[ccp4bb] about partial plroteolysis

hello CCp4ers: Sorry about the off-topic question. My protein is a kinase which the predicted kinase domain is from 32aa to 319aa. But the protein sequence after partial proteolysis is from 52aa. what should I do? Are there any reasonable expanation? Thanks in advance! Xinghua Qin -- Xinghua

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Re: [ccp4bb] Flag tag vs. his-tag

My vote is for His-tag *unless* your protein is of the same general class as Zn-fingers, B-boxes, etc. (weak/multisite/uncertain metal binders) - these proteins often do not fare too well with His-tags because a) the tag residues can participate in artefactual metal-binding sites and b) the use of

Re: [ccp4bb] off topic: problematic protein

HI Savvas, We recently had a protein that showed two overlapping peaks on the disposable fast flow Q columns, so we decided to see if we could resolve them with a higher resolution Q media. It ended up having 7 distinct peaks, only one of which was free of contaminants. We have also noticed that

Re: [ccp4bb] off topic: problematic protein

I recently followed a protocol from Stephen Sligar's lab for the purification of his "nanodisc" protein, which has strong hydrophobic character as it associates with phospholipids. His protocol includes washes with 1% Triton X-100 and then with 50 mM cholate (both at pH 8 in the presence of 300

[ccp4bb] off topic: problematic protein

Dear colleagues We are working on a large bacterial protein (featuring a large number of repeats) that appears to copurify with a lot of other proteins after Ni-affinity chromatography and gel-filtration. We have tried adjusting the ionic strength of these runs and have gone to as high as 5M Na

Re: [ccp4bb] Flag tag vs. his-tag

Thanks everyone for your comments. The overall opinion appears to be that large-scale purification of flag-tag protein is expensive. I 100% agree and have thought about that plus the dilemma with the flag-tag elution difficulty (low pH etc) which some of you also pointed out. So I will proba

Re: [ccp4bb] Cluster Design

Hello Frank, Here at the CLS we have a 8-CPU AMD Quad-Core 2.9GHz machines (ie 32 cores per node). There are newer models capable of 48 cores. Each node has four Gb network ports bonded through Etherchannel. Our file server is a Sun 7310 storage server also with 4 bonded Gb network ports. We us

Re: [ccp4bb] General question about Heme Binding Proteins

On 04/19/11 14:18, Stephane Richard wrote: Dear CCP4 community, I have a question a bit off CCP4 topic, but that could use so expert input. While discussing about a bacterial secreted hemophore (heme scavenging protein) for which the apo form has been solved, it seems that attempts to obtain the

Re: [ccp4bb] Flag tag vs. his-tag

His-tag is close to neutral but flag-tag is acidic. Using flag-tag for affinity purification usually gives cleaner protein but it costs a fortune due to low binding capacity of commercially available anti-flag resins. --Chun From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Be

[ccp4bb] General question about Heme Binding Proteins

Dear CCP4 community, I have a question a bit off CCP4 topic, but that could use so expert input. While discussing about a bacterial secreted hemophore (heme scavenging protein) for which the apo form has been solved, it seems that attempts to obtain the Heme bound form are failing; in fact during d

[ccp4bb] Flag tag vs. his-tag

Hi all, I am trying to decide between using a N-term his-tag or an N-term flag tag to be expressed on a protein that I will eventually want to try and crystallize. I usually don't cleave my tags unless absolutely necessary and I want to avoid double tagging. I am leaning towards the flag since

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[ccp4bb] viewing and scoring diffraction using the PX Scanner

Dear Artem, Thanks for your very comprehensive reply. At the risk of reaping further criticism, I will now do little more than just reiterate my earlier comment: that from the feedback that we receive, the PX Scanner is much valued by the number and range of groups that are now using these

Re: [ccp4bb] Cluster Design

Hi Frank, sorry, my first response was not very specific for your situation! I studied your list and graphs and would just like to point out that in "2.1 Testing Filesystem Performance" you may be severely overcommitting the CPU resources, if this was for one machine only with 16 (=8+8) cores

Re: [ccp4bb] Cluster Design

Hi Frank, the following are some recommendation for increasing the processing speed of XDS. You can find them (and add to them !) at http://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/Performance . Only item 7 is specific for a cluster. In the order of effect: 1. XDS scales well

Re: [ccp4bb] viewing and scoring diffraction using the PX Scanner

Hi Marcus, I think you misinterpret my comments :) My first point is not that my specific proteins make it easy to distinguish salt from protein visually - it's rather that I am experienced enough and also know enough chemistry to make an educated conclusion about the contents of my drops. I expec

[ccp4bb] Cluster Design

Dear All, Here at NE-CAT, we make extensive use of XDS in a parallel environment. We are looking to purchase some new hardware, so I am soliciting your opinions. Our current cluster is made up of 16 nodes, each with 2 processors that have four cores, running at 2.2 GHz (I believe). We run with

Re: [ccp4bb] viewing and scoring diffraction using the PX Scanner

OK, I took the challenge. I got 7 out of 10. The three I missed were 2 questions about multi-well crystals which would be better (no problem) and the capillary (no problem either, because you can mount it)... I wouldn't be that snipe and braging (pun intended) if I would not agree with Klaus...

Re: [ccp4bb] viewing and scoring diffraction using the PX Scanner

Dear Marcus, I always feel a bit uneasy about the advertisement-like posts that Agilent (and others) place on this BB. Of course, there are interactions between users and suppliers on many fronts, not least the support you guys provide in the form of sponsorship to meetings and conference

Re: [ccp4bb] viewing and scoring diffraction using the PX Scanner

Dear Artem, Thanks for your reply. You raise a number of points. Immediately, I should comment that the price of the PX Scanner is very considerably less than the $400k that you mention. Whilst - with the proteins and crystallisation conditions that you may be working with, visual inspecti