Re: [ccp4bb] [EXTERNAL] [ccp4bb] Viewing samples under liquid nitrogen

[Bonsor, Daniel (NIH/NCI) [C]]

Clear Pyrex beaker/dish/bowl filled with liquid nitrogen on the microscope 
stand. Place pin on a wand and hold at an angle in the bath.
Probably best to do when the safety-officer is not around.

Can you mount on a home source with the cryo-stream on?

Best,

Dan

From: CCP4 bulletin board  on behalf of Matt McLeod 

Date: Friday, December 15, 2023 at 12:20 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [EXTERNAL] [ccp4bb] Viewing samples under liquid nitrogen
Hi all,

Has any heard of or has ideas on how to view a sample after being plunged in 
liquid nitrogen but while still under LN2?
Thanks!



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Re: [ccp4bb] Expi 293 (Met-) expression medium for selenomethionine labeling

[Bonsor, Daniel]

Hopefully, someone will be able to share 293 Met- media. But if not, could you 
not use DMEM media with FBS, then deplete with DMEM with no glutamine, no 
methionine, no cysteine (thermos fisher 21013024) but supplemented with 
glutamax and cystine?

The following paper describes it in the large scale protein production section 
at the end.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2242577/

Good luck with your search and finishing your PhD.

Best,
Dan


Daniel A Bonsor PhD.
Lu Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of LAURA DEL 
AMO MAESTRO
Sent: Tuesday, March 10, 2020 1:41 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Expi 293 (Met-) expression medium for selenomethionine 
labeling

Dear ccp4 community,

I am in my last 3 months of my PhD, working at the Structural Biology Unit at 
IBMB-CSIC in Barcelona. As my experimental phasing trials using various heavy 
metals failed so far, we ordered two months ago Expi 293 (Met-) expression 
medium for selenomethionine labeling (see the reference: 
https://www.thermofisher.com/order/catalog/product/A4096701#/A4096701. But we 
got now the notice that it will not be delivered until end of April.

I had been working so hard on this project, and this medium is essential to 
finished with the experimental part of my thesis. Thus, I was wondering if 
someone in the community has a bottle (1L) of this expression medium without 
methionine that I could borrow, and which I would return immediately once our 
medium arrives.

Also in case you have recommendations or other sources of a defined Se-Met 
medium for Expi293 cells, please let me know. With kind regards from Barcelona, 
and hope someone can help,

Laura

---
Laura del Amo Maestro - Proteolysis Lab
SBU-Structural Biology Unit, IBMB CSIC
Barcelona, Spain, Europe



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Re: [ccp4bb] ITC question -dimer vs monomer

[Bonsor, Daniel]

You may want to look at the following paper concerning Il8, dimerization, 
binding and ITC.

http://m.jbc.org/content/279/35/36175.full

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From: CCP4 bulletin board  on behalf of Bernhard Rupp 

Sent: Friday, October 4, 2019 5:06:10 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

Thanks to All for the extended & informative responses. If true thermodynamic 
equilibria
are realized, then I would agree that regardless of the pathway the endpoint 
(or integrated H)
should be the same.  The actual pathway and cooperativity probably will make 
this an
interesting problem. I may keep bugging selected victims off-board once I have 
the first data.

Many thanks again, BR

From: CCP4 bulletin board  On Behalf Of Barone, Matthias
Sent: Thursday, October 3, 2019 18:59
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer


As Reza already pointed out, ITC cannot tell you anything about the 
sub-processes that underlay an equilibrium, be it complicated like 2A+B2 <-> 
AB2 + A <-> A2B2, or with (virtually) no intermediate 2A+B2 <-> A2B2 due to a 
fast second forward reaction , or a simple one-to-one model A+B2 <-> AB2.

Given the lack of additional information, its probably good to assume a simple 
one-to-one model and titrate either partner to the other.

If you have two separate binding steps (with similar Kd for B2 for A as well as 
AB2 for A), you would measure an apparent affinity and would see the 
stochiometrics according to  the inflection point (be it around equimolar 
excess or at 0.5 or 2, depending on whether you titrate A or B). If the 
reaction is more complicated and the the affinities for B2 for A differ 
significantly much from the affinity of AB2 for A, then a simple one-to-one 
would leave some notable information in the residual standard deviations 
(meaning, the residuals would not spread normally around the Regression line, 
but should show a wavy pattern).

Sorry for the long mail..

Matthias



Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284
future.

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Bernhard Rupp
Sent: Thursday, October 3, 2019 11:06 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] ITC question -dimer vs monomer

Hi Fellows,

please let me ask the respective experts an ITC question: I have 2 proteins, 
stable and dialyzed in identical buffer.
A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer 
will form.

Intuitively, it should make a difference whether I titrate the dimer with the 
monomer or vice versa.
In the first case, a momomer would initially meet a lot of free dimers, and I 
would expect that randomly, a AB2 complex
is more likely to form than a A2B2 (let’s disregard any more complex 
colligative/cooperative effects).

If I drip the dimer into the monomer pool, it is quite likely that the B dimer 
meets 2 free As, and I get right away a higher population of A2B2s.

