Re: [ccp4bb] If it is a new structure?
Hi, Last couple of times I asked myself the same question (what does it look like?) I used ssm (or PDBeFold as seems to be called now). http://www.ebi.ac.uk/msd-srv/ssm/ HTH, Fred. Liu Zhao wrote: The structure of my protein is as shown as the purple one. Another one ,as shown as green,is homologous .But the structure of my protein can't be obtained by using molecular replacement. And both structures have much different, especially in B chain. If my structure is a new one? thank you for help.
Re: [ccp4bb] If it is a new structure?
You're welcome! Next time, irrespective of whether your structure is a new one or not, please upload your images to picasa, flickr, your university's file sharing server or some such thing and include the link in your email. Don't flood several thousand inboxes with megabytes of pixels. And compress bitmaps, for crying out loud. How hard can it be? Thanks. Andreas On 20/12/2010 10:49, Liu Zhao wrote: The structure of my protein is as shown as the purple one. Another one ,as shown as green,is homologous .But the structure of my protein can't be obtained by using molecular replacement. And both structures have much different, especially in B chain. If my structure is a new one? thank you for help. -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk
[ccp4bb] Postdoc position at IBS Grenoble, France
A post-doctoral position is available to join the group of Pr. E. Pebay-Peyroula at IBS (Institut de Biologie Structurale, Grenoble, http://www.ibs.fr). The group explores functional and structural aspects of membrane proteins mainly focussing on the mitochondrial carriers family. The research emphasis is in deciphering atomic details of these proteins combining X-crystallography with functional assays, biophysical characterizations, molecular dynamics simulations, spectroscopy and microscopy techniques. The candidate will be expected to participate in the characterization of the molecular mechanism and supramolecular arrangement of the carriers as well as in protein-protein interaction studies. This 18 months position is open starting early 2011. The candidate should hold a PhD in biochemistry, molecular biotechnology, physical chemistry, or related discipline. Prior knowledge and experience in optical spectroscopy and microscopy techniques (confocal and multiphoton, FRET...), and expertise in basic molecular biology techniques are mandatory. Experience in working with cell cultures would be an advantage and experience on membrane proteins will be considered as a plus but not essential. Applications are only considered from candidates with no more than five years of research experience including PhD and who have notresided in France the last years. Additional information and details regarding the laboratory or the project in question can be obtained by contacting Pr. Eva Pebay-Peyroula (eva.pebay-peyro...@ibs.fr) and Dr. Stephanie Ravaud (stephanie.rav...@ibs.fr mailto:stephanie.rav...@ibs.fr). If you are interested in joining our team at IBS, please send your CV and the names of two references to Pr. Eva Pebay-Peyroula (eva.pebay-peyro...@ibs.fr) and Dr. Stephanie Ravaud (stephanie.rav...@ibs.fr). -- --- Stéphanie RAVAUD Laboratoire des Protéines Membranaires (LPM) Institut de Biologie Structurale J.-P. Ebel 41, rue Jules Horowitz F-38027 GRENOBLE Cedex 1 phone: +33(0)4 38 78 68 01 fax: +33(0)4 38 78 54 94 e-mail: stephanie.rav...@ibs.fr
[ccp4bb] off topic: young scientists linkedin group
Dear CCP4 users: This email is to encourage you to join the Young Scientist Special Interest Linkedin Group, as part of the American Crystallographic Association. Anyone with an interest in promoting the professional development of young scientists in the field of crystallography is encouraged to join. Most activities occur during our annual meetings and include: New Attendee Orientation, Etter Award Symposium, Mentoring Dinner, Social Mixer and the Professional Odyssey (career panel). More details can be found on the website: http://www.amercrystalassn.org/2011-ys-insiders-guide.100100.content The primary objective of YSSIG is to offer useful career-related resources for young scientists at every stage of their training. Some of the unique resources we plan to provide throughout the year include: *E-Mentoring Discussions * Mapping your career trajectory * When/how to apply to grad school; When/How to apply for a postdoc Making the most of your training * How to be and stay productive in grad school/postdoc *Funding* Identifying funding opportunities - National and International *Grant writing* Tips on writing grants To join the YSSIG Linkedin Group - connect with me (Jamaine SC Davis) to receive an invitation - do a search for Young Scientist - ACA As the group grows, we hope to provide additional resources and corporate sponsorships to help promote career development in all industries (academic, gov't, industry). Thanks for your time! Best, Jamaine SC Davis ACA - YSSIG Chair, 2011 -- _ Jamaine SC Davis, Ph.D. CRTA Postdoctoral Fellow National Cancer Institute | Macromolecular Crystallography Laboratory 1050 Boyles Street, PO Box B, Bldg 539/145 | Frederick, MD 21702 Tel: 301.846.5326 | Email: *davij...@mail.nih.gov *website: *http://ccr.cancer.gov/staff/staff.asp?profileid=12829 *_
Re: [ccp4bb] If it is a new structure?
