Re: [ccp4bb] What could these crystals be?

[Kevin Jin]

I had the same issue before. PAS should be a decent polymer for
modification.

On Thu, Nov 9, 2023 at 7:04 PM sadik sattar  wrote:

> Hi Careina,
>
> I hope this email finds you well. I wanted to share my experience with a
> similar issue I faced in the past concerning one of my proteins, and I've
> attached a picture of my round crystals.
>
> Despite numerous attempts with various screens and additives, I
> consistently encountered these round crystals. Interestingly, PEG3350 as
> the precipitant seemed to be a common factor in most of these instances.
> The crystals would diffract to approximately 6 or 7A in a home source.
>
> To have a deeper understanding of the situation, I  extensively washed the
> crystals with the mother liquid and then ran them on the gel. The results
> revealed two distinct bands: one corresponding to the expected size of my
> protein and another smaller band. Subsequent Mass-spec confirmed that the
> smaller band represented a truncated version of my protein. It seemed that
> the protein was being cleaved during the course of crystallization and the
> cleaved protein is forming crystals with the intact protein.
>
> Unfortunately, despite my efforts, I couldn't prevent the unintended
> cleavage of the protein at that time, leading me to abandon the project. I
> wish I had a more definitive solution for you.
>
> Feel free to reach out if you have any questions or if there's anything
> else I can assist you with.
>
> Best regards,
> Sadik.
>
>
> On Nov 8, 2023, at 10:00 AM, careinaedgo...@yahoo.com <
> 02531c126adf-dmarc-requ...@jiscmail.ac.uk> wrote:
>
> Hi all
> We have been trying with no success to crystalize a protein. Recently we
> got these strange shape "crystals". They are hard and flat but they do not
> diffract at all. Any ideas as to what could cause this?
> Careina
>
> --
>
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>
>
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Re: [ccp4bb] What could these crystals be?

[Kevin Jin]

Dear Careina,

I usually see such kind of "Quasi-crystal" when the folding force was not
properly adjusted. Of course, you need to verify they are protein ppt
before any further modifications.

For these melon-seed-like crystals,  the sharp-end indicated that some
crystallization occurred. However, the condition needs to be further
modified.

1. For buffer, you need to try different buffers within the similar pH
range 7.0 ~ 7.8. For Tris, the temperature is important.  Different buffer
salts could give you crystals with different morphologies. In general,
sulphate salt is harder. Sodium salt or other cations from buffer salts
should also be counted for the total Sodium/cation concentration. PEG
usually could bring down the pH value.

2. For NaCl, you may try  NH4 salts, Li Salts, Na salts, K salts with
different anion combinations, including Cl, NO3, SO4.

3. For PEG3350, you may try PEG with different molecular weights, 1K -6K.
In your case, I will expect better crystallization with lower MW PEG, since
higher MW may introduce more hydrophobic factors resulting in oil-like
effects. Anyway, you need to try both for comparison.

Different polymers have different pH values, like MME 1.5K, PVP 1.5K, PAS
5.1K. For PAS 5.1K, the pH value is 7.0, and I obtained nice crystals of my
target which I could only get quasi-xtals under PEG 3k.

4. Try 5mM  ZnCl2, which may work too.

Well, there must be a lot of modification strategies ahead. All of my
suggestions here are to fine tune the ionic strength in the conditions
since you are very close to get xals.

Good luck !

Kevin.

PS. You may try Coca or Pepsi + PEG 3K too. I got a hit for DEAD-Box
complex before.






On Wed, Nov 8, 2023 at 7:18 AM stephen.c...@rc-harwell.ac.uk <
8f3604def7f0-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hi Careina,
>
> Without knowing what's in your protein buffer or crystallisation condition
> it's hard to comment.
>
> Best wishes,
> Steve
>
> Dr Stephen Carr
> Research Complex at Harwell (RCaH)
> Rutherford Appleton Laboratory
> Harwell Oxford
> Didcot
> Oxon OX11 0FA
> United Kingdom
> Email stephen.c...@rc-harwell.ac.uk
> tel 01235 567717
> --
> *From:* CCP4 bulletin board  on behalf of
> careinaedgo...@yahoo.com <02531c126adf-dmarc-requ...@jiscmail.ac.uk>
> *Sent:* 08 November 2023 15:00
> *To:* ccp4bb 
> *Subject:* [ccp4bb] What could these crystals be?
>
> Hi all
> We have been trying with no success to crystalize a protein. Recently we
> got these strange shape "crystals". They are hard and flat but they do not
> diffract at all. Any ideas as to what could cause this?
> Careina
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] seek your opion on this weird diffractio pattern

[Kevin Jin]

Hi Joseph,

It is a very beautiful crystal with neat diffraction patterns and
background, which may suggest a good cryoprotection. Here is my observation
from your images.

1. From your image taken at 30 degrees (first page), I believe it is a
protein crystal with a larger unit cell length on one dimension, rather
than an inorganic crystal. By measuring the distance between two spots in
line, you may be able to estimate the length of the unit cell two
dimensions.

2. According to the first crystal image, there is a line in the middle of
the crystal. If I am correct, it could be that two crystals grew  and
merged together. I used to have some cases like this. In my case, one half
of the crystal showed a hexahedron shape, and another half with a nearly
cubic shape. Eventually, the structure was determined in both P3 and P222
with refined conditions, respectively.  If this is true in your case, you
may have a multi crystal growth.

3. According to the 90 degree angle image on your second page, the
diffraction spots at right-bottom corner (4:30 O'Clock), those three spots
could be a protein crystal with a shorter length. There could be two
possibilities, very anisotropic growth or taking a longer exposures.

4. According to the outlooking of you crystal, it is very possible to carry
P3 or P6 symmetry. If this is true, it won't be an inorganic crystal.

5. If you still have crystals from the same drop, you may cut a piece from
the right side and take a long shoot. You may have a chance to index it.

Best,

Kevin

On Tue, Dec 8, 2020 at 7:47 AM Joseph Ho  wrote:

> Dear all:
> We recently worked on 8kDa protein crystal structure. We obtained
> crystals in Zinc acetate and PEG8000 condition. However, we observed
> this unusual diffraction patterns. I am wondering if anyone observed
> this and know how this can occur. The cryoprotectant is glycerol.
>
> Thank you for you help
>
> Joseph
>
> 
>
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-- 
Kevin Jin Ph.D

Sharing knowledge  is always joyful..A flash scientist in biology.

Website: http://www.jinkai.org/



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Re: [ccp4bb] Macports and Fink - failed building open source pymol on MacOS Catalina

[Kevin Jin]

My OS Version: Catalina 10.15.5;

brew install brewsci/bio/pymol

To verify, I just installed Pymol using brew 2 minutes ago. It works.

On Sat, Jun 13, 2020 at 4:45 PM Javier Gonzalez  wrote:

> Hello,
> I'm attempting to build pymol on a laptop running MacOS Catalina 10.15.5,
> Processor 2.8GHz Quad-Core Intel i7, Graphics Intel Iris Pro 1536 MB
> I downloaded the latest version of Xcode 11.1
>
> I tried from Fink (fink install pymol-py27) and Macports (sudo port
> install pymol), as indicated here:
> https://pymolwiki.org/index.php/MAC_Install
>
> Both scripts run to download all dependencies but at the end fail with
> different messages (see below). Any ideas? Is it doable or am I just
> following outdated instructions?
> Thanks in advance!
> Javier
>
> Fink: fails at compiling term-readkey-pm5184-2.37-1
>
> -
>
> -
> Failed: phase compiling: term-readkey-pm5184-2.37-1 failed
>
> Before reporting any errors, please run "fink selfupdate" and try again.
> Also try using "fink configure" to set your maximum build jobs to 1 and
> attempt to build the package again.
> If you continue to have issues, please check to see if the FAQ on Fink's
> website solves the problem.  If not, ask on one (not both, please) of
> these mailing lists:
>
> The Fink Users List 
> The Fink Beginners List ,
>
> with a carbon copy to the maintainer:
>
> Christian Schaffner 
>
> Note that this is preferable to emailing just the maintainer directly,
> since most fink package maintainers do not have access to all possible
> hardware and software configurations.
>
> Please try to include the complete error message in your report.  This
> generally consists of a compiler line starting with e.g. "gcc" or "g++"
> followed by the actual error output from the compiler.
>
> Also include the following system information:
> Package manager version: 0.45.1
> Distribution version: selfupdate-rsync Fri Jun 12 21:49:17 2020, 10.15,
> x86_64
> Trees: local/main stable/main
> Xcode.app: 11.5
> Xcode command-line tools: 11.5.0.0.1.1588476445
> Max. Fink build jobs:  8
>
> -
>
> -
> Macports: apparently there is an issue with py38-pyqt5
> --->  Computing dependencies for pymol
> The following dependencies will be installed:  py38-pyqt5
> Continue? [Y/n]:
> --->  Fetching archive for py38-pyqt5
> --->  Attempting to fetch py38-pyqt5-5.14.2_0.darwin_19.x86_64.tbz2 from
> https://packages.macports.org/py38-pyqt5
> --->  Attempting to fetch py38-pyqt5-5.14.2_0.darwin_19.x86_64.tbz2 from
> http://aus.us.packages.macports.org/macports/packages/py38-pyqt5
> --->  Attempting to fetch py38-pyqt5-5.14.2_0.darwin_19.x86_64.tbz2 from
> https://ywg.ca.packages.macports.org/mirror/macports/packages/py38-pyqt5
> --->  Building py38-pyqt5
> Error: Failed to build py38-pyqt5: command execution failed
> Error: See
> /opt/local/var/macports/logs/_opt_local_var_macports_sources_rsync.macports.org_macports_release_tarballs_ports_python_py-pyqt5/py38-pyqt5/main.log
> for details.
> Error: Follow https://guide.macports.org/#project.tickets to report a bug.
> Error: Processing of port pymol failed
>
> -
>
> -
> --
> Dr. Javier M. González
> Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
> Universidad Nacional de Santiago del Estero (UNSE)
> RN9, Km 1125. Villa El Zanjón. (G4206XCP)
> Santiago del Estero. Argentina
> Tel: +54-(0385)-4238352
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>


-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/



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Re: [ccp4bb] how to get protein crystal

[Kevin Jin]

Due the resolution of your images, it is a little bit difficult to identify
crystallization under these conditions. However, F3, F6 and F5 are
crystals, but the condition needs to be optimized.  Likely, you may try
different concentration of NaCl, instead of MgCl2 and KBr, respectively.
Good luck.

On Fri, Dec 27, 2019 at 5:31 AM amala mathimaran 
wrote:

> Dear all,
>
> Can you suggest me how to get protein crystal???
>
>
>
> I purified my target protein and concentrated to 12mg/ml (pI 6.04). Final
> purified protein contains 50mM HEPES pH 8.0 and 50mM NaCl. Then the initial
> screening was done using hanging drop method but no crystal. So 2mM NADP
> cofactor was added again screen with Hampton (PEG ion, PEG RX, crystal
> screen, Index) and Molecular dimensions conditions etc. I got precipitate
> like formation the image was attached below. From this formation what I can
> do… mean while I increase the protein concentration and did screening for
> that selected conditions again I got same kind of formations. I am new to
> protein crystallography kindly suggesting me. And how much concentration is
> suitable for protein crystallization?? How to find which concentration is
> enough for our target protein crystallization?? Thanks in advance
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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>


-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/



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Re: [ccp4bb] suggestions for cryoprotectant

[Kevin Jin]

Dear Firdous,

Probably other people already suggested above, and I don't know which kind
of protein in your case.  Here is my suggestion, you may try sugar solution
or polysaccharide solution as cryoprotectant. The whole idea is to provide
alternative H-bond network to support protein freezing. Indeed, some
startups are using similar ideas to develop cryoprotectants for medical
applications.

Best,

Kevin



On Fri, Oct 19, 2018 at 2:58 PM Firdous Tarique 
wrote:

> Dear members
>
> I have got beautiful crystal hits in SaltRx screens which are not
> diffracting to a good resoultion. All of them are salt based condition and
> I am not able to formulate a good cryoprotectant for these crystals. I also
> think that in my case the poor resolution is due to a poor cryoprotectant
> selection.
>
> The conditions are as follows:
>
> 1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0
> 2>0.5M KCN 100mM Tris pH8.5
> 3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0
> 4>4M Sodium Nitrate 100mM Tris pH8.5
> 5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6
>
> There are few more conditions but so far not able to see good diffraction
> with using lower peg and glycerol based cryoprotectants.
>
> Can anybody suggest me good cryos conditions for salt based
> crystallization conditions or anything good for SaltRx crystallization hits.
>
> Thanks
>
> Firdous
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>


-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/



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Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Electron density maps for Cryo-EM structures.

