Re: [ccp4bb] Issue with high Rfree (0.25) for a high-resolution dataset (1.05 Ang)
Hi Pavel, Obviously higher resolution typically means a more accurate atomic model of a crystal structure, but I also think that a 1.05 Ang structure is only going to provide you with a great deal more insight than a 1.1 or 1.2 Ang structure in very specific cases. Particularly if your stats aren't completely ideal , why not make sure you get the best possible stats out of your data by slightly cutting the resolution, which is still going to leave you with a VERY high resolution data set (at least I would consider 1.1 or 1.2 Ang as very high resolution)? Please forgive my ignorance, but I have to admit I haven't researched this topic. Is there something wrong with using TLS refinement coupled with anisotropic refinement in Refmac? I just checked and I have found that TLS still provides a small drop in the R factors when comparing anisotropic refinement to TLS + anisotropic refinement in the 1.2 Ang data set I'm currently working on. Ok, it's just under 0.5%, which I admit isn't a huge change, but shouldn't I take any improvement I can get? Cheers, Tony -- Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE e-mail: antonio.ar...@path.ox.ac.uk<mailto:antonio.ar...@path.ox.ac.uk> Tel: 00 +44 1865 285655 Links to my public profiles: ResearchGate<https://www.researchgate.net/profile/Antonio_Ariza> LinkedIn<https://www.linkedin.com/in/antonioariza1> GoogleScholar<https://scholar.google.co.uk/citations?user=9pAIKV0J=en> Twitter<https://twitter.com/DrAntonioAriza?lang=en> Check out my latest paper!!! Structural insights into the function of ZRANB3 in replication stress response<http://www.nature.com/articles/ncomms15847> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Issue with high Rfree (0.25) for a high-resolution dataset (1.05 Ang)
Hi Guto, I always feel some people are too "greedy" with the resolution they want to achieve. I mostly find that extremely high density is a pain to work with as it's usually accompanied by many dual, triple conformers, a lot of noise in the solvent phase that is often difficult to interpret, etc... Often you spend more time on a high resolution structure that clearly shows your protein, as opposed to a low resolution structure where it's difficult to interpret parts of the map. Also, in most cases you REALLY don't need a 1 Ang map to clearly show the overall structure of your protein, ligands, first shell of solvent molecules on the surface of your protein, etc... Your completeness is 90% in your high resolution shell, which is fine, but have you checked you can clearly see most reflections for h, k and l? Maybe you're missing many reflections for one of them. I would at least try cutting your data back to 1.1 or 1.2 Ang, as it might dramatically improve your R factors and still show everything you want to show. Also, did you try TLS refinement? Best regards, Tony -- Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE e-mail: antonio.ar...@path.ox.ac.uk<mailto:antonio.ar...@path.ox.ac.uk> Tel: 00 +44 1865 285655 Links to my public profiles: ResearchGate<https://www.researchgate.net/profile/Antonio_Ariza> LinkedIn<https://www.linkedin.com/in/antonioariza1> GoogleScholar<https://scholar.google.co.uk/citations?user=9pAIKV0J=en> Twitter<https://twitter.com/DrAntonioAriza?lang=en> Check out my latest paper!!! Structural insights into the function of ZRANB3 in replication stress response<http://www.nature.com/articles/ncomms15847> From: CCP4 bulletin board on behalf of Guto Rhys Sent: 09 October 2018 18:12 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Issue with high Rfree (0.25) for a high-resolution dataset (1.05 Ang) Hi all, I have a 1.05 Angstrom dataset that I was able to phase but the refined model only has an Rfree of approximately 0.25. The dataset includes 1800 images and, as the crystal did not suffer significantly from radiation damage, comprises all 360 deg. Auto-processing pipelines at diamond light source all suggest I222. I have also indexed the data in iMOSFLM, which has the highest-symmetry Laue group that is least penalised of I422. Subsequent scaling and merging in AIMLESS strongly indicates that I222 is the likely space group (see below). I have ran the refined model through ZANUDA, which has similar R values to lower symmetry space groups (see below). The output from Phenix Xtriage does not find any specific crystal pathologies and if twinning is present it is very low (2 to 4%, see below). The difference map suggests that the model accounts for nearly all the density. Any ideas or direction would be greatly appreciated. Best, Guto AIMLESS Summary Overall InnerShell OuterShell Low resolution limit 27.75 27.75 1.07 High resolution limit 1.05 5.75 1.05 Rmerge (within I+/I-) 0.050 0.078 0.466 Rmerge (all I+ and I-)0.051 0.080 0.536 Rmeas (within I+/I-) 0.055 0.086 0.591 Rmeas (all I+ & I-)0.054 0.085 0.613 Rpim (within I+/I-)0.023 0.034 0.359 Rpim (all I+ & I-) 0.017 0.028 0.288 Rmerge in top intensity bin0.049- - Total number of observations 107950 779 1972 Total number unique1131588 486 Mean((I)/sd(I)) 19.7 46.2 1.8 Mn(I) half-set correlation CC(1/2) 0.998 0.994 0.796 Completeness99.1 99.2 90.4 Multiplicity 9.5 8.9 4.1 Anomalous completeness 98.1 100.0 79.1 Anomalous multiplicity 5.0 6.4 2.2 DelAnom correlation between half-sets -0.067 0.286 0.097 Mid-Slope of Anom Normal Probability 0.789 - - Estimate of maximum resolution for significant anomalous signal = 1.14A, from CCanom > 0.15 Estimates of resolution limits: overall from half-dataset correlation CC(1/2) > 0.30: limit = 1.05A == maximum resolution from Mn(I/sd) > 1.50: limit = 1.05A == maximum resolution from Mn(I/sd) > 2.00: limit = 1.07A Estimates of resolution limits in reciprocal lattice directions: Along h axis from half-dataset correlation CC(1/2) > 0.30: limit = 1.06A from Mn
Re: [ccp4bb] conversion from pdb to cif format changes R factors ... why?