Hi Liu, If I understand your question correctly, youre asking how different do two structures need to be for one to be new'. If by new you mean a new fold, then the answer is NO. Your structure and the homolog have the same fold. However, if your structure is the first structure of a protein in a new class, then your structure is a new insight for that reason (e.g. it is the first structure of a Unobtainium-metalloprotease). If it is not the first structure of a protein from a new class, lets say a previous structure of Unobtainium-metalloprotease has been solved using H. sapiens' sequence, but your protein is the first D. melanogaster ortholog solved, then your structure is a new insight for that reason. So, in a nut-shell, I guess what I am saying is that your protein is not a new fold, but is almost certainly new by some qualification, and you will know best what that qualification is. I hope that helps, cheers and happy holidays~ ~Justin On 20/12/2010 10:49, Liu Zhao wrote: The structure of my protein is as shown as the purple one. Another one ,as shown as green,is homologous .But the structure of my protein can't be obtained by using molecular replacement. And both structures have much different, especially in B chain. If my structure is a new one? thank you for help.
Re: [ccp4bb] If it is a new structure?
On 12/20/10 05:49, Liu Zhao wrote: The structure of my protein is as shown as the purple one. Another one ,as shown as green,is homologous .But the structure of my protein can't be obtained by using molecular replacement. And both structures have much different, especially in B chain. If my structure is a new one? thank you for help. The structure appears to consist of 3 distinct domains, a central beta barrel and two mostly alpha helical domains. RE the failure of molecular replacement - you don't say how hard you tried. Given a starting structure like that, I would have tried searching separately with models for the various domains. I also cannot tell from two images how similar the helical domains are; is it just a simple rotation, or is the arrangement of helices substantially different? There are algorithms for comparing protein folds. This article addresses your need, but is a bit old: http://www.ncbi.nlm.nih.gov/pubmed/14696188 Evaluation of protein fold comparison servers. Novotny M http://www.ncbi.nlm.nih.gov/pubmed?term=%22Novotny%20M%22%5BAuthor%5D, Madsen D http://www.ncbi.nlm.nih.gov/pubmed?term=%22Madsen%20D%22%5BAuthor%5D, Kleywegt GJ http://www.ncbi.nlm.nih.gov/pubmed?term=%22Kleywegt%20GJ%22%5BAuthor%5D. Proteins. 2004 Feb 1;54(2):260-70. PMID: 14696188 [PubMed - indexed for MEDLINE] -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] REFMAC 5.2.0019 question
On Sun, 2010-12-19 at 18:58 -0500, Hailiang Zhang wrote: (1). CTLS=a, CC=b (2). CTLS=a, CC=0; followed by: CTLS=0, CC=b In (2), you are doing two separate runs, with the second not using TLS. If you want to combine TLS and positional refinement, you must have them included in the same refmac run. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] REFMAC 5.2.0019 question
I would try it with the latest version (5.6.x) in case there's a bug.
That's a very old version you're still using!
-- Ian
On Sun, Dec 19, 2010 at 11:58 PM, Hailiang Zhang zhan...@umbc.edu wrote:
Hi,
I am using REFMAC 5.2.0019 to run the following script:
***
refmac5 hklin a xyzin b eof
REFI TLSC ${CTLS}
REFI BREF OVERall
NCYC ${CC}
**
I thought this script will do CTLS cycles of TLS refinement followed by CC
cycles of verall B and geometry refinement. Then I did the following 2
tests:
(1). CTLS=a, CC=b
(2). CTLS=a, CC=0; followed by: CTLS=0, CC=b
***
The results are just very different from (1) and (2), and I am not sure why.
By the way, my system has a small region with identical ADPs. After doing
(2), the ADPs at this region becomes different; however, after doing (1),
these ADPs are still identical, although different from the original ADPs.
Thanks for any clarifications!
Best Regards, Hailiang
Re: [ccp4bb] REFMAC 5.2.0019 question
PS one other thought: in your run 2b you are not reading in (as TLSIN)
the TLSOUT file produced by run 2a. So run 2b is not starting from
the same point that it would have done as in run 1.
I.