Maybe at dilutions of ITC and with sufficient equilibration that is not an 
issue at all (again, absent any cooperative effects that might alter the first 
Kd vs. the second, despite the sites on the dimer are at least initially 
equivalent).

Can someone guide me towards literature about this or perhaps share some 
first-hand experience?

Many thanks, BR

--
Bernhard Rupp
http://www.hofkristallamt.org/
b...@hofkristallamt.org
+1 925 209 7429
+43 676 571 0536
--
Many plausible ideas vanish
at the presence of thought
--




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Re: [ccp4bb] Secrets of ZYMIT

[Bonsor, Daniel]

Could you take a solution of it, buffer exchange/size exclusion and then 
perform MS fingerprinting to identify the protease if you don't have any luck 
with the companies?

Best

Dan

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From: CCP4 bulletin board  on behalf of Jorg Stetefeld 

Sent: Sunday, September 8, 2019 6:01:49 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Secrets of ZYMIT


Hi everyone,


this is Joerg Stetefeld.


I would like to approach the community in a peculiar case of a specific 
proteolytic cleavage during crystallization attempts.

Performing several crystallization screens we figured out that ZYMIT 
(https://www.ipcol.com/cleaners/zymit-low-foam) is the reason for a highly 
specific, but undesireable cleavage of protein components in our setups. Most 
remarkably, the cleavage site of our crystallisation target is not described in 
any protease database, and is highly inaccessible.



According to a publication by Naschberger et al, 2014 
(doi:10.1107/S2053230X14026053) the authors very nicely describe the 
phenomenon. They also describe a very elegant protease removal protocol, which 
works in our hands very well.

At this point, however, I would like to know what is “behind” ZYMIT? 
Naschberger et al say “Zymit contains an unspecified‘protease enzyme’ as a 
trade secret (http://www.ipcol.com/pdfs/Zymit_msds.pdf)”.


Does anyone has more insight into the secrets of ZYMIT? Our attempts to contact 
several vendors here in Canada/ abroad failed and a further characterisation of 
its nature would help us to understand this particular phenomenon of 
proteolytic cleavage.



Thank you very much in advance- Your advise is appreciated.


js




Jörg Stetefeld


https://stetefeldlab.ca/

[X]



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Re: [ccp4bb] Questionable Ligand Density: 6MO0, 6MO1, 6MO2

[Bonsor, Daniel]

Would it be possible to add a public annotations section to the PDB, to allow 
us to potentially flag/warn whoever downloads that particular structure, there 
could be something wrong with it, such as wrong space group, no/poor density 
fitting for ligand. Something similar to PubPeer maybe?


Daniel A. Bonsor PhD
Institute of Human Virology,
University of Maryland at Baltimore
725 W. Lombard Street N571
Baltimore
MD 21201



From: CCP4 bulletin board  on behalf of Patrick Loll 

Sent: Friday, July 19, 2019 1:17 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Questionable Ligand Density: 6MO0, 6MO1, 6MO2

The idea of contacting the editor (and/or author) is an excellent one, and 
indeed the correct thing to do scientifically. However, I’m disillusioned: I’ve 
been down this path before with a high-profile vanity journal, and while the 
editors paid lip service to the notion that the record should be corrected, in 
reality they led me on for the better part of a year, and got me to write up 
detailed analyses of why the ligand positioning was not justified, before 
eventually saying “no, we don’t see any need to publish a correction.” I 
speculate that the journal prefers not avoid corrections, for fear that too 
many corrections will make the journal a less desirable destination.

> On 19 Jul 2019, at 11:23 AM, Bärbel Blaum  
> wrote:
>
> Hi Rhys,
> the reported B-factors for the “ligands” are all way below the reported 
> B-factors for the protein chains, with the worst of the three complexes 
> reporting unitless numbers 23.2 and 64.8, respectively, just to highlight 
> *one* striking feature of the data collection and refinement table. So even 
> with the limited info normally available to reviewers this table should have 
> raised a red flag. After the re-refinement suggested by others, i.e. your own 
> proper assessment of the crystallographic data, if you do not find noteworthy 
> density you may want to contact the article’s editor with your results. If 
> you work in this field, i.e. really care about this paper scientifically and 
> you are not afraid to confront the authors you could suggest writing a 
> comment/direct response article but in my opinion that would only make sense 
> if you can be sure beforehand that it will be linked visibly to the actual 
> paper, else it will be a waste of time. And don’t forget that just because 
> one or some of the authors did a bad job at the crystallographic end other 
> findings of the paper might still be solid – in collaborations often one 
> author is unable to critically evaluate another author’s contribution and 
> this would not be the first case were good synthetic or biological work is 
> presented along with a bad crystal structure.
> By the way and a bit ironically this protein may have suffered bad 
> crystallography/scientific practice before - I think it was one of the fake 
> Krishna Murthy structures, right? The associated (now retracted) article I 
> mean is here
> https://www.sciencedirect.com/science/article/pii/S002228360093924X?via%3Dihub
[https://ars.els-cdn.com/content/image/S00222836.gif]
RETRACTED: Crystal structure of dengue virus NS3 protease in complex with a 
bowman-birk inhibitor: implications for flaviviral polyprotein processing and 
drug design - ScienceDirect - ScienceDirect.com | Science, health and medical 
journals, full text articles and 
books.
www.sciencedirect.com
COMMUNIC Crystal Structure of Dengue Complex with a Bowman-Bir ro L. 1Center 
for Macromolecular C f A 8 U T MCLM 244, Birmingham AL 35294-0005, USA 
2Department of Biochemistry and Molecular Biology, Kansas University Medical 
Center 3901 Rainbow Boulevard Kansas City, KS 66160- 7421, USA Dengue viruses 
are members of the Flaviviridae and cause dengue fever Dengue fever and dengue 
hemorrhagic ...