[Kevin Jin]

Dear Garib,

Thank you! I will definitely check the website immediately.

Sorry, I forgot to mention that I have been a user for CCP4 since 1998.
CCP4 is a great tool and reliable for crystallography, love it!

Cheers,

Kevin


On Mon, Sep 10, 2018 at 10:09 AM Garib Murshudov 
wrote:

> Dear Kevin,
>
> You can have access to all these resources (And ccpem, ccp4). They are
> free for non-commercial users.
>
> (You can also have access to any papers you want via https://sci-hub.tw/
> <http://scihub.tw>, it is also free but …)
>
> If you want to see some of the maps then you can go to emdb at
> https://www.ebi.ac.uk/pdbe/emdb/, they have many cryoEM maps and tools to
> visualise them.
>
> If you want to generate maps from coordinates then refmac  from ccp4 and
> ccpem has an option to generate 3D electrostatic potential from
> coordinates. I can send a script for that.
>
> Regards
> Garib
>
> P.S. In spite of some unusual answers you should not be afraid asking
> questions on ccp4 bb. This bulletin board is exactly for this type of
> questions.
>
>
> On 10 Sep 2018, at 18:01, Kevin Jin  wrote:
>
> Dear Herman and all ccp4ers,
>
> Many thanks for the helps and education from all of you.
>
> I am a fresh beginner in Cryo-EM,  and have no access to those resource,
> like Chimera, Phenix, Rosetta and papers, etc. For me, any answer will be
> valuable.
>
> Please forgive me for asking such a naive question.  I highly appreciate
> your comments and education.
>
> Kevin
>
> On Mon, Sep 10, 2018 at 5:28 AM  wrote:
>
>> Hi Kevin, Pavel and others,
>>
>>
>>
>> Since it seems that so far nobody answered the primary question: “Is
>> there any sever available to create electron density maps for cryo-em
>> structures?” So I will do it. The answer is very simple: They do not need
>> to be created, they are available from the pdb!
>>
>>
>>
>> To give an example: On the RCSB pdb web-site, I searched for entry 5vai.
>> Then under the button “Download Files” I selected “Download EM Map” and
>> downloaded emd_8653.map.gz. As the name suggests, this file needs to be
>> unzipped, but this is trivial.
>>
>>
>>
>> Then in Coot in the “File” pull-down menu, I select “Open Map” to load
>> the map.
>>
>> Next, you may not see anything since to contour level might be too high
>> (when I load the map, the contour level is around 6 rmsd) so you have to
>> scroll down the contour level to see anything. As Marin and Ian pointed
>> out, the maps are very similar to regular electron density maps, probably
>> with the exception that the EM “electron density” maps are influenced by
>> the local charge density. However in coot they behave 100% identical and
>> you can scroll up and down the contour level as you like.
>>
>>
>>
>> Of course, if the authors did not deposit their EM-map, you cannot
>> download it, but the same is true for X-ray structure factors.
>>
>>
>>
>> Happy viewing!
>>
>>
>>
>> Herman
>>
>>
>>
>>
>>
>>
>>
>> *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag
>> von *Ian Tickle
>> *Gesendet:* Montag, 10. September 2018 12:58
>> *An:* CCP4BB@JISCMAIL.AC.UK
>> *Betreff:* [EXTERNAL] Re: [ccp4bb] Electron density maps for Cryo-EM
>> structures.
>>
>>
>>
>>
>>
>> Hi Marin
>>
>>
>>
>> I was about to comment on that too but then I realised that Pavel is
>> referring to the map _contours_  (which is what most people using a map
>> visualisation program like Coot actually see).  So the contoured map does
>> represent an iso-potential surface.  I'm sure Pavel is aware that the
>> original cryo-EM maps are 3-dimensional objects.
>>
>>
>>
>> Cheers
>>
>>
>>
>> -- Ian
>>
>>
>>
>> On Mon, 10 Sep 2018 at 10:49, Marin van Heel <
>> 057a89ab08a1-dmarc-requ...@jiscmail.ac.uk> wrote:
>>
>>
>> Unfortunately,
>>
>> The problem here lies primarily in the answer given,  not so much in the
>> question asked by a newcomer:
>>
>> "1) In cryo-EM maps are not electron density maps but surfaces
>> representing electric potential. "
>>
>> The answer appears to reflect the widespread misunderstanding that EM
>> images (and hence cryo-EM maps)  only show the surfaces not the internal
>> density of the complexes we study.
>> In my Imperial College/Leiden University  lecture notes, I have always
>> used the below slide to il

Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Electron density maps for Cryo-EM structures.

[Kevin Jin]

CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=oyq4_tjHvYKRzK3yN_rOpIQo6sflk9725vR5aINqpHg=A66BPdK7455wfEqkW7nlTOFIrLwyiZ0P6iRXbkPPZFs=>
>
>
>
> --
>
> ==
>
>
>
> Prof Dr Ir Marin van Heel
>
>
>
> Laboratório Nacional de Nanotecnologia - LNNano
>
> CNPEM/LNNano, Campinas, Brazil
>
>
>
> Skype:  Marin.van.Heel
>
> email:  marin.vanheel(A_T)gmail.com 
> <https://urldefense.proofpoint.com/v2/url?u=http-3A__gmail.com=DwMFaQ=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=oyq4_tjHvYKRzK3yN_rOpIQo6sflk9725vR5aINqpHg=dS2VEfbgNj0-C_rK4Gz-4eXTJsZj7sQEp5cuMvr1i5g=>
>
> marin.vanheel(A_T)lnnano.cnpem.br 
> <https://urldefense.proofpoint.com/v2/url?u=http-3A__lnnano.cnpem.br=DwMFaQ=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=oyq4_tjHvYKRzK3yN_rOpIQo6sflk9725vR5aINqpHg=yJ2hAZdRJw5ICBpLLolSUKM1Dp8cAemaHi6pNIR5Ua4=>
>
> and:mvh.office(A_T)gmail.com 
> <https://urldefense.proofpoint.com/v2/url?u=http-3A__gmail.com=DwMFaQ=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=oyq4_tjHvYKRzK3yN_rOpIQo6sflk9725vR5aINqpHg=dS2VEfbgNj0-C_rK4Gz-4eXTJsZj7sQEp5cuMvr1i5g=>
>
>
>
> --
>
> Emeritus Professor of Cryo-EM Data Processing
>
> Leiden University
>
> --
>
> Emeritus Professor of Structural Biology
>
> Imperial College London
>
> Faculty of Natural Sciences
>
> email: m.vanheel(A_T)imperial.ac.uk 
> <https://urldefense.proofpoint.com/v2/url?u=http-3A__imperial.ac.uk=DwMFaQ=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=oyq4_tjHvYKRzK3yN_rOpIQo6sflk9725vR5aINqpHg=MtApOPqE5MmHLkxoyVe7L2FShxxiDiX3rT_V0VZ-CQA=>
>
> --
>
>
>
> I receive many emails per day and, although I try,
>
> there is no guarantee that I will actually read each incoming email.
>
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Re: [ccp4bb] Electron density maps for Cryo-EM structures.

[Kevin Jin]

Great, thanks!


On Sun, Sep 9, 2018 at 9:49 PM Pavel Afonine  wrote:

> P.S.: all questions are welcome of course, no labeling. It's just some of
> them are so orthogonal to common sense that answers my be such as well.
> Best,
> Pavel
>
> On Sun, Sep 9, 2018 at 9:38 PM Pavel Afonine  wrote:
>
>> Hi,
>>
>> Is there any sever available to create electron density maps for cryo-em
>>> structures?
>>>
>>
>> The questions are nonsensical. Here is why:
>>
>> 1) In cryo-EM maps are not electron density maps but surfaces
>> representing electric potential.
>>
>> 2) Creating such a map is essentially carrying on from cryo-EM experiment
>> and obtaining the 3D reconstruction.
>>
>> Are you really sure about what you are asking for?
>>
>>
>>> Or, we should create the maps from mmCIF.
>>>
>>
>> mmCIF is a file format. It may contain representations of rabbits,
>> boysenberries or some diffraction data. So.. how you think it may be
>> related to cryo-EM, in your particular case?
>>
>>
>>> I am particularly interested in those cryo-em structures with high
>>> resolution, like 2.6~2.8A.
>>>
>>
>> Sure, all are excited about high-res cryo-EM!!!
>>
>>
>>> Please give me an education.
>>>
>>
>> Sure. One of available universities can do this.
>>
>> Cheers,
>> Pavel
>>
>>

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[ccp4bb] Electron density maps for Cryo-EM structures.

[Kevin Jin]

 Dear All,

Is there any sever available to create electron density maps for cryo-em
structures? Or, we should create the maps from mmCIF. I am particularly
interested in those cryo-em structures with high resolution, like 2.6~2.8A.

Please give me an education.

Thanks,

Kevin



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Re: [ccp4bb] Protein Purification- reg

[Kevin Jin]

Hi Amala,

1. Did you verify the sequence or the presence of the His-tag? If you were
not the person for cloning. I used to spend 14 months working on an clone
and eventually I was allowed to check the sequence and verify the
expression of His-tag by Western-Blot. There was no his-tag.
2. You can try ionic-exchange columns instead of Ni-Column.  In this case,
chaining the cationic column and anionic column in a linear way. You target
should either stay in 1) cation or 2) anionic or 3) flow through. You can
verify the presence by SDS page.
3. What's the temperature you used for purification? I.e. if the pH 7.5 of
tris was under RT, then it should be 8.3 in cold room.
4. Increasing the concentration of Na+ will increase protein solubility and
decrease non-specific binding ( possible langmuir adsoprtion). Normally,
people use 200-300 mM NaCl. If the Tris-tri sodium is used, the
concentration may need to controlled < 50mM. For instance, if 50mM Tris-tri
Na was used, then 150mM NaCl could be introduced in the buffer system
additionally.
5. 10mM Imidazole is used for removing non-specific adsorption in this
case.
6. In some cases, everything looks normal, but the protein just does like
to bind to the Ni(Co)- column. In this case, using ionic-columns will be an
alternative method before you try another vector or expression system.

The advantage of ionic-columns is you can buy the beads (+ and -) from
sigma with much less cost. You even don't need to use an AKTA system.

I hope this will be helpful and see your publication soon.

Kevin



On Mon, Aug 13, 2018 at 6:50 AM amala mathimaran 
wrote:

> Dear All
>
> I am working with HIS – tag protein in N-terminal (hexa histidine). The
> protein from Prokaryotic origin cloned into pET30a+ vector and expressed in 
> *E.coli
> *BL21 cells. The expression was good. I am trying to purify a protein
> using Ni-affinity column equilibrate with Buffer A 50mM Tris pH7.5, 50mM
> NaCl, 15mM imidazole, 3mM BME, 5%glycerol and eluted with Buffer B ( same
> as buffer A but 250mM imidazole). I eluted the protein in step wise
> gradiant. the protein was less binded in Ni affinity IMAC column because
> eluted fraction contain less amount and the protein remain present in the
> Flow through. Can any one suggest how to increase the binding affinity of
> the protein and how to purify the protein. The protein PI was 6.33
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

[Kevin Jin]

I just give an example of oil. Indeed, paraffin is also a very good option.
In some cases (<=5% alcohol).  Here is the difference I observed between
paraffin and paratone oil.
1. Paraffin oil has low viscosity. Paratone oil is too sticky with high
viscosity. Sometimes, I mix them with different ratio.
2. In the crystallization with high concentration of alcohol (>10%), I
prefer paratone oil, which can form a bulky cover immediately. Paraffin oil
may spread out and could not cover the drop completely.

The basic idea is to proved an effective shell to prevent solvent
evaporation.



