Re: [ccp4bb] clashscore question

2019-01-16 Thread Xiaodi Yu
Thank you all for the input. I think my problem got solved.

The structure was determined by cryo-EM at 3.2 A. I used 
phenix.real_space_refine to refine the structure using the model with no 
hydrogen. the statistic report directly from phenix was pretty good but not 
from pdb validation server (especially the clashscore changes a lot). As John 
and Sharan mentioned here that the PDB server calculates the clashscore with 
the hydrogen added and that makes the difference.

What I did is adding the hydrogen first and followed by one run of phenix 
refinement. Finally, the problem got solved, the clashscore was dropped to 2 
reported from both the phenix and PDB server. I do consider the alternative 
conformation and symmetry. While I think adding the hydrogen is the key to my 

Thank you again for your kindly suggestion. I wish my case may serve the people 
who have the similar problem in the future.


From: CCP4 bulletin board  on behalf of John Berrisford 

Sent: Wednesday, January 16, 2019 12:06 PM
Subject: Re: [ccp4bb] clashscore question

Dear Xiaodi

The wwPDB validation service does indeed add hydrogens using Reduce before 
calculating clashes.

For more details of what processes are run when generating the wwPDB validation 
report see the on-line help

If you would like us to look into this in more detail please can you email us 
directly on<>

with details of your validation session.

This email address is also listed on the wwPDB validation help page



From: CCP4 bulletin board  On Behalf Of Sharan Karade
Sent: 16 January 2019 04:28
Subject: Re: [ccp4bb] clashscore question

Dear Yu,

I think pdb validation server add hydrogen to the structure and then calculate 
the clashscore. The other methods you mentioned calculate clashscore without 
adding hydrogen to the structure.



On Tue, Jan 15, 2019, 11:23 PM Xiaodi Yu> wrote:

Hi All:

I have a pdb file and the clashscores reported from both phenix and MolProbity 
were around 5, while the clashscore from the pdb validation server is 17. I 
wonder what may cause the difference? Any suggestion is appreciated.

Thank you.


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[ccp4bb] clashscore question

2019-01-15 Thread Xiaodi Yu
Hi All:

I have a pdb file and the clashscores reported from both phenix and MolProbity 
were around 5, while the clashscore from the pdb validation server is 17. I 
wonder what may cause the difference? Any suggestion is appreciated.

Thank you.


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Re: [ccp4bb] model bias

2015-04-29 Thread Xiaodi Yu
Hi Aleks:

Maybe you can try CNS ( Initial refinement by simulated annealing) also. It may 
help to get rid of the model bias and takes short time to run.


 Date: Wed, 29 Apr 2015 13:52:53 +0200
 Subject: Re: [ccp4bb] model bias
 I would certainly try the usual approaches (map coefficients that are 
 less sensitive to model-bias, i.e. Sigmaa; OMIT maps - there are several 
 of these which can be calculated). In addition, you may have a look at 
 the approach of Liu and Xiong (2014, J. Mol. Biol. 426, 980-993). It is 
 a well known technique (applying a negative temperature factor to 
 amplitudes, for map sharpening) but this paper describes a systematic 
 study indicating improvement whatever the situation.
 As usual in macromolecular crystallography, confidence is gained by the 
 use of several approaches.
 On 29/04/15 13:16, Aleksandar Bijelic wrote:
  Dear CCP4 users,
  I am currently solving a structure (2.8-2.9 A resolution) of a protein 
  complexed with a ligand using MR with the apo-form of this protein as 
  model (resolution of the model is 2.4). After MR-phasing I performed a 
  regular autobuild run giving me good outputs and thus I refined the 
  best pdb leading to good values according to R-values and geometry, 
  however, the denstiy doesn´t look well (but I think it´s due to the 
  moderate resolution). Now I want to get sure if the side chains which 
  are involved in the ligand binding are correctly positioned. However, 
  the active site is suspicously similar to the active site of the model 
  (apo-form) and so I am afraid that this could be due to model bias.  
  My question is how to check and to get rid of the bias (if present) at 
  this stage (after several refinements). I read the publication of 
  Terwilliger about iterative-build OMIT maps but since I am a bloody 
  novice in this field I didn´t really understand it. I originally 
  thought iterative-build OMIT maps are performed to compare the output 
  map with one´s map in order to detect uncertainties, but what to do 
  next? Or should I start from the beginning but how to proceed than, 
  what should I do (I am using Phenix via GUI) ... Is it possible and 
  reasonable to run autobuild with iterative omit map option? Or is it 
  only reasonable if experimental phases are available? I didn´t run 
  iterative-build OMIT maps yet because I am not sure how to run it 
  correctly (what method is the best?) and at my institute the run will 
  take more than 1 day and I don´t want to block one computer until I am 
  not sure if it is reasonable. I hope you can give me some advice and 
  help me. Thank you in advance.
 Fred. Vellieux (B.Sc., Ph.D., hdr)
 Campus EPN
 71 avenue des Martyrs
 CS 10090
 F-38044 Grenoble Cedex 9
 Tel: +33 457428605
 Fax: +33 476501890