Hi Christian, Yes, I used TLS refinement for all the structures I deposited … but the TLS parameters were included in the deposition task, so I assumed the programme would know that it needs to take these into account. Anyway … I guess that in order to correct the remarks in the headers of the curated coordinates in the PDB, I'll have to alter all the cif files manually and then resubmit them to the deposition server. Cheers, Tony -- Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE e-mail: antonio.ar...@path.ox.ac.uk<mailto:antonio.ar...@path.ox.ac.uk> Tel: 00 +44 1865 285655 Links to my public profiles: ResearchGate<https://www.researchgate.net/profile/Antonio_Ariza> LinkedIn<https://www.linkedin.com/in/antonioariza1> GoogleScholar<https://scholar.google.co.uk/citations?user=9pAIKV0J=en> Twitter<https://twitter.com/DrAntonioAriza?lang=en> Check out my latest paper!!! Structural insights into the function of ZRANB3 in replication stress response<http://www.nature.com/articles/ncomms15847> From: Christian Roth Sent: 02 October 2018 20:52 To: Antonio Ariza; CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] conversion from pdb to cif format changes R factors ... why? Hi Tony, is that for all structures or just some? I recall there was a problem when one has used TLS refinement. The deposition task runs a 0 cycle refmac job to recreate the statistics. If there is a difference, e.g no TLS included that might explain the difference. Cheers Christian Am 02.10.2018 um 19:25 schrieb Antonio Ariza: Hi all, I'm wondering if anyone else has noticed this ... or is there something I am missing here? One of the reviewers of my latest manuscript just pointed out that the R factors in my validation reports are significantly different (i.e. worse) than those I stated in the paper. So, I checked and indeed they are. I tracked the difference in the numbers down to the point where I used "Prepare files for deposition" in ccp4i2. This is the first time I used this programme to convert my pdb and mtz files into cif files, instead of trying to deposit the pdb and mtz files as I've always done. The conversion, however, changed the R factors ... and seemingly nothing else as the coordinates of both files are still the same. Any idea why the programme does this? Is there another programme I can use that doesn't do this? It's annoying because now I have to redeposit the data for the 10 structures I've submitted for this paper. Here are the remarks of the pdb file and the remarks of the resulting cif file, as you can see they are the same except for the R factors. PDB: REMARK 3 DATA USED IN REFINEMENT. REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) : 1.67 REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) : 49.13 REMARK 3 DATA CUTOFF(SIGMA(F)) : NONE REMARK 3 COMPLETENESS FOR RANGE(%) : 97.26 REMARK 3 NUMBER OF REFLECTIONS : 35463 REMARK 3 REMARK 3 FIT TO DATA USED IN REFINEMENT. REMARK 3 CROSS-VALIDATION METHOD : THROUGHOUT REMARK 3 FREE R VALUE TEST SET SELECTION : RANDOM REMARK 3 R VALUE (WORKING + TEST SET) : 0.20233 REMARK 3 R VALUE(WORKING SET) : 0.20068 REMARK 3 FREE R VALUE : 0.23658 REMARK 3 FREE R VALUE TEST SET SIZE (%) : 4.7 REMARK 3 FREE R VALUE TEST SET COUNT : 1759 REMARK 3 REMARK 3 FIT IN THE HIGHEST RESOLUTION BIN. REMARK 3 TOTAL NUMBER OF BINS USED : 20 REMARK 3 BIN RESOLUTION RANGE HIGH :1.670 REMARK 3 BIN RESOLUTION RANGE LOW:1.714 REMARK 3 REFLECTION IN BIN (WORKING SET) : 2221 REMARK 3 BIN COMPLETENESS (WORKING+TEST) (%) :82.65 REMARK 3 BIN R VALUE (WORKING SET) :0.392 REMARK 3 BIN FREE R VALUE SET COUNT : 113 REMARK 3 BIN FREE R VALUE:0.390 CIF: REMARK 3 DATA USED IN REFINEMENT. REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) : 1.67 REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) : 49.13 REMARK 3 DATA CUTOFF(SIGMA(F)) : NONE REMARK 3 COMPLETENESS FOR RANGE(%) : 97.26 REMARK 3 NUMBER OF REFLECTIONS : 35463 REMARK 3 REMARK 3 FIT TO DATA USED IN REFINEMENT. REMARK 3 CROSS-VALIDATION METHOD : THROUGHOUT REMARK 3 FREE R VALUE TEST SET SELECTION : RANDOM REMARK 3 R VALUE (WORKING + TEST SET) : 0.24316 REMARK 3 R VALUE(WORKING SET) : 0.24133 REMARK 3 FREE R VALUE : 0.28140 REMARK 3 FREE R VALUE TEST SET SIZE (%) : 4.7 REMARK 3 FREE R VALUE TEST SET COUNT : 1759 REMARK 3 REMARK 3 FIT IN THE HIGHEST RESOLUTION BIN. REMARK 3 TOTAL NUMBER OF
[ccp4bb] conversion from pdb to cif format changes R factors ... why?