> Kind regards, Bärbel
> ---
> Bärbel Blaum, PhD
> Inthera Bioscience AG
> Einsiedlerstrasse 34
> CH-8820 Waedenswil
> Switzerland
> E-Mail: baerbel.bl...@intherabio.com
> Phone: +41 43 477 94 72--
>
>
>
> Von: CCP4 bulletin board  im Auftrag von "Manfred S. 
> Weiss" 
> Antworten an: "Manfred S. Weiss" 
> Datum: Freitag, 19. Juli 2019 um 16:03
> An: 
> Betreff: Re: [ccp4bb] Questionable Ligand Density: 6MO0, 6MO1, 6MO2
>
> Hi Rhys,
>
> all three structures are at modest resolution and they don't seem to
> be properly refined. At least they are all below average. I wonder
> how this paper made it past the referees.
>
> I haven't checked the paper, but there are ways and means how to
> deal with weakly bound ligands in the best possible way. One aspect
> is to improve the phases as much as possible without having the ligand
> present. This was obviously NOT done. Another way is to use the
> PANDDA approach, which relies on having many data sets available.
> I suppose 

Re: [ccp4bb] Fo-Fc density close to cysteine residue

[Bonsor, Daniel]

Have you mass-speced the protein before crystallization to make sure it wasn’t 
derivatized during expression and/or purification, or compared the mass spec of 
the crystals verses purified protein? Any fancy reagents or other reductants 
used during purification?
What about S-Acetyl-cysteine (3-letter code: SCY).

Best,

Dan

Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Lumbini 
Yadav
Sent: Tuesday, July 09, 2019 5:22 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Fo-Fc density close to cysteine residue

Dear all,

We have found a huge Fo-Fc density close to cysteine residue (see attached 
image) in the structure with resolution of 1.2A. In the crystallization 
condition, we have PEG 3350, Potassium phosphate monobasic, glycerol and 
protein was in Tris and NaCl. Before freezing the crystals were soaked in 
mother liquor containing sodium dithionite.

I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD, 
CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in all the 
screenings we do see some part of Fo-Fc density unaddressed at 3 sigma.

Does anyone have an idea about what this density could be? Covalent 
modification?

Thanks.



Kind regards,
Lumbini



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Re: [ccp4bb] W. Friedrich's thesis title

[Bonsor, Daniel]

According to the German Wikipedia page it is correct.
https://de.m.wikipedia.org/wiki/Walter_Friedrich_(Biophysiker)

Maybe it wasn't deposited in the library until 1912.

Dan

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From: CCP4 bulletin board  on behalf of Harry Powell 
<193323b1e616-dmarc-requ...@jiscmail.ac.uk>
Sent: Monday, June 10, 2019 6:18:30 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] W. Friedrich's thesis title

Hi folks

I've found a thesis dated 1912 with the title "Intensitätsverteilung der 
X-Strahlen, die von einer Platinaantikathode ausgehen" - could anyone confirm 
if that's the one? All the other references I've found say it should be 1911, 
and a year in science (particularly at that time, when things were moving very 
quickly!) can be a very long time!


On 10 Jun 2019, at 11:07, Harry Powell wrote:

> Hi folks
>
> I've been trying to track down the title of Friedrich's (of Friedrich and 
> Knipping fame) 1911 thesis at Ludwig-Maximilians Universität, München - all I 
> can find is a translation into English. I don't want my personal (or google 
> translate or similar) back in to German, so I was wondering if anyone out 
> there actually knows what it was?
>
> I've had a search on the LMU library website, but can't find the thesis 
> indexed there...
>
> The English translation is "Emission by a platinum target".
>
> Harry
> --
> Dr Harry Powell
>
>
> To unsubscribe from the CCP4BB list, click the following link:
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>
> 
>
>
> To unsubscribe from the CCP4BB list, click the following link:
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>



Harry
--
Dr Harry Powell




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Re: [ccp4bb] A Max Perutz question

[Bonsor, Daniel]

https://www.nature.com/articles/449144a

Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Zachary 
A. Wood
Sent: Wednesday, April 10, 2019 10:30 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] A Max Perutz question

