[ccp4bb] Mac M2 chip
Dear All, Has anyone encountered problems with CCP4i/Phenix/XDS/etc on Macs with the M2 chip? I saw a post from 2020 on the M1 chip, but was curious about if there are any issues between the M2 chip and our crystallographic software. Thanks for any information. Dan To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] PISA server
Thanks for all the replies. Like I said, it eventually did upload the structure after 20 minutes and I had no problems in analyzing the interface. I did try two different computers, my home computer on Friday night and the lab one on Saturday afternoon. At home it failed to load the structure, but it did eventually work at work. Dan
[ccp4bb] PISA server
Is the PISA server having problems? I can not seem to upload any structure. Thanks Dan
Re: [ccp4bb] PISA server
It has eventually loaded but has taken nearly 20minutes.
Re: [ccp4bb] Supplementary density
If the protein is His-tagged, load the purified protein on to a Nickel column and place the column in a water bath above the protein's melting temperature. Recycle 5mls of water through the column to collect the peptide and then dry freeze or speed vac the sample to concentrate the peptide. I have seen this done for peptidoglycan fragments: Peptidoglycan Recognition by Pal, an Outer Membrane Lipoprotein Biochemistry, 2006, 45 (7), pp 2122–2128 I cannot see why it should not work for peptides. Don't forget if you are going to run a gel of the peptide to use tricine gels. Dan
Re: [ccp4bb] E. coli mutant strains
If you want a ClpP minus strain you can get it from the Keio strains. http://cgsc.biology.yale.edu/index.php http://www.shigen.nig.ac.jp/ecoli/strain/top/top.jsp For expression with the T7 promotor, you will need to use the λDE3 Lysogenization Kit or use a Arabinose induction plasmid instead.
Re: [ccp4bb] E. coli mutant strains
Factor Xa will work depending on the exact sequence. If your sequence starts MRS... and the start methionine is important and not removed then yes it will work when you clone it into a Factor Xa site. If however the start sequence starts RS... and the start methionine is actually removed, when you clone the gene minus the start Met, Factor Xa will not cleave IEGR/RS. Have you done Mass spec on the protein to confirm that the start methionine is removed/present? Dan Dear Jerry, Whats about an N-terminal His-Tag with an Xa factor cleavage site behind it... After IMAC you only get protein with the entire N-Terminus (His-Tag), afterward digest the protein with Xa factor...it won’t leave any additional amino acids C-terminally! You’ll get your protein! ;) Cheers, Christian
Re: [ccp4bb] N-terminal sequencing
We have used Alphalyse (http://www.alphalyse.com/picknpost.html). Dan
Re: [ccp4bb] pH dependent conformational change
I would like to point out that HSQC could still be applied even in such a large protein. TROSY-HSQC has been successful in improving peaks in spectra of large protein. Typically the sample would need to be deuterated to see the full effect of TROSY, but even a partial deuteration can improve signal. We have run TROSY-spectra of a complex (75kDa) with no deuteration and that spectrum is much better than a normal HSQC spectrum. Dan
Re: [ccp4bb] protein interaction
Producing the proteins in cell free system or bacterial expression can affect the removal of the start methionine. Although the same rules of a small amino acids next to the start methionine apply, different methionine aminopeptidases tolerate certain small ones better. Depending on the which cell-free kit you use, you may have not removed the methionine, which may be important in protein binding. Protein N-Terminal Processing: Substrate Specificity of Escherichia coli and Human Methionine Aminopeptidases Biochemistry 2010, 49, 5588–5599 A second important consideration is that in cell-free systems other post-translational modifications can be a hit or miss depending on the cell-free kit and/or type of modification. For example acetylation occurs in transdirect insect line N-Terminal protein modifications in an insect cell-free protein synthesis system and their identification by mass spectrometry PROTEOMICS Volume 6, Issue 16, No. 16 August 2006, Pages: 4486–4495 but not wheat-embryo Sequence specificity and efficiency of protein N-terminal methionine elimination in wheat-embryo cell-free system Protein Expression and Purification 52 (2007) 59–65 though it can be achieved by the addition of Acetyl-CoA.
