[ccp4bb] dissolving peptides !
Hello everybody, I have a 16 mer peptide which is predicted to be positively charged alpha helix and has 50% of its sequence hydrophobic. Could any one recommend the best way to dissolve it, and then it can be used for crystallization trials. thanks -- rashmi
Re: [ccp4bb] Fwd: creating a metal plane model to fit a protein.
Well - try the coordinate utility recommended by Martyn Winn to convert cif to pdb coord_format xyzin ./1ivo.cif xyzout 1ivo.pdb eof END eof Then pdbset xyzin 1ivo.pdb xyzout 1ivo-sym.pdb symgen -x,y,z (or whatever sym op you want) end This will generate symmetry copy of your coordinates as you want and you can edit the two files together. or pdbset xyzin 1ivo.pdb xyzout 1ivo-sym.pdb symgen P222 (say) to generate all symmetry.. Eleanor noam grimberg wrote: Dear all, I have a coordinates CIF file of an interested metal from CRYSTMET Toth Materials Toolkit with the atoms coordinates. I want to create a metal plane according to the cell symmetry of the metal and to convert the coordinates so it be used in a pdb format file for exploring the protein fit to the metal surface. Has someone can recommend on a program that can create the symmetry and duplicate it to a metal plane, and to convert it into a pdb format file? thanks.
[ccp4bb] Scaling question
Dear CCP4bb, I was wondering if someone could tell me how mosflm and scala deal with overloaded reflections. From my understanding mosflm extrapolates the overloaded peaks but then scala throws them out completely - is this right? If so am I right to not worry about contamination from extrapolated peaks when combining high and low resolution datasets from the same crystal? Thanks Simon
Re: [ccp4bb] Scaling question
Hello Simon, if I remember correctly, mosflm only estimates overloads if you explicitly ask for it - which you should NOT do especially since you do have a low resolution pass. (from the documentation at http://www.mrc-lmb.cam.ac.uk/harry/mosflm/mosflm_user_guide.html : Note that you MUST include keywords PROFILE OVERLOAD in order to estimate the intensities of overloaded reflections by profile fitting.) Tim On Mon, Jun 07, 2010 at 03:09:18PM +0100, Simon Kolstoe wrote: Dear CCP4bb, I was wondering if someone could tell me how mosflm and scala deal with overloaded reflections. From my understanding mosflm extrapolates the overloaded peaks but then scala throws them out completely - is this right? If so am I right to not worry about contamination from extrapolated peaks when combining high and low resolution datasets from the same crystal? Thanks Simon -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: Digital signature
[ccp4bb] GPU computing
Hi All, I am wondering anyone is looking into GPU computing on structural refinement? I see VMD supports GPU computing already, how about other programs such as CCP4 and Phenix Thanks, Tongqing Tongqing Zhou, Ph.D. Staff Scientist Structural Biology Section Vaccine Research Center, NIAID/NIH Building 40, Room 4607B 40 Convent Drive, MSC3027 Bethesda, MD 20892 (301) 594-8710 (Tel) (301) 793-0794 (Cell) (301) 480-2658 (Fax) ** The information in this e-mail and any of its attachments is confidential and may contain sensitive information. It should not be used by anyone who is not the original intended recipient. If you have received this e-mail in error please inform the sender and delete it from your mailbox or any other storage devices. National Institute of Allergy and Infectious Diseases shall not accept liability for any statements made that are sender's own and not expressly made on behalf of the NIAID by one of its representatives. **
[ccp4bb] Pcube User meeting
Dear Colleagues, I would like to draw your attention to the upcoming Pcube-user meeting (September 8-9, 2010 in Grenoble). You are probably aware that the EU-founded Pcube project provides open access to several supportive technologies for macromolecular crystallography, ranging from fragment screening to advanced microscopy technologies. The best way to learn more about these technologies would be to visit *http://www.p-cube.eu/* and sign up for the meeting. Participation is free of charge but registration is mandatory and the deadline is approaching very quickly. Best regards, Peer
Re: [ccp4bb] Scaling question
Mosflm integrates them (profile-fitted overloads) but flags them. Pointless uses them for systematic absence tests. Scala by default ignores them, but you can include them if you want: this is not normally recommended since they are pretty inaccurate (look in the Excluded data tab of ccp4i/Scala) If you are merging strong weak datasets it should do the right thing, I think. Phil On 7 Jun 2010, at 15:09, Simon Kolstoe wrote: Dear CCP4bb, I was wondering if someone could tell me how mosflm and scala deal with overloaded reflections. From my understanding mosflm extrapolates the overloaded peaks but then scala throws them out completely - is this right? If so am I right to not worry about contamination from extrapolated peaks when combining high and low resolution datasets from the same crystal? Thanks Simon
