Re: [ccp4bb] Methylation of macromolecular complexes

Hi All, Many thanks for your reply. I am quite sure that my crystals are the complex. I made the complex by 1:1.1 ratio with the small component excess, then did the methylation, followed by gel filtration to remove the small reductant. The SDS-PAGEs for gel filtration fractions and crystals

Re: [ccp4bb] Help request: Failed MR using the same molecule as model

A few things to try (or double-check): 1. If you ran phaser with SGALTERNATIVE ALL, make sure the mtz file you gave to refmac has the same screw axes as the MR solution. If this is off, it'll lead to higher R-factors. 2. As Fred mentioned, try with a single copy of the protein, and check th

[ccp4bb] Bad geometry for alt. conformation refined in Refmac5

Hello All, We are trying to refine ARG residues with two conformations in Refmac5, and the refined atom positions in the output PDB file are all over the place, as if the geometry restraints are not well defined. We've tried several different formats for the input file, based on previous postings

Re: [ccp4bb] Bad geometry for alt. conformation refined in Refmac5

Could you please try the version from York: www.ysbl.york.ac.uk/refmac/latest_refmac.html I think probkem you mention is related with compilation or something. At least I cannot repat it on my computer regards Garib On 25 Nov 2009, at 16:18, John Pascal wrote: Hello All, We are trying to

Re: [ccp4bb] Help request: Failed MR using the same molecule as model

I would also try Open-EPMR as well as Phaser. It can sometimes find solutions that Phaser or Amore cannot, especially for multiple chains and low resolution. EPMR is especially good at handling high copy numbers of search models. Cheers. Pete Meyer wrote: A few things to try (or double-check

Re: [ccp4bb] Help request: Failed MR using the same molecule as model

also try MOLREP (CCP4i) - we had a case where AMORE and PHASER did not work (in our hands), but MOLREP did, using the internal MOLREP sequence alignment/unequal amino acid trimming feature. This was a case of a P1 cell with 12 copies of the same molecule, 2.3A resolution, not very high-qualit

Re: [ccp4bb] Help request: Failed MR using the same molecule as model

To complicate things some more, a couple of things could be going on. 1. You could have pseudosymmetry, where you true SG is I4 and the additional NCS operators make the crystal look I422. 2. You could have twinning. If merohedral, could be I4. If pseudomerohedral, could be any of the subgrou

Re: [ccp4bb] Help request: Failed MR using the same molecule as model

Actually, another thing could be going on as well. You show a large off-origin peak in the Patterson in I422 so you may have pseudotranslation going on and you processed in the supercell. You could probably try to reindex choosing fewer spots and get your P422 cell. I am sure there is some law

Re: [ccp4bb] decrease of background with distance?

Thank you all for your informative responses! While examining the effects of unusual beam profiles on data collection due to capillary optics, I had collected a wedge of data on a large, high-quality lysozyme crystal at 8 different sample to detector distances. I restricted the analysis of

Re: [ccp4bb] decrease of background with distance?

Richard Gillilan wrote: I like Jim Plufgrath's way of looking at the problem: reflections and background scatter have different effective source sizes and distances. For a reflection, the source is the same as the beamline source (some place way upstream). For solvent the source is the sample

[ccp4bb] Looking for open_EPMR

Thanks for your replies! Does anyone know where to find free open_EPMR software? It seems that website is no longer reachable. Chao

Re: [ccp4bb] Looking for open_EPMR

On Wednesday 25 November 2009 13:56:38 Chao Quan wrote: > Thanks for your replies! > > Does anyone know where to find free open_EPMR software? It seems that > website is no longer reachable. The web site looks perfectly normal from here: http://www.epmr.info/ -- Ethan A Merritt Biom

[ccp4bb] A dimer as a MR search model?

Dear CCP4 community: I am trying to solve a structure using Molecular Replacement. We obtained several crystal forms of the same molecule, which is a hetero-trimer(18KD+16kD+15kD). We have solved the structure of one crystal form(form_1), which has space group P 42 22, 1 molecule per Asymmetr

[ccp4bb] Truncation of first two residues "rescued" an insoluble protein?

Dear all, Sorry for the off-topic question, but I am really curious why two terminal residues could make such huge difference. I am working on a all helical domain starting from residue 1 to about 130. It expressed well with N-terminal His tag in both E.Coli and insect cell, but stayed in

Re: [ccp4bb] Truncation of first two residues "rescued" an insoluble protein?

Dear Tiancen, This is perhaps a more extreme example of what many of us have experienced over and over - namely that for some proteins very small changes make a huge difference in expression, solubility, activity or all three :) Long and rambling reply follows - don't read if you are easily bored