Re: [ccp4bb] Off Topic - Nickel Column
I wish to thank everyone. I did try shifting to a higher pH and flushing 3l of salt solution over the column which did not work. I tried 20ml 2M Urea on the column and a stepwise shift to no urea that showed removal of the protein. I will try binding studies to see if I did not denature the His-tagged protein. Thanks for your suggestions. Dan
[ccp4bb] Off Topic - Nickel Column
I have a His-tagged protein which I am coexpressing with it's binding partner to prevent proteolysis. Once on the Nickel column I can remove 80% of the partner by flushing 2l of 1.3M NaCl solution buffered at pH 8.5 overnight. However the last 20% is difficult to remove, even if I reload the Nickel column and flush a further 2l of salt solution. I am wondering if I can increase the pH to 9.0 or 9.5. It should not effect the binding of His for the Nickel as the His-tag has to be deprotonated to bind, though will it causing stripping of the Nickel? Thanks Dan
Re: [ccp4bb] Glutathione sepharose
I am using the glutathione sepharose 4B beads from GE. I had noticed that the capture efficiency does decrease with usage, though I have successfully regenerated the beads at least 20 times and I am still using them. I regenerate using 2CV of 6M guanidine after each run and every 5 runs with 70% ethanol as well. I have also used 5CV of 1M NaCl and noticed elution of proteins. I perform these experiments using the gravity flow method with a glass column. I have noted that the beads do clump together. A gentle re-suspension using a pipette seems to be good at unclumping them and improve capture. Also the diameter of the column seems to be important. I have noticed that using 5ml of beads on a 1cm diameter column captures far less than 5ml of beads on a 2.5cm diameter column. Hope this helps!
Re: [ccp4bb] Additional band on gel due to his-tag: any references?
THERMATOGA MARITIMA IscU IS A STRUCTURED IRON-SULFUR CLUSTER ASSEMBLY PROTEIN June 14, 2002 The Journal of Biological Chemistry, 277, 21397-21404. His-tagged Iron cluster that runs as a doublet. Mass-spec show they are the same species. They concluded that the protein binds SDS in two stoichiometries and therefore runs as a doublet as seen for the OmpA protein (references are in the paper). Hope that helps. Dan
Re: [ccp4bb] Cys auxotroph
Have you looked at the Keio collection. Though they are not compatible with T7 promoters, you can use the λDE3 Lysogenization Kit to add the T7 polymerase. A possible source is from Yale http://cgsc.biology.yale.edu/Auxotrophs.php though of course there are others. Dan
[ccp4bb] Reindexing scaled data
Hi all, This maybe a simple/stupid question. I collected data at the synchrotron, integrated and scaled the data in P222 using HKL2000, though I should of scaled the data as P212121. When I try to reindex using Reindex I get a message of You are changing the symmetry of merged data are you SURE you know what you are doing WARNING: ** Symmetry change of merged data ** I am unsure of what I am doing in this case. Currently I cannot reprocess the data as I do not have a working version of HKL2000. So my question is can I reindex scaled data and what I should be doing with the GUI of reindex? Thanks in advance for advice. Just in case it is important my cell dimensions are 109.9280 160.3030 186.2200 90. 90. 90. and the resolution is 2.7 Angstrom.
Re: [ccp4bb] Removing a tight binding ligand
It refolds properly according to the CD spectra but it some how manages to hold on to trace amounts of ligand despite serial dialysis (500ml to 5ml of sample) in 8M, 6M, 4M, 2M 1M urea followed by 50mM Tris. I also have a homolog that abjectly refuses to refold in either urea or guanidine, though it does turn the dialysis tubing into a lovely snow globe. I am assuming that the protein is tagged in someway and that you add your purified protein to Urea/Guanidine and refold by dialysis. If you protein is His-tagged, I would unfold it on the column and flush a large excess of Binding buffer (containing denaturant) to remove the ligand and elute the protein in elution buffer (containing denaturant) and then refold, to ensure that no ligand is present during refolding. If this is what you have done, ignore this. For the protein that cannot be refolding, have you investigated thermal denaturation. I have removed a 1kDa ligand by placing a nickel column in a water bath set near to the Tm of the protein. After flushing an excess of prewarmed buffer through, the water bath was switched off, allowed to cool to RT and then protein eluted off. Around ~95% was refolded. The unfolded protein was then separated by size-exclusion. Though I have no evidence for this and I am just thinking out aloud (please could someone correct me if I am wrong, have evidence to the contrary or both), if the ligand is not present in the periplasm, and the protein is targeted through the sec pathway (which recognizes unfolded proteins), purification from the periplasm could circumvent refolding if you are lucky and you do not get cytoplasmic contamination. All the best Dan
Re: [ccp4bb] difficult P1 crystal
There are a couple of papers... Acta Cryst. (2010). F66, 346-351 Crystallization and X-ray diffraction studies of cellobiose phosphorylase from Cellulomonas uda The space group was originally P21. During collection the crystal moved out of the beam (and possibly the cyrostream). Upon recentering, the space group was found to be P212121 Acta Cryst. (1998). D54, 448-450 Crystallization and preliminary X-ray analysis of thiaminase I from Bacillus thiaminolyticus: space group change upon freezing of crystals At room temperature the space group was P212121 but upon freezing the space group changes to P21212 Hope this helps.