On Tue, Aug 14, 2018 at 12:56 PM Patrick Loll  wrote:

> I second (third?) what Tommi and Kevin said about using an oil to cover
> the drop to slow evaporation (I like paraffin for this—not too viscous).
> Here’s an additional nuance: Saturate the oil with the alcohol first,
> before using it to cover the drop.
>
> > On 14 Aug 2018, at 2:58 PM, Thomas Krey 
> wrote:
> >
> > Dear crystallization experts,
> >
> > We have 3D protein crystals grown from a microseed matrix screening
> vapor diffusion experiment in either
> >
> > 15% (v/v) Reagent alcohol
> > HEPES Na pH 7.5
> > 0.2 M MgCl2
> >
> > or in
> >
> > 27% Isopropanol
> > 0.18 M MgCl2
> > 90 mM HEPES Na pH 7.5
> > 10% Glycerol
> >
> > Upon opening the corresponding wells these crystals move quite a bit –
> presumably due to the volatility of the alcohols. Does anyone have a good
> suggestion to stabilize the swirling movements? Does anyone have
> experience, whether these conditions alone can serve as cryo-protectant
> (i.e., do we really have to fish, move into cryo solution and fish again)?
> > Any suggestion or input would be highly welcome.
> >
> > Thank you very much in advance.
> >
> > Thomas
> >
> >
> > Prof. Dr. Thomas Krey
> > Hannover Medical School
> > Institute of Virology
> > Structural Virology Group
> > Carl-Neuberg-Str. 1
> > D-30625 Hannover
> > phone: +49 (0) 511 - 532 4308
> > email: krey.tho...@mh-hannover.de
> >
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> >
>
>
> ---
> Patrick J. Loll, Ph. D.
> Professor of Biochemistry & Molecular Biology
> Drexel University College of Medicine
> Room 10-102 New College Building
> 245 N. 15th St., Mailstop 497
> Philadelphia, PA  19102-1192  USA
>
> (215) 762-7706
> pjl...@gmail.com
> pj...@drexel.edu
>
> 
>
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Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

[Kevin Jin]

Hi Thomas,

This is usual when high volatile solvent is used in crystallization
(membrane or glycoproteins). The crystal may looks very nice with sharp
edges. When you open the cover glass, you may see a very thin film formed
on the hang-on drops. Once you touch the drop, then crystals move very
quickly like flying and start cracking.

Here is what you may try:
1. When you open the cover glass, using a big drop of Paratone oil to cover
(wrap) the drop immediately and completely.
2. Then, inject the cryoprotectant into the drop wrapped by paratone oil.
3.  When you wash the crystal in cryoprotectant and fish the crystal out,
please always keep all of the operation in a large paratone oil drop.
4. Make sure there is always a layer of paratone oil on the loop when you
freeze it in LN2.

I hope this will be helpful.

Regards,

Kevin





On Tue, Aug 14, 2018 at 12:00 PM Thomas Krey 
wrote:

> Dear crystallization experts,
>
>
>
> We have 3D protein crystals grown from a microseed matrix screening vapor
> diffusion experiment in either
>
>
>
> 15% (v/v) Reagent alcohol
>
> HEPES Na pH 7.5
>
> 0.2 M MgCl2
>
>
>
> or in
>
>
>
> 27% Isopropanol
>
> 0.18 M MgCl2
>
> 90 mM HEPES Na pH 7.5
>
> 10% Glycerol
>
>
>
> Upon opening the corresponding wells these crystals move quite a bit –
> presumably due to the volatility of the alcohols. Does anyone have a good
> suggestion to stabilize the swirling movements? Does anyone have
> experience, whether these conditions alone can serve as cryo-protectant
> (i.e., do we really have to fish, move into cryo solution and fish again)?
>
> Any suggestion or input would be highly welcome.
>
>
>
> Thank you very much in advance.
>
>
>
> Thomas
>
>
>
>
>
> Prof. Dr. Thomas Krey
>
> Hannover Medical School
>
> Institute of Virology
>
> Structural Virology Group
>
> Carl-Neuberg-Str. 1
>
> D-30625 Hannover
>
> phone: +49 (0) 511 - 532 4308
>
> email: krey.tho...@mh-hannover.de
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>


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Re: [ccp4bb] Crystal structure of an unknown protein

[Kevin Jin]

HI Jiyong,

If you still have protein left, you may try to sequence it. In some cases,
even N-terminal digestion is very helpful. In my previous, I always got a
90KDa protein, which was very close to my target kinase. Based on the
protein sequencing, we identified it was one of those chaperones.

Best,

Kevin

On Sun, Dec 17, 2017 at 11:39 PM, Jiyong Su <sujy...@nenu.edu.cn> wrote:

> Dear Pedro Matias,
>
> Thanks for you advice.
>
> After I manually changed the side chain of the residues, I got a
> "artificial" primary structure. I did a blast by using this primary
> structure.
> Finally, I found the amino acid sequence of this protein. The electron
> density could perfectly match the sequence.
> BTW, this protein is from a lovely weird bacteria which was cultured by a
> student.
>
> Best regards,
>
> Jiyong
>
>
>
>
> --
> Yours Sincerely,
>
> Jiyong Su
>
> The School of Life Sciences
> Northeast Normal University
> Changchun 130024, China
> Email: sujy...@nenu.edu.cn
> Tele: + 0086 13244318851
>
>
>
>
> 在 2017-12-14 21:08:57，Pedro Matias <mat...@itqb.unl.pt> 写道：
> >Hello,
> >
> >Welcome to the club of unexpected results!
> >
> >You don't provide a lot of background, but based on what you wrote you can:
> >
> >1. Do a BLAST search using a known part of your sequence to find whether
> >this sequence has been deposited.
> >
> >2. Assign the different residues based on the chemical environment and
> >electron density and refine the structure.
> >
> >I'm sure you can submit the refined structure to the PDB even from an
> >unknown protein.
> >
> >Regards,
> >
> >Pedro Matias
> >
> >
> >Às 12:08 de 14/12/2017, Jiyong Su escreveu:
> >> Dear CCP4bb,
> >>
> >> In 2014, I collected a high quality data set from a crystal. But I
> >> could not solve the structure of that crystal because this protein is
> >> a contaminate.
> >> Recently, I used StruBE's Contaminer and fortunately got the solution.
> >> Thanks ContaMiner!!!  This protein is a contaminate protein.
> >>
> >> However, I found this protein is an unknown protein (about 180
> >> residues) whose amino acid sequence is not totally same as E.coli.
> >> There are about 20 point mutation sites comparing to the E.coli
> >> protein. This means this protein may be from an unknown bacteria.
> >>
> >> The space group of this crystal is new. There is also a new ligand in
> >> this protein.
> >>
> >> My question is how could I found the primary structure of this protein
> >> and how to deposit this protein in PDB.
> >>
> >> Best regards,
> >>
> >> Jiyong
> >>
> >
> >--
> >
> >Industry and Medicine Applied Crystallography
> >Macromolecular Crystallography Unit
> >___
> >Phones : (351-21) 446-9100 Ext. 1669
> > (351-21) 446-9669 (direct)
> > Fax   : (351-21) 441-1277 or 443-3644
> >
> >email : mat...@itqb.unl.pt
> >
> >http://www.itqb.unl.pt/research/biological-chemistry/industry-and-medicine-applied-crystallography
> >http://www.itqb.unl.pt/labs/macromolecular-crystallography-unit
> >
> >Mailing address :
> >Instituto de Tecnologia Quimica e Biologica António Xavier
> >Universidade Nova de Lisboa
> >Av. da República
> >2780-157 Oeiras
> >PORTUGAL
> >
> >ITQB NOVA, a great choice for your PhD
> >https://youtu.be/de6j-aaTWNQ
> >
> >Master Programme in Biochemistry for Health
> >https://youtu.be/UKstDCFjYI8
>
>
>


-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/

Re: [ccp4bb] Difficult purification with imac columns

[Kevin Jin]

I had a similar case before. I could not find notes for the details.

1. During the lysis, add protein amine sulfate and spin down with the speed
up to 10K rpm (normally 8k) for 30 mins.
2. adding polysaccharide (1~ 5 %) as additive to adjust nonspecific bind.
For polysaccharides, I tried several kinds of them ordered from Sigma. I
forgot which one was the best (sorry).
3. use ionic columns ( anionic + cationic columns, chained them together)
at 4C.
4. Then, used Co-Column (not, NTA,) for further purification.
...

The final purity could reach 95%. However, l lost a lot of protein during
purification.



On Fri, Sep 15, 2017 at 4:53 AM, Narayanan Ramasubbu <
ramas...@sdm.rutgers.edu> wrote:

> Hi. We are working on a periplasmic protein that breaks naked glycans in
> peptidoglycans. There is truncated structure available but our target is
> the full length protein. The difficulty us that it strongly binds to the
> resin with or without his.tag. Changing the resin to acrylamide did not
> help.
> Has anyone come across similar problem and how was it resolved.
> The pdb structure is the catalytic domain and mussing a region that, in my
> opinion, binds to the resin.
> Thank you in advance
> Sent from my iPhone




-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/

Re: [ccp4bb] Help needed finding hit condition

[Kevin Jin]

Hi Jonathan,

 Old buffer may have slow evaporation with some side reactions. The
concentration of each component may increase a little bit.  In this case,
 I would consider the condition as the origin for further optimization.
Each component may need to be considered separately.

For 150mM Tris pH 8.000,
The pH and concentration may  have a slight change (increase ?).  I will
try the concentration of 150mM, 155 mM and 160mM. For pH,  pH 8.1 and pH
8.2...

For KBr, the concentration may be 80, 85, 90mM or higher.

For 47.1% w/v PEG1K (Pretty high concentration),
As the result for ring-opening epoxide reaction, it is not very stable
anyway. The reaction always continued. Basic condition made the case even
worse. In this case, the Mn (MW) of PEG1K may not be that average anymore.
It is very possible that the polymer chain is elongated. Of course, the
concentration of PEG is increased too. In the meanwhile, the presence of
KBr may cause further chemical modification on the PEG chains.  You may try
PEG 95--1050 (Sigma P3515), PEG 1305-1595 (Sigma 202136), PEG3K or PEG
3350, etc.


Best,

Kevin

P.S. If you take a look from the top of your old solution in the tube,
what's the color? Slight Yellow? You can use a tube with dd water a
reference.


On Mon, Jul 31, 2017 at 5:34 AM, Jonathan Bailey <baile...@tcd.ie> wrote:

> Dear CCP4bb community
>
>
> I apologies for the slightly off topic post.
>
>
> We have recently had success crystallizing a membrane protein (diffraction
> > 3 Å at a synchrotron source) using the *in meso* method, the hit
> condition was from the Jena Bioscience screen Pi-minimal condition number
> #57.
>
>
> Hit condition – 47.1 % w/v PEG1000, 150 mM Tris pH 8.0, 80 mM Potassium
> bromide
>
>
> The screen is old and expired 12/20/2013 (lot # JBS00013133), we have
> tried to reproduce the crystals using homemade optimization screens around
> the hit condition but have not had any success. We have tried reproducing
> the hit using a new (not expired) Pi-minimal screen but had no success. We
> are only able to reproduce the crystals using the expired screen and we do
> not have much of it left.
>
>
>
> We went back and tested the pH of the condition that had given crystals,
> the expected pH was 7.9 but we found it to be 6 – 6.5 using a pH indicator
> strip. We believe the drop in pH is caused by oxidative degradation of the
> PEG1000 resulting in the formation of carboxylic acid species.
>
>
> We have contacted Jena Bioscience to try and get some of the old screen
> stock but unfortunately they do not have any.
>
>
> My question is does anyone out there happen to have any expired screen
> stocks of this Pi-minimal condition (#57), ideally from the same lot (lot #
> JB200013133), that they would be willing to send us.
>
>
>
> Does anyone have any advice as to how to reproduce the condition? We’ve
> considered bubbling oxygen through and heating the sample to accelerate the
> oxidation process.
>
>
>
> King Regards
>
>
> Jonathan Bailey (PhD student)
>
>
> Professor Martin Caffrey Lab MS group Trinity College Dublin
>



-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/

Re: [ccp4bb] Protein rapidly precipitates when off ice

[Kevin Jin]

Hi Chris,

In your Ni-NTA buffer, the total [Na+] could be greater than 530mM after
titration. Likely, your protein likes higher concentration salt for surface
charge stabilization. Adding Glycerol may further modify the charge
distribution and make the protein happy in the solution. For further
verification, I usually try the buffer without Glycerin or replace it with
small PEG (PEG 100) for +/- impacts, respectively. Indeed, it is pretty
good starting point for crystallization.

