[ccp4bb] Differentiate salt and protein crystals
Hi all. I set up some trays of membrane protein remotely in cubic phase. I don't have ready access to them so I can't shoot/pick the crystals. Under polarising lights, some crystals appears coloured across many conditions, making me think these are salt. Is there some knowledge of inorganic chemistry that be relied to prioritise some for reproductions at my lab? Can detergents crystallise and produce colours under polarising light? Thank you. Theresa
[ccp4bb] Opinion on automation
Dear crystallographers I would like to get some opinion. For someone beginning to learn basic crystallography including indexing, scaling ..., should I start with automated tool like Xia2? Or is manual method for each step better for learning? Thank you. Theresa
[ccp4bb] Choice of wavelength
Hi all. When collecting data, is there a specific wavelength to be chosen at synchrotron source? Does it make difference between 0.9 and 1.5 A, for example? I know it is important for SAD/MAD but how about MIR? Thank you. Theresa
Re: [ccp4bb] Freezing crystal
A little off from the original question. Why don't small crystals dissolve to make a bigger crystal, especially when the small ones grow on top of each other? Can the clustered 3D crystals (I think it is called macroscopic twin) be used for full data collection? Again, thank you. Theresa
Re: [ccp4bb] Freezing crystal
Hi all Thanks for all the suggestions which I will try soon. How do the crystallization condition (PEG vs. salts like ammonium sulfate) affect the croyprotectant condition? Do factors like presence of low concentration of high molecular weight PEG ( 2000) mean PEG is better? Do buffers and salts in protein also important? Trying to rationalize it :) Theresa
[ccp4bb] Freezing crystal
Hi all Is there a list of conditions to be tried *first* for cryoprotectant? My crystals diffract at room temperature capillary but no in 30% PEG 400. Crystals are from 2 M ammonium sulfate. Thank you. Theresa
[ccp4bb] Off-topic: DPI and SAXS
Hi all. Does anyone have experience in using dual polarization interferometry (DPI) to study conformation change of protein when binding ligand? How do this compare to SAXS? I exclude NMR because the protein is large 90 kDa and no crystal structure is available. Thank you. Theresa
[ccp4bb] No diffraction
Dear crystallographers I have a protein of 90 kDa forming dimers. Crystals formed with microbatch and vapor diffusion method in 24 hours but no diffraction at home source. Dissolved crystals was confirmed to be the protein with mass spec. Any suggestions to improve diffraction would be welcome. Thanking you in advance. Theresa
[ccp4bb] Off topic: His-tag purification
Hi all I have a His-tagged soluble protein (8 His residues added to 90 kDa protein) that do not bind to IMAC column based on flowthrough showing up with Western blott. Do you have suggestions to improve the binding? Binding condition is 50 mM Tris-HCl 8.0, 300 mM NaCl, 10 mM imidazole pH to 8.0. Thank you. Theresa
Re: [ccp4bb] Off topic: His-tag purification
On Sun, 15 Jan 2012 23:12:46 +, Xiaodi Yu uppsala@hotmail.com wrote: Another thing you can try is using Cu ion instead of Ni ion. It will fasten the binding. Can I know what is difference in binding chemistry of Ni, Cu, Co and Fe? Is there specific rule for binding affinity versus purity? Good luck Yu Xiaodi Theresa
Re: [ccp4bb] Sub-angstrom resolution
Thank you for the interesting replies so far. Please let me ask a related question - at what resolution should we stop efforts to get better diffracting crystals? Are there *biological* questions that a model with 1.8-2.0 A resolution (with combination of complementary methods like spectroscopy) cannot answer than a model with 1 A? Theresa
[ccp4bb] Sub-angstrom resolution
Dear crystallographers A theoretical question - can sub-angstrom resolution structures only be obtained for a limited set of proteins? Is it impossible to achieve for membrane proteins and large complexes? Theresa
