derived from a database of observed bonds of similar type found in highly accurate structures, like small molecules. So, that makes this a 3-sigma outlier. Assuming the distribution of deviations is Gaussian, that's a pretty unlikely thing to happen. You expect 3-sigma deviates to appear less than 0.3% of the time. So, is that significant? But, then again, there are lots of other bonds in the structure. Lets say there are 1000. With that many samplings from a Gaussian distribution you generally expect to see a 3-sigma deviate at least once. That is, do an "experiment" where you pick 1000 Gaussian-random numbers from a distribution with a standard deviation of 1.0. Then, look for the maximum over all 1000 trials. Is that one > 3 sigma? It probably is. If you do this "experiment" millions of times it turns out seeing at least one 3-sigma deviate in 1000 tries is very common. Specifically, about 93% of the time. It is rare indeed to have every member of a 1000-deviate set all lie within 3 sigmas. So, we have gone from one 3-sigma deviate being highly unlikely to being a virtual certainty if you look at enough samples. So, my question is: is a 3-sigma deviate significant? Is it significant only if you have one bond in the structure? What about angles? What if you have 500 bonds and 500 angles? Do they count as 1000 deviates together? Or separately? I'm sure the more mathematically inclined out there will have some intelligent answers for the rest of us, however, if you are not a mathematician, how about a vote? Is a 3-sigma bond length deviation significant? Or not? Looking forward to both kinds of responses, -James Holton MAD Scientist To unsubscribe from the CCP4BB list, click the following link: https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1__;!!H9nueQsQ!59F_VEEPv4VzGZgCp3sN73Rj4vKyZBcIOY9TwFRyr0U47aUiTqIIkUg_5ib8dT46tiKEAgQxf378QBG7$ This message was issued to members of https://urldefense.com/v3/__http://www.jiscmail.ac.uk/CCP4BB__;!!H9nueQsQ!59F_VEEPv4VzGZgCp3sN73Rj4vKyZBcIOY9TwFRyr0U47aUiTqIIkUg_5ib8dT46tiKEAgQxf28joQL8$ , a mailing list hosted by https://urldefense.com/v3/__http://www.jiscmail.ac.uk__;!!H9nueQsQ!59F_VEEPv4VzGZgCp3sN73Rj4vKyZBcIOY9TwFRyr0U47aUiTqIIkUg_5ib8dT46tiKEAgQxfwxM1QmH$ , terms & conditions are available at https://urldefense.com/v3/__https://www.jiscmail.ac.uk/policyandsecurity/__;!!H9nueQsQ!59F_VEEPv4VzGZgCp3sN73Rj4vKyZBcIOY9TwFRyr0U47aUiTqIIkUg_5ib8dT46tiKEAgQxf1Mbwaqi$ To unsubscribe from the CCP4BB list, click the following link: https://w
Re: [ccp4bb] [EXTERNAL] [ccp4bb] problems in Rfree-flags when refining with REFMAC and processing with STARANISO
Vera, I had a similar case and I solved it using the PDB tool pdb_extract from: https://pdb-extract.wwpdb.org/ PDB Extract is a pre-deposition service for assembling structure files for wwPDB OneDep deposition . You have a pdb and you have a mtz file, then you should be able to convert them to the latest mmcif format. After that you can deposit your files in: https://deposit-1.wwpdb.org/deposition/ https://validate-rcsb-2.wwpdb.org/ Since you used anisotropic data and different software, the final R/Rfree will likely be different of the estimated during PDB validation, and you will have the opportunity to explain it to the person from the PDB that will help you during your deposition. Finally, if you are using PHENIX, BUSTER, REFMAC/CCP4, you can also run a last refinement step with the latest version of the software. The latest version will ensure that the mmcif format is compatible with the PDB. Sometimes older versions of the software will give you that type of error. Good luck, Joao From: CCP4 bulletin board On Behalf Of Vera Pfanzagl Sent: Monday, July 11, 2022 10:52 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [EXTERNAL] [ccp4bb] problems in Rfree-flags when refining with REFMAC and processing with STARANISO Hello, I am trying to get a structure with a bit of a history in terms of programs I have used into a deposit-able form. However I have massif problems with the mtz labels, specifically the Rfree flags and cannot figure out where my problem is derived from. Specifically I have collected diffraction data on a very anistropic crystal (3 A in one direction and 1.4 in the other) which I processed using AUTOPROC because I wanted to make use of the inbuild STARANISO processing. I then went to Phenix for molecular replacement and refined a few rounds with Phenix. However one of my co-factors (a twice covalently linked heme) did not refine properly, which is why i switched the refinement to CCP4 and used REFMAC. What I initially did not know is that REFMAC and STARANISO do not go well together when it comes to the Rfree-flags in the diffraction cut-off regions. I went back and tried to use SFtools by CCP4 as described on https://staraniso.globalphasing.org/test_set_flags_about.html<https://urldefense.com/v3/__https:/staraniso.globalphasing.org/test_set_flags_about.html__;!!H9nueQsQ!