I noticed that HEPES is also slightly temperature dependent. In most of the
cases, the gap could be ignored. It may be helpful. In my previous cases, I
tried PBS and Bis-tris with the same pH. The stability was improved great.

Can you keep every thing same but different kind of buffer?

Hope it would be helpful.

Sincerely.



On Thu, Jul 13, 2017 at 3:40 PM, Chris Fage <fage...@gmail.com> wrote:

> Dear CCP4BB Community,
>
> This week, I purified a nicely overexpressing protein by Ni-NTA followed
> by gel filtration. In a 4 C centrifuge, I concentrated my gel filtration
> fractions to ~1 mL, transferred the spin filter to ice, and then collected
> 2 uL for measurement on the Nanodrop. Sadly, the protein precipitated
> heavily in the pipet tip before I could dispense it onto the Nanodrop
> pedestal, directly adjacent to my ice box. This effect seems to be abated
> at 4 C, as the protein remained stable in cold room-chilled pipet tips.
> However, the protein also precipitated heavily when overnight at 4 C in 1
> mL gel filtration buffer (150 mM NaCl, 10 mM HEPES pH 7.5), but not
> overnight at 4 C in 10 mL Ni-NTA buffer (500 mM NaCl, 30 mM HEPES pH 7.5,
> 10% glycerol) prior to gel filtration. Has anyone experienced and resolved
> a similar issue before? Do any useful additives come to mind?
>
> Things I have tried with the gel filtration sample:
> -Exchanging buffer to restore the salt concentration to Ni-NTA levels
> (e.g. 500 mM).
> -Exchanging buffer to add 10% glycerol.
> -Simply diluting the protein in gel filtration buffer to rule out
> concentration dependence.
>
> In each case, the protein precipitates to a milky solution within about a
> minute of removal from ice (I am working with 20-50 uL volumes in PCR
> tubes).
>
> Many thanks for any suggestions!
>
> Best,
> Chris
>



-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/

Re: [ccp4bb] off topic: Alternatives to GE HiTrap columns

[Kevin Jin]

Sorry, the company was CloneTech, now it is Takara (not Takeda). You may
talk with Dr. Gia Jokhadze, who is the director of protein chemistry
department. He would give you more information.  You can find his profile
on linkedin. He is very nice person. Probably, you can ask some free sample
from him.

The advantage of their clone: first, high capacity and better column
performance(plate numbers).

On Fri, Feb 17, 2017 at 9:33 AM, Kevin Jin <kevin...@gmail.com> wrote:

> Probably you can try His-Column from Clonetech (Takeda ?). They modified
> the column packing using nylon membrane, which offered better flow-through
> than that of traditional resin-gel column.
>
>
>
> On Wed, Feb 15, 2017 at 8:57 AM, Markus Seeliger <
> markus.seeli...@gmail.com> wrote:
>
>> Dear all,
>> we are happy users of all that GE offers around their FPLC system, but I
>> am getting a little tired of feeling monopolized. Are any of you aware of
>> either empty columns or prepacked columns (e.g. metal affinity or ion
>> exchange resins) from other companies?
>> Thanks for your advice
>>
>> Markus
>>
>
>
>
>
>



-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/

Re: [ccp4bb] off topic: Alternatives to GE HiTrap columns

[Kevin Jin]

Probably you can try His-Column from Clonetech (Takeda ?). They modified
the column packing using nylon membrane, which offered better flow-through
than that of traditional resin-gel column.



On Wed, Feb 15, 2017 at 8:57 AM, Markus Seeliger 
wrote:

> Dear all,
> we are happy users of all that GE offers around their FPLC system, but I
> am getting a little tired of feeling monopolized. Are any of you aware of
> either empty columns or prepacked columns (e.g. metal affinity or ion
> exchange resins) from other companies?
> Thanks for your advice
>
> Markus
>

Re: [ccp4bb] Unknown blob extended from catalytic serine residue

[Kevin Jin]

According to your first fig. The Ser may carry a dual conformation. If
then, the occ ration could be ~ 7:3

According to your 1st, 2nd and 3rd figures, the geometry of the density  is
tetrahedral. Can it be PO4 with the same occ (~70%) ?

If there were more figures available, the geometry of the unknown density
could be more clear.

Can You try Glucose phosphate withe occ of 70%?

On Sat, Feb 4, 2017 at 7:03 AM, sharifah nur hidayah syed mazlan <
shn.hida...@gmail.com> wrote:

> Dear All,
>
> I am working on a structure with an unknown blob extended from the gamma O
> of the catalytic serine residue. The resolution of the dataset is 1.38 A. I
> have no idea whether the residue is modified or the blob belongs to other
> molecule.
>
> The protein was expressed in Rosettagami (DE3), purified using
> Ni-Sepharose (affinity chromatography) and Q-Sepharose (Anion exchange
> chromatography). The crystallization formulation used contain 15% PEG 8000,
> 0.2 M Ammonium sulphate, 0.1 M sodium cacodylate trihydrate pH 6.5; and the
> buffer composed of 50 mM Sodium chloride and Phosphate buffer pH 8. No
> protease inhibitor was used (eg: PMSF)
>
> I have tried to fit in diethylene glycol as shown in one of the attached
> figures, but as observed, it is not really fit and the molecule is in
> incorrect conformation.
>
> Kindly help me with this matter.
>
>
>
> Thanks and regards,
>
> Sharifah Nur Hidayah
> Universiti Putra Malaysia,
> Malaysia
>
>
>


-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/

Re: [ccp4bb] Need suggestion for protein solubility

[Kevin Jin]

Likely, the protein is pH sensitive.

You may chain the anion and cation columns together for protein separation,
then you don't need to use Imidazole.

P.S. Tris pH 7.5 (RT) is ~ pH 8.2 in cold room



On Sat, Dec 24, 2016 at 2:52 AM, Praveen Tripathi <
tripathipraveen2...@gmail.com> wrote:

> Dear all,
> I am graduate student working on a functional protein which i have cloned
> in pET-28a vector for recombinant protein production in E.coli expression
> system.
> The expressed protein is purified on Ni-NTA resins with Imidazole
> gradient. Surprisingly, i am getting distinct visible white precipitate in
> pure fractions in eluted fractions itself.
> Please suggest how to make it soluble or how to prevent the precipitation.
> On concentrator the precipitate ration is very much increasing. The
> protein is pure in soluble as well as precipitate.
> Buffer condition- 50mM Tris(7.5), 500mM NaCl, 10% Glycerol. Elution buffer
> has varying concentration of imidazole varying from 10mM to 300mM.
>  Any kind of suggestion will be highly appreciated.
> My project requires structure determination.
>
> Thanks in advance.
>
> Regards
> Praveen
>

Re: [ccp4bb] very uninformative

[Kevin Jin]

Cool!  You are a good tutor for Greek culture.



Orcus blesses you.


^_^
On Tue, Apr 3, 2012 at 10:07 PM, Bernhard Rupp (Hofkristallrat a.D.) 
hofkristall...@gmail.com wrote:

 Ok Kevin,

 ** **

 thank you for your response. You got it, and that is good, and I am sure
 we’ll hear from you again and that is 

 good too. But let me explain the Orcus (however, keep in mind, I am only a
 single contributor and almost

 always do not represent the majority of CCP4BB users’ opinions. So that
 alone should be some comfort).

 ** **

 The title of Orcus means that you have earned yourself a nickname.
 Nicknames are a brutal invention, common in Western 

 civilization, almost always addressing some personal idiosyncrasy, in
 general politically incorrect, but nevertheless they stick*).

 ** **

 So let me explain:

 St. Orcus is the patron saint of trolls, hobgoblins and troglodytes, and
 the defender of off-topic posters and otherwise chastised

 contributors (just like the Hofkristallrat sitting in his Hofkristallamt
 is the defender of structures collected from 

 real data. That is for example why I do not get invited to modelers’
 conferences. Everything has its price). 

 So you are now in the unique position to evaluate the orcness of a
 contribution – perhaps first by making sure that

 your own contributions are not orcish - and exercise your right to
 identify any contributions you consider orcward.

  

 Experiencing a new culture can be a confusing and upsetting experience. If
 I may offer some comforting example

 relating to your blogs, and coming from a different planet myself, I once
 considered it a shocking calamity that 

 protein-ligand structures are published that do not contain a ligand. I
 have mellowed a lot since and prevented a few 

 cardiac events and assassination attempts by accepting the editorial
 indifference towards such orcward orcness. Maybe 

 you’ll get there too, and maybe you’ll become a Hofkristallamtsapprentice.
 

 ** **

 But let me tell you, if you are serious about correcting poor science,
 you’ve got to be ready to take a lot more flak 

 to get there than being beatified on the BB. Oh, and by the way, no
 academic career.   

 ** **

 Wingardium Leviosa!

 ** **

 Over and out, B

 ** **

 *) Like Kim Jong-il probably means something like Gold Upright Sun. Just
 to demonstrate how poor those things translate into reality….

 ** **

 PS: Orcward ligand orcs, the Amt is watching! 

 ** **

 PPS: it is still ok to ride a trolley.

 ** **

 *From:* Kevin Jin [mailto:kevin...@gmail.com]
 *Sent:* Tuesday, April 03, 2012 5:23 PM
 *To:* b...@hofkristallamt.org
 *Cc:* CCP4BB@jiscmail.ac.uk
 *Subject:* Re: [ccp4bb] very informative - Trends in Data Fabrication

 ** **

 Thanks of your education. I got it.

  

 By the way, what does Orcus mean here?

  

 Regards,

  

 Kevin

 On Tue, Apr 3, 2012 at 5:11 PM, Bernhard Rupp (Hofkristallrat a.D.) 
 hofkristall...@gmail.com wrote:

 ** **




-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/

Re: [ccp4bb] very informative - Trends in Data Fabrication

[Kevin Jin]

Dear All,
 Here may be another example for the importance of  image storage.

http://www.jinkai.org/DERA/DERA_1O0Y_3R12.html

Regards,

Kevin

Re: [ccp4bb] very informative - Trends in Data Fabrication

[Kevin Jin]

Thanks of your education. I got it.

By the way, what does Orcus mean here?

Regards,

Kevin

On Tue, Apr 3, 2012 at 5:11 PM, Bernhard Rupp (Hofkristallrat a.D.) 
hofkristall...@gmail.com wrote:

 Orcus,

 ** **

 if you put yourself persistently into the face of guys who play hard, you
 need to learn to

 take a few hits and shake it off. Maybe a little retrospection on why your
 postings might

 perhaps possibly maybe perceived as somewhat self-promoting and ungracious
 could be helpful.

 ** **

 The skill of presentation is at least as important in Science as being
 right.

 ** **

 Best, BR 

 ** **

 *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
 *Kevin
 Jin
 *Sent:* Tuesday, April 03, 2012 3:34 PM

 *To:* CCP4BB@JISCMAIL.AC.UK
 *Subject:* Re: [ccp4bb] very informative - Trends in Data Fabrication

 ** **

 Dear All, 

  Here may be another example for the importance of  image storage. 

  

 http://www.jinkai.org/DERA/DERA_1O0Y_3R12.html

  

 Regards,

  

 Kevin

 ** **




-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/

Re: [ccp4bb] very informative - Trends in Data Fabrication

[Kevin Jin]

“I hope and believe that this is not the case.  Even basically-trained
crystallographers should be able to calculate andinterpret difference
maps of the kind described by Bernhard.  And with the EDS and PDB_REDO
server, one does not even need to know how to make generate a difference map
...”

*You are right*!

Actually, I am not an experienced protein crystallographer. I have learnt a
lot from CCP4BB. I may have paid too much attention to bonding angle and
bond length, like in small molecule. This may be an example to share with
you.

When I worked on those nitroreductase complexed with FMN in 2009 (?), I
always observed that the flavin ring presented a strange geometry after
refinement. Indeed, I had used the definition of FMN from CCP4 library all
the time.

In some cases, the methyl group at position of either 7a or 8a was bent off
the aromatic ring, if the whole the rest of flavin was restrained in a flat
plane.  According to my limited knowledge from organic chemistry, carbon of
7 and 8 on the flavin ring is sp2 hybridized in a coplanar manner. How
could those methyl groups be bent as sp3 hybridization? Any chemistry
behind?