Re: [ccp4bb] GPU computing
I looked at it and concluded that our FFTs are on the whole too short for it to be worthwhile, and a lot of calculations aren't FFT bound anyway. An awful lot of our stuff is simply not very slow. Also, GPU computing at the moment is crippled for many problems by the bandwidth bottleneck and multitasking limitations. Both of those problems will go away with GPU features integrated onto the CPU. At that point the level of work required to implement it will drop significantly, and the range of application of the technology will increase significantly. So I'm waiting for now. Zhou, Tongqing (NIH/VRC) [E] wrote: Hi All, I am wondering anyone is looking into GPU computing on structural refinement? I see VMD supports GPU computing already, how about other programs such as CCP4 and Phenix…. Thanks, Tongqing *Tongqing Zhou, Ph.D. * Staff Scientist Structural Biology Section Vaccine Research Center, NIAID/NIH Building 40, Room 4607B 40 Convent Drive, MSC3027 Bethesda, MD 20892 (301) 594-8710 (Tel) (301) 793-0794 (Cell) (301) 480-2658 (Fax) */**/*/ / */The information in this e-mail and any of its attachments is confidential and may contain sensitive information. It should not be used by anyone who is not the original intended recipient. If you have received this e-mail in error please inform the sender and delete it from your mailbox or any other storage devices. National Institute of Allergy and Infectious Diseases shall not accept liability for any statements made that are sender's own and not expressly made on behalf of the NIAID by one of its representatives. /* */**/*
Re: [ccp4bb] insect cell media
We have happily made a transition last year from using Invitrogen's SFM medium and cellfectin to Insect-Xpress (Lonza) and polyethyleneimine for transfection. We are moving several protein targets to large-scale cultures and would consider cost-cutting alternatives. For example, Invitrogen and Thermo Sci both offer media in powder form at an attractive price. Has anyone made a systematic comparison of these media? Any particular recommendations regarding powder media? Last I checked, no manufacturer offered serum-free powedered medium. Fetal calf serum is very expensive - once you count in its cost, the overall price of the media becomes comparable. If your protein is secreted, serum-containing media are not even a realistic choice. If it is intracellular, buying dry medium and adding FBS work well: is a lot more hassle, is about 20% cheaper, and usually gives slightly higher titers and expression levels. Of all the common media we compared, TNM-FH works best for us with Sf9 cells. Switching to dry medium also means some initial investment: bottles, filtration equipment and filters. So it all pays off only of you need large scale cultures long-term. If you are aware of a dry serum-free insect cells medium, please let me know - I'd love to try it. Dima
Re: [ccp4bb] insect cell media
Hi, well, actually this is availalbe as powder (HYClone), as it says on the page.. (we have it made in our media kitchen on a regular basis, but not for huge scale... so i dont know about the prices..) https://www.thermoscientific.com/wps/portal/ts/products/detail?navigationId=LA11074__10347categoryId=82051productId=11960716 HTH, Tommi On Jun 7, 2010, at 5:52 PM, Dima Klenchin wrote: We have happily made a transition last year from using Invitrogen's SFM medium and cellfectin to Insect-Xpress (Lonza) and polyethyleneimine for transfection. We are moving several protein targets to large-scale cultures and would consider cost-cutting alternatives. For example, Invitrogen and Thermo Sci both offer media in powder form at an attractive price. Has anyone made a systematic comparison of these media? Any particular recommendations regarding powder media? Last I checked, no manufacturer offered serum-free powedered medium. Fetal calf serum is very expensive - once you count in its cost, the overall price of the media becomes comparable. If your protein is secreted, serum-containing media are not even a realistic choice. If it is intracellular, buying dry medium and adding FBS work well: is a lot more hassle, is about 20% cheaper, and usually gives slightly higher titers and expression levels. Of all the common media we compared, TNM-FH works best for us with Sf9 cells. Switching to dry medium also means some initial investment: bottles, filtration equipment and filters. So it all pays off only of you need large scale cultures long-term. If you are aware of a dry serum-free insect cells medium, please let me know - I'd love to try it. Dima Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
Re: [ccp4bb] dissolving peptides !
