[ccp4bb] metal ion dominating density!
Hi all, I recall there was a post a while back, but silly me can't remember the details - How does one stop metal ions dominating real space refinement in COOT? D. Dean Derbyshire Senior Research Scientist [cid:image001.jpg@01CF80A0.6F20B190] Box 1086 SE-141 22 Huddinge SWEDEN Visit: Lunastigen 7 Direct: +46 8 54683219 www.medivir.comhttp://www.medivir.com -- This transmission is intended for the person to whom or the entity to which it is addressed and may contain information that is privileged, confidential and exempt from disclosure under applicable law. If you are not the intended recipient, please be notified that any dissemination, distribution or copying is strictly prohibited. If you have received this transmission in error, please notify us immediately. Thank you for your cooperation.
Re: [ccp4bb] metal ion dominating density!
On 05/06/14 08:27, Dean Derbyshire wrote: Hi all, I recall there was a post a while back, but silly me can’t remember the details – How does one stop metal ions dominating real space refinement in COOT? sphere refine? Paul.
Re: [ccp4bb] metal ion dominating density!
Or, if for whatever reason sphere refine isn't an option, fix the metal and all of its ligands Sent on a Sprint Samsung Galaxy S® III div Original message /divdivFrom: Paul Emsley pems...@mrc-lmb.cam.ac.uk /divdivDate:06/05/2014 3:51 AM (GMT-05:00) /divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: Re: [ccp4bb] metal ion dominating density! /divdiv /divOn 05/06/14 08:27, Dean Derbyshire wrote: Hi all, I recall there was a post a while back, but silly me can’t remember the details – How does one stop metal ions dominating real space refinement in COOT? sphere refine? Paul.
[ccp4bb] PhD position in structural biology at Lund University
Doctoral student in molecular biophysics Type of employment: Limit of tenure, Four years Extent: 100 % Location: Department of Chemistry, Division of Biochemistry and Structural Biology, Lund First day of employment: 2014-10-01 Research assignments The PhD student will work with structural studies of protein-ligand interactions, using the carbohydrate-binding protein galectin-3 as a model system. The doctoral work is part of a larger project involving seven research groups at LU that aims at a detailed dissection of the roles of enthalpy and entropy role in protein-ligand interactions. The student will determine the structures of a large number of complexes of galectin-3 with novel synthetic ligand using X-ray crystallography at ultra-high resolution, as well as a smaller number of neutron crystal structures of selected complex. The work will include the purification of proteins, production of crystals (of normal size for X-ray diffraction and very large crystals for neutron studies), X-ray and neutron data collection and processing and modelling. The project will involve interaction with research groups engaged in organic synthesis, biophysical characterisation, in vitro and cell-based assays and quantum mechanics calculations. Eligibility requirements Students with basic eligibility for third-cycle studies are those who have completed a second-cycle degree, have completed courses of at least 240 credits, of which at least 60 credits are from second-cycle courses, or have acquired largely equivalent knowledge in some other way, in Sweden or abroad. Special eligibility requirements comprise knowledge from first-cycle studies or the equivalent, but can also refer to special professional experience. Additionally, sufficient knowledge of the subject area is required for third-cycle studies. Students meeting special eligibility requirements are those who have at least 60 higher education credits within the same subject area as the third-cycle studies, of which in-depth work of at least 30 higher education credits of relevance to the subject area, a Swedish medical degree or Swedish certification as a doctor. Special requirements for this position: The candidate must have Bachelor's degree in an appropriate subject (preferably Chemistry) and will ideally have previous experience in structural biology, both theoretical and practical. The candidate should also be proficient in physical chemistry, especially thermodynamics. Ability to work as part of a team that spans multiple disciplines, but at the same time to take responsibility for their own work and drive it forward is fundamental, as the research group is involved in a variety of other projects. Document to include with the application: A summary (1/2-1 page) motivating why you wish to perform a PhD education in molecular biophysics at Lund University, and how the present research project matches your own research interests and scientific background. Doctoral candidates may be required to work with educational tasks and administration in addition to research, up to a level of approximately 20%. See here for more details and instructions on how to apply: http://www.lu.se/lediga-anstallningar-available-jobs?x=0Dnr=615182Type=E For further details you can also contact Derek Logan, Senior lecturer derek.lo...@biochemistry.lu.se Esko Oksanen, Adjunct lecturer esko.oksa...@esss.se