Re: [ccp4bb] Regarding quality of protein for crystallization
I assume that the loss of the peptides that you observe for the smaller protein is at the other terminus from the tag? If it is at the terminus where the tag was it would suggest that removal of the tag using proteolysis is the most likely cause. Though saying that Factor Xa/thrombin is not 100% accurate and still maybe causing the slight degradation. You do not say if you see the two different species before cleavage. If you do, have you tried adding protease cocktail inhibitors before disruption of the cells? As you know which peptides are missing, you know roughly the region that is deleted. Does this cause a change in the pI of the smaller protein? If so you may try ion exchange chromatography to separate the two species. For crystallography purposes, two different species may effect crystallization through disruption of crystal packing, though in situ proteolysis crystallization can generate a mixture of different species with one of the fragments being more readily crystallizable. There is only one way to find out... Daniel A. Bonsor, Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472 USA
Re: [ccp4bb] protein turns brown
According to Pierce TCEP is more tolerant of nickel and cobalt. However, TCEP is inactivated by other metals, namely copper, magnesium, silver and zinc. Dan Daniel A. Bonsor, Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472 USA
Re: [ccp4bb] Protein melting temperatures
There is a nice paper Comput Biol Chem. 2009 Dec;33(6):445-50. Epub 2009 Oct 20. Predicting melting temperature directly from protein sequences. Ku T, Lu P, Chan C, Wang T, Lai S, Lyu P, Hsiao N. They have a list of 35 different proteins with their Tms with the references from where they obtained their data. Hope this aids in your work. Dan
Re: [ccp4bb] Crystallizing a membrane associated protein
It behaves very well. No precipitation/cloudiness during purification. Secondary structure estimation by CD is ~50% alpha helical, 25% beta-sheet and 25% random coiled. I screened at 2, 4, 6 and 8mg/ml (8mg/ml around about 60:40% precipitation). If any other information is needed, just ask. Thanks Dan
Re: [ccp4bb] Crystallizing a membrane associated protein
By size exclusion, it eluted where a 30kDa protein would be expected to elute. As for functional assay, I am currently trying to express its binding partner, though it shows a lot of degradation. But when I mix both proteins and run by size exclusion it causes the peaks of its binding partner to elute earlier suggesting that they are interacting.
[ccp4bb] Rsym problems...maybe???
Hello again. At first I was not worry but maybe now I am. I have completed a structure and submitted to the PDB. They queried my Rsym value in the highest resolution bin, 2.5-2.37A (may I dare say it 100%). I was not worried at the time as I had: 99.4% completeness Mean(I/sdI) of 2.5 and a redundancy of 11 (which would explain the high Rsym) Space group I422 My Rpim in this shell is 30%. Should I reduce the resolution and start from scratch again or is everything fine and dandy and I should stop worrying?
[ccp4bb] Follow up to TLS, NCS and refinement
Hello again! Following my previous question, there was something wrong with the staring model for molecular replacement. Now that is sorted, I have 8 complexes in the ASU. After a few rounds of refinement with NCS and isotropic Bfactors, both the Rfactor and Rfree get stuck at 30% and 36%, respectively. I have only just noticed that I am trying to model ~43000 atoms with ~95000 unique reflections (98% complete, at 2.58 Angstrom resolution). Is this the reason why the R factors are stuck and I should start the refinement again from scratch using overall Bfactors with NCS and switch to TLS once my Rfactor is less than 30% and possibly reduce the number of reflections used for Rfree (currently using the standard CCP4 5%) Any input would be greatly appreciated! Dan
[ccp4bb] TLS, NCS and refinement
Hello again... I have a 2.7A resolution data set. Spacegroup P212121 as suggested by Pointless and best space group when run through Phaser. Unit cell is 110.02x160.49x186.55. Mathew's suggest 11 complexes in ASU. Both Molrep and Phaser can only find 6. This seems to be a typical observation with the complexes that we solve (high solvent content). Due to the low(ish) resolution I wish to apply TLS and NCS during refinement. My question(s) is when to apply TLS and NCS, the order in which it should be done, and when to switch off NCS (if at all) during the stages of refinement. I have used a homologue complex (Protein A -100% identical, Protein B - 80% identical) as a model. I am open to any and all suggestions on the matter. Thanks in advance Dan
[ccp4bb] Full B Factor
How do I convert the B-factors from my final structure to full B factors if I did not use TLS refinement? I have been refining isotropic B-factors. Do I switch to overall B-factor refinement, do something else, or have I missed the point altogether? It may be a dumb question but best to be safe than sorry.
