Re: [ccp4bb] hydrophilic protein going to aggregate
Hi Anita, Try to lower the ph of binding buffer if your protein allows also you may lower the concentration of salt like nacl if adding into the buffer also try to reduce the imidazole concentration of your eluted fraction gradually by performing serial dialysis before SEC. Alternatively, you can try cobalt IMAC instead of nickel which may have lower affinty to proteins his tag. Also it may possible that your trucated construct may have lesser stability. You can also try to add some additives like glycerol to improve its stability along with some reducing agent like DTT or TCEP. Hope this may help your protein. Shivendra On Feb 25, 2015 9:13 AM, Anita P crystals...@gmail.com wrote: Hello Crystallographers, I am trying to express and purify a soluble domain of a membrane protein for crystallization. The amino acid content is as below Ala (A) 12 13.8% Arg (R) 10 11.5% Asn (N) 2 2.3% Asp (D) 8 9.2% Cys (C) 1 1.1% Gln (Q) 1 1.1% Glu (E) 4 4.6% Gly (G) 16 18.4% His (H) 3 3.4% Ile (I) 0 0.0% Leu (L) 3 3.4% Lys (K) 1 1.1% Met (M) 1 1.1% Phe (F) 2 2.3% Pro (P) 6 6.9% Ser (S) 11 12.6% Thr (T) 1 1.1% Trp (W) 1 1.1% Tyr (Y) 1 1.1% Val (V) 3 3.4% I could purify the protein using IMAC to 95% purity, how ever, I had to elute with very high concentration of imidazole 2 M, still some protein is attached to the beads as I could observe on the SDS PAGE. I concentrated the protein to 25 mg/ml of 3 mls and on performing SEC, I found the protein to be in the Void fraction of SEC 200. Any expert advice on how to optimize this purfication and why it is still attached to the beads? thanks in advance Anita
[ccp4bb] PEG dependent structural changes in crystal structure
Dear All, I have been working on a protein which initially got crystallised in condition having PEG1500 as precipitant. The space group was P21 and got solved with reasonable Rfree. Analysis of its structure showed large deviation and very distinct active site architecture along with disorderedness in one of its long loop (no density) in comparison with the expected result, based on related homologous structures. The structure does not seem to be active with one of its active site residue moved apart from other catalytic amino acids. Also the substrate entry tunnel looks distorted. The purified enzyme used for crystallisation showed optimum activity in vitro. This led us to screen it again for some other crystallisation condition and got another crystal hit in condition having PEG3350 as precipitant. Rest of the components of crystallisation cocktail were same. The data belonged to P212121 space group. The regions which were disordered or distorted in earlier case were observed to be ordered and in their expected orientation and position. The enzyme is not reported to be in different structural or functional states as observed. I am wondering how the protein from the same batch showed two distinct structural organizations in conditions with varying PEGs. What may cause it to follow such transition. Whether it has some significant functional aspect or just a result of improper crystal packing. Thanks. Shiv
[ccp4bb] how to ascertain the different ligand species in average electron density
Dear All, I am solving a 2.6A protein complex data and found the density for ligand but due to resolution constrains, it is not so sharp but clearly suggesting the fitting of ligand. The ligand is not so potent and tight binding inhibitor of enzyme and mimics the substrate and may exist in covalently linked acylated (single ring), hydrolyzed (single ring) and non-covalent (double ring) forms. Although deacylation rate of inhibitor is very slow in comparison to substrate therefore enzyme-inhibitor complex is expected to be in covalently linked state but the presence of other tow species in small proportion is also inevitable. The density is continuos through catalytic Ser residue to the carbonyl carbon atom of ligand which was expected to participate in covalent bond formation with enzyme. Although while fitting the ligand, it is always moving beyond the C-O covalent bond distance and forcing it by fixing atom resulted in distorted ligand geometry after refinement. The density around the side chain of serine is bit wide but the placement of its side chain closer to the carbonyl carbon of ligand was reverted back after refinement. The placement and fitting of other two (hydrolized and non-covalent) species seem to be optimum and no negative density was observed after refinement. These ligand species varies slightly in atomic composition due to addition or deletion of few atoms by virtue of acylation and deacylation reaction but the density at this resolution does not allow us to identify the exact species. There are four mol/ASU and the orientation of some of the atoms of ligand are different among them. I am very much eager to get some insights for few of my quarries: 1- How can I ascertain the that which of the ligand species is actually present in my structure? 