[ccp4bb] Clashes in Phaser

2018-11-10 Thread D Bonsor
I am currently trying to solve two datasets of two different protein-protein complexes. One of the proteins is common to both complexes and there is a deposited structure in the PDB (100% identical). The other proteins currently have not been solved and share ~50% identity between them. Both

Re: [ccp4bb] Crystallization with no precipitant

2017-03-03 Thread D Bonsor
Thanks for the all the comments on/off board. Especially for papers describing this method as it may be handy if the structure is solved. The crystals are reproducible. They have started to appear after 4 days. I can accelerate it to a day using just 1.5M NaCl as a reservoir solution, though I

[ccp4bb] Crystallization with no precipitant

2017-02-28 Thread D Bonsor
Dear All, I have produced crystals from a robotic screen (API) in two conditions. However, I have not been able to reproduce them. They originally grew in 2.5 days (over the weekend, though it could have been sooner). I noticed that when I first set up the tray, these two wells were clear but

[ccp4bb] Anisotropy and temperature

2017-01-19 Thread D Bonsor
A PhD student asked me what causes diffraction anisotropy. Quoting from the Diffraction Anisotropy Server webpage that it is caused by whole-body anisotropic vibration of unit cells. He asked whether a colder cyrostream could improve anisotropy. My answer would be yes, as colder temperatures

[ccp4bb] Phenix

2017-01-14 Thread D Bonsor
Is the phenix website down? Anyone know when it will be back up?

[ccp4bb] Inclusion Bodies

2016-11-21 Thread D Bonsor
We have been expressing a family of human proteins as inclusion bodies in E. coli, which we can be refolded and crystallized. However, three members show no expression either as inclusion bodies or as soluble proteins. The genes were ligated into the pE21d vector with either a His-tag or no

[ccp4bb] Reindexing lattice

2015-07-01 Thread D Bonsor
Hi, I am trying to reindex a P212121 lattice to change the axis order from 32.62, 64.89, 103.16 to 64.89, 103.16, 32.62. Looking at the REINDEXING manual I can't find how to reindex the lattice. Does anyone know how I should do this and an example of the execute script that I should use?

Re: [ccp4bb] 3BDN, 16.5% Ramachandran Outliers!!!!!

2015-04-23 Thread D Bonsor
They have, it is call PDB_REDO. You can download the maps and refined structure there. http://www.cmbi.ru.nl/pdb_redo/ Dan

Re: [ccp4bb] Cleaved peptide density!

2015-04-20 Thread D Bonsor
First of all, you don't say how long it took to first set up crystals, for them to grow, harvest, freeze and collect data on. Secondly how long did leave the peptide/substrate for your SDS PAGE experiment? If they are of a different time scale e.g. 6 hours v.s. 30 days, it may be that your

Re: [ccp4bb] RMSD of dimers

2015-03-04 Thread D Bonsor
Hi all, Just writing an update, as I have had two people contact me asking if I had found a solution and if I could share. I would like to apologize first for not phrasing my question to describe the situation simply. I had several suggestions that Coot, and PISA could do it but actually can

[ccp4bb] RMSD of dimers

2015-02-24 Thread D Bonsor
I have a family of homodimers (denoted A1B1, A2B2, A3B3...) which I have superimposed using Chain A. Several programs will produce the RMSD of Chain A2, A3, A4... to Chain A1. However, I would like to know the RMSDs of Chain B2, B3, B4... to Chain B1 when I have superimposed the structures

[ccp4bb] Wrong Space Group?

2013-12-13 Thread D Bonsor
Dear all, I have collected ~160 degrees of data on a new crystal form of a protein which has already been solved. Data was processed with XDS and reindex, scaled and truncated with Aimless. Both XDS and Pointless suggested a Laue group of P6/mmm with a possible space group of P6122 or P6522.

Re: [ccp4bb] A photograph of the Arndt-Wonacott rotation camera?

2013-10-30 Thread D Bonsor
Bob Sweet has a powerpoint presentation but it took a long time to download the file

Re: [ccp4bb] Philosophical question

2013-03-19 Thread D Bonsor
You may want to read: Evolutionary conservation of codon optimality reveals hidden signatures of cotranslational folding Nature structural molecular biology VOLUME 20 NUMBER 2 FEBRUARY 2013 237 Here they suggest that the codon bias is such it allows translation to pause and folding of the

Re: [ccp4bb] Off Topic GST-tag Protein Purification

2013-02-20 Thread D Bonsor
Protein A must be co-expressed as it does not express well alone. Have you tried separating A+B on the Nickel column by flowing 2M urea to help tickle off protein B. The amount will vary depending on the affinity. In my case (~50nM) requires about 1000-1700ml of 20mM Tris, 2M urea pH 7.5. Then