Hi all, I'm wondering if anyone else has noticed this ... or is there something I am missing here? One of the reviewers of my latest manuscript just pointed out that the R factors in my validation reports are significantly different (i.e. worse) than those I stated in the paper. So, I checked and indeed they are. I tracked the difference in the numbers down to the point where I used "Prepare files for deposition" in ccp4i2. This is the first time I used this programme to convert my pdb and mtz files into cif files, instead of trying to deposit the pdb and mtz files as I've always done. The conversion, however, changed the R factors ... and seemingly nothing else as the coordinates of both files are still the same. Any idea why the programme does this? Is there another programme I can use that doesn't do this? It's annoying because now I have to redeposit the data for the 10 structures I've submitted for this paper. Here are the remarks of the pdb file and the remarks of the resulting cif file, as you can see they are the same except for the R factors. PDB: REMARK 3 DATA USED IN REFINEMENT. REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) : 1.67 REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) : 49.13 REMARK 3 DATA CUTOFF(SIGMA(F)) : NONE REMARK 3 COMPLETENESS FOR RANGE(%) : 97.26 REMARK 3 NUMBER OF REFLECTIONS : 35463 REMARK 3 REMARK 3 FIT TO DATA USED IN REFINEMENT. REMARK 3 CROSS-VALIDATION METHOD : THROUGHOUT REMARK 3 FREE R VALUE TEST SET SELECTION : RANDOM REMARK 3 R VALUE (WORKING + TEST SET) : 0.20233 REMARK 3 R VALUE(WORKING SET) : 0.20068 REMARK 3 FREE R VALUE : 0.23658 REMARK 3 FREE R VALUE TEST SET SIZE (%) : 4.7 REMARK 3 FREE R VALUE TEST SET COUNT : 1759 REMARK 3 REMARK 3 FIT IN THE HIGHEST RESOLUTION BIN. REMARK 3 TOTAL NUMBER OF BINS USED : 20 REMARK 3 BIN RESOLUTION RANGE HIGH :1.670 REMARK 3 BIN RESOLUTION RANGE LOW:1.714 REMARK 3 REFLECTION IN BIN (WORKING SET) : 2221 REMARK 3 BIN COMPLETENESS (WORKING+TEST) (%) :82.65 REMARK 3 BIN R VALUE (WORKING SET) :0.392 REMARK 3 BIN FREE R VALUE SET COUNT : 113 REMARK 3 BIN FREE R VALUE:0.390 CIF: REMARK 3 DATA USED IN REFINEMENT. REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) : 1.67 REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) : 49.13 REMARK 3 DATA CUTOFF(SIGMA(F)) : NONE REMARK 3 COMPLETENESS FOR RANGE(%) : 97.26 REMARK 3 NUMBER OF REFLECTIONS : 35463 REMARK 3 REMARK 3 FIT TO DATA USED IN REFINEMENT. REMARK 3 CROSS-VALIDATION METHOD : THROUGHOUT REMARK 3 FREE R VALUE TEST SET SELECTION : RANDOM REMARK 3 R VALUE (WORKING + TEST SET) : 0.24316 REMARK 3 R VALUE(WORKING SET) : 0.24133 REMARK 3 FREE R VALUE : 0.28140 REMARK 3 FREE R VALUE TEST SET SIZE (%) : 4.7 REMARK 3 FREE R VALUE TEST SET COUNT : 1759 REMARK 3 REMARK 3 FIT IN THE HIGHEST RESOLUTION BIN. REMARK 3 TOTAL NUMBER OF BINS USED : 20 REMARK 3 BIN RESOLUTION RANGE HIGH :1.670 REMARK 3 BIN RESOLUTION RANGE LOW:1.714 REMARK 3 REFLECTION IN BIN (WORKING SET) : 2221 REMARK 3 BIN COMPLETENESS (WORKING+TEST) (%) :82.65 REMARK 3 BIN R VALUE (WORKING SET) :0.426 REMARK 3 BIN FREE R VALUE SET COUNT : 113 REMARK 3 BIN FREE R VALUE:0.429 Cheers, Tony -- Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE e-mail: antonio.ar...@path.ox.ac.uk<mailto:antonio.ar...@path.ox.ac.uk> Tel: 00 +44 1865 285655 Links to my public profiles: ResearchGate<https://www.researchgate.net/profile/Antonio_Ariza> LinkedIn<https://www.linkedin.com/in/antonioariza1> GoogleScholar<https://scholar.google.co.uk/citations?user=9pAIKV0J=en> Twitter<https://twitter.com/DrAntonioAriza?lang=en> Check out my latest paper!!! Structural insights into the function of ZRANB3 in replication stress response<http://www.nature.com/articles/ncomms15847> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] I used up all CCP4 projects ... seemingly ...
