[ccp4bb] MIND in shelxd
Dear all How to decide for minimum distance between heavy atoms in shelxd esp w.r.t. S-SAD. Thanx in advance Monica
[ccp4bb] shelxd
Dear all I am naive in phasing experiments. CAn anyone please guide how to do the following: In a S-SAD for *SHELXD* searches, try various high-resolution cutoffs for example 2.5 to 5 Å in steps of 0.1 Å. Based on these attempts, use a high-resolution cutoff at for example 3.8 Å for substructure determination with an *E* min value of for example 1.6 to search for X no. of protein sulfur sites. Thanx in advance Monica
[ccp4bb] AW: [ccp4bb] coot problems to decrease R FREE
Dear Peter, First a common misconception: your goal should be to get the best possible fit of your model to your electron density maps. Rfree is only an indicator, telling you whether you are moving in the right direction. So in coot, you should look for places where your model does not fit the electron density very well and correct them. Things to look for are peaks (positive and negative) in the electron density maps. Here I use the default of +/- 3 sigma. You should also look for parts of your model with weak or bad electron density, as well as uninterpreted electron density. Good parts usually have density above the 1.5 sigma level, but for weak parts, you could go down to ~0.6 sigma. Below that, you get into the noise/artefact level. Things to do are fitting side chains in different orientations, flipping peptides, removing disordered loops with no or very bad density, fitting extra residues where there is extra density etc. In certain cases, you may have to rebuild complete loops. You are done with your refinement when you see nothing any more that could be improved. Good luck! Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von ?? Gesendet: Montag, 21. April 2014 02:34 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] coot problems to decrease R FREE Dear all, I'm a novice of coot and ccp4. Now I'm doing refinement using both refmac5 and coot.Here are some problems I'm facing. Really hope you can give me some suggestions. 1、THE RESOLUTION OF THE DATA IS 2.5 angstrom. After first refinement of refmac5 I got R factor which is 0.26 and R FREE which is 0.31. My question is what the final R factor and R FREE should be after several rounds of refinement by refmac5 and coot. 2、At which map level（e/A3 or rmsd）should I refine the data by coot? 3、Can you give me some tips and strategies about how to use coot to decrease R free? now I just use some basic tricks such as fit density and Ramachandran plot to refine the data. Best regards, Peter Chen
Re: [ccp4bb] shelxd
Dear Monica, if you are new to phasing I recommend you first use the ``standard'' values in shelx c/d/e, either using one of my tutorials or, even better, the GUI hkl2map, available at http://webapps.embl-hamburg.de/hkl2map/. If your native data are about 2A or better, and your structure is a protein, shelxe should be able to trace a major part of it even from very poor starting phases, making it unnecessary to try the below hint. If you do, shelxd is steared by an input file containing various keywords. One is the SHEL command with the upper and lower resolution limit. If you prepare 26 input files (all with different names) and vary the second number in SHEL from 2.5 2.6 2.7 ... 5.0 you scan the different resolution cut-offs. However, shelxd has become less sensitive to the resolution cut-off, in particular in combination with auto-tracing in shelxe. The second part, Emin, refers to the keyword ESEL. Its default value is 1.5. If you lower the number to 1.2, say, shelxd uses more data for locating the substructure, but since this way you may also include noise, the exact number is difficult to tell in advance. Best wishes, Tim On 04/22/2014 10:13 AM, Monica Mittal wrote: Dear all I am naive in phasing experiments. CAn anyone please guide how to do the following: In a S-SAD for *SHELXD* searches, try various high-resolution cutoffs for example 2.5 to 5 Å in steps of 0.1 Å. Based on these attempts, use a high-resolution cutoff at for example 3.8 Å for substructure determination with an *E* min value of for example 1.6 to search for X no. of protein sulfur sites. Thanx in advance Monica -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
[ccp4bb] Dehydration
Dear all, Right now, we are try to crystallise an protein complex. By all kinds of efforts, the resolution of the crystal was about 8 angstrom. So we would like to try dehydration. The precipitant that our crystal grew was ethanol. Since the evaporation of the ethanol, our crystal is unstable at the room temperature or 4 degree when it was exposed to the air directly. Do any people have some suggestions about the dehydration way of our crystal? Best Yahui
Re: [ccp4bb] shelxd
I think what one uses will depend on what one expects to be in the structure and the resolution and the quality of their diffraction data. I usually start with 1.8 Å resolution data in case there is chance of having disulfides. Then 1.9, then 2.4, then 2.0, then 2.6, then 2.2, then 1.85, then 1.75, …. If there are disulfides, then there is the DSUL option if the resolution of the diffraction data is not conducive to separating the individual sulfur atoms. One should also change the number of sites that SHELXD is looking for, perhaps in a systematic way. For example, one might be looking for sulfurs based on amino acid sequence, but there may be calcium, zinc, manganese, or other strong anomalous scattering atoms in the crystal that would make searching for sulfurs moot or at least problematic. Jim From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Monica Mittal [monica.mitta...@gmail.com] Sent: Tuesday, April 22, 2014 3:13 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] shelxd Dear all I am naive in phasing experiments. CAn anyone please guide how to do the following: In a S-SAD for SHELXD searches, try various high-resolution cutoffs for example 2.5 to 5 Å in steps of 0.1 Å. Based on these attempts, use a high-resolution cutoff at for example 3.8 Å for substructure determination with an E min value of for example 1.6 to search for X no. of protein sulfur sites. Thanx in advance Monica