Re: [ccp4bb] protein cleavage

2012-11-04 Thread D Bonsor
You do not mention what buffer you are trying to do your cleavage in. You need a reducing agent for TEV to work (e.g. reduced gluthionine, DTT, mercaptoethanol). EDTA (0.5-1mM) is recommended as TEV is a cysteine protease and the presence of divalent metal ions will/eventually inhibit TEV. TEV

Re: [ccp4bb] off-topic: detergents for the stabilisation of water-soluble proteins

2012-10-12 Thread D Bonsor
The following paper (which can be found at www.wolfson.huji.ac.il/purification/PDF/Literature/Bondos2003.pdf Detection and prevention of protein aggregation before, during, and after purification. Sarah E. Bondos and Alicia Bicknell (2003) Analytical Biochemistry, 316, 223-231 contains a

Re: [ccp4bb] Overlapping transparent surface representations in Pymol

2012-10-02 Thread D Bonsor
I am assuming you are after something like a difference map of the two surfaces. http://www.pymolwiki.org/index.php/Map_set Map_set command can average, copy, difference, maximum, minimum, sum and unique Hope this is what you are after. Dan

Re: [ccp4bb] co-express two proteins in E.coli

2012-07-16 Thread D Bonsor
I have been using the Duet system from Novagen (or whatever it is called these days), specifically the pETDuet-1 and pRSFDuet-1. Co-expression of my proteins did not work in either vector. Either, one protein expressed or the other. I played around with the promotors (they are both T7) by

Re: [ccp4bb] Off-topic His-Antibody

2012-06-28 Thread D Bonsor
Dear All, Thanks for all the replies on and off-board. I received around twenty replies and the majority have spoken in favor of the QIAgen BSA-free anti-5His mAb from QIAGEN. Not to be bias, a couple of people recommended the one from Abcam as well. Thanks again, Dan

[ccp4bb] Off-topic His-Antibody

2012-06-25 Thread D Bonsor
Are there any good antibodies for His-tags on the market. I have never used one and I heard several stories that they were poor and not worth the money, over the past few years. The only post I could find on the BB was from 2008. Have His-Antibodies improved or are they still rubbish.

Re: [ccp4bb] about point mutation

2012-02-24 Thread D Bonsor
Phusion requires that the primers are phosphorylated for mutagensis to work, unlike Pfu. If you cannot phosphorylate them use Pfu as recommended by Charlotte Dan

Re: [ccp4bb] Merging data collected at two different wavelength

2012-01-18 Thread D Bonsor
Isn't it true that we cannot even agree on what MAD stands for? Is the following right? M = Multiple-wavelength. I think everyone agrees to this, although I believe I've seen the occasional (and sometime non-sensical) variant A = Anomalous (I think everyone agrees, although this term should

Re: [ccp4bb] raw data deposition

2011-10-27 Thread D Bonsor
Why should we store images? From most of the posts it seems to aid in software development. If that is the case, there should be a Failed Protein Databank (FPDB) where people could upload datasets which they cannot solve. This would aid software development and allow someone else to have ago

Re: [ccp4bb] should the final model be refined against full datset

2011-10-14 Thread D Bonsor
I may be missing something or someone could point out that I am wrong and why as I am curious, but with a highly redundant dataset the difference between refining the final model against the full dataset would be small based upon the random selection of reflections for Rfree?

Re: [ccp4bb] Off topic_protein degradation.

2011-08-19 Thread D Bonsor
If you are unsure about whether the disulfides have formed treat a small amount of protein with N-ethylmaleimide. If the disulfides have not formed, when you perform mass spec on the protein you should see an increase of mass of 125Da for each exposed cysteine.

Re: [ccp4bb] Off topic - Streptactin Column Summary

2011-07-12 Thread D Bonsor
A couple of people asked why the GST/His steps. The only way I can expressed this protein is by co-expression. Co-protein is GST tagged and my protein of interest is N-term His and C-term StrepTag. Even with the coexpression, there is a major degradation product, the reason why for the strep

[ccp4bb] Could Biological Negative Results be published?

2011-07-11 Thread D Bonsor
There are least two types of negative results 1) Contradiction of previously published results. Negative results of this kind is either they are wrong, you are wrong or it depends on the differences within the experimental methods used. An example of the latter case would be SPR vs ITC. SPR

[ccp4bb] Off topic - Streptactin Column

2011-07-11 Thread D Bonsor
Does anyone know how many times roughly you can re-use a Streptactin column? I know that contamination with biotin will destroy the column but the protein that I am using has gone through both a GST and Nickel purification steps before seeing the Streptactin column and I think lately that I