With increased resolution (1.6 ~ 1.8 Ang), I observed that the electron
density map was a bent along the N5-N10 axis. The bend angle was around ~16
degree.   Again, I questioned myself why it was bent? Should this be
correct?

According to my limited knowledge in chemistry, N10 should be sp3
configuration even if FMN is in its oxidization form, in which the flavin
ring should be bent. A quick “google” immediately gave me a link to a very
nice paper published by David W. Rodgers in 2002.

http://www.jbc.org/content/277/13/11513.full.pdf+html

According to this paper, Yes!  “*In the oxidized enzyme, the flavin ring
system adopts a strongly bent (16°) conformation, and the bend increases
(25°) in the reduced form of the enzyme*,…”

When I reported this in the group meeting, I was laughed and told that this
is just a model bias. It was over interpreted.  Nobody has such sharp
vision on electron density map.  If this was correct, why nobody could find
this and report to CCP4 within last 7 years?

Eventually, a senior team member emailed to CCP4 about this issue. Since
then, the definition of FMN was updated, according to my suggestion.

I was asked “how did you find it?”……. “why you believed you are so right?”
 I really don’t how to answer.

*Je pense donc je suis*

Kevin


On Sun, Apr 1, 2012 at 8:09 AM, Paul Emsley paul.ems...@bioch.ox.ac.uk
wrote:
 On 31/03/12 23:08, Kevin Jin wrote:


 I really wish PDB could have some people to review those important
 structures, like paper reviewer.


 So do the wwPDB, I would imagine.

 But they can't just magic funding and positions into existence...

 If the coordinate is downloaded for modeling and docking, people may not
 check the density and model by themself. However this is not the worst
case,
 since the original data was fabricated.


 1. All of data was correct and real,


 Hmmm...

  It will be very difficult for people to check the density and coordinated
 if he/she is not a well-trained crystallographer.


 I hope and believe that this is not the case.  Even basically-trained
 crystallographers should be able to calculate and interpret difference
maps
 of the kind described by Bernhard.  And with the EDS and PDB_REDO server,
 one does not even need to know how to make generate a difference map...

 Paul.





-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/

Re: [ccp4bb] very informative - Trends in Data Fabrication

[Kevin Jin]

Nice paper !

I really wish PDB could have some people to review those important
structures, like paper reviewer. If the coordinate is downloaded for
modeling and docking, people may not check the density and model by
themself. However this is not the worst case, since the original data was
fabricated.

The worst case is:

1. All of data was correct and real,

2. The key structural evidence ( 1%) was not presented or excluded in the
discussion.

3. Then the paper was reviewed by some famous people, and then published on
those top level journals.

4. Eventually, it is cited in our textbook for years.

For the most of cases, reviewer and reader will mainly rely on graphics
displaying only. It will be very difficult for people to check the density
and coordinated if he/she is not a well-trained crystallographer.

Recently, I have seen several stories like this. Here is an open letter to
Nature.

http://www.jinkai.org/AAD/AAD_letter_2_nature.html

I hope you experts will also check the coordinate files for verification.

Sincerely,

Kevin

On Sat, Mar 31, 2012 at 8:26 AM, Bosch, Juergen jubo...@jhsph.edu wrote:

 really fascinating, bringing back the discussion for a repository for your
 collected frames.

 Jürgen


 *Acta Cryst.* (2012). F*68*, 366-376
 doi:10.1107/S1744309112008421http://dx.doi.org/10.1107/S1744309112008421
 *
 *
 Detection and analysis of unusual features in the structural model and
 structure-factor data of a birch pollen allergenB. 
 Rupphttp://scripts.iucr.org/cgi-bin/citedin?search_on=nameauthor_name=Rupp,%20B.

 *Abstract:* Physically improbable features in the model of the birch
 pollen structure Bet v 1d (PDB entry 
 3k78http://pdb.pdb.bnl.gov/pdb-bin/opdbshort?3k78)
 are faithfully reproduced in electron density generated with the deposited
 structure factors, but these structure factors themselves exhibit
 properties that are characteristic of data calculated from a simple model
 and are inconsistent with the data and error model obtained through
 experimental measurements. The refinement of the 
 3k78http://pdb.pdb.bnl.gov/pdb-bin/opdbshort?3k78model
 against these structure factors leads to an isomorphous structure different
 from the deposited model with an implausibly small *R* value (0.019). The
 abnormal refinement is compared with normal refinement of an isomorphous
 variant structure of Bet v 1l (PDB entry 
 1fm4http://pdb.pdb.bnl.gov/pdb-bin/opdbshort?1fm4).
 A variety of analytical tools, including the application of Diederichs
 plots, *R*[image: [sigma]] plots and bulk-solvent analysis are discussed
 as promising aids in validation. The examination of the Bet v 1d structure
 also cautions against the practice of indicating poorly defined protein
 chain residues through zero occupancies. The recommendation to preserve
 diffraction images is amplified.
 ..
 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
 http://web.mac.com/bosch_lab/







-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/
sigma_rmgif.gif

Re: [ccp4bb] very informative - Trends in Data Fabrication

[Kevin Jin]

Keeping Bernard's book as reference, it is the best way.

Kevin

On Sat, Mar 31, 2012 at 4:56 PM, Tom Peat tom.p...@csiro.au wrote:
 Bernard went to a lot of work to verify that this structure was wrong, so we 
 should also thank him for his efforts.
 It is good to see someone who has a hunch follow that up and let the rest of 
 us know about it.
 Thanks Bernard!


 Tom Peat
 Biophysics Group
 CSIRO, CMSE
 343 Royal Parade
 Parkville, VIC, 3052
 +613 9662 7304
 +614 57 539 419
 tom.p...@csiro.au
 
 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Anastassis 
 Perrakis [a.perra...@nki.nl]
 Sent: Sunday, April 01, 2012 7:59 AM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] very informative - Trends in Data Fabrication

 Reading the paper from Dr. Hofkristallrat a.D. and the editorial in ActaF, I 
 must say that besides the rather reasonable demand for journals to include 
 crystallography experts as referees, Table 1 would have fooled me as 
 referee. A validation report of the VTF style might not had helped either in 
 refereeing - in this case. Alarm bells could had rung possibly if the PDB was 
 re-refining all submitted structures and look for 'too good to be true' 
 improvements (sorry Robbie ... we are not there yet to improve things SO 
 much!). Saving the images in a repository would had been equally unlikely to 
 have helped (they would had submitted some data ... unless these were 
 systematically validated and cross-matched to the CRYST data cards no alarm 
 bells either - even if running PDB_REDO in all submissions appears a tad 
 unrealistic, re-processing all images and matching them to CRYST records 
 seems more troublesome at the present moment).

 A thing that could had helped, would had been if our biology colleagues who 
 want a structure for their story would had valued more the structural 
 contribution by scrutinising the data (a corresponding author must scrutinise 
 all data before accepting responsibility - and not when questioned throw the 
 hands up waving 'it was not me ...'). Maybe ourselves as a community could 
 also help by making our colleagues aware that crystallographic work is a tad 
 more than 'and the author in the middle of the paper just contributed a 
 structure' and explain them that if they want to be using structures for 
 their publications they should be always prepared to engage in close and real 
 collaborations where both sides accept responsibility for the data of each 
 other, as it happens in many fruitful collaborations between biologists and 
 crystallographers (such as these I had the privilege to engage with 
 collaborators that criticised my data, as I did theirs ...).

 regards to all -

 Tassos

 (and please, no 1st April joke with fraud cases !)



-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/

[ccp4bb] a small trick for protein and organic compound cocrystallization.

[Kevin Jin]

Dear All,

Here is way I have used for protein and hydrophobic organic compound
cocrystallization. Via this method, less amount of DMSO will be used
to aviod protein ppt.

I hope you can use for your research.

http://www.jinkai.org/Xtal.html

Regards,

-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/

Re: [ccp4bb] need help for crystallization

[Kevin Jin]

My pleasure !
Since this may be help for other people, I  also cc it to CCP4BBS.

According to your email, I guess the buffer is phosphate buffer at pH
7.4.  You can do a quick buffer exchange before crystallization.

Since PO4 is a competitor in this case, I will also avoid PO4 and
Cocodylate buffer from screen kits.

Here is what you can do,
1. concentrate your protein using an amicro centrocon with 3KD cutoff.
2. Measure how much buffer has been spin down, then add equal amount
of Tris with same pH back to the top.
3. Then, spin down again.
4. Repeat several times, 5 times may be good enough.
5. Most of Phosphate buffer should be removed.

That's what I used before.

Let me know if it works.

Best,

Kevin Jin





On Fri, Mar 30, 2012 at 9:40 AM, Afshan Begum afshan...@yahoo.com wrote:

  Dear Kevin

 I have seen your website and come to you to discuss my problem actually i
 want to co-crystallized my enzyme with inhibitor but problem is that the
 phosphate ion come to the active site and occupied the cavity and my
 inhibitor not bind to the active center i purchase this enzyme from a
 commercial source and they purify  with phosphate buffer that's why also
 phosphate is occupied the cavity how can i get ride of it from the active
 center could you kindly help me regarding this i would be really thankful to
 you.


 Best Regards

 AFSHAN
 ===
 Dr. Afshan Begum
 University of Hamburg
 Institute of Biochemistry and Molecularbiology
 Laboratory for Structural Biology of Infection and Imflammation
 c/o DESY, Build. 22a
 Notkestr. 85
 22603 Hamburg
 Germany
 Home phone: +49 40 22888618
 Fax:      +49 40 8998-4747
 E-mail: afshan...@yahoo.com




-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/

Re: [ccp4bb] dea all

[Kevin Jin]

I guess you already run SDS-PAGE to check the pellets before and after
sonication. Not only the media.

On Tue, Mar 27, 2012 at 7:35 AM, rana ibd rna19792...@yahoo.com wrote:
 Dear all
 I am expressing a 6xHis tagged in a dHBx protein in E.coli BL21 using LB
 madia, I am having problems with the expression which shows small amount of
 the protein , I also have problems with purification using NI-NTA by also
 having small amount even after extensive buffer exchange , Is it likely due
 to the small amount of protein in the medium , should I use a different kind
 of media, any sugestions or any kind of details or a paper that might help I
 will be thankful

 Best Regards
 Rana



-- 
Kevin Jin

Candle always burns itself out to light up the tunnel .

Website: http://www.jinkai.org/

[ccp4bb] a small trick for SDS-Gel storage

[Kevin Jin]

Dear All,

Maybe most of you already knew. I just put my understanding of SDS
page gel storage here, if you want to make gel yourself and safe some
money.

http://www.jinkai.org/SDS_page.html

If I am wrong, please let me know.
Sharing knowledge each other is always joyful.

-- 
Kevin Jin

Protein crystallographer and Biopolymer chemist

Personal Website: http://www.jinkai.org/

[ccp4bb] structure refinement and analysis work

[Kevin Jin]

Dear All,

Do you need some help for structure refinement or structure analysis?
I will be very happy to work for you, while I am looking for next
position.

To me, solving structure is a puzzle game and for fun.

Regards,

Kevin Jin

Re: [ccp4bb] My protein precipitates at r.t and dissolves at 4 oC

[Kevin Jin]

Hi Min,

Please  try this way if you use your protein for crystallization.

1. collect the needle and run SDS page or FPLC to verify the presence
of protein. Make sure it is not a buffer salt.

2. You don't need to do dialysis to remove b-ME, otherwise it will
take too long and you may lose some protein.

Here is what I did before:
1. Kee you stock protein solution with 2mM b-ME on ice-bath.

2. Use those eppendoff like tubue (~ 500ul) with membrane, which we
use to concentrate protein with reasonable MW cut-off (I usually used
8KD).

3. Add protein solution with b-ME (2mM) in the tube, spin down (10K,
eppendoff centrifuge) for 2mins,

4. Use your pipette to measure how much solution has been filted into
the bottom tube.

5. add equal amount fresh buffer solution without b-ME to the top tube,

Repeat 3~5 times, and make the fine concentration of b-ME to 0.5mM.

Then use the protein immediately for crystallization.

Actually, I used the same way for buffer exchange, instead of
dialysis. Based on this way, I crystallized thiopurine
methyltransferase with 2.0 Ang after folks' 5 years effort.