Hi all and sorry ... (I thought it best to apologise pre-emptively for this rather long question). Ok ... so, I know I should have changed over to CCP4i2 a LNG time ago, but a student in my lab had trouble transferring his projects from CCP4i to CCP4i2, so I decided to postpone it ... and I kept postponing it ever since. I have now run into a problem with the old CCP4i gui, which means I might have to change over to CCP4i2. It seems I have run out of projects! Over the last 4 years I have created 251 projects. After I created the last one and worked on it for a while, I went and revisited an older project. Now I can't access the last project I created from the gui anymore. The last project I created would, alphabetically speaking, also be the last project in the list that appears when you press the "Change Project" button at the top of the gui. However, the list that appears seems to only have enough spaces for 250 project names, which means it shows the first 250 projects (alphabetically) and the last project isn't on it. When I press the "Directories" button I can see I did indeed create the project that I'm looking for and when I check the computer, all the files for this project are in their corresponding directory. I guess, if I were to create a 252nd project with a title that would fall towards the beginning of the alphabetical project list, the gui would let me do that, but I would then lose what is currently the last project I can see on the list, which means I would have 2 projects I can't access via the gui. If I start using CCP4i2 and copy all my old projects across, will it have the same problem? Has anybody else with a large number of projects run into this problem? Should I re-create only the 251st project and set up all new projects in CCP4i2 and keep the old projects for use in CCP4i instead? Cheers, Tony ------ Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE e-mail: antonio.ar...@path.ox.ac.uk<mailto:antonio.ar...@path.ox.ac.uk> Tel: 00 +44 1865 285655 Links to my public profiles: ResearchGate<https://www.researchgate.net/profile/Antonio_Ariza> LinkedIn<https://www.linkedin.com/in/antonioariza1> GoogleScholar<https://scholar.google.co.uk/citations?user=9pAIKV0J=en> Twitter<https://twitter.com/DrAntonioAriza?lang=en> Check out my latest paper!!! Structural insights into the function of ZRANB3 in replication stress response<http://www.nature.com/articles/ncomms15847> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] Post Doc - University of Oxford - Ivan Ahel Lab (with correct link this time)
Postdoctoral Research Assistant – Electron Microscopy Sir William Dunn School of Pathology, South Parks Road, Oxford Grade 7: £31,604 - £38,833 p.a. A joint postdoctoral position is available for Dr Ivan Ahel’s and Dr Dragana Ahel’s groups at the Sir William Dunn School of Pathology, University of Oxford to study the structure of novel DNA repair complexes. The project will involve structural biology techniques, largely electron microscopy. For structural studies, our labs have regular access to several synchrotron facilities (Diamond, ESRF, SOLEIL, and DESY), as well as to the new CryoEM facility in our Department that includes the FEI Titan Krios instrument. The post is available for 2 years in the first instance. Applicants should have a PhD in biology or a related subject. A high level of competence in structural biology is required and relevant experience demonstrated by first author publications in high-profile journals. The ideal candidate should be organised, highly motivated and able to work independently as well as a part of a team. Informal enquiries should be directed to Dr Ivan Ahel: ivan.a...@path.ox.ac.uk<mailto:ivan.a...@path.ox.ac.uk>, or Dr Dragana Ahel: dragana.a...@path.ox.ac.uk<mailto:dragana.a...@path.ox.ac.uk>. If you are interested in this role, and have the skills and experience we are looking for, please apply online by using the link below. You will be required to upload a CV and supporting statement as part of your online application. The closing date for applications is 12.00 midday on Friday 29 June 2018. Interviews will be held as soon as possible thereafter. https://www.recruit.ox.ac.uk/pls/hrisliverecruit/erq_jobspec_details_form.jobspec?p_id=135155 -- Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE e-mail: antonio.ar...@path.ox.ac.uk<mailto:antonio.ar...@path.ox.ac.uk> Tel: 00 +44 1865 285655 Links to my public profiles: ResearchGate<https://www.researchgate.net/profile/Antonio_Ariza> LinkedIn<https://www.linkedin.com/in/antonioariza1> GoogleScholar<https://scholar.google.co.uk/citations?user=9pAIKV0J=en> Twitter<https://twitter.com/DrAntonioAriza?lang=en> Check out my latest paper!!! Structural insights into the function of ZRANB3 in replication stress response<http://www.nature.com/articles/ncomms15847> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] Postdoctoral Research Assistant – Oxford University, UK - Ivan Ahel Lab
Postdoctoral Research Assistant – Electron Microscopy Sir William Dunn School of Pathology, South Parks Road, Oxford Grade 7: £31,604 - £38,833 p.a. A joint postdoctoral position is available for Dr Ivan Ahel’s and Dr Dragana Ahel’s groups at the Sir William Dunn School of Pathology, University of Oxford to study the structure of novel DNA repair complexes. The project will involve structural biology techniques, largely electron microscopy. For structural studies, our labs have regular access to several synchrotron facilities (Diamond, ESRF, SOLEIL, and DESY), as well as to the new CryoEM facility in our Department that includes the FEI Titan Krios instrument. The post is available for 2 years in the first instance. Applicants should have a PhD in biology or a related subject. A high level of competence in structural biology is required and relevant experience demonstrated by first author publications in high-profile journals. The ideal candidate should be organised, highly motivated and able to work independently as well as a part of a team. Informal enquiries should be directed to Dr Ivan Ahel: ivan.a...