Re: [ccp4bb] Inclusion bodies centrifugation

agree with Michael, use low concentration of detergent. I normally resuspend the cell in buffer and sonicate, spin down at 6000 g., resuspend again with buffer containing small amount of detergent and resonicate, spin down again at 6000 g. finally was with buffer again (resuspend and spin down). B

Re: [ccp4bb] data reduction

Alex, With regard to scalepack, be sure you use NO MERGE ORIGINAL INDEX, not just NO MERGE. Marian Szebenyi MacCHESS > Dear all: > > I am trying to solve a structure from apparently a hexagonal crystal. > I indexed and scaled data in P6 in Scalepack (with merging) then used > Scalepack2mtz (wit

Re: [ccp4bb] Problem with TLS

Hi Matthias, a few suggestions: - use TLSMD server to define TLS groups; - before using TLSMD do some group ADP refinement with one or two refinable ADP per residue (you can do it in phenix.refine), so you get less biased ADPs from previous restrained B-factor refinement; - just out of curiosi

Re: [ccp4bb] data reduction

Dear Eleanor, Even if Denzo, Scalepack and HKL suite have been extensively developed, their file formats have not changed in many years. However, there are intermediate programs between Scalepack and structure solution ones that can potentially be a source of problem. For example, ctruncate for so

Re: [ccp4bb] Problem with TLS

I dont understand it either but I have also found there are problems with starting again from the previous end point.. If you have run TLSANL after the refinement, then the TLSOUT would be totally inappropriate to start from.. The TLS records n the PDB file and the ANISOU records are completely

Re: [ccp4bb] Duet vectors

Dear Silvia, we have cloned genes in pETDuet, pACYCDuet and pCDFDuet without any problems. What we always do, with any plasmid, is transform bacteria freshly before any miniprep or protein preparation - we do not believe in stocking plasmids "on plate" or as glycerol stocks. Glycerol stocks

[ccp4bb] Problem with TLS

Dear BB! I have a strange problem with TLS refinement and refmac. In my AU there are 4 monomers of the same protein. Based on structural subdomains I defined 3 TLS groups for each of those monomers so that the overall TLS group number is 12. When I do TLS and restrained refinement from the GUI u

Re: [ccp4bb] Duet vectors

Hi, This is not typical. Try different cell lines for propagation and also add more sugar and glycerol to your medium - this helps. Artem > Dear all, > > We are attempting to clone into pETDuet, pACYCDuet, pCola1Duet and > pCDFDuet > and we are encountering numerous difficulties. > > We are awar

Re: [ccp4bb] Duet vectors

This is strange. I've used the pET-Duet and pACYCDuet vectors extensively and the others once or twice and I've never seen behavior like that. The pET-Duet has the same replication origin as "normal" pET vectors and has never presented me with any more difficulty than they do. I typically

[ccp4bb] Correlative Microscopy Workshop

This may be of interest to some of you. We are in the process of putting together what we hope to be an exciting program of talks with plenty of opportunity for discussion. Liz Dr. Liz Duke Principal Beamline Scientist Diamond Light Source Harwell Science and Innovation Campus Chilton OX11 0DE

[ccp4bb] Duet vectors

Dear all, We are attempting to clone into pETDuet, pACYCDuet, pCola1Duet and pCDFDuet and we are encountering numerous difficulties. We are aware that most of them are low copy number, but even taking this into account, we find it puzzling that whereas the first preps of the vectors gave low

Re: [ccp4bb] data reduction

Can you sed a bit of your scalepack unmerged data - that would allow us to check format and pointless behavior.. It sounds a bit like a scalepack problem though.. Eleanor Alexandra Deaconescu wrote: Dear all: I am trying to solve a structure from apparently a hexagonal crystal. I indexed an