Re: [ccp4bb] ITC with unfolded proteins

2014-03-14 Thread Xiaodi Yu
I have done once by using two proteins, one is disordered, the other is very 
well folded. The result I got is the baseline drift. The baseline goes up upon 
each injection. The reason I thought at that time is the heat capacity changed 
dramatically in the system. The disordered protein may form some degree of 
structure after interacting with its partner. And we did find some 
precipitation after the experiment.


Xiaodi.yu@childrens.harvard edu

Sent from my iPad

 On Mar 14, 2014, at 9:57 AM, Anita P wrote:
 That is a very interesting question, which I would request the seniors out 
 there to give their insights on.
 I was imagining that a recombinant purification of an unfolded partner would 
 aggregate which would cause trouble in ITC. Am I correct in this theory?
 Would love to have more insights.
 thanks in advance
 On Fri, Mar 14, 2014 at 7:18 PM, wrote:
 I think the experiment is doable, but how would you decouple
 protein-protein interaction from folding of the unfolded
 protein due to protein interaction?
 Reza Khayat, PhD
 Assistant Professor
 The City College of New York
 Department of Chemistry, MR-1135
 160 Convent Avenue
 New York, NY  10031
 Tel. (212) 650-6070
  Original message 
 Date: Fri, 14 Mar 2014 18:07:48 +0530
 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf
 of Anita P
 Subject: [ccp4bb] ITC with unfolded proteins
Hello everyone,
I have a query for the scientists working on
protein-protein interaction.
It is known that some proteins exist in unfolded or
molten globule state and attain structure on
interaction with other folded proteins.
Many a times, it is difficult to obtain the
structure of these complexes.
Is it possible to quantitatively determine the
thermodynamics of interaction between an unfolded
protein and a folded protein using ITC? Later may be
perform an alascan to determine the residues of the
unfolded partner involved in the interaction.
Please share your ideas

Re: [ccp4bb] A question on protein microheterogenity for crystalization

2013-12-14 Thread Xiaodi Yu
Hi Acoot:

Can your protein form some kind of dynamic self oligomers? When you test by 
using the gel-filtration column, if the peak is symmetry? Sometime if the 
target protein can have week self interaction, you can observe a tailed peak. 
If the protein can have a strong self interaction, maybe you can observe 
isolated peaks, each peak is corresponding to different amount of the 
oligomersation state of the protein. You also can test your protein by using 
native or IEF gels.


Date: Sat, 14 Dec 2013 19:01:09 +0100
Subject: Re: [ccp4bb] A question on protein microheterogenity for crystalization

Dear Brett, We have seen this behaviour several times for different adenovirus 
fibre head proteins and don't really have an explanation for it. We have always 
set the peaks up separately when we had enough protein.
For this purification, as you don't have enough protein to pool them 
separately, I would pool them together and do a first crystallisation screen. 
But I would also immediately start a larger scale purification so you in the 
next crystallisation trial you can set the peaks up separately. If you are 
lucky, by the time you have done the second prep, from the first screen you may 
have some conditions to optimise. If not, the first screen should at least give 
some ideas about which precipitants, pHs and perhaps additives are most 
On 14 Dec 2013, at 13:16, Acoot Brett wrote: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 
 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.