@path.ox.ac.uk<mailto:ivan.a...@path.ox.ac.uk>, or Dr Dragana Ahel: dragana.a...@path.ox.ac.uk<mailto:dragana.a...@path.ox.ac.uk>. If you are interested in this role, and have the skills and experience we are looking for, please apply online by using the link below. You will be required to upload a CV and supporting statement as part of your online application. The closing date for applications is 12.00 midday on Friday 29 June 2018. Interviews will be held as soon as possible thereafter. https://www.recruit.ox.ac.uk/pls/hrisliverecruit/erq_jobspec_version_4.display_form -- Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE e-mail: antonio.ar...@path.ox.ac.uk<mailto:antonio.ar...@path.ox.ac.uk> Tel: 00 +44 1865 285655 Links to my public profiles: ResearchGate<https://www.researchgate.net/profile/Antonio_Ariza> LinkedIn<https://www.linkedin.com/in/antonioariza1> GoogleScholar<https://scholar.google.co.uk/citations?user=9pAIKV0J=en> Twitter<https://twitter.com/DrAntonioAriza?lang=en> Check out my latest paper!!! Structural insights into the function of ZRANB3 in replication stress response<http://www.nature.com/articles/ncomms15847>
[ccp4bb] Postdoctoral Research Assistant - University of Oxford - Ivan Ahel Lab
Postdoctoral Research Assistant in Biochemistry Sir William Dunn School of Pathology, South Parks Road, Oxford, UK Grade 7: £31,604 - £38,833 p.a. A postdoctoral position is available in Dr Ivan Ahel’s group at the Sir William Dunn School of Pathology, University of Oxford to study the role of ADP-ribosylation signalling in microbial pathogenicity (Rack et al, Mol Cell, 2015; Jankevicius et al, Mol Cell, 2016). The project will involve a combination of biochemistry and structural biology techniques. The post is available for 2 years in the first instance. Applicants should have a PhD in biology or a related subject. A high level of competence in biochemistry and/or structural biology is required and relevant experience demonstrated by first author publications in high-profile journals. The ideal candidate should be organised, highly motivated and able to work independently as well as part of a team. Informal enquiries should be directed to Dr Ivan Ahel: ivan.a...@path.ox.ac.uk<mailto:ivan.a...@path.ox.ac.uk>. If you are interested in this role, and have the skills and experience we are looking for, please apply online. You will be required to upload a CV and supporting statement as part of your online application. The closing date for applications is 12.00 midday on Monday 5 March 2018. Interviews will be held as soon as possible thereafter. https://www.recruit.ox.ac.uk/pls/hrisliverecruit/erq_jobspec_details_form.jobspec?p_id=133257 -- Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE e-mail: antonio.ar...@path.ox.ac.uk<mailto:antonio.ar...@path.ox.ac.uk> Tel: 00 +44 1865 285655 Links to my public profiles: ResearchGate<https://www.researchgate.net/profile/Antonio_Ariza> LinkedIn<https://www.linkedin.com/in/antonioariza1> GoogleScholar<https://scholar.google.co.uk/citations?user=9pAIKV0J=en> Twitter<https://twitter.com/DrAntonioAriza?lang=en> Check out my latest paper!!! Structural insights into the function of ZRANB3 in replication stress response<http://www.nature.com/articles/ncomms15847>
[ccp4bb] question regarding sequence numbering
Hi all, Here's a problem I haven't come across before. I'm working on a structure whose expression plasmid was designed to remove the first 9 amino acids from the protein of interest and to which an N-terminal tag was added. After cleaving the tag I am left with 3 amino acids (GPM) followed by the original sequence. Obviously the residues of interest should follow the numbering of the original sequence (i.e. 10, 11, 12, ...). What numbers would you assign to the first 3 residues (GPM)? 7, 8, 9? -2, -1, 0? Cheers, Tony -- Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE e-mail: antonio.ar...@path.ox.ac.uk<mailto:antonio.ar...@path.ox.ac.uk> Tel: 00 +44 1865 285655 Links to my public profiles: ResearchGate<https://www.researchgate.net/profile/Antonio_Ariza> LinkedIn<https://www.linkedin.com/in/antonioariza1> GoogleScholar<https://scholar.google.co.uk/citations?user=9pAIKV0J=en> Twitter<https://twitter.com/DrAntonioAriza?lang=en> Check out my latest paper!!! Structural insights into the function of ZRANB3 in replication stress response<http://www.nature.com/articles/ncomms15847>
Re: [ccp4bb] protein precipitation reg
If I remember correctly, Triton X-100 (or any other surfactant for that matter) is a bad idea for protein intended for crystallography. I can't remember the paper, but I'm sure I read that somebody showed it's basically impossible to remove all of the surfactant molecules from the protein no matter how you dialyse it. These large floppy molecules stick to your protein molecules and interfere with methods such as mass spec and can hinder the crystallisation process. I'm not sure this is your case, but it might help. If your protein has a very high (or low) PI, it will probably need a strongly ionic environment to be stable. I work with nucleic acid binding proteins and in general I find they do better in buffers with concentrations > 500 mM NaCl until all contaminating proteins have been removed. Only after SEC do I lower the NaCl concentration to between 50 and 150 mM NaCl (you'll need to test which final concentration works best for your protein). In any case, some precipitation is normal and you can easily remove it by centrifugation. An ultracentrifuge works best, but even a benchtop centrifuge for eppendorfs can remove most of the precipitate from your dialysed solution. Best Regards, Tony -- Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE e-mail: antonio.ar...