[ccp4bb] Manually setting 96 wells plates with lower volume samples!

Hi, I apologize in advance as it is not a ccp4 related question, but over the years, CCP4bb is synonymous with protein crystallographers virtual university, at least for me. Ok, now I do not have an easy access to crystallization robot, so I was hoping if someone here have ever used the 96 well pl

Re: [ccp4bb] Efficient way of showing residue conservation

I input the alignment in ESPript. Add the template PDB file and it makes a Bcol.pdb file where temperature factors are replaced my sequence similarity. I open this file in Pymol in Surface and color B-Factors as rainbow. Ivan On 12/7/11, Yuri Pompeu wrote: > I once saw a figure showing the prot

[ccp4bb] Crystal gel band

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 no

Re: [ccp4bb] Crystal gel band

> samples then. > > Kind regards, > > Kenneth Verstraete > L-PROBE > Ghent University > Belgium > > > Citeren "xaravich ivan" : > > > Hi everyone, >> I have grown some crystals after micro-seeding starting from thin-small >> needles from ne

[ccp4bb] Fab:antigen complex crystallization!!!

Hi everyone, I have been trying to crystallize Fab:antigen complex( 50kda:90kDa) complex and initially got needle clusters which after microseeding gave me single crystals but they are very small and I could not repeat the results. I have been using HEPES buffer at pH 6.8 to do the final SEC purifi

Re: [ccp4bb] Fab:antigen complex crystallization!!!

Thanks everyone, I got my crystals with PEG 8000 at first and after micro-seeding aith PEG 3350.Now I would work with all your suggestions and references with new vigor. ivan On Thu, Jul 28, 2011 at 9:43 AM, Patrick Shaw Stewart wrote: > Ed (and Ivan) > > Peter Sun and colleagues published two

[ccp4bb] anomalous difference fourier maps

Hello, I wanted to make an anomalous difference fourier map of a structure with zinc bound to it. However I have not been successful in making the map and I would really appreciate your help if anyone could suggest me where I am going wrong. I solved this zinc bound structure, by molecular repla

[ccp4bb] RE-anomalous difference fourier maps

Thank you guys, All of your suggestions were great. It worked. I can see the electron density of zinc, but now the problem is that it is not where I was expecting but at a very near but not superimposable position with the calcium. So I am wondering what would be the best way to rectify my structu

Re: [ccp4bb] PEG interaction with Trp in active site

Is this the active enzyme or an inactive mutant? does your substrate have any similarity with PEG (size, conformation, bulk etc?) would you assume that if there was a substrate bound to the active site and say a few waters, and/or metal ions it would probably fill the space which in this case PEG i

Re: [ccp4bb] anomalous difference fourier maps

ok I think I should say something here.For some reason I was unable to find REPLY-ALL button and my reply did not go to everyone first so I had to write another message.I got the answer to my original query and I used CCP4 (CAD) and coot as suggested by Jan. Having said that I did not know that you

Re: [ccp4bb] anomalous difference fourier maps

Hi Everyone, I hope everyone gets this email. Below are the two answers I got on how to solve my problem using ccp4.I actually emailed another person who wanted to know how I did it. So I got to transfer what I learned immediately. But his email was offline not through the forum. The answers

Re: [ccp4bb] Does the substrate has access to the active site?

Hi, I am assuming the enzyme is not active, or by substrate you do not mean the actual substrate, may be an analogue. The substrate might be converted into product and the leave through the channel and you will not find anything bound to it. But I think you have taken care of that. On Fri, Apr 9

[ccp4bb] THANKS Cryo Shipping!!

Thanks everyone for letting me know its canister. I had a different idea of a canister. I really appreciate all your replies.Instead of replying individually, I am sending this common email. Thanks again, Ivan

Re: [ccp4bb] X-Ray films

Right you are, Chayne, I think it should be shown to every " Protein crystallographer "graduate student on their first day in the lab. ivan On Thu, Apr 15, 2010 at 11:42 AM, Chayne Piscitelli wrote: > The documentary "Naturally Obsessed: The Making of a Scientist" > is definitely a must-see fi

[ccp4bb] Arp Waters vs coot find waters!