[ccp4bb] Contact Surface Area
I am trying to calculate the contact surface area of a loop. Using ArealMol I only get the overall contact surface area per residue. Is there any way to get it per atom or does anyone know of a program (online/software) which will perform this task. Thanks in advance Dan Daniel A. Bonsor, Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472 USA Tel: +1 617.658.7845
Re: [ccp4bb] Contact Surface Area
Sorry I should of made this clearer in my original post. Thanks anyway to people who have responded thus far. I am trying to calculate the buried surface area of a loop which folds from a disordered to an ordered state. I am looking for a program that will allow me to calculate; (1) the buried surface area per atom (not residue) or (2) something that returns the buried surface area of apolar and polar atoms. It has to be per atoms and not per residues. Thanks again in advance. Dan
[ccp4bb] Molecular Replacement of a protein complex
My first time posting to the CCP4BB board and I am very sure it will not be my last. I have a general and specific question concerning molecular replacements of protein-protein complexes. I have a good dataset (2.5A, 98% complete), which has been integrated and scaled. Pointless suggested the pointgroup P222, in agreement with imosflm and HKL2000. For the spacegroup it could not decide between P2221 or P21221. Spacegroup TotProb SysAbsProb Reindex Conditions P 2 2 21 ( 17)0.889 0.900 00l: l=2n (zone 3) P 21 2 21 ( 18)0.889 0.900 h00: h=2n, 00l: l=2n (zones 1,3) .. P 2 2 2 ( 16)0.057 0.057 P 21 2 2 ( 17)0.057 0.057 h00: h=2n (zone 1) .. P 2 21 21 ( 18)0.039 0.040 0k0: k=2n, 00l: l=2n (zones 2,3) P 21 21 21 ( 19)0.039 0.040 h00: h=2n, 0k0: k=2n, 00l: l=2n (zones 1,2,3) I reindexed to both space groups and proceeded with Mathews Coeff. Assuming a 1:1 complex had formed (MW = 39000) it predicts 1 complex per ASU. MolRep using the larger protein (MW=27000) found the protein with a contrast of 3.33 (p2221) and 2.61 (p21221). However repeating MolRep for the smaller protein in both spacegroups failed to locate it. Contrast was low, 1.11 (p2221) and 1.10 (p21221). I reran Mathews Coeff in case the second protein was not their (though gels of crystals show both proteins) and it predicted 2 proteins per ASU. MolRep looking for 2 of the larger protein generated contrasts of 1.99 (p2221) and 2.27 (p21221). Using Refmac5, I performed a rigid body refinement of all 4 cases with details summarised below p2221 1proteinper ASU - Proteins are arranged in layers separated by a 55Angstrom space. Some electron density is present but nothing that could be generate a polypeptide. Perhaps two or three amino acids here and there. Density fits the protein well. Rfac, 0.527, Rfree 0.523 p21221 1proteinper ASU - Proteins are again arranged in layers separated by a 55Angstrom space. Again some electron density is present that could accommodate two or three amino acids. Density also fits the protein well. Rfac, 0.527, Rfree 0.512 p2221 2proteinper ASU - No layers are observed. Density fits the proteins very poorly. Rfac, 0.541, Rfree 0.546 p21221 2proteinper ASU - No layers are observed. Density fits the proteins very poorly. Rfac, 0.540, Rfree 0.547 My questions are this; In general what is the best procedure for molecular replacement and refinement of protein-protein complex where the Kd is 1uM and weaker and it is difficult to observe one of the proteins; In this case I believe the second protein is their, though either the starting model is poor (80% identical) or a gross conformational change of the protein has taken place (which is unlikely based upon similar complexes that have been solved). I am also concerned about Rfree being smaller than Rfac. Should I continue with these two data set and try modeling alanine in the electron density I can observe and see if further density is generated on further refinement? Or should I go back and look at alternative space groups? Thanks in advance for you opinions, suggestions and help Daniel A. Bonsor, Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472 USA