On Sun, Dec 19, 2010 at 11:58 PM, Hailiang Zhang zhan...@umbc.edu wrote:
Hi,
I am using REFMAC 5.2.0019 to run the following script:
***
refmac5 hklin a xyzin b eof
REFI TLSC ${CTLS}
REFI BREF OVERall
NCYC ${CC}
**
I thought this script will do CTLS cycles of TLS refinement followed by CC
cycles of verall B and geometry refinement. Then I did the following 2
tests:
(1). CTLS=a, CC=b
(2). CTLS=a, CC=0; followed by: CTLS=0, CC=b
***
The results are just very different from (1) and (2), and I am not sure why.
By the way, my system has a small region with identical ADPs. After doing
(2), the ADPs at this region becomes different; however, after doing (1),
these ADPs are still identical, although different from the original ADPs.
Thanks for any clarifications!
Best Regards, Hailiang
[ccp4bb] Bfactor is zero?
Hi All, Recently I solved a structure in which some water molecules have Bfactors at 0 and overall wilson Bfactor is 0.654 based on PHENIX refinement. Is it possible? Bill Lu
Re: [ccp4bb] Bfactor is zero?
On Mon, Dec 20, 2010 at 7:34 AM, Zhibing Lu billz...@gmail.com wrote: Recently I solved a structure in which some water molecules have Bfactors at 0 and overall wilson Bfactor is 0.654 based on PHENIX refinement. Is it possible? Very unlikely - it might be worth trying the latest nightly build and seeing if the problem persists. You should email b...@phenix-online.org or h...@phenix-online.org for further assistance. -Nat
Re: [ccp4bb] REFMAC 5.2.0019 question
Dear Hailiang,
I regularly separate TLS refinement from restraint refinement to spead things
up when I try to optimise the restraint weights. Using Refmac 5.6, I do the
following:
- Do TLS Refinement in refmac
- Use TLSanl to change the total B-factors to residual B-factors.
- Load the output TLS file from the first Refmac run as static TLS tensors and
do restrained refinement. Use ISOtropic B-factors!
See if this works for you. Keep in mind that there still may be small
differences in (free) R-factor.
Cheers,
Robbie
Date: Mon, 20 Dec 2010 14:44:34 +
From: ianj...@gmail.com
Subject: Re: [ccp4bb] REFMAC 5.2.0019 question
To: CCP4BB@JISCMAIL.AC.UK
PS one other thought: in your run 2b you are not reading in (as TLSIN)
the TLSOUT file produced by run 2a. So run 2b is not starting from
the same point that it would have done as in run 1.
I.
On Sun, Dec 19, 2010 at 11:58 PM, Hailiang Zhang zhan...@umbc.edu wrote:
Hi,
I am using REFMAC 5.2.0019 to run the following script:
***
refmac5 hklin a xyzin b eof
REFI TLSC ${CTLS}
REFI BREF OVERall
NCYC ${CC}
**
I thought this script will do CTLS cycles of TLS refinement followed by CC
cycles of verall B and geometry refinement. Then I did the following 2
tests:
(1). CTLS=a, CC=b
(2). CTLS=a, CC=0; followed by: CTLS=0, CC=b
***
The results are just very different from (1) and (2), and I am not sure why.
By the way, my system has a small region with identical ADPs. After doing
(2), the ADPs at this region becomes different; however, after doing (1),
these ADPs are still identical, although different from the original ADPs.
Thanks for any clarifications!
Best Regards, Hailiang
Re: [ccp4bb] REFMAC 5.2.0019 question
Thanks Ian, but I was using the output from 2a for 2b running. Results are
still different between 2 and 1. More curious is more second question, the
region with identical ADPs still ended up with identical ADPs (although
different from before running) after 1, and that's why I also tried 2.
Hailiiang
PS one other thought: in your run 2b you are not reading in (as TLSIN)
the TLSOUT file produced by run 2a. So run 2b is not starting from
the same point that it would have done as in run 1.
I.
On Sun, Dec 19, 2010 at 11:58 PM, Hailiang Zhang zhan...@umbc.edu wrote:
Hi,
I am using REFMAC 5.2.0019 to run the following script:
***
refmac5 hklin a xyzin b eof
REFI TLSC ${CTLS}
REFI BREF OVERall
NCYC ${CC}
**
I thought this script will do CTLS cycles of TLS refinement followed by
CC
cycles of verall B and geometry refinement. Then I did the following 2
tests:
(1). CTLS=a, CC=b
(2). CTLS=a, CC=0; followed by: CTLS=0, CC=b
***
The results are just very different from (1) and (2), and I am not sure
why.