_IB8G3WAOxxFQn5h4jDFwAmN1RVYVhI2LKrKWB5_94y6bVx9BkPLOZgonHnWVltzEZXXeqtezxw6x39nT1tLrXv_1w$> to generate a staraniso_mtz suitable for refinement in REFMAC, phased it again with my initial model and ran a refinement cycle, because I thought skipping a program might be a good idea. However I do not know if the mtz file I get has the appropriate column labels and also I do not know how to convert it to an mmcif that includes the data from processing. i have never before tried to combine AUTOPROC-processed data with REFMAC data and am not very familiar with REFMAC to begin with. I have tried the sftool server to validate the mtz from REFMAC processing but I get an error message Converting SF file to mmcif format ... Warning: (data block=1): all status (80493) are assigned as 'x' (changed! 'x'->'o') Error: File () has no mandatory items 'F/I/F+/F-/I+/I-' (data block=1) I tried to use Phenix (as it automatically converts the mtz to an mmcif file) by running a "mock"-refinement with fixed coordinates but the mmcif file I get out of this seems to contain some wrongly labeled data fields in block 1 when I try to upload it to PDBs validation server. Essentially it seems to contain some 3 data points in block 1 that have a ? This is the error message I receive: Warning: (data block=1): 30301 reflections have status assigned as '?' (changed to 'x') Warning: (data block=1): 30301 reflections have wrong status. Not in list(o,f,x,-,<,h,l) Warning: No wavelength value was found in SF file (data block=1). which is essentially the same as above, so I guess it is derived from the same problem. I would be very grateful for any help. Cheers and thanks, Vera To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1<https://urldefense.com/v3/__https:/www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1__;!!H9nueQsQ!_IB8G3WAOxxFQn5h4jDFwAmN1RVYVhI2LKrKWB5_94y6bVx9BkPLOZgonHnWVltzEZXXeqtezxw6x39nT1tvNXKb7A$> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Questions regarding MTZFIX and EDSTATS
Dear Ian, I'm trying to use the edstats.pl script but I'm having problems with the column labels. mtzdmp can read the mtz file, but edstats.pl gives me a message saying that mtzfix cannot find the labels. I'm using a mtz file from a refinement job in Phenix. Best regards, Joao Ramos Ian Tickle escreveu no dia terça, 16/06/2020 à(s) 09:39: > Hi João > > In cases like this it's always a good idea to check whether you can read > the same MTZ file with mtzdmp. If that fails with the same error message > it means that the pathname/filename was indeed incorrectly specified, or it > could possibly be a permissions issue (the file needs to be given read > permission). > > Cheers > > Ian > > > On Mon, 15 Jun 2020, 19:09 Ian Tickle, wrote: > >> >> Hi Joao >> >> The top of the page was cut off in your screenshot so I can't see the >> command line you typed. It would be better to copy/paste the entire text, >> including the input command line and the output, into the email rather than >> taking a screenshot. 'Cannot open file' most likely means that the input >> filename was not typed correctly or the path was not specified correctly if >> it's in another directory. Anyway you really shouldn't be calling the >> MTZFIX or EDSTATS programs directly. Using the Perl script 'edstats.pl' >> http://www.ccp4.ac.uk/dist/checkout/edstats/edstats.pl is much easier. >> It should be in your path after running the CCP4 setup script. >> >> Cheers >> >> -- Ian >> >> >> On Mon, 15 Jun 2020 at 18:41, Joao Ramos >> wrote: >> >>> Dear Ian, >>> >>> Thanks for the reply! >>> >>> I've sourced the setup script, but now I get a 'cannot open file' error >>> message (screenshot attached) when trying to run MTZFIX. >>> >>> Best regards, >>> Joao Ramos >>> >>> Ian Tickle escreveu no dia segunda, 15/06/2020 à(s) >>> 16:57: >>> >>>> >>>> Hi Joao >>>> >>>> For all CCP4 programs you need to source your CCP4 setup script. >>>> >>>> Cheers >>>> >>>> -- Ian >>>> >>>> >>>> On Mon, 15 Jun 2020 at 15:29, Joao Ramos >>>> wrote: >>>> >>>>> Dear CCP4bb, >>>>> >>>>> I'm trying to obtain RSC metrics using the CCP4 program EDSTATS for a >>>>> Phenix refinement job, in order to have a more reliable analysis of model >>>>> quality. From what I read, one should go through the program MTZFIX and >>>>> then create the FFT maps (density and difference maps). However, while >>>>> attempting to run the Phenix output mtz file in MTZFIX, I receive an error >>>>> message saying: "Cannot open environ.def". >>>>> >>>>> My questions are: >>>>> 1) Is the way to go from the Phenix refinement output files to EDSTATS >>>>> described earlier correct? Are there alternative programs included in >>>>> Phenix? >>>>> 2) How to fix the error and run MTZFIX? >>>>> >>>>> I'm currently using the new ccp4 v7.1, in macOS v10.14.5. >>>>> >>>>> Best regards, >>>>> Joao Ramos >>>>> >>>>> -- >>>>> >>>>> To unsubscribe from the CCP4BB list, click the following link: >>>>> https://www.jiscmail.ac.uk/cgi-bin/wa.exe?SUBED1=CCP4BB=1 >>>>> >>>> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] Questions regarding MTZFIX and EDSTATS