Rashmi, You can try 1%DMSO and also acetonitrile but the former is preferable. Gauri On Mon, Jun 7, 2010 at 3:04 AM, rashmi panigrahi rashmi.panigrah...@gmail.com wrote: Hello everybody, I have a 16 mer peptide which is predicted to be positively charged alpha helix and has 50% of its sequence hydrophobic. Could any one recommend the best way to dissolve it, and then it can be used for crystallization trials. thanks -- rashmi
Re: [ccp4bb] Scaling question
Dear Simon, mosflm does indeed estimate the intensity of overloaded reflections, and these are rejected by default in SCALA, but you can choose to include them using the appropriate keywords (ACCEPT OVERLOADS). The number of overloads in the MTZ file, and the number accepted for output, are reported by SCALA in the logfile. Andrew On 7 Jun 2010, at 15:09, Simon Kolstoe wrote: Dear CCP4bb, I was wondering if someone could tell me how mosflm and scala deal with overloaded reflections. From my understanding mosflm extrapolates the overloaded peaks but then scala throws them out completely - is this right? If so am I right to not worry about contamination from extrapolated peaks when combining high and low resolution datasets from the same crystal? Thanks Simon
[ccp4bb] Ligand weak density in SHELXL
Dear all, I solved the structure of an enzyme at resolution of 1.1A with a bound substrate using Refmac5 and Coot for refinement/building. I have a very nice density for my ligand and a very good structure, as judged by Molprobity analysis. Now I'm using SHELXL to finish refinement but most of the density for my ligand disappears when I put it in, and it gets very high B factors. I though it could be some problem with the occupancy so I did a set of runs with different occupancies ranging from 0.2 to 1 and I got the best R factors when using 0.4/0.5 for the occupancy. However, I still have most of the ligand outside electron density… In Coot I get no ligand with “Find ligand” even when I reduce the cluster sigma level to 0.8. What am I missing here? Just another question, how to reduce the acceptable fit fraction in Coot “Find ligand”? Thanks for any advice, Fátima
Re: [ccp4bb] GPU computing
Most of what Kevin says applies equally well to Phenix - refinement is a relatively complex series of calculations, much less inherently parallel than what VMD is using GPUs for. There are many other things we could do to speed up calculations that would probably be more useful and more effective. -Nat On Mon, Jun 7, 2010 at 7:47 AM, Kevin Cowtan cow...@ysbl.york.ac.uk wrote: I looked at it and concluded that our FFTs are on the whole too short for it to be worthwhile, and a lot of calculations aren't FFT bound anyway. An awful lot of our stuff is simply not very slow. Also, GPU computing at the moment is crippled for many problems by the bandwidth bottleneck and multitasking limitations. Both of those problems will go away with GPU features integrated onto the CPU. At that point the level of work required to implement it will drop significantly, and the range of application of the technology will increase significantly. So I'm waiting for now. Zhou, Tongqing (NIH/VRC) [E] wrote: Hi All, I am wondering anyone is looking into GPU computing on structural refinement? I see VMD supports GPU computing already, how about other programs such as CCP4 and Phenix…. Thanks, Tongqing *Tongqing Zhou, Ph.D. * Staff Scientist Structural Biology Section Vaccine Research Center, NIAID/NIH Building 40, Room 4607B 40 Convent Drive, MSC3027 Bethesda, MD 20892 (301) 594-8710 (Tel) (301) 793-0794 (Cell) (301) 480-2658 (Fax) */**/*/ / */The information in this e-mail and any of its attachments is confidential and may contain sensitive information. It should not be used by anyone who is not the original intended recipient. If you have received this e-mail in error please inform the sender and delete it from your mailbox or any other storage devices. National Institute of Allergy and Infectious Diseases shall not accept liability for any statements made that are sender's own and not expressly made on behalf of the NIAID by one of its representatives. /* */**/*