The images is available here:
http://www.jinkai.org/Crystal_imgs.html

Best,

Kevin






On Tue, Mar 13, 2012 at 8:36 AM, Min-Kyu Cho min-kyu@live.com wrote:
 Thank you Kevin,

 I will try to remove b-ME as you suggested.

 Min-Kyu

  | -Original Message-
  | From: Kevin Jin [mailto:kevin...@gmail.com]
  | Sent: Monday, March 12, 2012 5:50 PM
  | To: Min-Kyu Cho
  | Subject: Re: [ccp4bb] My protein precipitates at r.t and dissolves at 4
  | oC
  |
  | Hi Min,
  |
  | I need to look back my note. Here is from my old memory:
  |
  | 1. The protein was loaded in FPLC column at 4 degree C and the collection
  | was clear.  In this case, i did not use Tris Buffer, 2. When the protein
  | was warmed up in room temperature, ppt appeared.
  | 3.In the buffer, only beta-mercaptoethanol problem was added, in addition
  | to PBS buffer.
  |
  | In my case, I removed beta-mercaptoethanol just before assay and
  | crystallization, it worked and no ppt.
  |
  | I could not remember the detail.
  |
  | I hope this would be helpful.
  |
  | Kevin
  |
  |
  |
  |
  | On Mon, Mar 12, 2012 at 3:42 PM, Min-Kyu Cho min-kyu@live.com
 wrote:
  |  Hi Kevin,
  | 
  |  Could you tell me more detail about beta-mercaptoethanol problem?
  | 
  |  Min-Kyu
  | 
  |   | -Original Message-
  |   | From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf
  |  Of
  |   | Kevin Jin
  |   | Sent: Monday, March 12, 2012 3:32 PM
  |   | To: CCP4BB@JISCMAIL.AC.UK
  |   | Subject: Re: [ccp4bb] My protein precipitates at r.t and dissolves
  |  at 4
  |   | oC
  |   |
  |   | I remember I saw the similar problem caused by beta-mercaptoethanol.
  |   |
  |   |
  |   | Kevin
  |   |
  |   |
  |   | On Mon, Mar 12, 2012 at 1:29 PM, Artem Evdokimov
  |   | artem.evdoki...@gmail.com wrote:
  |   |  Could be one of those weird behaviors displayed by detergents
  |  where
  |   |  cloud point anomalously changes with temperature...
  |   | 
  |   |  Artem
  |   | 
  |   |  On Mar 12, 2012 1:11 PM, Min-Kyu Cho min-kyu@live.com
  | wrote:
  |   | 
  |   |  I am using KPi buffer at pH 5.5, 100mM KCl, 2mM
  |  beta-mercaptoethanol,
  |   |  0.02% NaN3.
  |   | 
  |   |  Yes, I agree I should check CD melting curve to see temperature
  |   |  preference of my protein.
  |   | 
  |   |  Min-Kyu
  |   | 
  |   |   | -Original Message-
  |   |   | From: Kevin Jin [mailto:kevin...@gmail.com]
  |   |   | Sent: Monday, March 12, 2012 11:16 AM
  |   |   | To: Min-Kyu Cho
  |   |   | Cc: CCP4BB@jiscmail.ac.uk
  |   |   | Subject: Re: [ccp4bb] My protein precipitates at r.t and
  |  dissolves
  |   |  at 4
  |   |   | oC
  |   |   |
  |   |   | Which kind of buffer you use? If it is Tris, then temperature
  |   |  change will
  |   |   | cause pH change.
  |   |   |
  |   |   | Actually, this is a good way for crystallization.
  |   |   |
  |   |   | Kevin
  |   |   |
  |   |   | On Mon, Mar 12, 2012 at 9:02 AM, Min-Kyu Cho
  |   |  min-kyu@live.com
  |   |  wrote:
  |   |   |  Hi all,
  |   |   | 
  |   |   |  I have a homotetrameric coiled-coil domain sample with 45aa
  |  per
  |   | each.
  |   |   |  While I store this sample at 4oC, the sample looks clear
  |  w/o any
  |   |   |  particles. But when I took out the sample to my bench at
  |  r.t, I
  |   |  can
  |   |   |  see there are precipitates (as stack of needle like
  |  particles)
  |   |  at the
  |   |   |  bottom of the tube after several hours. Interestingly, when
  |  I
  |   |  put it
  |   |   |  back into 4oC fridge, the precipitates disappeared and the
  |   |  solution
  |   |   | turned into clear again.
  |   |   | 
  |   |   |  Does anyone have knowledge of such behavior of any protein?
  |  I
  |   |   |  appreciate any information related.
  |   |   | 
  |   |   |  Min-Kyu

Re: [ccp4bb] My protein precipitates at r.t and dissolves at 4 oC

[Kevin Jin]

Min,

I forgot one more thing.

If you try to grow crystall of membrane protein and get tiny crystal,
you may try to add 5~10% IPA + 5mM NH4SO4 to addjuct the surface
charge of your protein.

The crystall may get larger.  Folks like this trick.

Good luck.

Kevin





On Tue, Mar 13, 2012 at 10:38 AM, Kevin Jin kevin...@gmail.com wrote:
 Hi Min,

 Please  try this way if you use your protein for crystallization.

 1. collect the needle and run SDS page or FPLC to verify the presence
 of protein. Make sure it is not a buffer salt.

 2. You don't need to do dialysis to remove b-ME, otherwise it will
 take too long and you may lose some protein.

 Here is what I did before:
 1. Kee you stock protein solution with 2mM b-ME on ice-bath.

 2. Use those eppendoff like tubue (~ 500ul) with membrane, which we
 use to concentrate protein with reasonable MW cut-off (I usually used
 8KD).

 3. Add protein solution with b-ME (2mM) in the tube, spin down (10K,
 eppendoff centrifuge) for 2mins,

 4. Use your pipette to measure how much solution has been filted into
 the bottom tube.

 5. add equal amount fresh buffer solution without b-ME to the top tube,

 Repeat 3~5 times, and make the fine concentration of b-ME to 0.5mM.

 Then use the protein immediately for crystallization.

 Actually, I used the same way for buffer exchange, instead of
 dialysis. Based on this way, I crystallized thiopurine
 methyltransferase with 2.0 Ang after folks' 5 years effort.

 The images is available here:
 http://www.jinkai.org/Crystal_imgs.html

 Best,

 Kevin






 On Tue, Mar 13, 2012 at 8:36 AM, Min-Kyu Cho min-kyu@live.com wrote:
 Thank you Kevin,

 I will try to remove b-ME as you suggested.

 Min-Kyu

  | -Original Message-
  | From: Kevin Jin [mailto:kevin...@gmail.com]
  | Sent: Monday, March 12, 2012 5:50 PM
  | To: Min-Kyu Cho
  | Subject: Re: [ccp4bb] My protein precipitates at r.t and dissolves at 4
  | oC
  |
  | Hi Min,
  |
  | I need to look back my note. Here is from my old memory:
  |
  | 1. The protein was loaded in FPLC column at 4 degree C and the collection
  | was clear.  In this case, i did not use Tris Buffer, 2. When the protein
  | was warmed up in room temperature, ppt appeared.
  | 3.In the buffer, only beta-mercaptoethanol problem was added, in addition
  | to PBS buffer.
  |
  | In my case, I removed beta-mercaptoethanol just before assay and
  | crystallization, it worked and no ppt.
  |
  | I could not remember the detail.
  |
  | I hope this would be helpful.
  |
  | Kevin
  |
  |
  |
  |
  | On Mon, Mar 12, 2012 at 3:42 PM, Min-Kyu Cho min-kyu@live.com
 wrote:
  |  Hi Kevin,
  | 
  |  Could you tell me more detail about beta-mercaptoethanol problem?
  | 
  |  Min-Kyu
  | 
  |   | -Original Message-
  |   | From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf
  |  Of
  |   | Kevin Jin
  |   | Sent: Monday, March 12, 2012 3:32 PM
  |   | To: CCP4BB@JISCMAIL.AC.UK
  |   | Subject: Re: [ccp4bb] My protein precipitates at r.t and dissolves
  |  at 4
  |   | oC
  |   |
  |   | I remember I saw the similar problem caused by beta-mercaptoethanol.
  |   |
  |   |
  |   | Kevin
  |   |
  |   |
  |   | On Mon, Mar 12, 2012 at 1:29 PM, Artem Evdokimov
  |   | artem.evdoki...@gmail.com wrote:
  |   |  Could be one of those weird behaviors displayed by detergents
  |  where
  |   |  cloud point anomalously changes with temperature...
  |   | 
  |   |  Artem
  |   | 
  |   |  On Mar 12, 2012 1:11 PM, Min-Kyu Cho min-kyu@live.com
  | wrote:
  |   | 
  |   |  I am using KPi buffer at pH 5.5, 100mM KCl, 2mM
  |  beta-mercaptoethanol,
  |   |  0.02% NaN3.
  |   | 
  |   |  Yes, I agree I should check CD melting curve to see temperature
  |   |  preference of my protein.
  |   | 
  |   |  Min-Kyu
  |   | 
  |   |   | -Original Message-
  |   |   | From: Kevin Jin [mailto:kevin...@gmail.com]
  |   |   | Sent: Monday, March 12, 2012 11:16 AM
  |   |   | To: Min-Kyu Cho
  |   |   | Cc: CCP4BB@jiscmail.ac.uk
  |   |   | Subject: Re: [ccp4bb] My protein precipitates at r.t and
  |  dissolves
  |   |  at 4
  |   |   | oC
  |   |   |
  |   |   | Which kind of buffer you use? If it is Tris, then temperature
  |   |  change will
  |   |   | cause pH change.
  |   |   |
  |   |   | Actually, this is a good way for crystallization.
  |   |   |
  |   |   | Kevin
  |   |   |
  |   |   | On Mon, Mar 12, 2012 at 9:02 AM, Min-Kyu Cho
  |   |  min-kyu@live.com
  |   |  wrote:
  |   |   |  Hi all,
  |   |   | 
  |   |   |  I have a homotetrameric coiled-coil domain sample with 45aa
  |  per
  |   | each.
  |   |   |  While I store this sample at 4oC, the sample looks clear
  |  w/o any
  |   |   |  particles. But when I took out the sample to my bench at
  |  r.t, I
  |   |  can
  |   |   |  see there are precipitates (as stack of needle like
  |  particles)
  |   |  at the
  |   |   |  bottom of the tube after several hours. Interestingly, when
  |  I
  |   |  put it
  |   |   |  back

Re: [ccp4bb] My protein precipitates at r.t and dissolves at 4 oC

[Kevin Jin]

Which kind of buffer you use? If it is Tris, then temperature change
will cause pH change.

Actually, this is a good way for crystallization.

Kevin

On Mon, Mar 12, 2012 at 9:02 AM, Min-Kyu Cho min-kyu@live.com wrote:
 Hi all,

 I have a homotetrameric coiled-coil domain sample with 45aa per each. While
 I store this sample at 4oC, the sample looks clear w/o any particles. But
 when I took out the sample to my bench at r.t, I can see there are
 precipitates (as stack of needle like particles) at the bottom of the tube
 after several hours. Interestingly, when I put it back into 4oC fridge, the
 precipitates disappeared and the solution turned into clear again.

 Does anyone have knowledge of such behavior of any protein? I appreciate any
 information related.

 Min-Kyu

Re: [ccp4bb] My protein precipitates at r.t and dissolves at 4 oC

[Kevin Jin]

I remember I saw the similar problem caused by beta-mercaptoethanol.


Kevin


On Mon, Mar 12, 2012 at 1:29 PM, Artem Evdokimov
artem.evdoki...@gmail.com wrote:
 Could be one of those weird behaviors displayed by detergents where cloud
 point anomalously changes with temperature...

 Artem

 On Mar 12, 2012 1:11 PM, Min-Kyu Cho min-kyu@live.com wrote:

 I am using KPi buffer at pH 5.5, 100mM KCl, 2mM beta-mercaptoethanol,
 0.02%
 NaN3.

 Yes, I agree I should check CD melting curve to see temperature preference
 of my protein.