Re: [ccp4bb] distinguish ligand binding sites within a protein

2013-11-18 Thread Xiaodi Yu
Hi Wei:

Based on the structure, you can calculate the binding surface between the 
protein and the ligand. Maybe the two binding pockets will give you two 
different numbers. And the larger one usually can have the higher binding 
affinity.  You also can analyse how the ligand interacts with the protein 
though hydrophobic or electrostatic interaction , etc?  the last, you may also 
compare the b factors of the ligand or the protein binding pocket regions after 
you refining the structure. These things may give you some hints about which 
binding site is more strong.


Date: Mon, 18 Nov 2013 22:45:58 -0500
Subject: Re: [ccp4bb] distinguish ligand binding sites within a protein

Thank you so much for the suggestions, Tomas! Yes, my ligand is a small 
molecule. I have the crystal structure of the ligands bound to the protein, do 
I still need to computationally dock the ligand to the two pockets, can I 
calculate the parameters of binding directly using the crystal structure? 


On Mon, Nov 18, 2013 at 9:03 PM, Tomas Malinauskas wrote:

Dear Wei Shi,

is your ligand a small molecule? If it is a small molecule, I would

try to computationally dock the small molecule to two pockets

separately using AutoDock, and look at the estimated free energies of


Best wishes,


On Mon, Nov 18, 2013 at 8:55 PM, Wei Shi wrote:

 Hi all,

 I got the crystal structure of a transcription factor, and every monomer

 binds two molecules of the same ligand in different binding pockets. And I

 also did the ITC experiment, titrating the ligand into the protein, and got

 a U-shaped curve. The binding affinity for the first binding site is higher

 than the second binding site.

 I am wondering whether I could computationally determine from the

 protein-ligand complex structure that which binding site has higher affinity

 for the ligand and correlate the binding sites with the parameters I got

 from ITC experiment.

 Thank you so much!




Re: [ccp4bb] Biacore/SPR

2013-10-28 Thread Xiaodi Yu
Another option is BLItz which is cheaper and uses interferometry.


Date: Mon, 28 Oct 2013 11:05:33 -0400
Subject: Re: [ccp4bb] Biacore/SPR

BiaCore 3000 or if you can afford the T200.Proteon XPR36 would be a cheaper 
Option and should work as well.Jürgen
On Oct 28, 2013, at 10:54 AM, Gang Dong wrote:We mainly measure protein-protein 
interactions (sometimes protein-small molecules). Thanks! _Gang From: Bosch, 
Juergen [] 
Sent: Monday, October 28, 2013 3:17 PM
To: Gang Dong
Subject: Re: [ccp4bb] Biacore/SPR Protein-protein interaction?protein-small 
molecule interactions ?Epitope mapping of mABs ?Could you specify what you 
would like to do, as different models are good for different things.Jürgen On 
Oct 28, 2013, at 10:08 AM, Gang Dong wrote:

Dear all, Could anyone tell me your experience with Biacore (any models) or a 
similar SPR-based technology for measuring interaction kinetics? I also want to 
know the price ranges to discuss with our department/facility managers. 
Thanks,Gang   ..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry  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

Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry  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


[ccp4bb] The binding between disordered and ordered proteins

2013-10-21 Thread Xiaodi Yu
Dear All:

I have a general question about protein- protein interactions. I have two 
proteins, A and B. A is a disordered protein while B is a well folded protein. 
The binding between A and B has been approved by GST-pull down assay 
previously. The strange thing is I cannot get them bind if protein A were just 
freshly prepared. However, if I kept these two proteins separately for one or 
two days at 4 degree and then did the GST-pull down assay again, I can observe 
very strong interaction between A and B. 

Protein A doesn't contain any cys residue. I have already test certain 
chemicals which might affect the interactions, for example, DTT and EDTA. These 
chemicals seems to have no effect on the binding. 

Although A is a disordered protein, does it need such long time to find its 
proper conformation?

Do any people have similar experience? Any suggestions are greatly appreciated.



[ccp4bb] Intra-molecular interactions

2012-11-03 Thread Xiaodi Yu

Dear All:

I have a quick question: how common it is that electrostatic interactions are 
involved in intra-molecular interactions, particularly in intrinsically 
disordered proteins? Is this interaction specific and any example?



Xiaodi Yu, Ph.D.
Boston Children's Hospital 
Dana-Farber Cancer Institute
Harvard Medical School
3 Blackfan
Boston, MA 02115

Re: [ccp4bb] no expression

2012-05-05 Thread Xiaodi Yu

Plus check the insoluble part or the whole cell.