Dear CCP4bb, I'm trying to obtain RSC metrics using the CCP4 program EDSTATS for a Phenix refinement job, in order to have a more reliable analysis of model quality. From what I read, one should go through the program MTZFIX and then create the FFT maps (density and difference maps). However, while attempting to run the Phenix output mtz file in MTZFIX, I receive an error message saying: "Cannot open environ.def". My questions are: 1) Is the way to go from the Phenix refinement output files to EDSTATS described earlier correct? Are there alternative programs included in Phenix? 2) How to fix the error and run MTZFIX? I'm currently using the new ccp4 v7.1, in macOS v10.14.5. Best regards, Joao Ramos To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/wa.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] Protein crystallographer position at Heptares Therapeutics
I would like to draw your attention to the fact that Heptares Therapeutics is hiring a protein crystallographer. Please send your application in pdf format, to: h...@heptares.commailto:h...@heptares.com quoting reference number 2014/2B. The closing date for applications is 18th April 2014. Protein Crystallographer Heptares is a Drug Discovery Company located in Hertfordshire UK, working on G protein-coupled receptors (GPCRs) involved in CNS and metabolic diseases. Our unique structure-based approach to discovering therapeutic agents for these diseases relies on a proprietary approach to stabilise GPCRs, enabling their purification for structural and biophysical studies. We are currently seeking an experienced protein crystallographer to join the crystallography group working on GPCR structure determination to enable drug discovery efforts. Applicants should have a PhD in protein crystallography or related discipline and be familiar with purification of proteins from recombinant expression systems such as E. coli or baculovirus/insect cells, setting up crystallisation experiments including Lipidic Cubic Phase crystallisation, X-ray data collection, data processing, structure solution and refinement of novel structures and follow on complexes. Experience working with GPCRs or other membrane proteins would be an advantage. Experience of working in a drug discovery environment where structural studies are used to drive compound design and medicinal chemistry would also be of benefit. The successful candidate will be expected to work independently and within a highly successful group to: Express and purify GPCRs for structural and biophysical methods; Set up and optimise vapour diffusion and LCP crystallisation experiments; Collect X-ray diffraction data at synchrotron sources; Solve and refine structures; Assist with interpretation of structural results for the design of new compounds; Present results (written and oral) to internal and external audiences. This is an exceptional opportunity to participate in pioneering science with an Industry-leading Drug Discovery company. Applicants must be able to demonstrate proof of the right to work in the United Kingdom. Further information can be found on our website (www.heptares.comhttp://www.heptares.com). Applications should include a covering letter with curriculum vitae including the names and contact details for two referees. Please send your application in pdf format, to h...@heptares.commailto:h...@heptares.com quoting reference number 2014/2B. The closing date for applications is 18th April 2014. No agencies, please. Thanks, Joao Dr Joao Dias Principal Scientist Heptares Therapeutics Ltd BioPark, Broadwater Road, Welwyn Garden City, Herts, AL7 3AX UK This document contains company confidential and/or proprietary information. It is intended for the exclusive attention of the addressee(s) above and should not be copied or disclosed to any other. If you have received this transmission in error, please make no use of its contents and contact the sender. [www.heptares.com]www.heptares.comhttp://www.heptares.com The information in this e-mail is confidential and may be legally privileged. It is intended for the exclusive attention of the addressee stated above and should not be copied or disclosed to any other. If you have received this transmission in error, please make no use of its contents and contact the sender. Contact and registered office address: Heptares Therapeutics Limited, BioPark, Broadwater Road, Welwyn Garden City, Hertfordshire, AL7 3AX. Company No: 06267989 inline: d8a2d3.png