Hi everyone, I am in the process of refining my structure. I have built almost the whole protein. Now its time to add waters. Should I do refinement in refmac5 with (adding waters running coot) or should I use Arp/wArp waters or both?. I have 2.15 Angs near data and about 1200 residues. Does it m

[ccp4bb] Ramchandran outliers

Hi everyone, I have a few residues sharing hydrogen bonding and salt bridges and they tend to be fitting the density perfectly, but are Ramchandran outliers, irrespective of the cycles of refinement. It seems to me that it might be expected as these residues have some kind of strained geometry bec

Re: [ccp4bb] Advice on Over-expressing and Purifying Metalloproteins

I have found that if you give a cold shock ( 4 degrees for 30 mins-1 hr) before low temperature induction it helps to keep proteins soluble. Ivan

[ccp4bb] delta delta G color coding

Hi, Apologies for a non CCP4 questions but this one is definately for crystallographers, I have a set of delta delta Gs for there different protein ligand interactions.I want to show them in pyMOL. Is there any program or script that I could run such that the output will be read by Pymol and color

Re: [ccp4bb] please recommend a crystallization incubator

I have been using the ecotherm incubators and it is fine.heres the link. https://www.torreypinesscientific.com/products/incubators/echotherm-in30-in35-in40-and-in45-bench-top-incubators Ivan On Fri, May 28, 2010 at 3:09 PM, lei feng wrote: > hello everyone > > can anyone recommend an affordabl

[ccp4bb] Softwares for Protein-Protein docking!

Dear CCP4 users, Though this is not directly linked to ccp4, i bet many of you have solved crystal structures of the ligand and receptor separately and tried to dock it. is there any program that docks two protein molecules. We have an overall idea where the protein will bind to the receptor. Is th

Re: [ccp4bb] Softwares for Protein-Protein docking!

m.sertch...@weizmann.ac.il> wrote: > See the list in the following link > http://bip.weizmann.ac.il/toolbox/structure/binding.htm#pp > > Good Luck > Rotem Sertchook > > > On 8 Jun, 2010, at 21:59, xaravich ivan wrote: > > Dear CCP4 users, >> Though this is not

[ccp4bb] Multiple ligand refinement

Hi all, How can I refine multiple ligands( metall ions and other organic molecules) and in the same structure? I guess Refmac automatically generates restraints for common metal ions, but how could I put multiple pdbs and cif files of molecules in my Refmac cycles other than just metal ions. In my

Re: [ccp4bb] Multiple ligand refinement

, Jun 22, 2010 at 11:10 PM, Tim Gruene wrote: > Dear Ivan, > > > > On Tue, Jun 22, 2010 at 11:06:08AM -0700, xaravich ivan wrote: > > Hi all, > > How can I refine multiple ligands( metall ions and other organic > molecules) > > and in the same structure? I

Re: [ccp4bb] Density changes from Positive to negative after ligand addition and refinement

your unknown atom has a B factor similar to its > neighbors, you are probably at about the right occupancy. > > -James > > > On 6/29/2010 6:35 PM, xaravich ivan wrote: > >> Dear CCP4BB, >> >> I have come across something that might be pretty obvious to experienc

[ccp4bb] anisotropic/isotropic

Dear CCP4bb, Can I refine anisotropic ADPs for macromolecule only, while isotropic ADPs for water, simultaneously in ccp4? I have a 1.1.5 Angs data and when I refine anisotropically the rfactor/rfree difference is 6. Is it true that if I could refine the macromolecule anisotropically and the water

Re: [ccp4bb] anisotropic/isotropic

anisou records for all > the atoms and then doing something like "egrep -v 'ANISOU|HOH'" on the > pdb file. Mixed refinement will then refine only the atoms with > pre-existing anisou records (e.g. non-waters) anisotropically. > > On Thu, 2010-07-15 at 10:06 -0

Re: [ccp4bb] FT of fo-fc.coeff

Hi Vandu, I would advise you to contact CNS guys instead of CCP4 bb in this case. Although I know that many could tell you how to do it, its not a great idea to use ccp4bb to ask question about other programs, when they have relevant bulletin boards/user groups. If you do not get a satisfactory an

Re: [ccp4bb] Fab:Peptide complex crystallization

Hi Christine Do you know the conditions for crystallization of the Fab only ? You might start using the Pro-complex screens to start with and then use DMSO solubilized peptide in and around that solution. Thanks, Ivan On Mon, Jul 19, 2010 at 10:37 AM, Harman, Christine < christine.har...@fda.hhs.