Date: Sat, 5 May 2012 16:30:06 -0400
Subject: Re: [ccp4bb] no expression

Some very stupid questions from my side:- it is an expression vector and you 
are in frame with your gene of interest ?- you are using E. coli strains 
suitable for expression ?- have you tried adding glucose to the media to avoid 
leaky expression in case your protein is toxic to E. coli ?- the wild type 
expresses well ? In the same vector  strain ?- how do you determine if it was 
expressed ? Western blot of tag ?

Sent from my iPad
On May 5, 2012, at 16:17, Jahan Alikhajeh wrote:

Dear friends,

I am sorry for off-topic though it may be related indirectly!
I mutated a 60kDa protein (changing from X--Pro). After doing all the steps, I 
sequenced the expression vector. It seems everything is fine, even with 
promoter, but, I can't express it. I tried different Tempratures, IPTG (e 
with new Stock), media, but they didnot make any difference. Any suggestion is 
highly appreciated.


Re: [ccp4bb] Desalting columns

2012-02-27 Thread Xiaodi Yu

Hi Sangeetha:

If you just want to check which buffer is good for your protein, maybe you can 
try to set up a crystallization screen, keeping your protein concentration just 
3 mg/ml. You can observe (after several days) which conditions give you a clear 
drop, and maybe you can find a clue which buffer is better for your protein. If 
you want to speed up the whole processes, you can also add glycerol into the 
drops to grasp the water molecules. 

Yu Xiaodi

Date: Mon, 27 Feb 2012 11:01:33 -0500
Subject: [ccp4bb] Desalting columns

Dear bb users,

I am trying to crystallize a ~320 kDa protein that crashes out if concentrated 
past about 3 mg/mL. 

I would like to try to exchange it into various buffer-salt-additive 
combinations to see which buffer works. For a starting point, I'd like to use 
desalting colums.

Does anyone have suggestions for good buffer exchange and sample recovery? I 
woud like to load about 250 uL onto each column.

Thanks a lot!

Best regards,


Re: [ccp4bb] about point mutation

2012-02-24 Thread Xiaodi Yu

Hello Arun:

Actually, I am not sure about  i couldn't get my desire point mutation. You 
mean you didn't get the pcr product or you could get the pcr product but there 
is no mutation. If you didn't get the PCR,Just lower the annealing temperature 
to 55 degree. And try extension temperature 72 or 68 degree. You can get it. 
After PCR, using DpnI to treat your PCR product to get rid of the template 
Yu Xiaodi

 Date: Fri, 24 Feb 2012 09:05:40 +
 Subject: [ccp4bb] about point mutation
 can any one help me in suggesting that what mistake i have did in my 
 mutagenic pcr . actually my problem is my primer annealing temperature is 
 81degree. im using phusion pol enzyme. i have made many trial, i.e., made 
 annealing at 68 and then followed 2 step pcr method and then added 1micro lit 
 of dmso to 50micro lit of pcr mix etc.. but till now i couldn't get my desire 
 point mutation. my primer length is about 33 and the mutation id at the 
 centre of the primer.
can anyone help me what i can improve to get result or what mistake i had 
 thank you all the members in advance, 

Re: [ccp4bb] protein degradation

2012-02-15 Thread Xiaodi Yu

Hi Sivasankar:

Are you sure it is due to the protein degradation? Maybe you can try to do a 
western blot or others to check if it is the product of degradation. By the 
way, where you put the 6 histag, N- or C-terminal? If it is at the N terminal, 
maybe it is the truncation version of your protein. 
After looking at the gel, it seems your sample was over-load or had lots of 
unspecific binding to the column. Maybe you can add salt (250 mM NaCl, final 
concentration) and  small amount of imidazle in the sample before you load onto 
the column (for example, 20 mM Imidazole final concentration). 
One small trick you can try is wash the cell with the buffer containing PMSF 
once before lysising the cell. 