2- If more than one species is present and observed density is the average of them then how to find out the proportion of each of the ligand species? 3- As mentioned earlier that I was able to fit all the forms of ligand with ocuupancy-1 in density without any negative density, why so? 4- How can we decide the occupancy factor for different species of same ligand? Based on assumption or is there any way to decide this? 5- If finally I could model the ligand in any of the form than how can we validate its correctness? 6- Is there any way to sharpen the density at lower sigma by excluding noise. Thank you. Shiv
[ccp4bb] How to ascertain the different ligand species in average electron density
Dear All, I am trying to solve a 2.6 A protein complex data and found the density for ligand but due to resolution constrains, it is not so sharp but clearly suggesting the fitting of ligand. The ligand is not so potent and tight binding inhibitor of enzyme and mimics the substrate and may exist in covalently linked acylated (single ring), hydrolyzed (single ring) and non-covalent (double ring) forms. Although deacylation rate of inhibitor is very slow in comparison to substrate therefore enzyme-inhibitor complex is expected to be in covalently linked state but the presence of other tow species in small proportion is also inevitable. The density is continuos through catalytic Ser residue to the carbonyl carbon atom of ligand which was expected to participate in covalent bond formation with enzyme. Although while fitting the ligand, it is always moving beyond the C-O covalent bond distance and forcing it by fixing atom resulted in distorted ligand geometry after refinement. The density around the side chain of serine is bit wide but the placement of its side chain closer to the carbonyl carbon of ligand was reverted back after refinement. The placement and fitting of other two (hydrolized and non-covalent) species seem to be optimum and no negative density was observed after refinement. These ligand species varies slightly in atomic composition due to addition or deletion of few atoms by virtue of acylation and deacylation reaction but the density at this resolution does not allow us to identify the exact species. There are four mol/ASU and the orientation of some of the atoms of ligand are different among them. I am very much eager to get some insights for few of my quarries: 1- How can I ascertain the that which of the ligand species is actually present in my structure? 2- If more than one species is present and observed density is the average of them then how to find out the proportion of each of the ligand species? 3- As mentioned earlier that I was able to fit all the forms of ligand with ocuupancy-1 in density without any negative density, why so? 4- How can we decide the occupancy factor for different species of same ligand? Based on assumption or is there any way to decide this? 5- If finally I could model the ligand in any of the form than how can we validate its correctness? 6- Is there any way to sharpen the density at lower sigma by excluding noise. Thank you. Shiv
Re: [ccp4bb] Crystal gel band
Hi ivan, The detection senstivity of proteins depends on staining technique, You have not mentioned about staining, whether silver or Coomassie. The silver staining is more sensitive than coomassie, typically you can see 10-50 ng of a protein in silver staining. It does vary, however, with the glycosylation and physical properties of the protein. Shivendra On 2 November 2010 08:20, xaravich ivan xaravich.i...@gmail.com wrote: Hi everyone, I have grown some crystals after micro-seeding starting from thin-small needles from needle-clusters. These crystals are larger in size than the needles but are comparable to the shape and don't look like salt crystals. But I cannot see the bands( its a complex) in the SDS-PAGE.I do not have a home source,handy and would like to send these to the synchrotron. Is it possible to NOT see a band of protein crystals in SDS-PAGE, if, say, the amount of protein is 1uG? Has anyone experienced such a thing (no band in gel, but crystal diffracts)? It would be nice if I get observations/suggestions. ivan