By the way, my system has a small region with identical ADPs. After
doing
(2), the ADPs at this region becomes different; however, after doing
(1),
these ADPs are still identical, although different from the original
ADPs.
Thanks for any clarifications!
Best Regards, Hailiang
Re: [ccp4bb] Bfactor is zero?
some water molecules have Bfactors at 0 B-factors refining towards zero may be an indication of heavier molecules, e.g. SO4. You have to model them manually. Ralf
Re: [ccp4bb] REFMAC 5.2.0019 question
On Mon, 2010-12-20 at 13:11 -0500, Hailiang Zhang wrote: Thanks Ian, but I was using the output from 2a for 2b running. It's not enough to use the pdb output - you have to make the proper tls input file for refmac to incorporate the tls correction -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] If it is a new structure?
Hi Liu Looks like (on the images you show) that you have a nice conformational difference for some of the helices between the 2 structures... Happy Hols Gina -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Justin Hall Sent: Monday, December 20, 2010 8:57 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] If it is a new structure? Hi Liu, If I understand your question correctly, youre asking how different do two structures need to be for one to be new'. If by new you mean a new fold, then the answer is NO. Your structure and the homolog have the same fold. However, if your structure is the first structure of a protein in a new class, then your structure is a new insight for that reason (e.g. it is the first structure of a Unobtainium-metalloprotease). If it is not the first structure of a protein from a new class, lets say a previous structure of Unobtainium-metalloprotease has been solved using H. sapiens' sequence, but your protein is the first D. melanogaster ortholog solved, then your structure is a new insight for that reason. So, in a nut-shell, I guess what I am saying is that your protein is not a new fold, but is almost certainly new by some qualification, and you will know best what that qualification is. I hope that helps, cheers and happy holidays~ ~Justin On 20/12/2010 10:49, Liu Zhao wrote: The structure of my protein is as shown as the purple one. Another one ,as shown as green,is homologous .But the structure of my protein can't be obtained by using molecular replacement. And both structures have much different, especially in B chain. If my structure is a new one? thank you for help. Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] Mg2+ or water
Mg2+ is (almost) aways octahedrally coordinated, usually by oxygen atoms, with distances of ~2A. Phil On 20 Dec 2010, at 21:16, jlliu liu wrote: Hi All, I am refining a structure and encountered a problem of modeling a difference density as water or Mg2+, and would like to hear opinions from the community. It has the following coordinations (attached): the water/Mg2+ forms salt bridge/H-bonding interaction with a carboxylate group from the ligand, it also forms salt bridge/H-bonding interaction with a Glu residue from the protein, it is also within hydrogen bonding distance to the main chain N of another protein residue. In provious publication, it was modelled as a Mg2+ and the author reasoned the dual salt-bridge stabilizes the liganding binding, also the Mg2+ is present in the protein solution for crystallization. For my case, I have no Mg2+ present in the protein buffer, also modelling it with water refines perfectly with no indication of positive difference density even at 2.0 sigma cut off. Should I modelled this density as water or as Mg2+. Your opinions are appreciated. JL test.png.odp
Re: [ccp4bb] Mg2+ or water
what is the .odp file extension? JPK On Mon, Dec 20, 2010 at 3:48 PM, Robbie Joosten robbie_joos...@hotmail.com wrote: Dear jlliu liu, Also note that Mg2+ is significantly smaller than water. It fits in places where water cannot go. This doesn't look like a magnesium site on first glance. If you can (privately) give the PDBid of the previous publication, I can have a look in 3D. Cheers, Robbie Date: Mon, 20 Dec 2010 21:31:58 + From: p...@mrc-lmb.cam.ac.uk Subject: Re: [ccp4bb] Mg2+ or water To: CCP4BB@JISCMAIL.AC.UK Mg2+ is (almost) aways octahedrally coordinated, usually by oxygen atoms, with distances of ~2A. Phil On 20 Dec 2010, at 21:16, jlliu liu wrote: Hi All, I am refining a structure and encountered a problem of modeling a difference density as water or Mg2+, and would like to hear opinions from the community. It has the following coordinations (attached): the water/Mg2+ forms salt bridge/H-bonding interaction with a carboxylate group from the ligand, it also forms salt bridge/H-bonding interaction with a Glu residue from the protein, it is also within hydrogen bonding distance to the main chain N of another protein residue. In provious publication, it was modelled as a Mg2+ and the author reasoned the dual salt-bridge stabilizes the liganding binding, also the Mg2+ is present in the protein solution for crystallization. For my case, I have no Mg2+ present in the protein buffer, also modelling it with water refines perfectly with no indication of positive difference density even at 2.0 sigma cut off. Should I modelled this density as water or as Mg2+. Your opinions are appreciated. JL test.png.odp -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Mg2+ or water
