[ccp4bb] DNA prep for protein-DNA complex crystallization

loops. 2. Should I visualize it on the agarose gel and then try to get the dsDNA band? Sorry for these queries as I have little idea about this. Many thanks in advance. Cheers -- Amit Sharma, Ph.D. Postdoctoral Fellow, Department of Biophysics, Johns Hopkins University, Baltimore, MD21218

[ccp4bb] crystal contacts

interaction between the tyrosine residues is not a consequence of crystal contacts. I guess the fact that the molecule occurs as a dimer in solution strongly suggests so. Also, any directions towards literature showing similar cases would be of great help. Many thanks in advance -- Amit Sharma

[ccp4bb] Need isolated crystals

Dear all, I am getting protein crystals in clusters. How can I achieve isolated crystals. Thanks Amit Sharma

[ccp4bb] measuring volume of cavity

Dear All, I want to measure the volume of a cavity in my structure and look to see if it can accommodate a water molecule. Could someone please suggest me program(s) that would do this? Many thanks, -- Amit Sharma, Postdoctoral Fellow, Department of Biophysics, Johns Hopkins University

[ccp4bb] heavy atom soak

time is generally used in either case. The crystals came up in pH 6.0 buffer and the protein contains 1Cys, 3Met and 3His residues. I would appreciate any advice or link to the literature. Many thanks in advance Amit Sharma, Postdoctoral Fellow, Department of Biophysics, Johns Hopkins University

[ccp4bb] microseeding SeMet drops with native xtal seeds

alright to microseed my SeMet protein drops with seeds from the native xtal. I would be grateful if someone could shed light on this. thanks in advance, -- Amit Sharma, Postdoctoral Fellow, Department of Biophysics, Johns Hopkins University, Baltimore, MD21218

[ccp4bb] to back-soak or not to back-soak

, -- Amit Sharma, Postdoctoral Fellow, Department of Biophysics, Johns Hopkins University, Baltimore, MD21218

[ccp4bb] pH-ing Jeffamine ED-2001

Dear CCP4ers, Apologies for a non-CCP4 query. I intend to set up an optimization grid around one of the crystallization conditions (containing Jeffamine ED-2001pH 7.0 and HEPES pH 7.0) by varying the buffer pH from 5.5 to 7.5. In such a case, is it recommmended that the pH of Jeffamine ED-2001(pH

[ccp4bb] optimization of needle custers

Dear All, Apologies for a non-CCP4 query. I have clusters of needles growing for one of my proteins. It would be of great help if I could be enlightened on how to optimize them further. Additionally, could I please be directed to some literature pertaining to the same. Thanks in advance. Regards

[ccp4bb] mutation to cysteines

Dear All, Sorry for a non-CCP4 question. I intend to mutate a couple of residues to cysteines(so that they form a disulphide linkage) in a certain region of my protein. Could I please be directed to program(s) that would reliably let me do that, prior to primer designing? Thanks in advance, Amit

[ccp4bb] streak seeding in the presence of Ammonium sulfate

Dear CCP4ers, Apologies for a non-CCP4 question. I have been trying to streak seed one of my protein crystals(clusters of needles) grown in the presence of Ammonium sulphate. I set up the drops for equilibration overnight. When I opened the coverslips to begin streak seeding (the next day), within

[ccp4bb] small xtals even after streak seeding

Dear All, I had initially got some clusters of needles for one of my proteins. I then tried to streak seed them. However, I still seem to get quite a large number of nucleations, hence smaller xtals. I have tried streak seeding in in drops carrying additives such as ethanol, isopropanol, ethylene g

[ccp4bb] weak protein complexes

Dear CCP4BBers, I am trying to crystallize a protein-protein complex with Kd in the range of ~40uM. I think that if I hike the concentration of the complex by 10-fold(~400uM), I would still have ~25%of uncomplexed material present. Is it possible that I add some crowding agent such as PEG to bring