[ccp4bb] solvent flattening with resolve - phenix
Dear all, I am trying to get a sharper map with RESOLVE in Phenix and I provide the .mtz or high resolution data as well as the mtz corresponding to experimental data. However I get the following message, and I don't really know how to fix it even though it looks quite silly. Might be that today I am not inspired, :-P Please identify the column 'PHIB' in your input data file. You can specify all columns to use in 'input_labels' like this: FP SIGFP PHIB FOM HLA HLB HLC HLD where you have to put something in each of these 8 spots (use None for missing ones). You currently have: FP SIGFP None FOM hltofom.ABCD.A hltofom.ABCD.B hltofom.ABCD.C hltofom.ABCD.D Possibilities are... FreeRflag FP SIGFP FC PHIC FC_ALL PHIC_ALL FWT PHWT DELFWT PHDELWT FOM FC_ALL_LS PHIC_ALL_LS hltofom.ABCD.A hltofom.ABCD.B hltofom.ABCD.C hltofom.ABCD.D None Any suggestions? Thank you very much in advance. Best wishes, from sunny Göttingen! Almudena -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
Re: [ccp4bb] solvent flattening with resolve - phenix
Dear Almudena, i think your problems com from the Flag in your input.mtz . You have to check if your mtz has the correct column name. And you have to designate the one which correspond to PHIB. It depend from which programm com your input.mtz . Hope to help. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Almudena Ponce Salvatierra [maps.fa...@gmail.com] Envoyé : jeudi 5 juin 2014 11:43 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] solvent flattening with resolve - phenix Dear all, I am trying to get a sharper map with RESOLVE in Phenix and I provide the .mtz or high resolution data as well as the mtz corresponding to experimental data. However I get the following message, and I don't really know how to fix it even though it looks quite silly. Might be that today I am not inspired, :-P Please identify the column 'PHIB' in your input data file. You can specify all columns to use in 'input_labels' like this: FP SIGFP PHIB FOM HLA HLB HLC HLD where you have to put something in each of these 8 spots (use None for missing ones). You currently have: FP SIGFP None FOM hltofom.ABCD.A hltofom.ABCD.B hltofom.ABCD.C hltofom.ABCD.D Possibilities are... FreeRflag FP SIGFP FC PHIC FC_ALL PHIC_ALL FWT PHWT DELFWT PHDELWT FOM FC_ALL_LS PHIC_ALL_LS hltofom.ABCD.A hltofom.ABCD.B hltofom.ABCD.C hltofom.ABCD.D None Any suggestions? Thank you very much in advance. Best wishes, from sunny Göttingen! Almudena -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
Re: [ccp4bb] AW: [ccp4bb] possible twinning issue in P4212 / I422
There are cases of different crystal forms of the same thing turning up with similar lattice dimensions... But if if the cell dimensions are the same, and one form is P4i with 4 molecules in the cell, and just enough space for one mol/asymm unit, and the 2nd form is I422 then isnt the asymm volume much too small in that form? Eleanor On 4 June 2014 20:23, Bjørn Panyella Pedersen bj...@msg.ucsf.edu wrote: Dear All, Thanks for all the suggestions, on and off the board! Summary: Some have asked for the L-statistic in P 4 21 2: Mean |L| :0.397 (untwinned: 0.500; perfect twin: 0.375) All programs tried (xtriage, truncate, pointless) agree that the 4Å data is likely twinned P4 with an estimated twin-fraction ranging from 0.38 to 0.48. People seem to agree (as was my initial understanding) that twinning by itself cannot change a primitive lattice to a body-centered lattice. Thus this change in my low-resolution dataset must be caused by something else. Pseudo body centering has been suggested as the likely explanation for the primitive to body-centered lattice change in the low-resolution dataset. - As suggested, I have looked at the self-patterson for the 4Å dataset and see no peaks, at 1/2,1/2,1/2 or elsewhere. - As suggested, I have looked at