[ccp4bb] Tenure track position in Protein Chemistry, Leiden University, NL
Dear all, Leiden University, The Netherlands has a tenure track position available in its Protein Chemistry department. For details, please see the following site: http://werkenbij.leidenuniv.nl/vacatures/wetenschappelijke-functies/14-118-vacature-universiteit-leiden-tenure-track-position-in-protein-chemistry.html This message was posted on behalf of Marcellus Ubbink (Head of the Protein Chemistry department) and Jaap Brouwer (Head of the Chemistry institute). Best wishes, Navraj
Re: [ccp4bb] Non-catalytic Rossmann fold domain
Dear Jack, In this recent review paper (http://www.biomedcentral.com/1472-6807/13/6), I read about non-catalytic Rossmannn fold proteins which include mitochondrial transcription factor B (sc-mtTFB) (http://www.ncbi.nlm.nih.gov/pubmed/12897151) and a t-RNA (1-methyladenosine) MTase from Saccharomyces cerevisiae (http://www.ncbi.nlm.nih.gov/pubmed/11684083). Best wishes, Avinash Punekar Uppsala University
Re: [ccp4bb] Non-catalytic Rossmann fold domain
DprA, from the Streptococcus pneumoniae transformasome, is one, I believe: http://www.ncbi.nlm.nih.gov/m/pubmed/22904190/ On 22 Apr 2014, at 14:26, Tanner, John J. tanne...@missouri.edu wrote: Does anyone know of a protein that has a Rossmann dinucleotide binding domain that does not participate in cofactor/substrate binding? Thanks. Jack Tanner John J. Tanner Professor of Biochemistry and Chemistry University of Missouri-Columbia 125 Chemistry Building Columbia, MO 65211 email: tanne...@missouri.edu phone: 573-884-1280 fax: 573-882-2754 http://faculty.missouri.edu/~tannerjj/tannergroup/tanner.html
Re: [ccp4bb] Trouble Refining Ligand in Phenix
Actually, that was the issue. I manually reset the occupancies of the problematic atoms in the PDB to unity. This solved my problem. Thanks to Nat and Tom. Best, Chris On 4/21/14, tom.p...@csiro.au tom.p...@csiro.au wrote: Hello Chris, This probably isn't your problem, but I once put some atoms into difference density (in Coot) and for some reason the occupancy was set to zero, so although everything was refined, the density was the same as before (massive difference density). You might want to just check. cheers, tom Tom Peat Biophysics Group CSIRO, CMSE 343 Royal Parade Parkville, VIC, 3052 +613 9662 7304 +614 57 539 419 tom.p...@csiro.au From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Chris Fage [cdf...@gmail.com] Sent: Tuesday, April 22, 2014 8:21 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Trouble Refining Ligand in Phenix Hi Everyone, I am trying to refine a structure with a phosphorylated amino acid. After refining in Phenix, the Fo-Fc density (green) overlaps the 2Fo-Fc density for all atoms of the derivatized amino acid in Coot, almost as if I had not built in the residue. I am loading a .cif for the derivative when I run phenix.refine. I have also tried ReadySet, but when I click the Run in phenix.refine button, I see the message Error interpreting command line argument as parameter definition: refine_65-coot-2.metal.edits RuntimeError: Unexpected end of output. Am I just seeing noise, or is Phenix not actually refining this portion of the model? I would appreciate any suggestions. Best, Chris
[ccp4bb] Problem with making a continuous nucleic acid chain
Dear all: I am facing a display issue in pymol. In my structure, there is a nucleic acid chain with some monomers which are modified nucleotides built in scketcher of ccp4 suite. However, When I showed the structure in pymol as cartoon, the nucleic acid chain was discontinuous where the monomers are placed. I could not get a intact nucleic acid chain except setting the cartoon_nucleic_acid_mode value to 1, with the backbone passing through ribose C3' atoms. I tried to add SEQRES lines into the pdb file according to the pdb instruction, but it doesn't work if I want the backbone to pass through phosphorus atoms. May I get some suggestions from you? Although it's not a big problem, it's really painful. Thanks a lot and best, Xiaoming
[ccp4bb] 2nd Australian Advanced Methods in Crystallography Workshop - New registration deadline
Dear all, The registration deadline to the 2nd Australian Advanced Methods in Crystallography Workshop has been extended. Registration deadline: 6th May 2014 Workshop dates: 22-25th June 2014 Website: https://events.synchrotron.org.auhttps://events.synchrotron.org.au/ Contact: xtalworkshop2...@synchrotron.org.aumailto:xtalworkshop2...@synchrotron.org.au Best regards, Sofia Caria (by the organising committee) [cid:7A5AF159-4877-40FE-AF2E-F4EFD9468592] Organising committee: Daniel Eriksson- Australian Synchrotron David Aragão - Australian Synchrotron Jade Forwood - Charles Sturt University Mihwa Lee - La Trobe University Sofia Caria - La Trobe University Stephanie Gras - Monash University