Re: [ccp4bb] Crystallogrphy today

Also, the Proceedings of the CCP4 study weekends (Acta D) have been of great help in me understanding some key concepts. It is indeed quite exciting to be able to understand the concepts that seem to be difficult initially. Cheers, Amit Sharma, Department of Biology, University of York, United K

[ccp4bb] Quick-soak

. I have no prior experience in doing this. So, it would be of great help if I could be directed towards literature/protocols pertaining to this. Any advice/suggestions would be greatly appreciated. Thanks in advance -- Amit Sharma

[ccp4bb] precipitates

could optimize the conditions further? thanks in advance, -- Amit Sharma

Re: [ccp4bb] precipitates

Dear All, Many thanks for the suggestions. Cheers, Amit 2008/10/1 Chavas Leo <[EMAIL PROTECTED]> > Dear Amit -- > On 30 Sep 2008, at 14:16, amit sharma wrote: > > I am trying to crystallize a protein with a peptide attached to it via a > 9aa linker. I am mostly ge

[ccp4bb] storing crystal seeds

the seed stock take multiple(2-3) freeze-thaw cycles? I would be grateful if anybody could shed light on this. Many thanks, in advance -- Amit Sharma

[ccp4bb] putting in methionines for SeMet crystals

/crystallizability of the protein is not compromised. Any suggestions would be of great help. Thanks in advance, -- Amit Sharma, Ph.D. Research Associate, Department of Biology, University of York, YO10 5DD UK

[ccp4bb] modelling oligomers

the docking programs, but it obviously failed as I was giving the same coordinates for the two molecules to be docked. Could someone please direct me to program(s) or online server(s) that could possibly model the trimer. Many thanks, in advance -- Amit Sharma

Re: [ccp4bb] modelling oligomers

for the replies. Amit On 10/03/2009, Ed Pozharski wrote: > > I assume that the structural homologue does not form a trimer? If it > does, than the obvious solution is to superpose your model on top of > that trimer. > > On Tue, 2009-03-10 at 10:39 +, amit sharma w

[ccp4bb] soaking with Samarium chloride

can I use and what conc. of Samarium chloride? My protein is 10 kDa , has no cysteines/methionines and pI for the molecule is 4.6. Also, could somebody suggest what other heavy atom soaks could be performed in this case? Any suggestions would be appreciated. Many thanks in advance -- Amit Sharma

[ccp4bb] high number of lysines

lysines or remove a few of those by mutagenesis. I would be grateful if somebody could share their experience in crystallization of proteins with a high number of lysines. Also, any inputs on strategies to crystallize such molecules would be greatly appreciated. Many thanks in advance. -- Amit

[ccp4bb] shape complementarity

Dear All, Could someone please suggest me program(s) to get the shape complementarity index for a protein complex structure of mine? Many thanks -- Amit Sharma, Ph.D. Research Associate, Department of Biology, University of York, YO10 5DD UK

[ccp4bb] kinetic parameter prediction

Dear All: I want to generate homology-modelled structures for some proteins(>80% Seq ID), with mutations mainly in the loops. Subsequently, I'd like to use these structural models to predict kinetic parameters(association/dissociation rates) for protein-protein interactions with cognate/non-cognat

[ccp4bb] Help with importing density fit bar plots from Coot

Hi, Density fit analysis in Coot plots the real space R-factor for each residue. Is it possible to import these values for each residue? Thank You, Amit

[ccp4bb] Wilson B-factors recorded for all PDB's to 3.5Å

Hi, What is the average Wilson B-factor for all single crystal protein structures deposited in PDB at 3.5Å? Is there any software or a publication which could provide such an estimate? Thanks, Amit

[ccp4bb] Scaling problem with multiple crystals

Dear All, I have been trying to scale (using SCALA) the diffraction data set collected from multiple crystals. Data was indexed using mosflm but fails to scale with an error saying "negative scale factors" when scaling was tried in a constant mode whereas it fails to scale with an error saying