the truncate output of the table Analysis of mean intensity by parity for reflection classes in the h+k+l column, but see no differences from h+k+l=2n to h+k+l=2n+1 in the 4Å dataset. - As suggested I have processed the 4Å data at 8Å to see if pseudo body centering was breaking down at higher resolution. At 8Å there was still not sign of pseudo body centering. Thus the 4Å data does not not support pseudo body centering, as far as I can tell. Some has suggested that the crystals are simply two different things. This might be, but since the low-resolution dataset has exactly the same unit-cell parameters as the 4Å dataset, it seems unlikely to me that the crystal packing is significantly different, or that the content of the crystals differ. It seem likely that both crystals contain the same thing in approx. the same type of packing. So no clear explanation for the observed behavior so far, but most likely the low resolution is obscuring the real problem in this particular instance. As is almost always the case, the way forward is to get more and better data to understand the problem. As one suggested offboard (tongue-in-cheek): Why not find crystals that are not twinned, probably with higher resolution? I will do that, and thanks again for your input! All the best, -Bjørn On 06/04/2014 03:09 AM, Eleanor Dodson wrote: It helps to look at the output from the truncate step quite critically. First is there a non cryst translation of 1/2,1/2,1/2 indicated in the P4 2i2 data set? If so then the I centring at lower resolution might just be approximate.. If there is NC translation then other twinning statistics are distorted and I find the only semi-reliable one is the L test. But if you say there is no room for your protein with that translation and 4/mmm symmetry then there must be twinning or you have crystallised something else! Eleanor On 4 June 2014 08:48, herman.schreu...@sanofi.com mailto:herman.schreu...@sanofi.com wrote: Dear Bjørn, I guess the first step to enlightment is to recognize that we as mere mortals are not able to deduce the space group from diffraction data alone. All Aimless, XDS etc. can produce are educated guesses what the space group might be. Especially when twinning is involved, the crystal packing may not heed the rules and classifications that we humans try to impose. In many cases, one might have to go down to P1 and solve the structure in P1 to find out what the true space group is. Here are some comments to your questions: -the same protein under the same crystallization conditions and even in the same drop may produce crystals with very different crystal packings, even with the same unit cell, so your 4 and 7.5Å crystals may be different. -If there is no way to fit the protein in the asymmetric unit that is a very strong indication that you do have twinning. -There have been some discussions in the CCP4BB, but I do not believe that twinning can generate body centering. -You might be barking at the wrong tree and the twinning axis might be parallel to the 4-fold axis, or even generating the 4-fold. You may even have 4-fold twinning. -You may have pseudo body centering, which is perfect at low resolution, but breaks down at higher resolution. As a test, you could process your 4Å data only to 7.5Å and see what the statistics would look like. What I would do: If you have more crystals, collect data on them all, maybe there is one which is not or not perfectly twinned. If there is a model which could be used for molecular
[ccp4bb] : MTZ for a protein fragment and R factors, Masking and Maps
Hi, First off I am pretty new to CCP4/X-ray crystallography so please bear with me as I try to explain my question. I was looking at a protein structure from the PDB (let's say 1aho.pdb). I have the corresponding MTZ file. I wanted to calculate the R-factor for some selected residues (lets say 17-40). I can calculate the R factor for the whole protein using the (MTZ + pdb file) SFCheck tool. Is there a way to calculate the R-factor for a segment of the protein? I can generate a masked map using COOT as follows: Extensions-- Maps-- Mask Map by Atom Selection (inverse) But the result is a map and not a MTZ file (the format I need for the next step SFCheck) I tried using NCSMask but it did not work out. Could someone suggest where I am going wrong in trying to calculate R for a small part of the protein? Thank you, George
[ccp4bb] Diamond Light Source MX Bag Training 15-16 July
