Re: [ccp4bb] [External] Re: [ccp4bb] protein DNA complex structure and extension of DNA structure

, 2022 11:01 AM To: Srivastava, Dhiraj <mailto:dhiraj-srivast...@uiowa.edu>; CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> <mailto:CCP4BB@JISCMAIL.AC.UK> Subject: [External] Re: [ccp4bb] protein DNA complex structure and extension of DNA structure I would try the t

Re: [ccp4bb] [External] Re: [ccp4bb] protein DNA complex structure and extension of DNA structure

@JISCMAIL.AC.UK Subject: [External] Re: [ccp4bb] protein DNA complex structure and extension of DNA structure I would try the tools on this site - http://web.x3dna.org/index.php/protein From: CCP4 bulletin board on behalf of "Srivastava, Dhiraj" Reply-To: "Srivastava, Dhiraj

Re: [ccp4bb] protein DNA complex structure and extension of DNA structure

I would try the tools on this site - http://web.x3dna.org/index.php/protein From: CCP4 bulletin board on behalf of "Srivastava, Dhiraj" Reply-To: "Srivastava, Dhiraj" Date: Monday, December 27, 2021 at 8:32 PM To: "CCP4BB@JISCMAIL.AC.UK" Subject: [ccp4bb] pro

[ccp4bb] protein DNA complex structure and extension of DNA structure

Hi I solved the structure of protein DNA complex where the DNA molecule is bent. I want to extend the DNA computationally so I can model two DNA binding proteins together on same DNA molecule. is there a way by which I can extend the DNA in both direction? its easy to do it with ideal DNA mo

Re: [ccp4bb] Protein-DNA complex crystallization

mar farook >) >Subject: [ccp4bb] Protein-DNA complex crystallization >To: CCP4BB@JISCMAIL.AC.UK > > Dear All, > I have been trying to crystallize protein DNA > complex, but all the time i end up with DNA > crystals. Even i changed the length of DNA many > times

Re: [ccp4bb] Protein-DNA complex crystallization

Sent from my Verizon Wireless Phone - Reply message - From: "umar farook" Date: Mon, Nov 21, 2011 10:59 am Subject: [ccp4bb] Protein-DNA complex crystallization To: Dear All, I have been trying to crystallize protein DNA complex, but all the time i end up with DNA crysta

Re: [ccp4bb] Protein-DNA complex crystallization

Curious, how did you assess that your crystals only have DNA? F On Nov 21, 2011, at 8:59 AM, umar farook wrote: > Dear All, > > I have been trying to crystallize protein DNA complex, but all the time i end > up with DNA crystals. Even i changed the length of DNA many times but still > no comp

[ccp4bb] Protein-DNA complex crystallization

Dear All, I have been trying to crystallize protein DNA complex, but all the time i end up with DNA crystals. Even i changed the length of DNA many times but still no complex, DNA only crystallizes! Does anybody has idea, why do DNA crystallize by itself ? My protein behaves very nicely, Dynamic L

Re: [ccp4bb] Protein-DNA complex crystallisation

Hi Jhon, Here are a few hints for protein-DNA crystallization: 1.) Starting point for protein:DNA ratio is 1:1.2 for a tight binder. In your case it would be protein complex to DNA 1:1.2. I like to run size-exclusion chromatography (if applicable) to determine an optimal ratio with a slight excess

[ccp4bb] Protein-DNA complex crystallisation

Hello all I am novice to this field and trying to crystallize a protein-protein complex verses DNA i.e a ternary complex of protein complex and DNA, which stochiometry is 2:!. I am wondering 1-what should be the DNA and protein ratio for the ternary complex like this typically for binary complex i

[ccp4bb] protein DNA complex

Dear all, I am working on protein-DNA complex of which I finally got crystals, which diffract to at about 2 A. Unfortunately I wasn't able (so far) to solve the structure by MR, since the sequence identity to known homologues is very low (below 25%). By now I have successfully produced SeMe

Re: [ccp4bb] Protein/DNA complex in Buccaneer

Yes, this can be done, but you need buccaneer 1.4, which I haven't released yet. We've seen significant benefits to preserving the heavy atoms through to the refinement, although the code has been written so that it can preserve DNA or any other know structure features as well. I'll try and pu