If it's more than likely Mg and it's easy to grow crystals, try soaking/cogrowing BaCl2/BaOAc. At least in the RNA world, Mg sites can easily be displaced by Ba. The latter of course has anomalous signal on the home sources. Place your divalents using the anomalous diff map. Have no clue what this .odp file ext is so I can't view the png. F On Dec 20, 2010, at 2:16 PM, jlliu liu wrote: Hi All, I am refining a structure and encountered a problem of modeling a difference density as water or Mg2+, and would like to hear opinions from the community. It has the following coordinations (attached): the water/Mg2+ forms salt bridge/H-bonding interaction with a carboxylate group from the ligand, it also forms salt bridge/H- bonding interaction with a Glu residue from the protein, it is also within hydrogen bonding distance to the main chain N of another protein residue. In provious publication, it was modelled as a Mg2+ and the author reasoned the dual salt-bridge stabilizes the liganding binding, also the Mg2+ is present in the protein solution for crystallization. For my case, I have no Mg2+ present in the protein buffer, also modelling it with water refines perfectly with no indication of positive difference density even at 2.0 sigma cut off. Should I modelled this density as water or as Mg2+. Your opinions are appreciated. JL test.png.odp - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
Re: [ccp4bb] Mg2+ or water
On 20/12/10 21:48, Robbie Joosten wrote: Also note that Mg2+ is significantly smaller than water. It fits in places where water cannot go. This doesn't look like a magnesium site on first glance. I tend to agree with Robbie. I wonder what WASP would say... (if you use Coot, you can try the Highly Coordinated Waters validation test - a symmetry-enhanced implementation of the Nayal Di Cera (1996) algorithm). Paul.
Re: [ccp4bb] Mg2+ or water
Sorry, the attachment is in here. Doesn't look like Mg2+ at all. Distances are too long, Mg is never coordinated by amides and if it were Mg you would have seen waters around it. Looks like tightly bound water to me. - Dima On Mon, Dec 20, 2010 at 4:16 PM, jlliu liu mailto:jlliu20022...@gmail.comjlliu20022...@gmail.com wrote: Hi All, I am refining a structure and encountered a problem of modeling a difference density as water or Mg2+, and would like to hear opinions from the community. It has the following coordinations (attached): the water/Mg2+ forms salt bridge/H-bonding interaction with a carboxylate group from the ligand, it also forms salt bridge/H-bonding interaction with a Glu residue from the protein, it is also within hydrogen bonding distance to the main chain N of another protein residue. In provious publication, it was modelled as a Mg2+ and the author reasoned the dual salt-bridge stabilizes the liganding binding, also the Mg2+ is present in the protein solution for crystallization. For my case, I have no Mg2+ present in the protein buffer, also modelling it with water refines perfectly with no indication of positive difference density even at 2.0 sigma cut off. Should I modelled this density as water or as Mg2+. Your opinions are appreciated. JL Content-type: image/png; name=367-mgtest.png Content-disposition: attachment; filename=367-mgtest.png X-Attachment-Id: f_ghy0k5e31
Re: [ccp4bb] Bfactor is zero?
On 12/20/2010 10:34 AM, Zhibing Lu wrote: Hi All, Recently I solved a structure in which some water molecules have Bfactors at 0 and overall wilson Bfactor is 0.654 based on PHENIX refinement. Is it possible? Bill Lu Hi Bill, What resolution are you working with here? An overall Wilson B 1 is sort of odd... If the resolution is too low - the slope in the Wilson plot has a lot of play - and might be indetermiate I would use phenix.xtriage and see if there are any indications of something interesting happening with your data... Ezra
Re: [ccp4bb] Bfactor is zero?
Hi Bill, if you put a water oxygen in place where a heavier atom is, then water oxygen's B-factor will refine to a value close to zero. This is the feature that we currently use as one of many criteria to develop automatic identification and building of metals. Overall Wilson B-factor of 0.6A**2 tells that there is something weird about the data. What is the resolution? Pavel. PS As Nat mentioned, PHENIX related questions are best to send to PHENIX (and not CCP4) mailing list: http://www.phenix-online.org/ On Mon, Dec 20, 2010 at 7:34 AM, Zhibing Lu billz...@gmail.com wrote: Hi All, Recently I solved a structure in which some water molecules have Bfactors at 0 and overall wilson Bfactor is 0.654 based on PHENIX refinement. Is it possible? Bill Lu