Re: [ccp4bb] insect cell media
I mentioned to to Chris already, but we use nothing but HyClone SFX-Insect powder. We make 20-30 L batches, sterilize with a large peristaltic pump and a disposable Millipak filter from Millipore. We never have contamination problems that are due to preparing our own media from powder. We buy 10L bottles of powder. This media works great for baculovirus, stable cells, Sf9, Sf21, Tn5. We prefer it over Invitrogen (why have separate media for Sf9 and Tn5 cells?), and it's cheaper too! We no longer use serum media for anything (virus production and amplification, all goes fine in serum free. Serum helps storing viral stocks for long periods). It's ~$26 per L, plus $1-$2 in filter costs (a $30 filter will sterilize ~30 L of media). So it comes to about ~$28/L, I haven't priced out alternative products recently, but I think liquid from Invitrogen runs around $50/L? We also use the peristaltic pump for tangential flow filtration, it's pretty heavy duty (tubing is like 5/8 thick). I think that help make the filtering efficient. Bottle top filters won't work because they foul to quickly, same is try of 4.5 cm filter discs that you can insert into an in-line filter, they clog after 1 L or so. You need the 'asymmetric' filter material offered by Millipore in the Millipak or Steripak filter (the later is the filter you see attached to a MilliQ water dispenser, it can be autoclaved 3X they claim) Hope that helps, Nat On Mon, Jun 7, 2010 at 10:59 AM, Tommi Kajander tommi.kajan...@helsinki.fi wrote: Hi, well, actually this is availalbe as powder (HYClone), as it says on the page.. (we have it made in our media kitchen on a regular basis, but not for huge scale... so i dont know about the prices..) https://www.thermoscientific.com/wps/portal/ts/products/detail?navigationId=LA11074__10347categoryId=82051productId=11960716 HTH, Tommi On Jun 7, 2010, at 5:52 PM, Dima Klenchin wrote: We have happily made a transition last year from using Invitrogen's SFM medium and cellfectin to Insect-Xpress (Lonza) and polyethyleneimine for transfection. We are moving several protein targets to large-scale cultures and would consider cost-cutting alternatives. For example, Invitrogen and Thermo Sci both offer media in powder form at an attractive price. Has anyone made a systematic comparison of these media? Any particular recommendations regarding powder media? Last I checked, no manufacturer offered serum-free powedered medium. Fetal calf serum is very expensive - once you count in its cost, the overall price of the media becomes comparable. If your protein is secreted, serum-containing media are not even a realistic choice. If it is intracellular, buying dry medium and adding FBS work well: is a lot more hassle, is about 20% cheaper, and usually gives slightly higher titers and expression levels. Of all the common media we compared, TNM-FH works best for us with Sf9 cells. Switching to dry medium also means some initial investment: bottles, filtration equipment and filters. So it all pays off only of you need large scale cultures long-term. If you are aware of a dry serum-free insect cells medium, please let me know - I'd love to try it. Dima Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