Hello Fellow Structural Enthusiasts,

My apologies for the slightly off-topic question. I am trying to track down a 
higher resolution image of the jpg that I have attached. I use this photo of 
Max Perutz when I am teaching about protein folding, and have always wanted a 
better quality one. I believe it is credited to Nature, and I am trying to find 
out what issue, but I am hoping that one of you may have more information or 
perhaps even a better photo. Thanks for any help, and for those of you who may 
never have seen this photo before, I hope you enjoy it. I like to imagine that 
Perutz is considering the challenges associated with folding that chain after 
he determined the crystal structure. If you have never read the discussion in 
his famous Nature paper, I will leave you with a relevant quote of him 
referring to the structural similarity between horse hemoglobin and sperm whale 
myoglobin, in which he predicts the thermodynamic hypothesis (Anfinsen’s dogma):

“How does this arise? It is scarcely conceivable that a three-dimensional 
template forces the chain to take up this fold. More probably, the chain, once 
synthesized and provided with a haem group around which it can coil, takes up 
this configuration spontaneously, as the only one which satisfies the 
stereochemical requirements of its amino acid sequence.”

Thank you for any help you may be able to offer!

 Best regards,

Z

***
Zachary A. Wood, Ph.D.
Associate Professor and Graduate Coordinator
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
***
[cid:image001.jpg@01D4EF89.4B618FB0]



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Re: [ccp4bb] off topic: Membrane protein "into" soluble protein

[Bonsor, Daniel]

In theory yes, you can fuse the termini to restrain the protein. You could use 
T4 lysozyme and insert in the loop which they use for crystallizing GPCRs (many 
references, including https://www.ncbi.nlm.nih.gov/pubmed/25450769). The 
potential problems are that you have no idea where the termini are 
distance-wise to each other in your receptor. Maybe trying some modelling with 
Phyre or Rosetta) You would probably have to make several constructs with 
varying lengths of flexible linkers and then there is no guarantee the fusion 
protein will actually express or crystallize.

You say you have had no luck crystallize the construct you have at the moment. 
Is it His-tagged? Can you remove it? If C-terminal you could use 
Carboxypeptidase A (1:100 w/w ratio) or limited proteolysis in general? You 
said it is a receptor. Have you tried with the ligand/binding protein? Lysine 
methylation? Thermal Shift Assay to identify a storage buffer which increases 
the Tm of the protein?

You could make new constructs with trimmed termini. Surface entropy reduction 
of the protein (use the SER server, http://services.mbi.ucla.edu/SER/). You 
could try fusing it to MBP or surface entropy reduced MBP 
(https://www.ncbi.nlm.nih.gov/pubmed/20196072).

Hope something helps and it crystallizes.

Dan

Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Andrew 
Lovering
Sent: Wednesday, March 06, 2019 9:28 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] off topic: Membrane protein "into" soluble protein

Dear ccp4bb members,

A quick off topic question. I have a protein whose domain structure runs 
N-TM-receptor-TM-enzyme-C, i.e. is a two transmembrane-helix containing 
signalling protein. One would suspect that the receptor domain has ends that 
cluster, and that the enzyme (via homology with known systems) is activated 
when the two helices self-interact in a dimeric protein as a four helix bundle.

So...my question - I have tried expressing the receptor domain as a soluble 
protein, it works well, folds, purifies, but doesn't crystallize. Does anyone 
know of an example of taking an exposed membrane protein loop/domain and making 
a chimera with a soluble 4 helix bundle such that the ends of the loop are 
tethered and restrained nicely?

I was thinking that this must've been attempted for transmembrane alpha-helical 
signalling proteins, perhaps even "PDB output" successfully!

Best
Andy



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Re: [ccp4bb] KCl in SDS-PAGE workarounds?

[Bonsor, Daniel]

SDS is soluble, whereas the potassium salt of dodecyl sulphate is insoluble. 
You could try 18-Crown-6 ether to chelate the potassium, though I don't know if 
the crown ether would then affect the gel.


Dan


Daniel A. Bonsor PhD
Institute of Human Virology,
University of Maryland at Baltimore
725 W. Lombard Street N571
Baltimore
MD 21201



From: CCP4 bulletin board  on behalf of Keller, Jacob 

Sent: Saturday, March 2, 2019 9:52 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] KCl in SDS-PAGE workarounds?


Dear crystallographers,



It has been my experience that KCl does nasty things when loading SDS-PAGE 
gels. Does anyone have an easy workaround, perhaps TCA precipitation? Ideally 
this would be something nicely quantitative yet quick and easy….



Any suggestions appreciated.



All the best,



Jacob Keller



+

Jacob Pearson Keller

Research Scientist / Looger Lab

HHMI Janelia Research Campus

19700 Helix Dr, Ashburn, VA 20147

Desk: (571)209-4000 x3159

Cell: (301)592-7004

+



The content of this email is confidential and intended for the recipient 
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received this message by mistake, please reply to this message and follow with 
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From: CCP4 bulletin board  On Behalf Of Artem Evdokimov
Sent: Saturday, March 2, 2019 8:32 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ionic strength for extraction buffer of membrane proteins



Hi Alex,



In my experience you just have to experiment with these parameters (in however 
of a limited space that is afforded by your experimental set-up). If you have 
the right tools you can slog through semifactorial or DoE-based scouting of 
various parameters with relative ease. These parameters typically include pH, 
salinity, detergent(s), and other variables (e.g. the nature of the buffer and 
salt(s) - many cases exist where the 'standard' choices do not work well).