Dear all, Diamond Light Source will be holding the next training day for MX BAG Users on Tuesday 15th July and Wednesday 16th July 2014. The aim is to provide MX users with sufficient training to be able to operate any of the Diamond MX beamlines efficiently and to get the most benefit from their beamtime. The training will involve hands-on sessions on the suite of five operational MX beamlines (http://www.diamond.ac.uk/mx-home/) as well as offline software sessions. Sessions include: 15th July, Afternoon session: Hands on software Three tutorial sessions: * CCP4: MR and experimental phasing (CCP4 team) * Manual processing iMosflm (Harry Powell and Andrew Leslie) * Multi crystal analysis / BLEND (Pierre Aller) 16th July: * MX software: automation in data analysis * Mini-Kappa goniometry * Sample humidity control (HC1) * Microbeam crystallography and fast data collection * In-situ diffraction * Experiment database: new ISPyB Registration is free-of-charge with lunch provided on the 15th and 16th July, and accommodation and dinner for the night of the 15th July. Travelling expenses within the UK will also be provided. The training is targeted at all users, and is not limited to students and post docs. It is essential that each BAG sends at least one representative per calendar year. Individuals wishing to register should register here: http://www.diamond.ac.uk/Home/Events/2014/MX-Bag-Training.html Places are limited to twenty five and registration deadline is on 30th June. If you have any question, please feel free to contact me. Best wishes, Pierre Pierre Aller, Ph.D. Senior Support Scientist Diamond Light Source Ltd., Diamond House Harwell Science Innovation Campus Didcot, Oxfordshire OX11 0DE +44 (0) 1235 778183 pierre.al...@diamond.ac.uk -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
Re: [ccp4bb] : MTZ for a protein fragment and R factors, Masking and Maps
Hi George, The R-factor is an overall disagreement statistic, it can not be broken down in parts for bits of the structure. (in other words, you can not correlate parts of the model with a subset of the reflections in the mtz, as a non-crystallographer might think - all the electrons in the asymmetric unit contribute to the intensity of each reflection) what you can calculate for parts of the structure is how well it agrees with the corresponding part of the map, and some of the structure validation programs output statistics like this, i.e. map correlation per residue. And you could then average this for the residues of interest I suppose. Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 5 Jun 2014, at 17:10, George Devaniranjan wrote: Hi, First off I am pretty new to CCP4/X-ray crystallography so please bear with me as I try to explain my question. I was looking at a protein structure from the PDB (let's say 1aho.pdb). I have the corresponding MTZ file. I wanted to calculate the R-factor for some selected residues (lets say 17-40). I can calculate the R factor for the whole protein using the (MTZ + pdb file) SFCheck tool. Is there a way to calculate the R-factor for a segment of the protein? I can generate a masked map using COOT as follows: Extensions-- Maps-- Mask Map by Atom Selection (inverse) But the result is a map and not a MTZ file (the format I need for the next step SFCheck) I tried using NCSMask but it did not work out. Could someone suggest where I am going wrong in trying to calculate R for a small part of the protein? Thank you, George
Re: [ccp4bb] : MTZ for a protein fragment and R factors, Masking and Maps
On 05/06/14 16:10, George Devaniranjan wrote: Hi, First off I am pretty new to CCP4/X-ray crystallography so please bear with me as I try to explain my question. I was looking at a protein structure from the PDB (let's say 1aho.pdb). I have the corresponding MTZ file. I wanted to calculate the R-factor for some selected residues (lets say 17-40). I can calculate the R factor for the whole protein using the (MTZ + pdb file) SFCheck tool. Is there a way to calculate the R-factor for a segment of the protein? I can generate a masked map using COOT as follows: Extensions-- Maps-- Mask Map by Atom Selection (inverse) But the result is a map and not a MTZ file (the format I need for the next step SFCheck) I tried using NCSMask but it did not work out. Could someone suggest where I am going wrong in trying to calculate R for a small part of the protein? Try something like this: mapmask MAPIN coot-out-masked.map MAPOUT tmp.map ! AXIS Z X Y END ! sfall MAPIN tmp.map HKLOUT sfs-from-map.mtz ! MODE SFCALC MAPIN SYMM 1 ! You might not need the mapmask step. Paul.