Re: [ccp4bb] Ligand weak density in SHELXL
Dear Fatima, just a couple of things you may consider: - you can let shelxl refine the occupancy by assigning a free variable to the whole ligand - did you refine the structure anisotropically? At this resolution you certainly should. - maybe you made a mistake converting the files from refmac to shelxl, e.g. you might have used amplitudes where shelxl expects intensities or vice versa. Good luck, Tim On Mon, Jun 07, 2010 at 05:46:01PM +0100, Fatima Fonseca wrote: Dear all, I solved the structure of an enzyme at resolution of 1.1A with a bound substrate using Refmac5 and Coot for refinement/building. I have a very nice density for my ligand and a very good structure, as judged by Molprobity analysis. Now I'm using SHELXL to finish refinement but most of the density for my ligand disappears when I put it in, and it gets very high B factors. I though it could be some problem with the occupancy so I did a set of runs with different occupancies ranging from 0.2 to 1 and I got the best R factors when using 0.4/0.5 for the occupancy. However, I still have most of the ligand outside electron density… In Coot I get no ligand with “Find ligand” even when I reduce the cluster sigma level to 0.8. What am I missing here? Just another question, how to reduce the acceptable fit fraction in Coot “Find ligand”? Thanks for any advice, Fátima -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] Ligand weak density in SHELXL
Dear Tim, Thanks for your advice. I am refining my structure anisotropically. If, as you suggested, I did something wrong converting files to use in SHELXL I would expect that the whole structure to have problems or am I wrong? And how can I assign a free variable to the whole ligand? I've just started using SHELXL. Best, Fátima Em Mon, 7 Jun 2010 20:54:07 +0200 Tim Gruene t...@shelx.uni-ac.gwdg.de escreveu: Dear Fatima, just a couple of things you may consider: - you can let shelxl refine the occupancy by assigning a free variable to the whole ligand - did you refine the structure anisotropically? At this resolution you certainly should. - maybe you made a mistake converting the files from refmac to shelxl, e.g. you might have used amplitudes where shelxl expects intensities or vice versa. Good luck, Tim On Mon, Jun 07, 2010 at 05:46:01PM +0100, Fatima Fonseca wrote: Dear all, I solved the structure of an enzyme at resolution of 1.1A with a bound substrate using Refmac5 and Coot for refinement/building. I have a very nice density for my ligand and a very good structure, as judged by Molprobity analysis. Now I'm using SHELXL to finish refinement but most of the density for my ligand disappears when I put it in, and it gets very high B factors. I though it could be some problem with the occupancy so I did a set of runs with different occupancies ranging from 0.2 to 1 and I got the best R factors when using 0.4/0.5 for the occupancy. However, I still have most of the ligand outside electron density… In Coot I get no ligand with “Find ligand” even when I reduce the cluster sigma level to 0.8. What am I missing here? Just another question, how to reduce the acceptable fit fraction in Coot “Find ligand”? Thanks for any advice, Fátima -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A --- Maria Fátima da Fonseca PhD student CESAM Dep. Biologia Campus Universitário de Santiago, Universidade de Aveiro 3810-Aveiro, Portugal Fax: 234 372 587 E-mail: ffons...@ua.pt
[ccp4bb] MR on low resolution soaking data.