Literature-based analogies do help on occasion. For example, membrane-spanning 
p450-type proteins often prefer high salt, and often would do better in sodium 
acetate as opposed to chloride. I've worked with ion channels that preferred 3M 
NaCl, or 2M MgCl2 and other fairly weird conditions...



Artem

- Cosmic Cats approve of this message





On Fri, Mar 1, 2019 at 7:27 AM Alex Perálvarez Marín 
mailto:aperalva...@gmail.com>> wrote:

Dear all,

any reference as a guide for selecting the appropriate salt and
concentration for membrane proteins extraction buffer?

Best,

Alex

--
Alex Perálvarez-Marín, Ph.D.
Centre d'Estudis en Biofísica / Unitat de Biofísica
Edifici M
Universitat Autònoma de Barcelona
08193 Cerdanyola del Vallés
Barcelona
Spain
Phone: +34 93 581 4504
FAX:  +34 93 581 1907
e-mail: aperalva...@gmail.com
LinkedIn: 
es.linkedin.com/in/aperalvarez/



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Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

[Bonsor, Daniel]

I have had some luck with adding ~5-10ul of 60%-70% dilution of the reservoir 
to the drop. The larger volume of the drop appears to slow down the whizzing of 
the crystals and allows you to get a few crystals. Though it still occurs. You 
could also cool the area down or move into the cold room if your crystals 
survive the transfer as evaporation should be less at 4oC.

You can also spot several 1-2uL of neat reservoir solution around the drop to 
create a local vapor barrier to prevent evaporation of the drop. It can 
completely stop the movement of crystals for a few minutes.

Dan

Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Thomas 
Krey
Sent: Tuesday, August 14, 2018 2:59 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Fishing crystals from volatile solvent as precipitant

CAUTION: This message originated from a non UMB, UMSOM, FPI, or UMMS email 
system. Whether the sender is known or not known, hover over any links before 
clicking and use caution opening attachments.

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




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Re: [ccp4bb] efresol download?

[Bonsor, Daniel]

Is this not it?

http://www-ibmc.u-strasbg.fr/spip-arn/rubrique257?lang=en

At the bottom of the page.


Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Pavel 
Afonine
Sent: Thursday, October 26, 2017 11:54 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] efresol download?

Johan,

core functionality described in that paper is implemented in cctbx. Re the 
stand-alone program -- I'm cc'ing to the author.

Pavel

On Thu, Oct 26, 2017 at 8:39 AM, Hattne, Johan 
> wrote:
Dear all;

Would anybody know where I can find efresol (as detailed in 
http://dx.doi.org/10.1107/S0907444913016673) these days?  Googling lead me to a 
2014 post 
(https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind1406=ccp4bb===155751),
 but those links result in 403 Forbidden as of right now.

// Best wishes; Johan


  Research Specialist @ Gonen Lab

Janelia Research Campus * 19700 Helix Drive
Ashburn, VA 20147 * +1 (571) 209-4000 extension 
3376

Re: [ccp4bb] off-topic: negative thermal shift upon ligand binding

[Bonsor, Daniel]

What are you using to dissolve the compounds? If DMSO or other organics, you 
maybe witnessing unfolding due to the organics. Have you done a control of just 
the solution with the protein.

Some compounds may drastically change the pH of the buffer your protein is in. 
You maybe observing a change in the stability of your protein due to pH change.

You could also try the same compounds against BSA, lysozyme and other control 
proteins to see if you observe a similar effect.

Dan

Get Outlook for Android



From: megha abbey
Sent: Saturday, 8 July, 20:30
Subject: [ccp4bb] off-topic: negative thermal shift upon ligand binding
To: ccp4bb@jiscmail.ac.uk


Hello,


I am working on DSF to verify if some compounds bind to my protein. I see a 
negative shift of about 3-4 degrees upon ligand addition (dose-response) in 
comparison to the protein alone. I assume that this might be due to the binding 
of compound to the unfolded stated rather than folded protein.


In such a situation where compounds are to be screened with the aim of drug 
discovery, are these negative thermal shift compounds relevant and how can they 
be followed upon, or they should simply be discarded?


Thank you.

[ccp4bb] Arcimboldo, ShelxE, Windows

[Bonsor, Daniel]

Dear All,


Does Arcimboldo actually run on WIndows as I can see it is installed and so is 
ShelxE, but I have a warning in the GUI saying "ShelxE is not found and option 
to run on 'this machine' is disabled. Is this a bug, do I need alter something 
so get it to recognize ShelxE or it does not run because I am using WIndows and 
I should get a Mac or Linux.


Thanks for any input or solving this problem.


(This problem is seen in arcimboldo_lite, arcimboldo_borges and 
arcimboldo_shredder).


