[ccp4bb] solvent-flattened electron density map
Dear All, Will you please let me know what is the solvent-flattened electron density map and how to get it? Cheers, Acoot
[ccp4bb] A question on the diffrentiattion of the salt crystal and salt diffraction in the protein metal complex
Dear All, Suppose I have a crystal hit from the protein-metal complex, with the possibility of that the hit is a salt crystal. When I diffract it by X-ray, I got some metal (or salt) diffraction without the protein diffraction (maybe due to too low protein resolution). Will you please tell me how to know whether my diffraction was from a salt crystal or from the diffraction of the metal in my protein-metal complex? I am looking forward to getting your reply. Cheers, Acoot
[ccp4bb] A question on protein microheterogenity for crystalization
Dear All, When I purified my protein by ion exchange chromatography for crystallization, there were several peaks containing the target protein as analyzed by SDS-PAGE. All these peaks have the same MW as determined by gel filtration coupled MALLS. For crystallization purpose, can I merge the corresponding ion exchange chromatography peaks together? Otherwise the protein yield will be too low. And how to explain the heterogeneity by ion exchange chromatography in this situation? I am looking forward to getting a reply from you. Acoot
[ccp4bb] A question on crystal optimization
Dear All, I am optimizing a crystal. In one of the optimizing conditions I find the crystal is cubic-like shape (the crystal is not large, but absolutely not the traditional tiny crystal. The crystal has some kind of faces and edges but not so sharp, and it is absolutely not round). But after 1 day the crystal changed into sphere form (the cubic not obvious). Will you please introduce your experience on how to get the sharp face and sharp edge crystal for my situation)? There is source says if the crystal grows too faster, the sharpness would be lost. Will you please also let me know how to slow down the growth rate of the crystal? Cheers, Acoot
Re: [ccp4bb] DNA Protein co- Crystallization
Dear All, For the question, I think for a protein-DNA interaction, the protein may interact with any sequences of DNA, which will give a lot of combination of protein-DNA sequence for crystallization screening. Or do anyone regard to just try the crystallization of the protein-one specific sequence DNA fragment for the trial (for example the DNA sequence with the highest binding affinity)? In another word, does the easiness of the crystallization has relation with how strong the protein interacts with the DNA sequence? Acoot On Saturday, 4 January 2014 11:02 AM, rajakumara eerappa reera...@gmail.com wrote: My suggestions are 1 try complementary and non-complementary overhangs which can form Watson-crick and/ or Hoogstein base pairing. 2. If it binds self-complementary duplexes then try them also. 3. Peg conditions with slight acidic pH are more suitable. 4. Divalent cation salts (Ca, Mg, Mn) in crystallization. Wish you good luck Raj On Friday, January 3, 2014, venkatareddy dadireddy wrote: Hi, I'm working on DNA binding protein, looking to co-crystallize protein- DNA complex and have no previous experience. Your suggestion would be very precious on the following queries. 1. My protein is 646 amino acid long and it exists as homodimer. It is also having around 20 amino acid extra sequence from vector. Will vector sequence affect crystallization? 2. Its homologous protein shows good affinity for 31-mer. Shall I use same length of DNA for co- crystallization. 3. What is the length of DNA to be used? 4. What is purity of oligos to be used? Is it HPLC pure or normal desalted ones. I have read on CCP4 mails for screening purpose normal oligos are fine. Please comment on that. 3. Any other suggestions on Protein DNA co- crystallization. Thanks venkat
[ccp4bb] A question on protein-DNA complex crystallization
Dear All, I am now working on the crystallization of a complex of protein-16 bp DNA by co-crystallization. In the screening very small needle-like crystal occurs. If not salt crystal, is there a method to know it is not the crystal of the DNA? Cheers, Acoot
Re: [ccp4bb] Assays for protein-ligand interaction?
FRET， CD， Fluorescence， NMR chemical shift assay, isotope-labelled ligand interaction assay, protein melting temperature assay, gel filtration retention assay, gyration radius assay by Malls, native page gel analysis, etc. Acoot On Monday, 13 January 2014 8:51 PM, HJ Lee hojunle...@gmail.com wrote: Sorry for the off topic. I'm looking for a way to monitor protein-(potential) ligand interaction. The ligand is small molecule (mw~250) and we're looking for its potential interaction with couple human proteins. (We do not know this small molecule interacts with these human protein or not.) Is there any efficient way to quickly identify whether this ligand interacts with those human protein? We can buy some protein, but the amount of commercially available purified proteins is very little, making them hard to be analyzed by some good methods (e.g. ITC). That would be really great if anyone suggest any idea. Sorry for the off topic question again. Thanks!