Re: [ccp4bb] DDM
Kasia, A lot of people uses DDM to purify membrane proteins, not a lot of people crystallises them. If you want to crystallise a protein purified in DDM, then you should use LCP. If you go for vapour diffusion, you should exchange the DDM for a detergent with a smaller micelle size otherwise you might get crystals but it is difficult to get good diffraction. Try mixed micelles for example. Typically use 0.05% DDM during purification and use 100kDa cut-off membranes in order to prevent detergent concentration. For extraction it depends on your protein and expression system but you can see in the literature values between 0.5-2% being used successfully. Good luck. Cheers, Joao Joao Dias, Ph.D. Senior Scientist Heptares Therapeutics Ltd BioPark, Broadwater Road, Welwyn Garden City, Herts, AL7 3AX UK This document contains company confidential and/or proprietary information. It is intended for the exclusive attention of the addressee(s) above and should not be copied or disclosed to any other. If you have received this transmission in error, please make no use of its contents and contact the sender. [cid:image6c7082.GIF@43484746.4ab9695e]http://www.heptares.com The information in this e-mail is confidential and may be legally privileged. It is intended for the exclusive attention of the addressee stated above and should not be copied or disclosed to any other. If you have received this transmission in error, please make no use of its contents and contact the sender. Contact and registered office address: Heptares Therapeutics Limited, BioPark, Broadwater Road, Welwyn Garden City, Hertfordshire, AL7 3AX. From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Katarzyna Rudzka Sent: 26 March 2012 18:17 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] DDM Hi All, Has anyone had any luck purifying membrane proteins with DDM (n-dodecyl-D-maltoside) using concentration of detergent ~1-2 x CMC ? (Its CMC is very low: 0.009%). I would like to keep it as low as possible, so I don't have too much DDM around when I get to the crystallization step. I wonder If the amount of detergent sufficient for the protein extraction has to be determined experimentally for each protein or maybe there are some good rules of thumb. I appreciate your help. Thanks. Kasia Katarzyna Rudzka, Postdoctoral Fellow Department of Biophysics and Biophysical Chemistry Johns Hopkins University, School of Medicine Baltimore, Maryland 21205 USA inline: image6c7082.GIF
[ccp4bb] Structural Biologist/Protein Biochemist position at Heptares Therapeutics Ltd.
We are looking for an outstanding Structural Biologist/Protein Biochemist for our group at Heptares Therapeutics Ltd. Heptares is a Drug Discovery Company located in Hertfordshire UK, working on G protein coupled receptors involved in CNS and metabolic disease. We are using a unique structure based approach, which originated from work at the MRC Laboratory of Molecular Biology in Cambridge, to characterise this important family of receptors and develop novel innovative therapeutics for these diseases. We are now seeking enthusiastic and motivated scientists with experience in the expression, purification and crystallisation of proteins to assist Heptares in obtaining unique structures of GPCRs for drug discovery. This is an exceptional opportunity to participate in pioneering science whilst helping to grow an Industry-leading Drug Discovery company. Applicants should have a PhD and post-graduate experience in a research environment. Candidates should be familiar with protein expression and purification in a variety of expression systems such as E.coli, baculovirus and mammalian cells. We also wish to recruit candidates with experience in protein crystallisation who are interested in following proteins through purification to structural studies. Previous experience of working with GPCRs or other membrane proteins would be an advantage. Experience of working in a drug discovery environment where structural studies are used to drive medicinal chemistry would also be of benefit. Further information can be found on our website (www.heptares.com). To apply for this position, please send your application to h...@heptares.com, quoting reference number 2011/1B Best wishes, Joao Dias, Ph.D. Senior Scientist Heptares Therapeutics Ltd BioPark, Broadwater Road, Welwyn Garden City, Herts, AL7 3AX UK This document contains company confidential and/or proprietary information. It is intended for the exclusive attention of the addressee(s) above and should not be copied or disclosed to any other. If you have received this transmission in error, please make no use of its contents and contact the sender.