 Min-Kyu

  | -Original Message-
  | From: Kevin Jin [mailto:kevin...@gmail.com]
  | Sent: Monday, March 12, 2012 11:16 AM
  | To: Min-Kyu Cho
  | Cc: CCP4BB@jiscmail.ac.uk
  | Subject: Re: [ccp4bb] My protein precipitates at r.t and dissolves at 4
  | oC
  |
  | Which kind of buffer you use? If it is Tris, then temperature change
 will
  | cause pH change.
  |
  | Actually, this is a good way for crystallization.
  |
  | Kevin
  |
  | On Mon, Mar 12, 2012 at 9:02 AM, Min-Kyu Cho min-kyu@live.com
 wrote:
  |  Hi all,
  | 
  |  I have a homotetrameric coiled-coil domain sample with 45aa per each.
  |  While I store this sample at 4oC, the sample looks clear w/o any
  |  particles. But when I took out the sample to my bench at r.t, I can
  |  see there are precipitates (as stack of needle like particles) at the
  |  bottom of the tube after several hours. Interestingly, when I put it
  |  back into 4oC fridge, the precipitates disappeared and the solution
  | turned into clear again.
  | 
  |  Does anyone have knowledge of such behavior of any protein? I
  |  appreciate any information related.
  | 
  |  Min-Kyu

Re: [ccp4bb] FW: endotoxin removal

[Kevin Jin]

To Pius,

In my case, the protein/biopolymer is Lysine/amine riched.

1. Lys or Arg are mainly positively charged under pH 7.0 ~ 8.0, and
then provide positively charge to LPA. Of course, some metal cations
could also be involved in LPA binding.

2. Tris with the NH2 group could also be protonated and positively
charged under the same pH condition. This is why I use high
concentration Tris here.

3. Tris buffer could function as an ionic-exchange competitor for LPA
binding. If you look those commercial available Endotoxin assay kits,
they also use Tris buffer.

4. IPA could change the charge distribution on the surface of protein.
In usual, high% IPA could remove LPA efficiently but may cause protein
denature. In lot of cases, 5~10% IPA could help protein
crystallization to a larger size. As a tradeoff, it may also bring
down the packing quality.

In this case, I used it for endotoxin  removal.

5. NaCl (you can try other salt) would provide additional ion-strength
to Interfere (or weak) the ionic-paring between Lys/Arg and LPA.

6. If you can add EDTA to remove metal cation, it may be even better.
In my case, I could not use EDTA.

I tested the sample (1600EU/ml). After 3 days dialysis, it went down
to  5EU/ml.  The assay kit for LPA measurement was from Charles River
(?). The procedure is too long to present here. The good thing is that
your sample would not be dehydrated and refold again.

I am saying this will work for protein in all of case, but you try in
an eppendorf tube like amini-prep.

I guess this method may have been  patented.






















On Thu, Mar 8, 2012 at 7:16 AM, Pius Padayatti ppadaya...@gmail.com wrote:
 This is in response to a comment to this thread
 Kevin,
 Could you explain how that worked?
 How do you know your method worked?
 Did you estimate the lipopolysaccharide before and after the method?

 The method already mentioned here to wash using TritonX100 makes sense.
 by washing bound protein sample May able to phase separate into
 detergent micellar phase and get rid of endotoxins.

 Most widely accepted method to separate is by two phase micellar system.
 above the CMC endotoxins will be accomodated by micelles through non-polar
 interactions of alkyl chains of lipidA.

 Padayatti PS



 On Wed, Mar 7, 2012 at 9:50 AM, Jerry McCully
 for-crystallizai...@hotmail.com wrote:
o

 Dear All;

     We purified a His tag protein by Ni-NTA and gel-filtration from
 E.coli.

  We tried two endotoxin removal resins from Pierce. However, it is
 hard to remove the endotoxins in the purified protein because the protein
 bound to the resin as well.

  This protein contains quite a few aromatic residues and has a pI
 around 6.

  Any ideas to remove the endotoxins will be highly appreciated.

     best regards,

 Jerry McCully





 --
 Pius S Padayatti,PhD,
 Phone: 216-658-4528

Re: [ccp4bb] FW: endotoxin removal

[Kevin Jin]

In my case, this method was developed for large quantity sample
(~100g/batch) purification.

Under cGMP, I could not introduce EDTA or other chemicals like
Triton-X100 into the system. Otherwise, I just solve small problem but
bring into an even large problem to the manufacture line.

I hope you can test my strategy and improve it. If you can give me a
feed back, there will be great!


Kevin

On Thu, Mar 8, 2012 at 1:01 PM, Kevin Jin kevin...@gmail.com wrote:
 To Pius,

 In my case, the protein/biopolymer is Lysine/amine riched.

 1. Lys or Arg are mainly positively charged under pH 7.0 ~ 8.0, and
 then provide positively charge to LPA. Of course, some metal cations
 could also be involved in LPA binding.

 2. Tris with the NH2 group could also be protonated and positively
 charged under the same pH condition. This is why I use high
 concentration Tris here.

 3. Tris buffer could function as an ionic-exchange competitor for LPA
 binding. If you look those commercial available Endotoxin assay kits,
 they also use Tris buffer.

 4. IPA could change the charge distribution on the surface of protein.
 In usual, high% IPA could remove LPA efficiently but may cause protein
 denature. In lot of cases, 5~10% IPA could help protein
 crystallization to a larger size. As a tradeoff, it may also bring
 down the packing quality.

 In this case, I used it for endotoxin  removal.

 5. NaCl (you can try other salt) would provide additional ion-strength
 to Interfere (or weak) the ionic-paring between Lys/Arg and LPA.

 6. If you can add EDTA to remove metal cation, it may be even better.
 In my case, I could not use EDTA.

 I tested the sample (1600EU/ml). After 3 days dialysis, it went down
 to  5EU/ml.  The assay kit for LPA measurement was from Charles River
 (?). The procedure is too long to present here. The good thing is that
 your sample would not be dehydrated and refold again.

 I am saying this will work for protein in all of case, but you try in
 an eppendorf tube like amini-prep.

 I guess this method may have been  patented.






















 On Thu, Mar 8, 2012 at 7:16 AM, Pius Padayatti ppadaya...@gmail.com wrote:
 This is in response to a comment to this thread
 Kevin,
 Could you explain how that worked?
 How do you know your method worked?
 Did you estimate the lipopolysaccharide before and after the method?

 The method already mentioned here to wash using TritonX100 makes sense.
 by washing bound protein sample May able to phase separate into
 detergent micellar phase and get rid of endotoxins.

 Most widely accepted method to separate is by two phase micellar system.
 above the CMC endotoxins will be accomodated by micelles through non-polar
 interactions of alkyl chains of lipidA.

 Padayatti PS



 On Wed, Mar 7, 2012 at 9:50 AM, Jerry McCully
 for-crystallizai...@hotmail.com wrote:
o

 Dear All;

     We purified a His tag protein by Ni-NTA and gel-filtration from
 E.coli.

  We tried two endotoxin removal resins from Pierce. However, it is
 hard to remove the endotoxins in the purified protein because the protein
 bound to the resin as well.

  This protein contains quite a few aromatic residues and has a pI
 around 6.

  Any ideas to remove the endotoxins will be highly appreciated.

     best regards,

 Jerry McCully





 --
 Pius S Padayatti,PhD,
 Phone: 216-658-4528

Re: [ccp4bb] FW: endotoxin removal

[Kevin Jin]

Sure, we can talk about this off line.

-- Kevin

On Thu, Mar 8, 2012 at 7:48 PM, Pius Padayatti ppadaya...@gmail.com wrote:
 Kevin,

 thanks.Sine this is of not much interest to many here


 please feel free to go off line in the future.

 The method of dialysis is confusing here.
 How can one achieve this by buffer exchange?

 I wonder if an extensive wash of protein on a column would work
 instead.

 Pius

 On Thu, Mar 8, 2012 at 4:01 PM, Kevin Jin kevin...@gmail.com wrote:
 To Pius,

 In my case, the protein/biopolymer is Lysine/amine riched.

 1. Lys or Arg are mainly positively charged under pH 7.0 ~ 8.0, and
 then provide positively charge to LPA. Of course, some metal cations
 could also be involved in LPA binding.

 2. Tris with the NH2 group could also be protonated and positively
 charged under the same pH condition. This is why I use high
 concentration Tris here.

 3. Tris buffer could function as an ionic-exchange competitor for LPA
 binding. If you look those commercial available Endotoxin assay kits,
 they also use Tris buffer.

 4. IPA could change the charge distribution on the surface of protein.
 In usual, high% IPA could remove LPA efficiently but may cause protein
 denature. In lot of cases, 5~10% IPA could help protein
 crystallization to a larger size. As a tradeoff, it may also bring
 down the packing quality.

 In this case, I used it for endotoxin  removal.

 5. NaCl (you can try other salt) would provide additional ion-strength
 to Interfere (or weak) the ionic-paring between Lys/Arg and LPA.

 6. If you can add EDTA to remove metal cation, it may be even better.
 In my case, I could not use EDTA.

 I tested the sample (1600EU/ml). After 3 days dialysis, it went down
 to  5EU/ml.  The assay kit for LPA measurement was from Charles River
 (?). The procedure is too long to present here. The good thing is that
 your sample would not be dehydrated and refold again.

 I am saying this will work for protein in all of case, but you try in
 an eppendorf tube like amini-prep.

 I guess this method may have been  patented.






















 On Thu, Mar 8, 2012 at 7:16 AM, Pius Padayatti ppadaya...@gmail.com wrote:
 This is in response to a comment to this thread
 Kevin,
 Could you explain how that worked?
 How do you know your method worked?
 Did you estimate the lipopolysaccharide before and after the method?

 The method already mentioned here to wash using TritonX100 makes sense.
 by washing bound protein sample May able to phase separate into
 detergent micellar phase and get rid of endotoxins.

 Most widely accepted method to separate is by two phase micellar system.
 above the CMC endotoxins will be accomodated by micelles through non-polar
 interactions of alkyl chains of lipidA.

 Padayatti PS



 On Wed, Mar 7, 2012 at 9:50 AM, Jerry McCully
 for-crystallizai...@hotmail.com wrote:
o

 Dear All;

     We purified a His tag protein by Ni-NTA and gel-filtration from
 E.coli.

  We tried two endotoxin removal resins from Pierce. However, it is
 hard to remove the endotoxins in the purified protein because the protein
 bound to the resin as well.

  This protein contains quite a few aromatic residues and has a pI
 around 6.

  Any ideas to remove the endotoxins will be highly appreciated.

     best regards,

 Jerry McCully





 --
 Pius S Padayatti,PhD,
 Phone: 216-658-4528



 --
 Pius S Padayatti,PhD,
 Phone: 216-658-4528

Re: [ccp4bb] FW: endotoxin removal

[Kevin Jin]

I tried to use 250mM Tris pH 7.6, 1M NaCl and 5~10% 2-propanol for
dialysis. It works,



On Wed, Mar 7, 2012 at 6:50 AM, Jerry McCully
for-crystallizai...@hotmail.com wrote:


 Dear All;

     We purified a His tag protein by Ni-NTA and gel-filtration from
 E.coli.

  We tried two endotoxin removal resins from Pierce. However, it is
 hard to remove the endotoxins in the purified protein because the protein
 bound to the resin as well.

  This protein contains quite a few aromatic residues and has a pI
 around 6.

  Any ideas to remove the endotoxins will be highly appreciated.

     best regards,

 Jerry McCully

[ccp4bb] a story of Orotidine 5'-Monophosphate Decarboxylase

[Kevin Jin]

Dear All,

I wrote a story for  the catalysis of  Orotidine 5'-Monophosphate
Decarboxylase.

http://www.jinkai.org/Enzymology.html

I hope you will like it.

Regards,

Kevin

Re: [ccp4bb] Collecting small-molecule diffraction on a Macromolecular xtallography beam line

[Kevin Jin]

I collected GTP/Mg2+ crystal on  SSRL beamline 9-1 before. The images
was processed by Mosflm and structure was solved by Shelx as usual.

Kevin

On Wed, Feb 8, 2012 at 3:41 AM, Giorgio Giardina
giorgio.giard...@uniroma1.it wrote:
 Hello,
 I have some interesting small molecule xtals.
 I was wondering if it is possible to collect a small molecule data-set using 
 a sincrotron macromolecular  xtallography beam line, maybe with a very low 
 beam intensity and moving the detector as close as possible?
 Has anybody experienced that?
 And if I get the images back home,  can I process them using standard 
 macromolecular software or do I need ab-initio special programs?
 Will MR work for phasing?