Yu Xiaodi

Date: Wed, 15 Feb 2012 18:39:19 +0530
Subject: [ccp4bb] protein degradation

Dear All,
Can anybody suggest the tricks and trades of stabilizing a 133 kDa (multi 
domain) DNA binding protein, that we are expressing at 18 degree Centigrade in 
E. Coli.  The protein appears to degrade during purification; we have protease 
inhibitor cocktail (in the lysis buffer) as well as 2 mM PMSF, 1 mM EDTA and 
1mM DTT  throughout during purification ( right from lysis stage).   We handle 
the protein at 4 degree Centigrade.

Can you please suggest what precautions we can try to avoid such degradation ?  
 Please find the attached gel picture regarding protein
Sivasankar Putta


Re: [ccp4bb] pH optimisation for crystallisation

2012-02-08 Thread Xiaodi Yu

Hello Sreetama:
I think for crystallization, everything is hard to say. But if you find your 
crystal is sensitive to the pH, you certainly can optimize the pH value but it 
is better not to deviate a lot. For example you can make 0.2 unit interval (for 
example: pH value 4.5, 4.7, 4.9...etc which are closed to your original pH 
value ). For the buffer, you can change or not.  Another thing is that, you can 
also incorporate bis-tris in your last purification, since you find your 
crystal in this buffer. When you do additive screen, the drops which is clear, 
also can give you important information. You might find a compont which can 
inhibit crystal formation. You can use it to slow down the crystal formation 
to get a big or single crystal.However, you see, sometimes, this optimization 
is time consuming. I suggest you to try seeding. It can give you a big 
surprise, sometimes. 
Yu Xiaodi

Date: Wed, 8 Feb 2012 11:56:30 +0530
Subject: [ccp4bb] pH optimisation for crystallisation

Dear all,   I have a 17 KDa protein that gives crystals in a condition 
that has 0.1M bis-tris pH 6.5. The crystals are thin needle clusters and do not 
diffract. I have tried additives, but they haven't improved the crystals. I 
intend to vary the pH of the condition.   My questions are-1. should 
the buffer be kept the same or can it also be changed (as long as the desired 
pH is within the range of both the buffers)?2. in case of a different buffer, 
should its molarity be the same as that of the original one in the 
crystallization condition?

Re: [ccp4bb] Dye for protein affinity measurement

2012-02-08 Thread Xiaodi Yu

Hello Jiahong:

If I understand correctly that you want to test protein-protein interaction or 
inhibition study in solution, maybe you can try something like ELISA to test 
protein-protein interaction. Or if your B protein has 6 histag, you can use 
Ni-NTA agrose beads to test inhibition or binding depending on your purpose. 
And another option (a little dangerous ), is using radio active to label one 
of your protein. 

Yu Xiaodi

Date: Wed, 8 Feb 2012 14:17:47 +
Subject: Re: [ccp4bb] Dye for protein affinity measurement

Thermo sells a series of kits called DyLight Fluor for fluorescent labelling of 
antibodies or other proteins.  They have everything you need and they're very 
convenient and easy to use.  You can pick the excitation and emission 
wavelength.  If you label both A and B (or C) with different colors you will 
be able to see if both are in your crystals (assuming crystallization is part 
of your approach).

You need only label a small percentage of your protein or peptide to see 
whether the protein is present in a crystal.

Forsythe, E.L., Achari, A., and Pusey, Marc L. (2006),  Trace Fluorescent 
Labeling for High Throughput Crystallography, Acta Cryst. D62, 339-346. 
We used DyLight 350 NHS Ester to check we had protein crystals - see 
methods section of Cryst. Growth Des., 2011, 11 (8), pp 3432–3441

2012/2/8 Jiang Jiahong

 Dear all,
I am looking for some kind of dye for protein affinity comparison, but do not 
know which to choose.

I know  protein A can contact B to form a complex,now I hope to find something 
simiar with A to act as an inhibitor to block the process of A-B complex 
formation. Maybe a short peptide, a segment of protein A or even some organic 

Because here is a poor access to ITC nor Biacore, I can only rely on some dye 
to check the competence between A and inhibitor candidates.

If any one can offer any suggestions. That would be so grateful! Any way,thank 
kind-hearted people in advance!