Dan


Daniel A. Bonsor PhD
Institute of Human Virology,
University of Maryland at Baltimore
725 W. Lombard Street N571
Baltimore
MD 21201

Re: [ccp4bb] Se-Met and Se-Cys double labelling

[Bonsor, Daniel]

To perform double labelling on your protein in an NON auxotrophic strain may be 
difficult. For the seleomet side, you can shut down the biosynthesis of Met by 
the addition of lysine, phenylalanine, threonine, isoleucine and valine; 
various protocols exist online. However to shutdown biosynthesis of cysteine, 
you need to add cysteine (acts as a negative feedback inhibitor on itself ), 
which defeats the point. I cannot find online if selenocys can inhibit 
biosynthesis of cysteine, like cysteine can. You could perform a small test 
expression by adding all the amino acids (lysine, phenylalanine, threonine, 
isoleucine, valine, selenomet, selenocys) 30 mins before induction, do your 
typical expression and purification and confirm by mass spec of labelling.

If not you could trying sulfur SAD, the various derivatives suggested today, 
soaks with halides, magic triangle. Mutate leucine residues to methionine. 

Dan

Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457



-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Vito 
Calderone
Sent: Wednesday, June 21, 2017 11:47 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Se-Met and Se-Cys double labelling

I am working on a protein having 360 residues. In its sequence there are 3 Met 
and 5 free Cys.
I will need MAD to solve the structure since based on the sequence the closest 
homologue has 20% identity...I suppose MR would be very unlikely to work...so I 
would like to express a selenium derivative to exploit MAD.
Looking in the literature 1 Se-Met every 120 residues seems not to comply the 
threshold to get a good anomalous signal. For this reason I would like to 
exploit both Met and Cys so I would have 8 seleniums per 360 residues.
Could somenone suggest a reference to a protocol to express the double mutant 
protein in NON auxotrophic strains of E. coli which you have experienced 
working efficiently?
Thanks

Re: [ccp4bb] Problems with an exonuclease

[Bonsor, Daniel]

Several further notes after contemplation, lunch and a slow day. If the protein 
is His-tagged, you could stick the unfolded protein to Ni-Resin and extensively 
wash with 8M Urea to remove DNA/Triton-X-100, elute, and then refold. This may 
be easier than multiple sonications/centrifugations. Or you could stick the 
folded protein that you have purified to the Nickel resin and wash with high 
concentrations of salt to remove DNA/Triton-X-100.

To check if is really DNA-protein complex you have prepped, you can run two 
agarose gels. Stain one with ethidium bromide and the other one coomassie 
stain. The two bands should coincide with each other (protocol for native 
agarose gel electrophoresis can be found here 
http://www.sciencedirect.com/science/article/pii/S0003269700945986).

Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mohammad 
Khan
Sent: Wednesday, June 07, 2017 9:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Problems with an exonuclease

Dear all,

I am working with an exonuclease by refolding it from inclusion bodies (IBs). I 
tried various constructs and hosts, but couldn't get it in soluble form.

I lyse my cells using a cell disruptor and after solubilizing IBs with urea, I 
refold the protein by rapid dilution and get an aggregate and monomer peak of 
the same on GFC. and have checked CD as well as activity, both of which are 
good.

My issues is as follows:

I get a high 260 nm peak while purifying it on GFC. the 260/280 ratio can reach 
upto 2. I have tried all means to get rid of watever this contamination is: 
cleaned the IBs with 1% Triton X-100, 2 M NaCl, added Dnase prior to lysis. I 
have also used methods to remove the DNA from protein, if that is the 
contaminating agent.
I am trying to crystallize the protein with no success so far.
Moreover, my thermofluor assays give very low fluorescence. I use Sypro Orange 
as a fluorophore.

Suprisingly, a point mutation in the active site (His to Arg) gets rid of the 
issue of contamination and gives me good thermofluor curves. I purify the 
mutant also form IBs.

Can someone suggest what this "contamination" may be?

Thank you for your time.

Re: [ccp4bb] Problems with an exonuclease

[Bonsor, Daniel]

It will either be two things. DNA or residual Triton-X-100. When you say, 
cleaned the IBs, do you mean you sonicated the IBs, or just resuspended the 
pellet and then centrifuged again? If the latter, try sonication. I wash my IBs 
at least 4 times with the following buffers;

1. 20mM Tris, 500mM NaCl, 1% Triton-X-100, pH 7.5
2. 20mM Tris, 500mM NaCl, 1% Triton-X-100, pH 7.5
3. 10mM Tris, 1M NaCl
4. 20mM Tris, 500mM NaCl, pH 7.5

By resuspension and then sonication. This I find removes DNA and Triton-X-100.

Also, if the pellet is very large, you may need to increase the number of 
washes, volume and length of sonication or split the pellet up.

Other things to try…

1.   Change the wash salt to KCl and use more, (3M). I was informed that 
KCl is a better disrupter of DNA than NaCl (I stand to be corrected if this is 
wrong).

2.   At each wash stage, dissolve a small amount of IBs and measure the 
260/280. The ratio should decrease in the latter washes, if they are working.

3.   Does your exonuclease typically contain a divalent metal? You could 
try adding EDTA to the wash steps which may help in preventing DNA stick to 
your protein.

