Re: [ccp4bb] Mol Rep in high symmetry spacegroups

One major factor that increases the difficulty of finding a satisfactory MR solution (assuming a good search model) is the number of molecules that need to be placed in the ASU. Most programs can typically easily and quickly find a solution for one molecule per ASU. Placing three molecules per ASU

[ccp4bb] Mol Rep in high symmetry spacegroups

Hi All, I have a question- kinda general though. I've always heard of stories from people that molecular replacement is often difficult in high symmetry space groups, like R32, cubic, etc... I'm not quite sure I understand why that is the case? Is the molecular replacement difficult because in

Re: [ccp4bb] Superdex 200 PC columns

-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hi, You're right, there are two cell path sizes in AKTA systems: 2 mm and 10 mm. I don't know what path had the SMART. You definitely need the second one if you want to work with low protein concentrations. Miguel En/na Juergen Bosch ha escrit: > H

Re: [ccp4bb] Superdex 200 PC columns

Hi all, Isn't the flow cell volume the limiting factor for your detection limit ? I believe for the "old" Aktas (~5-8 years old) there were two cell sizes a laerger one and a smaller one, and I don't think it is the same size as in the SMART system. But I might be wrong. Jürgen [EMAIL PROTE

Re: [ccp4bb] Superdex 200 PC columns

Hi Robert, In my experience, there is no problem running a SMART column on an AKTA FPLC or Explorer. I have been using the same S75, S200, and Superose6 columns on all three systems depending on which one is available that day. I typically load 50 ul sample, between 10-0.01 mg/ml. The peak width I

Re: [ccp4bb] Low resolution model building

Hi Kolstoe, Kolstoe S.E. wrote: Dear ccp4bb, I am about to start model building a fairly low resolution structure (3A) with ten identical monomers in the asu, solved using MR. What I am thinking of doing is to build one monomer using some form of averaged NCS map, replicate and translate my

Re: [ccp4bb] S-tagged protein purification

Hello, This tag is very hard to release, as it forms an integral part of the RNAse A. It maybe released with 2M NaSCN but this often results in denaturation of the carrier protein. This is why the manufacturer strongly recommends having cleavage site(s) engineered in between the tag and the carrie

Re: [ccp4bb] Superdex 200 PC columns

It greatly depends on the state of your instrument - how well you took care of the optical elements, etc. It also depends on the initial concentration of the sample (and therefore the peak volume at detection time) and on the optical activity of the sample and the buffer. If the stars align and eve

[ccp4bb] S-tagged protein purification

Hi, I have a membrane protein with a C-terminal S-tag, and there is no cleavage site for the S-tag (novagen, e.g, thrombin, EK). I wonder whether I still can use this for purification without cleavage of the S-tag. Here are my questions: 1) How to release the protein bound to S-tag resin? I noti

[ccp4bb] Low resolution model building

Dear ccp4bb, I am about to start model building a fairly low resolution structure (3A) with ten identical monomers in the asu, solved using MR. What I am thinking of doing is to build one monomer using some form of averaged NCS map, replicate and translate my one built monomer into the other ni

Re: [ccp4bb] Superdex 200 PC columns

-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hi Robert, I have run those PC 2.3/30 with ~10 ug of protein (of course this depends on the protein) and still get a useful signal at 280 nm. If you don't have anything interfering at ~215 nm and your HPLC system can read at that wavelength, you can e

[ccp4bb] Superdex 200 PC columns

Sorry for perhaps off-topic question but I am writing to ask if anyone has experience in using Amersham Superdex 200 PC 3.2/30 columns in conjuction with the Precision Column holder on a standard (not SMART system) AKTA FPLC. At the moment using a Superdex 200 10/300 column on a standard AKTA I ca