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 Directors: Peter Baldock, Patrick Shaw Stewart
 Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034
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Re: [ccp4bb] [RESUME] [OFF-TOPIC] Site-Directed Mutagenesis [OFF-TOPIC]

2012-02-07 Thread Xiaodi Yu

Hi Fred:

For the mutated plasmids, it generates the nicked dna, so the transform 
efficiency will be lower compared to the parental plasmids. And that is the 
reason why people usually use super competent cell to transform these nicked 
plasmid. It seems ok to me to get 2 or 14 colonies from the nicked plasmid. You 
just need ONE. And usually for these mutagenesis PCR, the success rate is 
pretty high. I suggest you to sequence these colonies. And you can get it.

Yu Xiaodi

 Date: Tue, 7 Feb 2012 10:17:43 -0200
 Subject: Re: [ccp4bb] [RESUME] [OFF-TOPIC] Site-Directed Mutagenesis 
 Hi CCP4 list,
 Thank you very much for additional messages and references.
 Here goes the image of the PCR product before digested and after 
 digested and cleaned.
 The results of the transformation of 3 microL (90 ng) of 
 non-mutated/paretal plasmid gave hundreds of colonies; 3 microL of Dpn1 
 digested sample gave two colonies only; and transformation of 3 
 microL(90 ng) of cleaned product gave 14 colonies.
 So, if the amplification is not abundant, chances are that home made 
 competent cell will not be transformed with the digested product. Don't 
 want start another discussion but, is there any reason for differences 
 in the transformation efficiency between the parental and the mutated 
 (cleaned) plasmids?
 All the Best,
 Em 03-02-2012 16:14, Fred escreveu:
  Hi CCP4 list,
  Thanks everyone who have answered my post concerning to mutagenesis.
  From quick reading most of the answers, the following seems to be a 
  1) Do not concentrate your PCR product;
  2) Too much DNA and/or impurities like salts or whatever can inhibits 
  3) Purify your PCR product before transformation if possible or use 3 
  of 4 microL of it. This is more or less the amount of DNA showed in 
  the uploaded image.
  Kind regards,
  P.S.: I'll let you know the results.

Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Xiaodi Yu

Hi Deepak:

I think it is common for the residues which participate catalysis to have a Pka 
deviated from the reality pKa value especially for acid/base catalysis (acid 
base titration assay can help you to figure out the way of catalysis). Usually 
the pKa values of these kind of critical residues are affected by their local 
environment and this character is related to the enzyme's working mechanism.

I am sorry that I am not professional in enzyme, I cannot answer your questions 
for each questions.

Yu Xiaodi

Date: Tue, 7 Feb 2012 19:48:26 +0800
Subject: [ccp4bb] On pKa of Aspartic acid

Dear colleagues,

We have solved the crystal structure of a human enzyme. The pKa of a 
catalytically critical aspartic acid has increased to 6.44. It is hydrogen 
bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton 
during the catalysis. Can anybody help me a) interpret the significance of this 
increase in pKa of the aspartic acid from 3.8 to 6.44 in context with the 
catalysis? Is this advantageous or detrimental? b) How is pKa related to an 
amino acids’ ability to force a water molecule to donate a proton? c) At pH 
7.4, the aspartic acid would be de-protonated irrespective of whether the pKa 
is 3.8 or 6.44; isn’t that true? d) Have similar increase in pKa values 
observed for aspartic acids before? I would be grateful if anybody could 
explain or comment on the above queries.

Deepak Oswal  

Re: [ccp4bb] Off topic: His-tag purification

2012-01-15 Thread Xiaodi Yu

Hi Theresa:
If you can make sure that your target protein is expressed. You can first use 6 
M urea to denature the protein and then try to bind it to the column. If the 
denatured protein can bind to the column, it seems the histag is hided inside 
of the protein. It is not exposed enough to interact with the column. In this 
case, you can design a new construct, for example, put the tag to the other end 
of the sequence, or introduce a flexible linker between the tag and the 
protein. Another thing you can try is using Cu ion instead of Ni ion. It will 
fasten the binding.
Good luck
Yu Xiaodi

 Date: Sun, 15 Jan 2012 18:23:33 +
 Subject: [ccp4bb] Off topic: His-tag purification
 Hi all
 I have a His-tagged soluble protein (8 His residues added to 90 kDa protein) 
 that do not bind to IMAC column based on flowthrough showing up with Western 
 blott. Do you have suggestions to improve the binding?
 Binding condition is 50 mM Tris-HCl 8.0, 300 mM NaCl, 10 mM imidazole pH to 
 Thank you.