All the best!

Dan


Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mohammad 
Khan
Sent: Wednesday, June 07, 2017 9:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Problems with an exonuclease

Dear all,

I am working with an exonuclease by refolding it from inclusion bodies (IBs). I 
tried various constructs and hosts, but couldn't get it in soluble form.

I lyse my cells using a cell disruptor and after solubilizing IBs with urea, I 
refold the protein by rapid dilution and get an aggregate and monomer peak of 
the same on GFC. and have checked CD as well as activity, both of which are 
good.

My issues is as follows:

I get a high 260 nm peak while purifying it on GFC. the 260/280 ratio can reach 
upto 2. I have tried all means to get rid of watever this contamination is: 
cleaned the IBs with 1% Triton X-100, 2 M NaCl, added Dnase prior to lysis. I 
have also used methods to remove the DNA from protein, if that is the 
contaminating agent.
I am trying to crystallize the protein with no success so far.
Moreover, my thermofluor assays give very low fluorescence. I use Sypro Orange 
as a fluorophore.

Suprisingly, a point mutation in the active site (His to Arg) gets rid of the 
issue of contamination and gives me good thermofluor curves. I purify the 
mutant also form IBs.

Can someone suggest what this "contamination" may be?

Thank you for your time.

Re: [ccp4bb] protein precipitation reg

[Bonsor, Daniel]

Probably the price, HEPES is nearly 5-8 fold more expensive. PBS is fine, 
unless you have to add divalents. TRIS is your (cheap) friend!

Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, 
Jacob
Sent: Wednesday, March 29, 2017 11:33 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] protein precipitation reg

A bit off topic, but I’ve always wondered how TRIS got so popular what with 
it’s pKa of 8.3—does anyone know?

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger 
Rowlett
Sent: Wednesday, March 29, 2017 11:10 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] protein precipitation reg

What are you dialyzing against? Your storage solution should typically be 
buffered away from the pI and contain at least a small amount of kosmotropic 
salt, e.g. NaCl. Some proteins will require additional stabilizing/solubilizing 
agents such as glycerol or reducing agents. FYI, Tris-Cl, pH 7.5 has very 
little buffer capacity (about 15% of the total concentration in the acid 
direction). We typically use Tris-Cl pH 8.0, which is closer to the Tris pKa 
and has good buffer capacity for both acid and base. For pH 7.5 we would 
typically use HEPES as the storage buffer.

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu
On 3/29/2017 9:38 AM, Akilandeswari Gopalan wrote:
Dear all,
I am a PhD student doing structural studies on a few proteins from 
Mycobacterium tuberculosis. The gene encoding the proteins I work on are cloned 
into pet22b with c terminal His tag. the proteins are expressing well. upon 
purification I am getting good yield of protein but during dialysis, the 
proteins precipitate. Kindly suggest some solutions to avoid aggregation. pI of 
one protein is 9.7 and that of the other is 5.6
I am using 25mM Tris pH 7.5 and 100 mM NaCl buffer with 5mM 
beta-mercaptoethanol and 0.5% triton x 100 for lysis, the same buffer with 
20-30mM imidazole for washing and 300mM imidazole for eluting the proteins.

Thank you
Regards
Akila

--
Akilandeswari G

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

[Bonsor, Daniel]

You could try Goldbio.com. I have not used the His-columns from them, but have 
used the Protein A columns and they work as well as the Protein A from GE.

Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Markus 
Seeliger
Sent: Wednesday, February 15, 2017 11:57 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] off topic: Alternatives to GE HiTrap columns

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] Fwd: [ccp4bb] Remittance advice - Invitation to edit

[Bonsor, Daniel]

The Nigerian Prince wants your money.

Get Outlook for Android


From: CCP4 bulletin board  on behalf of Anindito Sen 

Sent: Sunday, December 11, 2016 12:45:16 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Fwd: [ccp4bb] Remittance advice - Invitation to edit

Guys

are you all receiving the same??? What on earth is this ?

Andy



Begin forwarded message:

From: Hai-fu Fan >
Subject: [ccp4bb] Remittance advice - Invitation to edit
Date: December 12, 2016 at 2:31:56 AM GMT+9
To: CCP4BB@JISCMAIL.AC.UK
Reply-To: Hai-fu Fan >

Please find attached for your review.
Regards.

Hai-fu Fan

Re: [ccp4bb] Off-topic: Expression of human single-pass membrane protein

[Bonsor, Daniel]

How are you targeting the protein to the membrane and to which one?
Which strain of E coli are you using? BL21(De3) may not be the best. Especially 
if the gene is not codon optimized. You could try Rosetta cells. Furthermore, 
you may get better expression with the Walker Strains (C41 and C43) which were 
developed for membrane proteins. 

Dan

Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457
http://www.sundberglab.org/Home.html

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mohamed 
Noor
Sent: Wednesday, July 13, 2016 4:52 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Off-topic: Expression of human single-pass membrane protein

Dear all

I am working on a single-pass MP (about 20 kDa) from human that is located in 
the mitochondrial membrane. I have been expressing it in E. coli but the yield 
seems low. Since the His-tag is at the N-terminus, there is also degradation on 
Western blot that very likely corresponds to truncation products (premature 
release from the mRNA??). Expression was induced with IPTG at 25 C in LB medium.