 Thanks in advance,
 Giorgio

Re: [ccp4bb] On pKa of Aspartic acid

[Kevin Jin]

As we know, the pKa of water is 15.7. Under pH 7.0, its protonation
should be 50/50.
In this case, we may need to consider water in two formats:

H2O vs. H3O+

When we say water as acid, it usually stands for H3O+ in chemistry. In
chemical equation, H+ represents H3O+.

In enzyme catalysis, water as a general acid sounds reasonable under
pH 7.0. In some famous paper, water has been concluded as the general
base (pKa 15.7) to deprotonate an alpha hydrogen (pKa ~ 22) or a
hydrogen from a sp3 hybridized carbon (pKa ~36). This logic may need
to be reconsidered.
.
Recently, I have read papers for pKa perturbation. I am also
interested in the general base of Asp and Glu in enzyme catalysis.


I will be very happy to read your paper in the future.

Regards,

Kevin Jin

On Tue, Feb 7, 2012 at 3:48 AM, Deepak Oswal deepos...@gmail.com wrote:
 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] On pKa of Aspartic acid

[Kevin Jin]

Maybe you would also be interested in

http://www.jinkai.org/AAD_history.html

Regards,

Kevin

On Tue, Feb 7, 2012 at 8:52 AM, Christian Roth
christian.r...@bbz.uni-leipzig.de wrote:
 Hi,

 you may also look into the papers of John A. Gerlt, who did a lot on
 protonabtraction reactions and the theory behind this. Esspecially the pKa
 disturbance and the match to the pkA of the substrate of the reaction.

 Best Wishes

 Christian

 Am Dienstag 07 Februar 2012 12:48:26 schrieb Deepak Oswal:
 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] On pKa of Aspartic acid

[Kevin Jin]

Oops, It should be: [H3O+]/[OH-]= 50/50

Kw = [H3O+][OH-],

pH = pKa +log ([OH-]/[H2O])

H3O+ concentration of pure water is 10^-7 mol/L

total H+ = 55.5M * 10^-7 = 5.55* 10^-6 mole. Is this right?

Regards,

Kevin


On Tue, Feb 7, 2012 at 12:13 PM, Zachary Wood z...@bmb.uga.edu wrote:
 Hi Kevin,

 Hate to point this out, but under pH 7.0, the protonation state of water is 
 not 50:50, and it is not a good acid.  The H30+ concentration of pure water 
 is 10^-7 Molar.  In pure water (assuming 55.5 M) only 1:555,000,000 water 
 molecules is in the protonated, charged state (H3O+).  This is why when an 
 enzyme uses water in its mechanism as a nucleophile, base, or acid, there is 
 usually an acid/base catalyst or  metal that protonates or deprotonates the 
 water to 'activate it'.


 Best regards,

 Z


 ***
 Zachary A. Wood, Ph.D.
 Assistant Professor
 Department of Biochemistry  Molecular Biology
 University of Georgia
 Life Sciences Building, Rm A426B
 120 Green Street
 Athens, GA  30602-7229
 Office: 706-583-0304
 Lab:    706-583-0303
 FAX: 706-542-1738
 ***






 On Feb 7, 2012, at 11:22 AM, Kevin Jin wrote:

 As we know, the pKa of water is 15.7. Under pH 7.0, its protonation
 should be 50/50.
 In this case, we may need to consider water in two formats:

 H2O vs. H3O+

 When we say water as acid, it usually stands for H3O+ in chemistry. In
 chemical equation, H+ represents H3O+.

 In enzyme catalysis, water as a general acid sounds reasonable under
 pH 7.0. In some famous paper, water has been concluded as the general
 base (pKa 15.7) to deprotonate an alpha hydrogen (pKa ~ 22) or a
 hydrogen from a sp3 hybridized carbon (pKa ~36). This logic may need
 to be reconsidered.
 .
 Recently, I have read papers for pKa perturbation. I am also
 interested in the general base of Asp and Glu in enzyme catalysis.


 I will be very happy to read your paper in the future.

 Regards,

 Kevin Jin

 On Tue, Feb 7, 2012 at 3:48 AM, Deepak Oswal deepos...@gmail.com wrote:
 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] No diffraction

[Kevin Jin]

For crystallization:

Your xtal may come out a little bit fast. If the condition contain
alcohol,  such as IPA, you may have to modify it.

If you let people know the condition, it may be more helpful.

Also, please check the purity of your protein.

Kevin

On Thu, Jan 26, 2012 at 7:33 AM, Theresa H. Hsu theresah...@live.com wrote:
 Dear crystallographers

 I have a protein of 90 kDa forming dimers. Crystals formed with microbatch 
 and vapor diffusion method in 24 hours but no diffraction at home source. 
 Dissolved crystals was confirmed to be the protein with mass spec.

 Any suggestions to improve diffraction would be welcome.

 Thanking you in advance.

 Theresa

Re: [ccp4bb] No diffraction

[Kevin Jin]

Maybe, you can adjust the ion strength of your condition.




On Thu, Jan 26, 2012 at 8:32 AM, Kevin Jin kevin...@gmail.com wrote:
 For crystallization:

 Your xtal may come out a little bit fast. If the condition contain
 alcohol,  such as IPA, you may have to modify it.

 If you let people know the condition, it may be more helpful.

 Also, please check the purity of your protein.

 Kevin

 On Thu, Jan 26, 2012 at 7:33 AM, Theresa H. Hsu theresah...@live.com wrote:
 Dear crystallographers

 I have a protein of 90 kDa forming dimers. Crystals formed with microbatch 
 and vapor diffusion method in 24 hours but no diffraction at home source. 
 Dissolved crystals was confirmed to be the protein with mass spec.

 Any suggestions to improve diffraction would be welcome.

 Thanking you in advance.

 Theresa

[ccp4bb] A story of acetoacetate decarboxylase

[Kevin Jin]

Dear All,

I wrote a story about the catalysis of Acetoacetate Decarboxylase. It
is available here:

http://www.jinkai.org/AAD_history.html
http://www.jinkai.org/AAD_catalysis.html

I wish you will like it.

Regards,

Kevin

Re: [ccp4bb] Autoreply: [ccp4bb] A story of acetoacetate decarboxylase

[Kevin Jin]

Dear All,

I got this email. Is my account blocked?

Thanks,

Kevin

2012/1/24  asch...@cipf.es:
 Esta cuenta de correo electrónico dejara de existir dentro de 6 meses 
 (25/05/2012).

 Si desea ponerse en contacto con el titular de este correo hágalo a través 
 del siguiente e-mail: annie_sch...@yahoo.de

 Todo el correo recibido está siendo redirigido a la cuenta indicada 
 anteriormente.

 Si desea contactar con el Centro de Investigación Príncipe Felipe puede 
 hacerlo por teléfono llamando al 963.289.680.

 Muchas gracias y disculpen las molestias.

Re: [ccp4bb] about alternate conformations

[Kevin Jin]

I did not make it clear. The protocol we used for protein expression always
limit the occ of MSE as 0.75. You may estimate your occ of MSE by ED. You
can check PDB for JCSG structures.

Cheers,

Kevin

On Fri, Sep 16, 2011 at 7:09 AM, Ming dongm...@udel.edu wrote:

 Hi all,


 I was using Refmac from CCP4 to refine a protein's crystal structure. The
 methionine has half selenium and half sulfur. I was trying to make alternate
 conformations and let refmac do the refinement. But it keeps giving me error
 message as follows:


 There is an error in the input coordinate file
 At least one the chains has 2 residues with the same number
 Check above to see error
 === Error: Problem with coordinate file
 BFONT COLOR=#FF!--SUMMARY_BEGIN--
  Refmac_5.5.0109:  Problem with coordinate file

 And here is my modified pdb file.

 HETATM  403  N  AMSE A 129  ***   N
 HETATM  404  CA AMSE A 129  ***   C
 HETATM  405  CB AMSE A 129  ***   C
 HETATM  406  CG AMSE A 129  ***   C
 HETATM  407 SE  AMSE A 129  ***  SE
 HETATM  408  CE AMSE A 129  ***   C
 HETATM  409  C  AMSE A 129  ***   C
 HETATM  410  O  AMSE A 129  ***   O
 ATOM411  N  BMET A 129  ***   N
 ATOM412  CA BMET A 129  ***   C
 ATOM413  CB BMET A 129  ***   C
 ATOM414  CG BMET A 129  ***   C
 ATOM415  SD BMET A 129  ***   S
 ATOM416  CE BMET A 129  ***   C
 ATOM417  C  BMET A 129  ***   C
 ATOM418  O  BMET A 129  ***   O

 I already confirmed with pdb that this is the right format for this case.
 But refmac doesn't work with it. I wonder if there is any other changes I
 should make for refmac?

 Thank you,

 Ming

Re: [ccp4bb] QC Server (was Re: [ccp4bb] Another paper structure retracted)

[Kevin Jin]

Chris,

I seriously think this sever should be available to folks. It is very
helpful for most students.

Will you and Abhinav keep improving it?

Kevin




On Fri, Aug 12, 2011 at 8:04 AM, Christopher Rife 
christopher.r...@danisco.com wrote:

 I think you'll find it works better if you use Fasta format:
 http://en.wikipedia.org/wiki/FASTA_format

 Chris

 ___

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   From: Phil Evans p...@mrc-lmb.cam.ac.uk To: CCP4BB@JISCMAIL.AC.UK Date: 
 08/12/2011
 01:16 AM Subject: Re: [ccp4bb] Another paper  structure retracted Sent
 by: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK
 --



 Can anyone get this server to work? For me it keeps complaining that my
 sequence file is not a PIR file. The file looks OK to me, but I've never
 really understood what a PIR file is

 Phil

 On 12 Aug 2011, at 01:39, Kevin Jin wrote:

 
  Should we really have some crystallographers to review and qc those
 structures before the formal releasing?  JCSG has set a very good mechanism
 for this issue.
 
  There is a sever for self check.
 
  http://smb.slac.stanford.edu/jcsg/QC/
 
 
 
 

Re: [ccp4bb] Another paper structure retracted

[Kevin Jin]

Phil,

The sever was developed by Chris. Currently, Abhinav is taking care of it.
If you have any questions, you may contact Abhinav Kumar at JCSG.

Kevin

On Fri, Aug 12, 2011 at 1:14 AM, Phil Evans p...@mrc-lmb.cam.ac.uk wrote:

 Can anyone get this server to work? For me it keeps complaining that my
 sequence file is not a PIR file. The file looks OK to me, but I've never
 really understood what a PIR file is

 Phil

 On 12 Aug 2011, at 01:39, Kevin Jin wrote:

 
  Should we really have some crystallographers to review and qc those
 structures before the formal releasing?  JCSG has set a very good mechanism
 for this issue.
 
  There is a sever for self check.
 
  http://smb.slac.stanford.edu/jcsg/QC/
 
 
 
 
 
 
  On Thu, Aug 11, 2011 at 4:58 PM, Jacob Keller 
 j-kell...@fsm.northwestern.edu wrote:
  I think they fudged the data in this paper...
 
  JPK
 
  On Thu, Aug 11, 2011 at 6:30 PM, David Schuller dj...@cornell.edu
 wrote:
   link: http://iai.asm.org/cgi/reprint/IAI.05661-11v1
  
   Ferric C. Fang  Arturo Casadevall
   Retracted Science and the Retraction Index
   Infec. Immun. doi:10.1128/IAI.05661-11
  
   Abstract: Articles may be retracted when their findings are no longer
   considered trustworthy due to scientific misconduct or error, they
   plagiarize previously published work, or are found to violate ethical
   guidelines. Using a novel measure that we call the “retraction index,”
 we
   found that the frequency of retraction varies among journals and shows
 a
   strong correlation with the journal impact factor.
   ...
  
   (with special attention to Figure 1, Retraction Index vs. Impact
 Factor)
  
  
   --
   ===
   All Things Serve the Beam
   ===
 David J. Schuller
 modern man in a post-modern world
 MacCHESS, Cornell University
 schul...@cornell.edu
  
 
 
 
  --
  ***
  Jacob Pearson Keller
  Northwestern University
  Medical Scientist Training Program
  cel: 773.608.9185
  email: j-kell...@northwestern.edu
  ***