Re: [ccp4bb] temperature after 30 minutes using microscopes ?
Jurgen, From your question I assume that you are having problems with the warming up of the crystal tray due to the microscope light. Did you consider the use of an external cold light source like the KL1500 LCD? I do not feel anything warming up. Which microscope are you using? The LED systems I have tried are not bright enough. Try them before you buy. Suggestion - request a demo to your supplier. Cheers, Joao João M. Dias, Ph. D. Ollmann Saphire Lab The Scripps Research Institute 10550 North Torrey Pines Rd. IMM-2 La Jolla, CA 92037 USA tel (858)784-8925 http://www.scripps.edu/~jmdias/ On Jan 21, 2009, at 2:50 PM, Jürgen Bosch wrote: Hi there, *warning, reading beyond this line might expose you to non CCP4 related topics* anybody out there who could do the following experiment: Turn on your microscope and measure the temperature after 30 minutes where you would place your precious crystal tray. In particular I'm interested in LED versus Halogen driven models. Is there anybody out there who would like to comment on LED driven microscopes for our purposes ? Thanks, Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Biochemistry and Molecular Biology, W8708 615 North Wolfe Street Baltimore, MD 21205 Phone: +1-410-614-4742 Fax: +1-410-955-3655
Re: [ccp4bb] Deglycosylation
Hi Eugenio, It will depend on the expressing system you are using: mamalian cells, insect cells, yeast. For enzymatic deglycosylation I suggest you to do first do a small scale test, using the following enzymes: PNGase F is extremely efficient. EndoH (you can try EndoHf which is cheaper and ) Neuraminidase to remove the sialic acid (specially useful for mammalian cells). You can check these enzymes at NEB or Prozyme (like Radu mentioned before. Check also his great paper Chang et al, 2007.). http://www.neb.com/nebecomm/default.asp You can also use glycosylation inhibitors like tunicamycin, kifunensine, swainsonine, etc... Another approach is site-directed mutagenesis. And yes, usually (this means not always...) it gives you better crystals which will diffract at higher resolution. Good luck, Joao João M. Dias, Ph. D. Ollmann Saphire Lab The Scripps Research Institute 10550 North Torrey Pines Rd. IMM-2 La Jolla, CA 92037 USA tel (858)784-8925 http://www.scripps.edu/~jmdias/ On Sep 25, 2008, at 1:40 PM, A. Radu Aricescu wrote: Dear Eugenio, I believe the paper by Chang VT et al 2007 (PMID: 17355862) might offer the answers you're after (assuming N-glycosylation is your main concern). For O-linked sugars, the situation is much more complex, reflecting their diversity: mucin-type can usually be dealt with by construct design (eliminate Ser/Thr/Pro-rich domains), for other types have a look at http://www.prozyme.com/pdf/ gk80110.pdf for example. Best wishes, radu -- A. Radu Aricescu, PhD University of Oxford Wellcome Trust Centre for Human Genetics Division of Structural Biology Roosevelt Drive, Oxford OX3 7BN United Kingdom Phone: +44-1865-287551 Fax: +44-1865-287547 Original message Date: Thu, 25 Sep 2008 11:30:24 -0700 From: Eugenio De la Mora [EMAIL PROTECTED] Subject: [ccp4bb] Deglycosylation To: CCP4BB@JISCMAIL.AC.UK Dear all, We have some troubles with the cristallization of glycosylated proteins and want to try to deglycosate them. We have never done this so we want to know what enzymes are the best (efficiency; less protein loss, ... ). And if it gaves better results in crystallization screens. Thank you Eugenio De la Mora Instituto de Biotecnologia Universidad NAcional Autonoma de Mexico
Re: [ccp4bb] multiple sequence alignment from multiple pairwise structural alignments