Re: [ccp4bb] Circular Dichoism 280nm range signal
''Signals in the region from 250-270 nm are attributable to phenylalanine residues, signals from 270-290 nm are attributable to tyrosine, and those from 280-300 nm are attributable to tryptophan. Disulfide bonds give rise to broad weak signals throughout the near-UV spectrum. '' http://www.ap-lab.com/circular_dichroism.htm From: JinSoo.Bae jiroz...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, 15 January 2014 9:41 AM Subject: [ccp4bb] Circular Dichoism 280nm range signal Dear all, Sorry for the off topic. We are comparing the high order structure between Fc engineered mAbs using CD methods. There are some different (noise ?) signal in the 280nm range each antibodies. Other region is almost close match with respect of peak shape and theta value. Is there any specific reason why the wavelength 280nm area have a little different signal ? That will be really thanks if someone explain the reason or give a some references papers. Thanks in advance. Jin Soo Bae 790-784 room 204, Dept of Life Science, POSTECH, san31, Hyoja-dong, Nam-gu, Pohang, Gyungbuk, Korea tel:82-54-279-8627 fax:82-54-279-8111
Re: [ccp4bb] Protein concentration form chromatograms
I don't think it possible for protein purification purpose. There would be no linear correlation between A280 and the protein concentration. For pure protein analysis, as for minor amount protein used, it is possible. Acoot From: Karel Chaz karel.c...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, 15 January 2014 11:09 PM Subject: [ccp4bb] Protein concentration form chromatograms Dear all, A question for the biochemistry-inclined folks in the bb; how do I calculate protein concentration of chromatography fractions, starting from Abs280 from the UV monitor? I know I could figure it out myself if I really tried, but why bother when I have access to so many brilliant minds Thanks to all, K
[ccp4bb] protein domain for crystalization contact
Dear All, After series of trial and even with the truncated forms, I find my protein has the difficulty to contact to form the crystallization, or lacking the nucleation center in the purified protein. Will you please introduce the domains with witch my protein fused, there will be more contact points for the crystallization formation, besides the traditionally known Fab fragment? Cheers, Acoot
Re: [ccp4bb] protein domain for crystalization contact
Dear Timothy, My is non-membrane protein. The proteins you recommended are also suitable, right? Cheera, Acoot On Thursday, 27 February 2014 7:07 PM, Timothy Craig tlmcra...@hotmail.com wrote: You may want to try cytochrome B 562 RIL (BRIL), T4 Lysozyme, rubredoxin, or amicyanin. These are commonly used for crystallizing difficult membrane proteins. The protein sequences can be found in the PDB. You can get the DNA from any gene synthesis vendor like dna2.0, geneart, or genscript. -Tim Craig Date: Thu, 27 Feb 2014 02:29:58 -0800 From: acootbr...@yahoo.com Subject: [ccp4bb] protein domain for crystalization contact To: CCP4BB@JISCMAIL.AC.UK Dear All, After series of trial and even with the truncated forms, I find my protein has the difficulty to contact to form the crystallization, or lacking the nucleation center in the purified protein. Will you please introduce the domains with witch my protein fused, there will be more contact points for the crystallization formation, besides the traditionally known Fab fragment? Cheers, Acoot
[ccp4bb] table 1 and scalar
Dear All, In the crystallography paper table 1, there is Values in parentheses are for the highest resolution shell. Is this highest resolution shell same as the OuterShell for the analysis result got by Scala? I am looking forward to getting your reply. Cheers, Acoot
Re: [ccp4bb] How to know if ADP exists in the ATP-binding site of bacterial expressed proteins
Ion exchange chromatography, this is the published method. Acoot From: Xinghua Qin xinghua...@126.com To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, 4 June 2012 2:51 PM Subject: [ccp4bb] How to know if ADP exists in the ATP-binding site of bacterial expressed proteins Deer CCP4ers, How to know if ADP exists in the ATP-binding site of bacterial expressed proteins? Check the UV spectrum at 280 nm is one way, but there is no Trp in my protein. Is there any other conveniet way to find out? Thanks in advance Best wishes Xinghua Qin -- Xinghua Qin State Key Laboratory of Plant Physiology and biochemistry College of Biological Sciences China Agricultural University No.2, Yuan Ming Yuan West Road Haidian District, Beijing, China 100193 Tel: +86-10-62732672 E-mail: xinghua...@126.com
[ccp4bb] a question on presenting 3-D crystal structure in the paper
Dear All, I want to use dash line to present the salt bridge in a crystal structure in the paper publication, for example the salt bridge formed between Glu (OE1 and OE2) and Lys (terminal N). Do you suggest I only draw a single dashed line between the terminal N of the Lys and the closer atom to N among the Glu OE1 and Glu OE2 in the final publication, or in the final publication I will have 2 dashed lines, one is between terminal N of Lys and OE1 of the Glu, the other is between terminal N of Lys and OE2 of the Glu, supposing the distance of both the 2 dash lines are within the acceptable range for salt bridge? I am looking forward to getting a reply from you. Cheers, Acoot
[ccp4bb] Salt bridge in crystallization
Dear All, A lot of 3-D crystal structures highlight the salt bridges in the structure, although some structures of them are got at high salt concentrations. Will you please explain to me why the protein salt bridge can still exist in the high salt concentration as used in the crystallization condition? I am looking forward to getting your reply. Acoot
[ccp4bb] inflluence of pH for crystallization on protein 3-D structure
Dear All, A protein crystal can be got at pH 5 or 8, or a pH with much extreme value. What will be the relatively extreme pH value to get the crystal on the protein structure solved based on the crystal got? I mean usually we regard the physiological pH as 7. If a crystal was got at pH 5, the structure solved may be different from the protein structure at pH 7. But it seems there is rarely analysis on the discrepancy of the protein structures when publishing 3-D structure with the protein crystal got at relatively extreme pH. I am looking forward to getting your comment on it. Cheers, Acoot
[ccp4bb] on NaCl and Tris for crystallization
Dear All, For the protein buffer for the crystallization purpose, if we buy from Sigma-Aldrich, which types of NaCl and Tris are much suitable? I am looking forward to getting your reply. Cheers, Acoot
[ccp4bb] Temperature and crystallization
Dear All, Will you please give a comment on how the temperature influences on the possibility to get protein crystal, and how the temperatures used to get the protein crystals of the same protein influences the protein 3-D structures of the same protein got based on the crystals of the same protein got at different temperatures? Cheers, Acoot
[ccp4bb] .4/7/2013 3:32:52 PM.
http://www.oborges.com/rxpou/sglkxqkvddy.yflv Acoot Brett