[ccp4bb] Fwd: [ccp4bb] : MTZ for a protein fragment and R factors, Masking and Maps
George, Remember that scattering from every point in the cell contributes to every reflection; the R-value is a global metric of agreement between the model and the data. Hence, calculating the R-value for a few selected residues is not a sensible thing to do, unless you want to ask how well your particular fragment explains the scattering from the entire crystal (which I'm guessing is not the case for a 23 amino-acid fragment). If you're interested in assessing the agreement between model and map for a particular fragment, you might want to consider instead the real-space R value and the related real-space correlation coefficient. You might also consider some reading to help bone up on the basics: http://www.amazon.com/Protein-Crystallography-Eaton-E-Lattman/dp/0801888085 (a primer, and a shameless plug) http://www.amazon.com/Biomolecular-Crystallography-Principles-Application-Structural/dp/0815340818 (the detailed stuff; essential) Cheers, Pat Loll On 5 Jun 2014, at 11:10 AM, George Devaniranjan wrote: Hi, First off I am pretty new to CCP4/X-ray crystallography so please bear with me as I try to explain my question. I was looking at a protein structure from the PDB (let's say 1aho.pdb). I have the corresponding MTZ file. I wanted to calculate the R-factor for some selected residues (lets say 17-40). I can calculate the R factor for the whole protein using the (MTZ + pdb file) SFCheck tool. Is there a way to calculate the R-factor for a segment of the protein? I can generate a masked map using COOT as follows: Extensions-- Maps-- Mask Map by Atom Selection (inverse) But the result is a map and not a MTZ file (the format I need for the next step SFCheck) I tried using NCSMask but it did not work out. Could someone suggest where I am going wrong in trying to calculate R for a small part of the protein? Thank you, George --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
[ccp4bb] Bfactors too high after TLS refinement
P { margin-bottom: 0.21cm; }
Hello,
I'm almost done refining a protein-DNA
complex at 3A, but when I apply TLS (Refmac+TLSanl to include the
ANISOU component) the B factors increase from 50-60 to 100-110 A2.
The crystal is a bit special since it has 80% of solvent and the
Wilson B-factor is 87 A2. When I compare my Bfactors with other
structures of similar resolution using Phenix Validation, my data is
clearly an outlier.
To perform the TLS refinement I have
generated .tlsin files for 2,4 and 6 groups with the TLSmd server and
used them in refmac jobs consisting of 20 cycles of TLS refinement+50
cycles of restrained refinement. This way the R-factor and R-free
values have drop 1-2% (before it was 22.3/24.1). The three different
options suffer from the same problem regarding high B-factors after
TLSanl. Am I doing something wrong during the TLS refinement? What
can I do to preserve the original Bfactors?
Thanks in advance,
Carles Palanca.
[ccp4bb] Postdoc and PhD positions in Structural Biology
Postdoc and PhD positions in Structural Biology A postdoc position and three PhD positions are available in the laboratory of Prof. Eva Wolf at the Johannes Gutenberg University (JGU) and Institute of Molecular Biology (IMB) in Mainz, Germany. The Wolf group is currently located at the IMB (www.imb-mainz.de/wolfhttp://www.imb-mainz.de/wolf). IMB is a Centre of Excellence for Life Sciences, which is funded by the Boehringer Ingelheim Foundation and located on the campus of the Johannes Gutenberg University, Mainz, Germany. Research in the Wolf group is focused on the structural, biochemical and biophysical analysis of circadian clock proteins, which generate circadian (24h) rhythms in physiology, metabolism and behaviour and synchronize them with the environmental light-dark cycle. Projects will include x-ray crystallography with ample support from other biochemical, molecular biology and spectroscopic techniques such as recombinant protein expression, protein purification, biochemical and biophysical protein-protein interaction analyses, UV/VIS- and CD spectroscopy (e.g. Czarna et al, Cell 2013;153(6):1394-405; Schmalen et al, Cell 2014;157(5):1203–1215). The successful postdoc candidate will hold a PhD in a relevant discipline and have solid practical experience in recombinant protein expression using E.coli and insect cells, protein purification and macromolecular X-ray crystallography. Additional experience with biochemical and biophysical protein-protein interaction analyses or UV/VIS spectroscopy is an advantage. For the PhD positions, candidates with a strong interest in structural biology and protein biochemistry and previous exposure to these research areas during their master education are encouraged to apply. To apply, please send a single PDF file containing your cover letter stating research and career interests, CV, scanned certificates and contact details of at least two references to sek...@uni-mainz.demailto:sek...@uni-mainz.de. Informal enquires should be addressed to Eva Wolf (evawo...@uni-mainz.demailto:evawo...@uni-mainz.de). PhD candidates also have to officially apply via IMB’s International PhD Programme before July 5, 2014 (www.imb-mainz.de/PhDhttp://www.imb-mainz.de/PhD).