Hi Stephen, I will add to the previously mentioned ones the MSD ssm server at EBI? http://www.ebi.ac.uk/msd-srv/ssm/cgi-bin/ssmserver You also have the Godzik tools if your proteins have flexible regions. FATCAT http://fatcat.burnham.org/ and POSA http://fatcat.burnham.org/POSA/ Good luck, Joao João M. Dias Ollmann Saphire Lab The Scripps Research Institute 10550 North Torrey Pines Rd. IMM-2 La Jolla, CA 92037 USA On Mar 4, 2008, at 11:13 AM, Stephen Graham wrote: Hi all, I would like to generate a structure-based multiple sequence alignment using 4 structures. I have already generated pairwise alignments for each 'pair' of structures (6 alignments in all). Is there a program out there that can take a number of aligned structures (or even just a number of pairwise sequence alignments) and calculate the 'best' multiple sequence alignment? Please note that there is absolutely no sequence conservation between these structures, making standard sequence-based alignment tools pretty useless. Thanks, Stephen -- Dr Stephen Graham Nuffield Medical Fellow Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549
[ccp4bb] Pymol installation
Dear all. We have had success installing Pymol on any 32 bits PC, but we are having some troubles with 64 bits. The OS is linux ubuntu kernel: 2.6.15-29-amd64-generic x86_64 The pymol version is: pymol-0_99rc6-bin-linux-x86-glibc23.tgz The error message is: freeglut (./pymol.exe): ERROR: Internal error Visual with necessary capabilities not found in function fgOpenWindow X Error of failed request: BadWindow (invalid Window parameter) Major opcode of failed request: 4 (X_DestroyWindow) Resource id in failed request: 0x0 Serial number of failed request: 16 Current serial number in output stream: 19 PyMOL: abrupt program termination. We have already installed the dependencies requested in the README file of the software (openGL, glutlibrary, python, tcl/tk and libpng). Can you provide any clues on how to proceed to get Pymol running? Thank you, João.
Re: [ccp4bb] Maps look different from auto-mtz vs EDS vs FFT in Coot or CCP4MG.
Dear Kendall, I would suggest you to run a simulated annealing omit map around the region you are interested. The model bias will be reduced and you will get a more clear answer. Ciao, Joao Joao M. Dias Ollmann Saphire Lab The Scripps Research Institute 10550 North Torrey Pines Rd. IMM-2 La Jolla, CA 92037 USA tel (858)784-8925 On May 24, 2007, at 9:57 AM, Kendall Nettles wrote: I’d like help in interpreting some mystery density in a structure. I’m writing a paper about soaking the apo-estrogen receptor with different ligands. The apo structure is already released, as pdb code 2B23. The question is whether there is a mystery molecule in the pocket of the apo receptor. If you superimpose 3ERD, you can see where the ligand binds. The problem is that with some maps the pocket appears completely empty, and with others, there appears to be something there. Protein looks essentially identical with the different maps. We have used a few different approaches to identify the compound with LC-MS, and are pretty sure there is nothing there. For example, we can bind our protein to beads, soak it with estradiol, wash extensively, elute with organic solvent and find a great peak for estradiol, but nothing for the apo protein. We have also tried non-denaturing MS. If you look at the 2mFo-DFc map from EDS in Coot or CCP4mg, you see mystery density in the pocket. If I use the MTZ, you see density in Coot, but not CCP4MG. I then downloaded the structure factors from the PDB and made an MTZ. The map in CCP4MG shows some density, but much less that with the map from EDS. When I used FFT to make a 2mF1-1nF2 map, there is no mystery density in either CCP4MG or coot. I was ready to submit the manuscript with a picture of the mystery density, but now I’m not sure if that is appropriate. Any suggestions, as far as how to interpret this mystery density would be greatly appreciated. Best Regards, Kendall -- Kendall W. Nettles, PhD Assistant Professor Department of Cancer Biology The Scripps Research Institute 5353 Parkside Dr. Jupiter Fl 33458 office 561-799-8851 fax 561-799-8805 cell 561-306-7566