[ccp4bb] Protein/DNA complex in Buccaneer

Hello, I am trying to use Buccaneer to improve the model of a protein/DNA complex which I solved recently. However, my initial attempt failed because the program deleted the DNA chains and rebuilt protein chains in their density. Is there a way to specify that the expected outcome should contain a

Re: [ccp4bb] Protein-DNA complex prepartion for crystallization

008 23:52:32 +0100 >From: E rajakumar <[EMAIL PROTECTED]> >Subject: [ccp4bb] Protein-DNA complex prepartion for crystallization >To: CCP4BB@JISCMAIL.AC.UK > >Dear All >Sorry for non-crystallography query. I am working on >DNA binding protein, while mixing DNA with p

Re: [ccp4bb] Protein-DNA complex prepartion for crystallization

Just wandering, is your DNA dissolved in a buffered solution or just water? I would suggest to buffer your DNA solution prior mixing with the protein. Otherwise I guess that your pH in the protein-DNA mix is lower then the expected pH7.0 and maybe your protein does not like it. Cheers, Djordje >

Re: [ccp4bb] Protein-DNA complex prepartion for crystallization

Dilute both the Protein and DNA before mixing them at the molar ratio you require - I would aim to have the protein component at around 1 mg/ml. Mix, then concentrate together, till you reach the concentration you want. Antony. On Wed, Oct 1, 2008 at 6:52 PM, E rajakumar <[EMAIL PROTECTED]

Re: [ccp4bb] Protein-DNA complex prepartion for crystallization

Hi Raj, I would definitely 10 mM MgCl2. I would also increases NaCl conc. as 200-250 mM. Also you can try 5 mM DTT. Good Luck Dev Devendra B. Srivastava Postdoctoral Associate Laboratory of Molecular Biophysics, The Rockefeller University 1230 York Avenue New York, NY-10021, USA Phone: 212-327-

[ccp4bb] Protein-DNA complex prepartion for crystallization

Dear All Sorry for non-crystallography query. I am working on DNA binding protein, while mixing DNA with protein for preparing Protein-DNA complex for crystallization, protein is precipitating. pI of the protein is 9.3 and in 15 mM HEPES 7.0, 150mM NaCl and 5% glycerol. Concentration of the protei

[ccp4bb] protein DNA complex screen Re: [ccp4bb] Query on program for creating random crystallization screen

Dear Rajakumara & all others interested in protein-DNA complex crystallization: I have an in-house sparse matrix screen (created with CRYSTOOL when it was still freeware) that is designed for protein-DNA complexes. It has been used in our lab successfully, with one case (so far) leading to public

Re: [ccp4bb] Protein DNA complex refinement

Hi all, The using of this library www.ysbl.york.ac.uk/refmac/data/refmac5.4_dictionary.tar.gz fixes the problem of refmac ! thanks Garib. Now Coot It seems that Coot does not use synonyms. Even so I have the same library for Refmac and Coot, I'm not abble to manipulate the DNA in Coot.

Re: [ccp4bb] Protein DNA complex refinement

It should. At the moment it is dealt with using synonyms. I have only done few tests. If it does not solve it then I have done something wrong need to correct it. If coot uses synonyms then it should be able to deal with these. Complete solution of this problem with backward compatablity require

Re: [ccp4bb] Protein DNA complex refinement

Also, the PDB now uses prime (') instead of asterisk (*) for the ribose numberings, and uses OP1 instead of O1P, and so forth. Do the new refmac dictionaries have that (I haven't been able to get it to work in the context of coot). Garib Murshudov wrote: > If you take new dictionary with new refm

Re: [ccp4bb] Protein DNA complex refinement

If you take new dictionary with new refmac it may solve this and some other naming problems. Try from the website: www.ysbl.york.ac.uk/YSBLPrograms/index.jsp Garib On 3 Jan 2008, at 16:53, Daouda TRAORE wrote: Hi all, I'm trying to refine a protein DNA complex using Refmac. Unfortunately t