@path.ox.ac.uk<mailto:antonio.ar...@path.ox.ac.uk> Tel: 00 +44 1865 285655 Links to my public profiles: ResearchGate<https://www.researchgate.net/profile/Antonio_Ariza> LinkedIn<https://www.linkedin.com/in/antonioariza1> GoogleScholar<https://scholar.google.co.uk/citations?user=9pAIKV0J=en> Twitter<https://twitter.com/DrAntonioAriza?lang=en> Check out my latest paper!!! The toxin-antitoxin system DarTG catalyzes reversible ADP-ribosylation of DNA<http://www.cell.com/molecular-cell/abstract/S1097-2765(16)30722-5> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Akilandeswari Gopalan [akilaibt2...@gmail.com] Sent: 30 March 2017 07:02 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] protein precipitation reg Dear all, I have used the following buffers for purification and dialysis. this is fyi. Lysis buffer: 25mM Tris pH 7 or 7.5 or 8 100-500mM NaCl (increase in salt concentration increased precipitation of the protein in the column itself) 5mM Beta mercaptoethanol 0.5% Triton X 100 I have tried with other buffers also. a. HEPES buffer pH7.5 b. Phosphate buffer pH 7.8 c. MOPS buffer pH 8 Wash and Elution Buffer: 25mM Tris pH 7 or 7.5 or 8 100-500mM NaCl 20 and 30mM Imidazole for wash 300mM for elution Dialysis Buffer: 1. Tris 25mM pH 7 2. Tris 25mM pH 7.5 3. Tris 25mM pH 8 4. Tris 25mM pH 7.5, 5% glycerol 5. Tris 25mM pH 7.5, 10% glycerol 6. Tris 25mM pH 7.5, 20% glycerol 7. Tris 25mM pH7.5, 50mM NaCl 8. Tris 25mM pH7.5, 100mM NaCl 9. Tris 25mM pH7.5, 1mM MgCl2 10. Tris 25mM pH7.5, 50mM NaCl, 50mM L-Arg, 50mM L-Glu In all these cases the protein precipitates. i have tried to do buffer exchange also. i can see precipitate sticking on the walls of the tube during the process.
Re: [ccp4bb] protein precipitation reg
I personally like TRIS for the first few steps of purification and then change to something else during my last dialysis step. I mostly work with bacteria and they often produce lysates that have pH's that are too acidic for good nickel affinity chromatography, which is why I use 100 mM TRIS pH > 7.8 at this point (if I remember correctly, histidines won't be properly charged and won't bind well to the nickel ions at pH < 7.6). 10 or 50 mM of most buffers might not actually buffer a fairly concentrated bacterial lysate and therefore produce a solution that is more acidic than expected. I also run size exclusion chromatography in 100 mM TRIS to reduce the cost as 1L of 100 mM HEPES is fairly expensive (pH can be lowered at this point depending on the protein's PI, but I like to have the solution strongly buffered). After SEC I will use 10 mM HEPES (or other appropriate buffers) for the final dialysis step. This reduces the cost and works well for me. BTW, TRIS is obviously not a good buffer choice for ion exchange chromatography (which relies on precise pH differences between two buffers) because the pH of a TRIS solution will change with even small fluctuations in temperature. Best regards, Tony -- Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE e-mail: antonio.ar...@path.ox.ac.uk<mailto:antonio.ar...@path.ox.ac.uk> Tel: 00 +44 1865 285655 Links to my public profiles: ResearchGate<https://www.researchgate.net/profile/Antonio_Ariza> LinkedIn<https://www.linkedin.com/in/antonioariza1> GoogleScholar<https://scholar.google.co.uk/citations?user=9pAIKV0J=en> Twitter<https://twitter.com/DrAntonioAriza?lang=en> Check out my latest paper!!! The toxin-antitoxin system DarTG catalyzes reversible ADP-ribosylation of DNA<http://www.cell.com/molecular-cell/abstract/S1097-2765(16)30722-5> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Chun Luo [c...@accelagen.com] Sent: 29 March 2017 22:15 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] [ccp4bb] protein precipitation reg In addition to price, the prevalence of Ni purification may be another reason for Tris popularity. Some His-tagged constructs don't bind to Ni well in HEPES. I wonder if anyone has similar experience or comments. --Chun
Re: [ccp4bb] secondary structure assignment to PDB file
I always use DSSP, which I believe has been the gold standard for secondary structure assignment from pdb files for many years. PYMOL also has a DSSP plugin that can be used to override its own secondary structure assignment, which is nowhere near as good as DSSP. Best Tony -- Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE Links to my public profiles: ResearchGate<https://www.researchgate.net/profile/Antonio_Ariza> LinkedIn<https://www.linkedin.com/in/antonioariza1> GoogleScholar<https://scholar.google.co.uk/citations?user=9pAIKV0J=en> ORCID<https://orcid.org/-0003-4364-823X> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of chemocev marker [jirivit...@gmail.com] Sent: 29 January 2017 10:41 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] secondary structure assignment to PDB file Hi Is there any tool that can assign secondary structure to the PDB file. The problem is if I used different modelling tools, there are regions in the protein which does not remain consistent and looks different in different application. best J. Vitali
Re: [ccp4bb] Protein Purification