[ccp4bb] Fab purification and crystallization

Hi CCP4bb, I have two questions regarding Fab purification and Fab-antigen complex crystallization and would really appreciate any input from the experienced board. 1) I have got some hits for Fab-antigen complex (150 kD) but they are all needle clusters. Whatever fine screen I formulate, it alwa

Re: [ccp4bb] Fab purification and crystallization

gt; > > > > -- > > For information and discussion about protein crystallization and > automation, please join > > our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en > > > > patr...@douglas.co.ukDouglas Instruments Ltd. > > Douglas

Re: [ccp4bb] Molecular replacement question

Two questions. What is the resolution of your data? what is the percentage sequence identity? even if you are confident of C2, try using PHASER with all space groups and searching for 2-4 monomers. Ivan On Mon, Sep 13, 2010 at 7:52 AM, Paul Holland wrote: > Hello fellow crystallographers, > >

Re: [ccp4bb] mol rep help needed

HI Dave, Have you tried PHASER. I think you might get all the four molecules in auto mode. PHASER does a great job and it should be already installed along with your ccp4i. Ivan On Fri, Oct 1, 2010 at 8:40 AM, David Roberts wrote: > Hi all, > > I'm relatively new to using CCP4 (I've done most

Re: [ccp4bb] yeast expression

Hi Alan, Is there a specific reason to choose Saccharomyces? I know a lot of labs use Pichia for protein production. I think you might look into it. On Mon, Aug 5, 2013 at 5:55 AM, Alan F Scott wrote: > Dear All, > > Sorry for the off-topic post. I am looking to overproduce a protein for > c

Re: [ccp4bb] Calcium soaking

Have you tried lower concentrations of Calcium soaking untli the crystals do not crack? Or does it crack even at very minute calcium concentration? On Thu, Feb 20, 2014 at 3:29 AM, Masaki UNNO wrote: > Dear all > > Apologies for the off-topic question: > We are studying an enzyme that is activat

[ccp4bb] Your top choice of crystal screens!

Dear Xtallographers, As the budget becomes tighter it is difficult to get hold of all the crystal screens one would want to try, to crystallize a protein. What in your opinion/experience is/are the first few commercial crystal screens you would try? ( or what are the ones you routinely start with

Re: [ccp4bb] Your top choice of crystal screens!

Thanks to all for your suggestions. I really appreciate your time. warm regards, Subhendu On Thu, Oct 16, 2014 at 9:57 AM, xaravich ivan wrote: > Dear Xtallographers, > > As the budget becomes tighter it is difficult to get hold of all the > crystal screens one would want to try, to

Re: [ccp4bb] Your top choice of crystal screens!

Hi everyone, Several of you wanted to know what kind of suggestions I received. Attached you will find all the replies. Thanks On Thu, Oct 16, 2014 at 9:57 AM, xaravich ivan wrote: > Dear Xtallographers, > > As the budget becomes tighter it is difficult to get hold of all the > cry

[ccp4bb] Strategies to bring out over-expressed protein from inclusion bodies to soluble fraction!!!

Dear cc4bb enthusiasts, This is slightly off topic but many protein crystallographers might be familiar with this problem. I have been trying to over-express a bacterial (non-E.Coli) protein in E.Coli and more than 80% goest to inclusion bodies. I tried the following Lowering the IPTG concentra

Re: [ccp4bb] The phase problem

Hi Giulliana, What is the percent identity of your best search model with your target? At what resolution does your crystal diffract? If you use PHASER without choosing the space group, it might help find you a solution in a different space group than you expect. There are also some tricks on mak

[ccp4bb] Plasmid for cloning scFV into Fab format and Bacterial expression for crystallography

Hi everyone, I have a few single chain antibodies (scFV) that I would like to express in a Fab format in bacteria for crystallization purposes. Could you suggest some plasmids that have success in such kind of projects? Are there commercial plasmids consisting of antibody constant regions ready fo

Re: [ccp4bb] molecular replacement with poor model

What is the resolution of your data? I have been able to get a solution for my protein with 30% identity but my resolution of data was 1.4 Angs. I believe to get a solution at 18% identity your search model has to be very close, like using Robetta to make the 3 mer and 9mer peptides and then work f

[ccp4bb] Protein precipitating at higher concentration!!