Re: [ccp4bb] Bfactors too high after TLS refinement
On Thursday, 05 June, 2014 17:51:33 Carles Palanca i García wrote:
P { margin-bottom: 0.21cm; }
Hello,
I'm almost done refining a protein-DNA
complex at 3A, but when I apply TLS (Refmac+TLSanl to include the
ANISOU component) the B factors increase from 50-60 to 100-110 A2.
The crystal is a bit special since it has 80% of solvent and the
Wilson B-factor is 87 A2. When I compare my Bfactors with other
structures of similar resolution using Phenix Validation, my data is
clearly an outlier.
To perform the TLS refinement I have
generated .tlsin files for 2,4 and 6 groups with the TLSmd server and
used them in refmac jobs consisting of 20 cycles of TLS refinement+50
cycles of restrained refinement. This way the R-factor and R-free
values have drop 1-2% (before it was 22.3/24.1). The three different
options suffer from the same problem regarding high B-factors after
TLSanl. Am I doing something wrong during the TLS refinement? What
can I do to preserve the original Bfactors?
Preserving the original B factors should not be your goal.
I can explain part of what you are seeing, but probably not the
entire effect.
When you refine using TLS, the various programs convert this back
to an equivalent isotropic B factor. Sometimes this is marked by
the programs as Beq, but it is placed in your output PDB
file simple as B with no warning that it is only an approximation.
But not only is Beq an approximation, it is invariably an overestimate of
the isotropic B. Beq can run as much as 40% larger than
the true isotropic B. A better estimate Best is available but so far
as I know none of the standard programs use it. Math, charts, and
example demonstrations of the too-large Beq are reported in
Some Beq are more equivalent than others. Acta Cryst. A67, 512-516.
E. A. Merritt (2011).
Ethan
Thanks in advance,
Carles Palanca.
--
Ethan A Merritt
Biomolecular Structure Center, K-428 Health Sciences Bldg
MS 357742, University of Washington, Seattle 98195-7742
Re: [ccp4bb] Bfactors too high after TLS refinement
TLS in phenix does seem to lead to high B factors in some of our cases. To check whether it was particular to our datasets , we rerefined some similar structures to ours from the PDB and found that their B factors increased to high values similar to our structure when run through the same phenix.refine protocol. Structure 1 Chain A original Average B 40.85 After Phenix with TLS 113.33 Chain B Original 50.1 After phenix TLS 121.11 Structure 2 Chain A original Average B 64.46 After Phenix with TLS 100.63 Chain B original 64.99 After Phenix TLS 103.5 Structure 3 Chain A original 46.44 After TLS 52.63 - so it does not always happen This of course only proved that our data was probably not exceptional it was the protocol. It does not explain what is going on. http://www.phenix-online.org/pipermail/phenixbb/2010-January/014374.html seems to offer some explanation of the TLS scaling in phenix. It may be the anisotropic component reduces the size of the actual B factor back to closer to the values before TLS? Best wishes Nick -- Prof Nicholas H. Keep Executive Dean of School of Science Professor of Biomolecular Science Crystallography, Institute for Structural and Molecular Biology, Department of Biological Sciences Birkbeck, University of London, Malet Street, Bloomsbury LONDON WC1E 7HX email n.k...@mail.cryst.bbk.ac.uk Telephone 020-7631-6852 (Room G54a Office) 020-7631-6800 (Department Office) Fax 020-7631-6803 If you want to access me in person you have to come to the crystallography entrance and ring me or the department office from the internal phone by the door
Re: [ccp4bb] Bfactors too high after TLS refinement
Carles, a few points to consider: - you might be comparing B-factors coming from local atomic vibrations alone (residual B, or whatever it's called), and the total atomic B-factor, that includes all components: Btotal = Btls + Bresidual +... . (for comprehensive review on this topic see: TLS from fundamentals to practice, Crystallography Reviews, (2013), 19:4, 230-270) - there may be a number of valid reasons for the mean B-factor to deviate from Wilson B. - as Ethan mentioned, obviously your goal is not to preserve the original B-factors. All the best, Pavel On Thu, Jun 5, 2014 at 8:51 AM, Carles Palanca i García cpala...@ibv.csic.es wrote: Hello, I'm almost done refining a protein-DNA complex at 3A, but when I apply TLS (Refmac+TLSanl to include the ANISOU component) the B factors increase from 50-60 to 100-110 A2. The crystal is a bit special since it has 80% of solvent and the Wilson B-factor is 87 A2. When I compare my Bfactors with other structures of similar resolution using Phenix Validation, my data is clearly an outlier. To perform the TLS refinement I have generated .tlsin files for 2,4 and 6 groups with the TLSmd server and used them in refmac jobs consisting of 20 cycles of TLS refinement+50 cycles of restrained refinement. This way the R-factor and R-free values have drop 1-2% (before it was 22.3/24.1). The three different options suffer from the same problem regarding high B-factors after TLSanl. Am I doing something wrong during the TLS refinement? What can I do to preserve the original Bfactors? Thanks in advance, Carles Palanca.