Hi Sajid, We need a bit more info to help you. 1) What sort of columns/techniques did you use? Affinity chromatography (nickel, GST, MBP, etc...), ion exchange (cation, anion ... heparin basically falls into this category as well), size exclusion, etc ...? 2) What were the exact compositions of the buffers? 3) Conditions of the runs (temperature, time between lysing the cells, first column and second colum, etc...) 4) What sort of protein are you trying to purify (give us a general type of enzyme/protein class, you don't need to be specific if you don't want to)? Many types of protein have certain needs/preferences that you might not know about but somebody on the bulletin board might point out for you. Finally, you should alway run "load", "flow through" and "elutate" samples from your column runs on SDS-PAGE. Did you do this and, if so, was your protein of interest present in the "load" sample (the pooled flow through sample you obtained from your first column)? It might have degraded or maybe it stuck to the filter/concentrator membrane if you filtered/concentrated the sample between columns. Regards, Tony ------ Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of sajid akthar [0e9cc00295de-dmarc-requ...@jiscmail.ac.uk] Sent: 18 January 2017 11:32 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Protein Purification Dear All I am purifying a protein from liver. In first step I spotted protein in Flow Through and could see intense band in SDS PAGE. I pooled the fraction and did second column. Surprisingly I could not see UV absorbance or even any band in the SDS. I used PMSF as protease inhibitor from begining of purification. First column I used Tris buffer (pH 8) and for second column I used MOPS buffer (pH 6). What could be the reason? Thanks in advance Sajid
[ccp4bb] CCP4 Study Weekend 2017
YEY! It's that time of the year again. I'm about to head off to Nottingham for the CCP4 Study Weekend. It's always a great opportunity to see some familiar faces and meet many new ones. See you there folks! Tony -- Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE
Re: [ccp4bb] Need suggestion for protein solubility
it to remove an aliquot and refreeze the rest of the sample, the sample will often precipitate badly when you thaw it again. So ... freeze small aliquots instead. I hope this helps. Best wishes, Happy Holidays, Merry Christmas (take your pick) and don't work too hard (or at all, if possible, LOL) until the New Year. Tony ------ Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Praveen Tripathi [tripathipraveen2...@gmail.com] Sent: 24 December 2016 10:52 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Need suggestion for protein solubility Dear all, I am graduate student working on a functional protein which i have cloned in pET-28a vector for recombinant protein production in E.coli expression system. The expressed protein is purified on Ni-NTA resins with Imidazole gradient. Surprisingly, i am getting distinct visible white precipitate in pure fractions in eluted fractions itself. Please suggest how to make it soluble or how to prevent the precipitation. On concentrator the precipitate ration is very much increasing. The protein is pure in soluble as well as precipitate. Buffer condition- 50mM Tris(7.5), 500mM NaCl, 10% Glycerol. Elution buffer has varying concentration of imidazole varying from 10mM to 300mM. Any kind of suggestion will be highly appreciated. My project requires structure determination. Thanks in advance. Regards Praveen
Re: [ccp4bb] Tenure Track Positions in UK
Dear Jakob, I'm trying to do this myself. So, this is my own experience of how things work around here ... if anybody disagrees or has any further input, please let me know as I'll appreciate any help I can get. In order to find an academic opening you can search for a lecturer (this is basically equivalent to assistant professor in the US) or professor position. The best site for this is JOBS.AC.UK. You will need to wade through the ads to find those that expect/allow you to spend time on research. Generally the universities with higher ranking will expect you to do both research and teaching, while the smaller universities mainly offer only teaching positions. The terms "group leader" and "principal investigator" are rarely used in job advertisements, but when you come across them it means they are looking for somebody who will mostly run a research group with only limited or no teaching obligations. The term "tenure track" is not used in the UK as, in the strictest sense, we never get tenure (i.e. a life-long position where you basically can't be fired). Even when you're offered a "permanent" position in the UK, you are still subject to passing periodic performance tests that check if you bring in enough grant money or produce enough high level publications to bolster the REF of your institution (a point system that grades British universities and their departments). I would say that in general new group leader positions in structural biology are VERY rarely offered in the UK and most new group leaders are initially appointed via fellowships. If you do well during your fellowship, then the university will grant you a "permanent" position as a senior lecturer or assistant professor (this is a higher position than the assistant professor position in the US) at the end of it. For the fellowship route you need to contact the department where you want to do your work, tell them what your research project will be about, give them a detailed 3-5 year project plan and tell them what fellowships/grants you will apply for. If they like you and your project, they will then offer you a place in the department subject to you obtaining the money first (they will help you with the application). If you've already held a fellowship and have some grant money you can take with you and therefore don't need the university to pay for your salary, overheads and research costs, then you can also contact most university departments and tell them about it. If you are at this level, then your chances are good. I hope this helps, Tony ---------- Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Keller, Jacob [kell...@janelia.hhmi.org] Sent: 07 October 2016 14:34 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Tenure Track Positions in UK Dear British Colleagues, I have been looking through the Nature/Science Jobs websites for tenure-track positions, but have seen proportionally very few notices for positions in the UK. Is there a hiring freeze due to Brexit, or is there some special way that these jobs are posted? How does one find openings in the UK? All the best, Jacob *** Jacob Pearson Keller, PhD Research Scientist HHMI Janelia Research Campus / Looger lab Phone: (571)209-4000 x3159 Email: kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org> ***