Dear colleagues, We are now trying to soak some ligands into a protein, which is about 60kd in size and the structure has been solved before. But the molecular replacement cannot give a right solution. Below is some contrast of the data: Native 2A P212121 monomer Soaked4A F222 monomer (more than 70% solvent) or dimer(more possible) I wonder if it is possible to find the ligand in the case of such low resolution, provided the ligand is not so small. What facts could probably lead to the failure of MR? Molrep gave a model of monomer but the rfree is as high as 0.7, while phaser could get no result. I tried phenix.explore_metric_symmetry to find the two spacegroups are not compatible, and the Rmerge of the data seems reasonable. One more question is: the wilson B of the data is lower than 20 from ccp4. Is it common for a 4A data? Since I donnot have the experience of handling this low resolution data yet. By the way, any suggestions about refinement methods in low resolution will be appreciated! Best wishes Yang
Re: [ccp4bb] MR on low resolution soaking data.
since the ligands bare being soaked into a known solved structure, why is MR necessary? Why not just start out with some rigid body refinement of the native structure to account for possible slight differences in the cell dimensions? -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu On 06/07/10 15:17, yang li wrote: Dear colleagues, We are now trying to soak some ligands into a protein, which is about 60kd in size and the structure has been solved before. But the molecular replacement cannot give a right solution. Below is some contrast of the data: Native 2A P212121 monomer Soaked4A F222 monomer (more than 70% solvent) or dimer(more possible) I wonder if it is possible to find the ligand in the case of such low resolution, provided the ligand is not so small. What facts could probably lead to the failure of MR? Molrep gave a model of monomer but the rfree is as high as 0.7, while phaser could get no result. I tried phenix.explore_metric_symmetry to find the two spacegroups are not compatible, and the Rmerge of the data seems reasonable. One more question is: the wilson B of the data is lower than 20 from ccp4. Is it common for a 4A data? Since I donnot have the experience of handling this low resolution data yet. By the way, any suggestions about refinement methods in low resolution will be appreciated! Best wishes Yang
Re: [ccp4bb] insect cell media
For large amount of media, it's actually cheaper to buy liquid media in bags when factor in the cost of labor and water. MilliQ water may not be low endotoxin. --Chun -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Nathaniel Clark Sent: Monday, June 07, 2010 11:00 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] insect cell media I mentioned to to Chris already, but we use nothing but HyClone SFX-Insect powder. We make 20-30 L batches, sterilize with a large peristaltic pump and a disposable Millipak filter from Millipore. We never have contamination problems that are due to preparing our own media from powder. We buy 10L bottles of powder. This media works great for baculovirus, stable cells, Sf9, Sf21, Tn5. We prefer it over Invitrogen (why have separate media for Sf9 and Tn5 cells?), and it's cheaper too! We no longer use serum media for anything (virus production and amplification, all goes fine in serum free. Serum helps storing viral stocks for long periods). It's ~$26 per L, plus $1-$2 in filter costs (a $30 filter will sterilize ~30 L of media). So it comes to about ~$28/L, I haven't priced out alternative products recently, but I think liquid from Invitrogen runs around $50/L? We also use the peristaltic pump for tangential flow filtration, it's pretty heavy duty (tubing is like 5/8 thick). I think that help make the filtering efficient. Bottle top filters won't work because they foul to quickly, same is try of 4.5 cm filter discs that you can insert into an in-line filter, they clog after 1 L or so. You need the 'asymmetric' filter material offered by Millipore in the Millipak or Steripak filter (the later is the filter you see attached to a MilliQ water dispenser, it can be autoclaved 3X they claim) Hope that helps, Nat On Mon, Jun 7, 2010 at 10:59 AM, Tommi Kajander tommi.kajan...@helsinki.fi wrote: Hi, well, actually this is availalbe as powder (HYClone), as it says on the page.. (we have it made in our media kitchen on a regular basis, but not for huge scale... so i dont know about the prices..) https://www.thermoscientific.com/wps/portal/ts/products/detail?navigationId= LA11074__10347categoryId=82051productId=11960716 HTH, Tommi On Jun 7, 2010, at 5:52 PM, Dima Klenchin wrote: We have happily made a transition last year from using Invitrogen's SFM medium and cellfectin to Insect-Xpress (Lonza) and polyethyleneimine for transfection. We are moving several protein targets to large-scale cultures and would consider cost-cutting alternatives. For example, Invitrogen and Thermo Sci both offer media in powder form at an attractive price. Has anyone made a systematic comparison of these media? Any particular recommendations regarding powder media? Last I checked, no manufacturer offered serum-free powedered medium. Fetal calf serum is very expensive - once you count in its cost, the overall price of the media becomes comparable. If your protein is secreted, serum-containing media are not even a realistic choice. If it is intracellular, buying dry medium and adding FBS work well: is a lot more hassle, is about 20% cheaper, and usually gives slightly higher titers and expression levels. Of all the common media we compared, TNM-FH works best for us with Sf9 cells. Switching to dry medium also means some initial investment: bottles, filtration equipment and filters. So it all pays off only of you need large scale cultures long-term. If you are aware of a dry serum-free insect cells medium, please let me know - I'd love to try it. Dima Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
Re: [ccp4bb] MR on low resolution soaking data.