Between switching the expression to a different host and doing codon 
optimization experiments for expression in E. coli, what is your overall 
experience in this situation?

The TM region is essential for function, so I cannot express just the soluble 
portion for activity assays.

Thanks.
Mohamed

[ccp4bb] Reindexing of lattice

[Bonsor, Daniel]

Hi,

I am trying to reindex a P212121 lattice to change the axis order from 32.62, 
64.89, 103.16 to 64.89, 103.16, 32.62. Looking at the REINDEXING manual I can't 
find how to reindex the lattice. Does anyone know how I should do this and an 
example of the execute script that I should use?

Thanks


Dan

Re: [ccp4bb] Interesting DNA contamination

[Bonsor, Daniel]

Several different approaches may help you to separate DNA from protein 
including;

1) When bound to the nickel column, wash the protein/beads with 1-2M NaCl or 
1-2M KCl for a few hours to overnight. Increase ionic strength should disrupt 
protein-DNA interactions.
2) Use a low concentration of urea (1.5-2M) or guanidine hydrochloride (1M) to 
partial unfold (increase breathing) to disrupt the interaction.
3) Combine salt with low concentrations of denaturants.
4) Try a couple of different restriction enzymes such as DpnI or FatI to see if 
you can break it into smaller fragments. If you can, you maybe able to clone 
into a cloning vector with compatable ends/blunt ligation to sequence and 
identify the region of host DNA that is causing the problem.

Dan




Daniel A. Bonsor PhD
Institute of Human Virology,
University of Maryland at Baltimore
725 W. Lombard Street N571
Baltimore
MD 21201

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Pramod Kumar 
[pramod...@gmail.com]
Sent: Thursday, June 25, 2015 5:23 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Interesting DNA contamination

Dear all

Sorry for off topic and lengthy post, but I came across a very unique DNA 
contamination during one membrane protein purification (a microbial external 
environment sensor/response protein)

Already done

* DNAse used as stranded protocol during cell break.
* Membrane extraction to Ni-Affinity and Ovenight TEV dig done with 0.5M NaCl
* Fluorescence size exclusion done with 0.1M NaCl
* Well stable peak and pure protein profile observed
* But final purified protein inherently contains specific 0.5 Kb DNA stretch 
visible through out purification (observed by running Agarose gel of protien 
sample @ each step)


Approach failed

* Buffer switched from HEPES to Na-PO4
* O.N. dialysis in presence of DNAse
* Using Heparin Column purification between Ni-Aff and SE (failed and protein 
starts deterioration)
* Since protein binds ATP (ATP and MgCl2 added after O.N. Dialysis/digestion)

Now... I need help for

* How to get rid of DNA without loosing active protein?
* What are best lipids to dope as -ve DNA replacement?
* Since protein is pure and ample, how likely I can get crystal hits with such 
big DNA attached?
* What longest possible DNA can be crystallize/ed with protein?
* How exclusive are some mem-spanign-proteins to provide anchorage of 
prokaryotic genomic stretch?



​Thanks in advance​ :)

Pramod Kumar

Re: [ccp4bb] Off topic - Mercury Lamp for Akta

[Bonsor, Daniel]

As per usual the CCP4BB community has offered several different on the board 
and directly to me. Companies suggested include;

Sonntek (www.sonntek.comhttp://www.sonntek.com) -- $695.
Kinesis 
(www.hplclampsandspares.kinesis.co.ukhttp://www.hplclampsandspares.kinesis.co.uk
 or http://kinesis-usa.com/) -- Stocks vary from country. None in the USA at 
the moment. Typically around $800.
Reflex (http://www.reflexusa.com/) -- $895.
Jelight (http://www.jelight.com/low-pressure-uv-lamps.html) -- This requires 
some filing and an external power supply bringing the cost to $470.

All cheaper than GE. I have just ordered from Sonntek.

GE does offer the lamp without the housing (28-9354-93) but is only $15 cheaper 
than the full bundle ($1330).

Artem did suggest rigging up a system with a UV LED. Unfortunately I am not 
electrically minded but I would be interested to know if anyone has done this 
and if it works. This would be so much cheaper and less hazardous than mercury 
lamps. We only use 280nm. I assume you would just place the LED so it sits next 
the hole in the housing unit and connect it up to a battery? 
Thoughts/suggestions on the type of 280nm LED I should use?

Thanks once again everyone.

Dan


Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457
http://www.sundberglab.org/Home.html

[ccp4bb] Off topic - Mercury Lamp for Akta

[Bonsor, Daniel]

The mercury lamp (28-4042-25) offered by GE Healthcare for Akta systems come 
with the housing and is priced at $1345. We have plenty of sparing housing 
units and are interested in just the lamp. Does anyone here have any cheaper 
alternatives/suggestions than buying the lamp and housing?

Thanks in advance.

Dan

Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457
http://www.sundberglab.org/Home.html