Re: [ccp4bb] Bfactors too high after TLS refinement
Dear Carles,
it used to be the case (and probably still is) that you had to specify
BRESID false
when using tlsanl with a PDB file from refmac5. It seems that 'TLSOUT
ADDU' specified for refmac5 allows you to use tlsanl with its default
value 'true'.
The incorrect mixture might lead to an artifical increase or decrease of
Biso.
Best,
Tim
On 06/05/2014 05:51 PM, Carles Palanca i García wrote:
P { margin-bottom: 0.21cm; }
Hello,
I'm almost done refining a protein-DNA
complex at 3A, but when I apply TLS (Refmac+TLSanl to include the
ANISOU component) the B factors increase from 50-60 to 100-110 A2.
The crystal is a bit special since it has 80% of solvent and the
Wilson B-factor is 87 A2. When I compare my Bfactors with other
structures of similar resolution using Phenix Validation, my data is
clearly an outlier.
To perform the TLS refinement I have
generated .tlsin files for 2,4 and 6 groups with the TLSmd server and
used them in refmac jobs consisting of 20 cycles of TLS refinement+50
cycles of restrained refinement. This way the R-factor and R-free
values have drop 1-2% (before it was 22.3/24.1). The three different
options suffer from the same problem regarding high B-factors after
TLSanl. Am I doing something wrong during the TLS refinement? What
can I do to preserve the original Bfactors?
Thanks in advance,
Carles Palanca.
--
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen
GPG Key ID = A46BEE1A
signature.asc
Description: OpenPGP digital signature
[ccp4bb] Deadline Approaches for Registration to the Northwest Crystallographic Workshop
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 (Pacific) Northwest Crystallography Workshop http://oregonstate.edu/conferences/event/nwcw2014/ June 20-22, 2014 Summer is fast approaching and so is the Northwest Crystal- lography Workshop to be held here in beautiful Corvallis Oregon. The last day to register is next Tuesday, June 10th. If you are planing to attend but have not registered, you'd best get to it! This workshop has been held at various locations in the Pacific Northwest since 1981. It has always proven to be a great venue to meet other researchers in the region who are interested both in using macromolecular crystallography to solve structures and in developing and enhancing methods. This will be a cozy meeting with lots to learn and plenty of networking opportunities. Dale and Andy - -- Dale E. Tronrud and P. Andrew Karplus Department of Biochemistry and Biophysics 2011 ALS Bldg Oregon State University Corvallis, OR 97331 -BEGIN PGP SIGNATURE- Version: GnuPG v2.0.22 (MingW32) Comment: Using GnuPG with Thunderbird - http://www.enigmail.net/ iEYEARECAAYFAlOQtYEACgkQU5C0gGfAG10MBgCgozEbT3eZohyTRBkwj7sN6Bwi 2gcAoLaKt9Xw+KAgs4HX6+fuNDTHBkLR =sVDs -END PGP SIGNATURE-
[ccp4bb] Beamtime @ SLS
=== SYNCHROTRON BEAM TIME FOR MACROMOLECULAR CRYSTALLOGRAPHY AT SLS === Proposal application deadline: Sunday, June 15, 2014 Periods: September 1, 2014 - December 31, 2014 (Normal / Test proposals) September 1, 2014 - August 31, 2016 (Long-term proposals) Proposal submission: http://www.psi.ch/sls/px-beamlines-call-for-proposals Travel support: http://www.psi.ch/useroffice/sls-elisa-biostruct PSI DUO application for iphone: http://itunes.apple.com/ch/app/psi-duo/id375328818?mt=8 X06SA Beamline features (http://www.psi.ch/sls/pxi/pxi) - Undulator beamline with flux of 2x10^12 photons/sec at 12.4 keV (1Å) and fully tunable from 6.0 to 17.5 keV (2.07 - 0.71 Å) - Focused beam size and divergency: HRD - 85x10 microns and 0.35x0.06 mrad; MD2 - 25x5 microns and 0.5x0.4 mrad - PILATUS 6M pixel detector at the High Resolution Diffractometer, allowing continuous, fine phi-sliced data acquisition (25 frames per second) with 20 bit dynamic range (see http://pilatus.web.psi.ch/ or www.dectris.com for further information) - MAR225 CCD at Micro-Diffractometer MD2, allowing data collection with a focussed beam size of 25 x 5 micrometers, and smaller beam size with triple-aperture assembly ( 5 x 5, 10 x 10, 20 x 20 micrometer). X06DA Beamline features (http://www.psi.ch/sls/pxiii/pxiii) - Super-bending magnet beamline with flux of 5x10¹¹ photons/sec at 12.4 keV (1Å) and fully tunable from 6.0 to 17.5 keV (2.07 - 0.71 Å) - Focused beam size and divergency: 80x45 microns and 2x0.5 mrad (with possibility to reduce horizontal divergency to 0.4 mrad) - Mini-hutch design for fast manual mounting - PILATUS 2M (60 Hz, 450 um Si sensor) - Multi-axis goniometer (PRIGO) for crystal re-orientation - New phasing protocols with energy interleaving and multi-orientation strategy - In-situ X-ray diffraction screening (with any SBS format plate) available during users shifts (R. Bingel-Erlenmeyer, et al., Crystal Growth Design 2011, 11, 916) X10SA Beamline features (http://www.psi.ch/sls/pxii/pxii) - Undulator beamline with flux of 2x10^12 photons/sec at 12.4 keV (1Å) and fully tunable from 6.0 to 20 keV (2.07 - 0.62 Å) - Focused beam size and divergency: 50x10 microns and 0.6x0.1 mrad - Micro-beam with apertures (10 x 10, 30 x 30 micrometer) - PILATUS 6M pixel detector Best regards, The MX group at SLS __ Meitian Wang Swiss Light Source at Paul Scherrer Institut CH-5232 Villigen PSI - http://www.psi.ch/sls/ Phone: +41 56 310 4175
[ccp4bb] DEADLINE EXTENDED: CALL FOR PROPOSALS: neutron beam time at the Protein Crystallography Station at LANL
Dear Colleagues, The Protein Crystallography Station (PCS) is a high performance neutron beamline located at the spallation neutron source at the Los Alamos Neutron Science Center at Los Alamos National Laboratory. The PCS has a long-standing history of serving the neutron crystallographic community. The PCS is equipped with a 3He detector, offering high sensitivity and an adjustable 2-theta arm, allowing for collection of ultra-high resolution data (up to 1.1 Å data has been collected) using crystals as small as 0.3 mm3. This is an excellent opportunity for traditional X-ray crystallographers interested in diverse aspects of structural biology such as enzyme mechanisms, protein dynamics, hydrogen bonding networks, and protonation states, to determine neutron derived structures of their target proteins. We also offer mail-in services for your protein crystals and routinely screen crystals using our in-house X-ray diffractometer. Full support for protein expression, purification, deuteration, and crystallization is also available, housed in a new laboratory space. We are accepting proposals for our upcoming neutron beam run cycle starting 7 October 2014 and running until 26 February 2015. The deadline for receipt of proposals has been extended to 13 June 2014. Proposals may be submitted by clicking on the “Apply for Beamtime” link in the left column of the Lujan home page: http://lansce.lanl.gov/lujan. Additional information about the PCS can be found at http://lansce.lanl.gov/lujan/instruments/PCS.shtml. We strongly encourage you to contact us for questions concerning sample preparation, instrument capabilities, sample environments, and/or proposal preparation. Julian Chen, che...@lanl.gov John Bacik, jba...@lanl.gov Clifford Unkefer, c...@lanl.gov