[ccp4bb] Protein DNA complex refinement

Hi all, I'm trying to refine a protein DNA complex using Refmac. Unfortunately the job fails because of the definition of nucleosides : In the pdb file I use for molecular replacement (and it's the same for all protein-DNA complex I found in the PDB) nucleosides are labelled DG, DT, DA , DC. In

Re: [ccp4bb] protein/dna complex crystal

Hi Steve, You should definitively follow Jürgen's suggestion and try to solve the structure. If you could get an idea about the crystal packing it would help you to optimize your DNA construct. This is often critical in protein-nucleic acid crystallization. You could try to change the length of you

Re: [ccp4bb] protein/dna complex crystal

W.M. B. wrote: Dear All: My protein/DNA complex crystal diffracts to 3.2 A ( in-house source). It crystalized in 20% PEG 8K, NaCaCo 6.5. I tried to add ATP, CaCl2 and spermidine. The diffaction doesn't improve. Would you please give me some advice? Thanks a lot, Steve How does

[ccp4bb] protein/dna complex crystal

Dear All: My protein/DNA complex crystal diffracts to 3.2 A ( in-house source). It crystalized in 20% PEG 8K, NaCaCo 6.5. I tried to add ATP, CaCl2 and spermidine. The diffaction doesn't improve. Would you please give me some advice? Thanks a lot, Steve

Re: [ccp4bb] Protein-DNA complex for crystallization

er to: Tan, S. , et. al JMB 2000,297(4), 947-59. Thanks, Debanu. From: CCP4 bulletin board on behalf of bputcha Sent: Mon 7/16/2007 9:01 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Protein-DNA complex for crystallization Hi, I am trying to crystallize a protei

Re: [ccp4bb] Protein-DNA complex for crystallization

Hi Kumar, 1) While you contemplate other ideas, have you tried the following already? - microseeding or macroseeding with the tiny crystals you have? - crystallization using sitting drop vapour diffusion under oil? Often, one gets a burst of nucleation with several tiny crystals. Not sure if thi

Re: [ccp4bb] Protein-DNA complex for crystallization

On Jul 16, 2007, at 12:01 PM, bputcha wrote: I am trying to crystallize a protein-DNA complex. I purify the protein finally using gel filtration. I purchase single stranded complementary oligos (desalting from idtdna.com), mix them up How can I purify the duplex DNA further? in my exper

[ccp4bb] Protein-DNA complex for crystallization

ssouri.edu/TannerGroup/tanner.html > From: <[EMAIL PROTECTED]> > Reply-To: <[EMAIL PROTECTED]> > Date: Mon, 16 Jul 2007 11:26:42 -0500 > To: > Subject: Re: [ccp4bb] Protein-DNA complex for crystallization > > Dear Kumar, > One often has to try duplexes with many differ

Re: [ccp4bb] Protein-DNA complex for crystallization

Kumar and Joe- I'm not sure if this helps but I always purified my oligos prior to crystallization. Originally, I would order them with the trityl-group on and purify then on a reverse phase HPLC column and used TFA to cleave on column. Although towards the end of the project, I just ordered the

Re: [ccp4bb] Protein-DNA complex for crystallization

Dear Kumar, One often has to try duplexes with many different ends before getting decent crystals (e.g. 18 for one project in my lab, even more for others). Depending on your Kd, you might find that your complex falls apart during gel filtration. How long are your oligos? Gel purification som

Re: [ccp4bb] Protein-DNA complex for crystallization

Hi Kumar, I also have the same issue. If you get any helpful response, could you forward me a copy? Thank you. P.S. Could anyone who has any comments or suggestions on this issue also forward the response to ccp4bb? Thank you in advance. Best, Joe On 7/16/07, bputcha <[EMAIL PROTECTED]>

[ccp4bb] Protein-DNA complex for crystallization

Hi, I am trying to crystallize a protein-DNA complex. I purify the protein finally using gel filtration. I purchase single stranded complementary oligos (desalting from idtdna.com), mix them up and make DNA duplex by heating to 95 degree C and cooling to room temperature. I mix protein and DNA,