You haven't given much detail to work with so I can only guess about your problem. A Wilson B of 20 for a 4 A data set is ridiculous, but the uncertainty in the Wilson B calculation at 4 A is enormous, so what might be a more reasonable statement would be to say your Wilson B calculates to 20 +_ 300 A^2 and the true value would be in that range. I don't think a precise Wilson B is important for MR so I wouldn't worry about it. An R value of .7 after MR is very large. Its size implies a systematic problem with your model - I would be looking for a second monomer. You haven't said anything about the structure of your monomer. Often a ligand will bind in the cleft between two domains, and the domains move relative to each other upon binding. You may have to perform separate searches for each domain or construct a range of trial models with different angles between the domains. Don't worry about the ligand until you solve the protein structure. Whether you see it in the end will depend on how big it is and how good your 4 A data are. Of course, it's possible that it doesn't bind at all. Dale Tronrud On 06/07/10 12:17, yang li wrote: Dear colleagues, We are now trying to soak some ligands into a protein, which is about 60kd in size and the structure has been solved before. But the molecular replacement cannot give a right solution. Below is some contrast of the data: Native 2A P212121 monomer Soaked4A F222 monomer (more than 70% solvent) or dimer(more possible) I wonder if it is possible to find the ligand in the case of such low resolution, provided the ligand is not so small. What facts could probably lead to the failure of MR? Molrep gave a model of monomer but the rfree is as high as 0.7, while phaser could get no result. I tried phenix.explore_metric_symmetry to find the two spacegroups are not compatible, and the Rmerge of the data seems reasonable. One more question is: the wilson B of the data is lower than 20 from ccp4. Is it common for a 4A data? Since I donnot have the experience of handling this low resolution data yet. By the way, any suggestions about refinement methods in low resolution will be appreciated! Best wishes Yang
[ccp4bb] New Version of the Protein Geometry Database Operational
A new version of the Protein Geometry Database (PGD) has just been released. This version includes - The ability to compose queries and analyze the behavior of side chain chi angles. - Structures released in the wwPDB up to April 8, 2010 consisting of roughly 18,000 nonredundant protein chains from crystal structures. That's over 1.8 million residues! The PGD enables users to easily and flexibly query information about the conformation alone, the backbone geometry alone, and the relationships between them. The capabilities the PGD provides are valuable for assessing the uniqueness of observed conformational or geometric features in protein structure as well as discovering novel features and principles of protein structure. So if you observe a certain conformation or geometric feature and wonder how unusual it is, the PGD may be able to provide the answer. Queries can be based on amino acid type, secondary structure, phi/psi/omega/chi angles, B factors and main chain bond lengths and angles. Queries for motifs of up to 10 residues in length can be made. Once a query has been made, plots can be drawn to show the relationship between any pair of conformational angles and/or main chain bond lengths or angles. In addition, the results of the query can be downloaded for local analysis. The PGD server is available at http://pgd.science.oregonstate.edu/ For more information please read http://pgd.science.oregonstate.edu/static/pdf/Berkholz-PGD-2010.pdf Happy hunting