Hi Xtallographers, I have been able to purify a protein that was initially going into inclusion bodies from the excellent suggestions that I got here. So my lysis buffer has 0.5M Guanidium Hydrochoride, 2% TritonX-100, 500mM NaCl, 5% Glycerol in 20 mM Tris-HCL pH 8.0 The problem is that the prote

[ccp4bb] Vanishing protein in coomasie stained SDS-PAGE gel

Hello everyone, I was wondering whether anyone has had such an experience!!! I loaded a 35 mg/ml concentrated protein (in a buffer containing 50 mM Arg and 50 mM Glu) for SEC in Superdex 200. I get a nice peak ~ 300mAu and have a volume 12 ml consisting of a few fractions included in the peak. H

[ccp4bb] Best (Suitable) Mac Laptop configuration for protein Xtallography

Hello everyone, I am planing to buy a new Mac laptop (price no bar) which will let me run all xtallographic (CCP4 and Phenix) and reasonable Rosetta Molecular Modelling (1000 to 1 decoys) softwares smoothly. What in your opinion is the best configuration. (RAM, memory, number and speed of pro

[ccp4bb] HPLC vs FPLC for protein purification

Hi CCP4eans, Do you guys have any preference in purifying a protein by SEC in FPLC system or using a solvent based HPLC system after the initial affinity column purification. Where would you prefer HPLC purification over standard FPLC? I have routinely seen HPLC based purification of organic molec

Re: [ccp4bb] HPLC vs FPLC for protein purification

Thanks everyone for your insights, I really appreciate it. Cheers, ivan On Mon, Apr 20, 2015 at 5:38 PM, Edward A. Berry wrote: > On 04/20/2015 06:44 PM, xaravich ivan wrote: > >> Hi CCP4eans, >> >> solvent based HPLC system? >> > > Do you mean like ace

[ccp4bb] Superdex vs Sephadex

Hi fabulous cc4bbs, I had recently asked you about using HPLC or FPLC for protein purification.Now I have another question relating to that. I am trying to buy a new SEC column for the FPLC for protein purification but am getting some resistance from some experienced people in the lab who have now

Re: [ccp4bb] imosflm errors

stry & Molecular Biology > Johns Hopkins Malaria Research Institute > 615 North Wolfe Street, W8708 > Baltimore, MD 21205 > Office: +1-410-614-4742 > Lab: +1-410-614-4894 > Fax: +1-410-955-2926 > http://lupo.jhsph.edu > > On Jun 28, 2015, at 12:47 PM, xaravich

Re: [ccp4bb] imosflm errors

e, MD 21205 > Office: +1-410-614-4742 > Lab: +1-410-614-4894 > Fax: +1-410-955-2926 > http://lupo.jhsph.edu > > On Jun 28, 2015, at 13:51, xaravich ivan wrote: > > Thanks Jurgen, > But there is no space in the last directory, I do not know where it is > com

Re: [ccp4bb] imosflm errors

om the command line, > for more information: > > http://xia2.sourceforge.net/using_xia2.html > > However this will not resolve your original iMosflm problem, which is the > subject of the post (just responding to your ccp4i problem) > > Best wishes Graeme > > On Mon, Jun

[ccp4bb] Single search Model input in Phaser!!!

Now that I am able to run ccp4i, I have a new problem. I generated a search model by chainsaw and I want to run Phaser in CCP4i with this search model as input, but it seems that I only can give an ensamble input and I cannot find a way to input a single chainsaw generated search model in Phaser. A

Re: [ccp4bb] imosflm errors

All of the above started to work On Mon, Jun 29, 2015 at 2:27 AM, Harry Powell wrote: > Hi Ivan > > When you say "it started to work" do you mean xia2, imosflm & ccp4i, or > just one of these?? > > :-) > > On 29 Jun 2015, at 08:38, xaravich ivan wrote: &

Re: [ccp4bb] AW: [ccp4bb] Single search Model input in Phaser!!!

abor > > On 2015-06-29 16:48, xaravich ivan wrote: > >> Hi Herman, CCp4bb, >> Attached you will find the inputs for running phaser with single pdb, >> and the error that is poping up indicating I have not set an ensamble. >> How can I set an ensamble if I have only one PDB? O

[ccp4bb] Phaser solution and solvent content for arp warp

Hi everyone, I think I have finally got a solution in Phaser (screen shot attached) as the TFZ is 10. However the solution PDB has 30 molecules in it as the search model was an NMR solution. As I have 0.944 Angs resolution data pretty complete, I thought of building the initial model in ArpWarp. I

Re: [ccp4bb] ANISOU in pdb and density improvement

4 Angstrom resolution is pretty low and there has to be other info associated with that to get more help from here. How big is your protein? How are you solving the phases? How complete is your data at that resolution? What kind of multiplicity are you getting? I think you have other issues that ar