Re: [ccp4bb] advice on anomoulous data collection strategy---low resolution, radiation damage, high mosaicity
Burning/frying the crystal is never the right strategy, at least not if you use a Pilatus detector. Burning/frying is a strategy that results from the widespread misunderstanding of using Rsym as a criterion of data quality. I apologise if I didn't make myself clear. The point I tried to make is that it is ok to strive to obtain higher resolution with native data sets ... and yes, fry is a poor choice of a word on my side as it implies damage to the crystal, which is not what I meant. I simply tried to emphasise that going for high resolution is ok when collecting native data sets but not when collecting anomalous ones. The rest of my message made a point of collecting data with high attenuation and fast shutter speed at a lower resolution to keep the anomalous signal high in as many images as possible before X-ray damage sets in lowers it. Tony -- Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE
Re: [ccp4bb] advice on anomoulous data collection strategy---low resolution, radiation damage, high mosaicity
Hi, I agree with Kay, try to fry your native crystals to get the highest overall resolution possible, but go for low resolution if your crystals decay rapidly, particularly when collecting anomalous data. A high overall resolution is always desirable, but during anomalous phasing you can potentially solve good data (no twinning, pseudo-translational NCS symmetry, etc...) even at an anomalous resolution (NOT overall resolution) as low as 5 to 6 Angstroms, though in practice you generally need at least 3 to 4 Angstroms of anomalous resolution for a straight forward solution. Once you have solved the substructure and have initial phases and an acceptable build, you can swap to your native data to build the structure using higher resolution data. Obviously, there are a number of factors that influence the result: If your crystals are stable, decay very slowly in the X-ray beam and you have a high symmetry space group, you can attempt MAD phasing. However, if your crystals decay rapidly and you have a low symmetry space group you should try SAD phasing. In this case it's best to collect a lot of data (360 to 720 degrees) close to the peak wavelength with high attenuation, fast exposure times and slightly larger oscillations than usual (1 to 2 degrees). My own experience with this has been that larger oscillations work better in this case than fine slicing, which doesn't seem logical. I've seen this with the last three structures I solved by anomalous phasing where 1 degree oscillations gave better anomalous signal that 0.2 degree oscillations with a Pilatus detector at 0.035 second exposures. Maybe it's to do with the fact that you collect more complete data (as in degrees collected) this way compared to fine slicing (where you will cover fewer degrees using the same dose before the crystal decays). You already tried merging several data sets collected from different positions of the same crystal, but if your crystals are isomorphous you can also merge the data from several crystals to improve your SAD phasing as the anomalous resolution often increases with high multiplicity (the overall resolution does not). For this you need to take into account that the heavy metal atoms will be damaged first at the wavelength at which they produce anomalous diffraction, so the best anomalous resolution will be provided by the images at the start of the data even if the overall resolution doesn't seem to decrease. So, check if you get better data by discarding the images to the end of each individual data set before you merge them. Merging and then scaling the data sets together might increase the overall error and not improve the high resolution data, but I have found this will boost the signal of week anomalous scatterers found in the same positions in all the crystals. I hope this helped. Tony -- Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE
Re: [ccp4bb] advice on anomoulous data collection strategy---low resolution, radiation damage, high mosaicity
Something else you could try is using a kappa goniometer. There were a couple of presentations at the last CCP4 meeting where they discussed that alignment along the appropriate axis using a Kappa goniometer often worked much better than the inverse beam mode. By aligning the crystal with the beam in such a way that one of the cell axes lies perpendicular to the beam, you can obtain diffraction images that have bilateral symmetry and where both parts of a Friedel pair are visible on the same image. This way the differences between each pair are easily measured and compared and, because they are measured at the same time, the differences cannot be due to X-ray damage, scaling or anisotropy that result when the two parts of a Friedel pair are measured from different images taken from different positions on the crystal at different times. At the Diamond Light Source they have started to implement these goniometers on some of the macromolecular beam lines. You simply take 2 to 4 test images between 0 and 90 degrees and the software automatically spits out the theta and kappa angles you need to orient the goniometer. After you align the crystal and collect the data set you should collect a second data set where you misalign all the crystal axes. This is useful since the first data set will have no data for the aligned axis and it will help determine the correct space group if there are systematic absences along that plane. By integrating both data sets and then merging and scaling them together you will also obtain much better completeness. Tony -- Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE
