[ccp4bb] Spam : Re: [ccp4bb] Off-topic: Attach His-tagged Protein to Coverslip

[Debasish Kumar Ghosh]

Assuming the protein is in solution, drop cast 2ul of solution onto a glass 
surface and spread evenly with a sharp needle. Air dry in dust free chamber and 
blow the surface with gentle stream of nitrogen gas. Proteins stick to surface 
quite well to be analyzed for various microscopy (AFM, SEM etc.). If the 
purpose is not to do any surface probe microscopy, coating the glass surface 
with Sigmacoat helps in better binding. Poly-lysine coated glass cover slips 
can be alternative but mostly not preferred for small proteins or peptides.

Hope that helps.

Best!!


Debasish

CSIR- Senior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group
Centre for DNA Fingerprinting and Diagnostics
Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab)
Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html



- Original Message -
From: Jacob Keller 
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wed, 05 Apr 2017 08:49:40 +0530 (IST)
Subject: [ccp4bb] Off-topic: Attach His-tagged Protein to Coverslip

Does anyone have a simple way to attach purified his-tagged protein solidly to 
a coverslip?

Thanks,

Jacob Keller

***
Jacob Pearson Keller, PhD
Research Scientist
HHMI Janelia Research Campus / Looger lab
Phone: (571)209-4000 x3159
Email: kell...@janelia.hhmi.org
***

[ccp4bb] Off-topic: Attach His-tagged Protein to Coverslip

[Keller, Jacob]

Does anyone have a simple way to attach purified his-tagged protein solidly to 
a coverslip?

Thanks,

Jacob Keller

***
Jacob Pearson Keller, PhD
Research Scientist
HHMI Janelia Research Campus / Looger lab
Phone: (571)209-4000 x3159
Email: kell...@janelia.hhmi.org
***

[ccp4bb] Postdoctoral position in Sydney, Australia

[Mary Christie]

Dear CCP4bb members,

We are seeking a recent PhD graduate for a postdoctoral position in structural 
biology. Applicants with experience in protein crystallography as well as 
mammalian cell culture, computational biology or RNA biology are particularly 
encouraged to apply. Please see link below for details.

https://www.seek.com.au/job/33144629?type=standout=no_tier=1=3000=cb3b40391f3562f29cc32f0db5fc087f-7329868=beta

All the best,

Mary

[ccp4bb] Accidental crystallization of E. coli protein

[Mohamed Noor]

During the crystallization of a totally unrelated protein from a different 
bacterium in E. coli, we managed to somehow crystallize an E. coli protein. It 
turned out to be only the catalytic domain of an enzyme. Two previous reports 
both used recombinant expression of this enzyme followed by limited proteolysis 
in order to crystallize this domain.

Has something like this been reported before? I know the stories of AcrB etc., 
but I am looking specifically for a 'naturally proteolyzed' crystallization.

Looking at our structure and the previously published one, there is not much 
difference, with an rmsd of about 0.3 A but our data resolution is a bit 
higher. This is perhaps unsurprising as the unit cells are very similar (in 
fact, I just did a quick RBR). Should we bother depositing and publishing this 
observation?

Thanks.
Mohamed

Re: [ccp4bb] E.coli strain, knock-out for SDH/FR

[Edward A. Berry]

People who would know (and know whether such a strain would even be viable)
are Gary Cecchini at UC San Francisco/VA and Bob Gennis at Univ of Illinois
at Urbana. In case you are not already talking with them.

It has been reported that an assembly factor (SDH5 or SDHE or SDHAF2)
is required for flavination of both proteins, without which you would have
no activity. So knocking out that one gene may be sufficient. It has
been reported to be a little leaky however, perhaps more so at
higher temperature.  Meaning that SDH5-independent flavination has
been seen in some cases. And fumarate reductase has activity with
non-covalently bound FAD, but only for fumarate reduction not succinate
oxidation, so if "any kind of Complex II activity" only includes the
forward reaction, you would be OK.
There's a group in New Zealand working n SDHE in bacteria- pubmed SDHE
and see what is their latest, and they may have the knockout (of SDHE).
eab.

On 04/04/2017 10:54 AM, Fulvio Saccoccia, Sapienza wrote:

Dear ccp4ers,

I was wondering if someone does know (and hence could let me know in
turn) an E.coli strain which lacks both  of the sdh (succinate
dehydrogenase) and frd (fumarate reductase) operon, so that bacteria do
no longer retain any activity from complex II or similar. I know the
question is off-topic but I am confident about your kindness.

Cheers

Fulvio


--
Fulvio Saccoccia, PhD
Institute of Cell Biology and Neurobiology (IBCN)
Consiglio Nazionale delle Ricerche
Via E. Ramarini, 32 - 00015 Monterotondo Scalo Roma (RM)
tel.+390690091244

Re: [ccp4bb] Helium-Temp Cryo-Cooling

[Jan Kern]

Hi Jakob,

There are several other examples where the effect of temperature on
radiation damage for metallo proteins was demonstrated, eg:
10.1074/jbc.M112.438796
10.1073/pnas.0505207102
10.1074/jbc.M509724200

The tricky part with liq He cryo streams is to get a good geometry that
provides stable temperature and prevents severe icing. Usually, for
spectroscopic experiments we used to mount our sample from the bottom with
the pin pointing vertically up (instead of mounting it with the pin
horizontally) in order to prevent disturbing of the He flow and reduce
icing. But of course this geometry is difficult to achieve in most standard
MX beam lines. I thought there was some initiative maybe 8-10 years ago at
APS and at ESRF to get a He cryo jet running at one of their MX beam lines
but I do not remember the details.

Greetings,

Jan

On Tue, Apr 4, 2017 at 2:14 AM, Colin Nave  wrote:

> Jacob
> Good question.
>
> One case  I know is for a metalloprotein where the photoreduction of the
> metal centre is decreased significantly at 40K compared with 110K.
> Acta Cryst. (2007). D63<
> http://journals.iucr.org/d/contents/backissues.html>, 951-960
> http://scripts.iucr.org/cgi-bin/paper?S0907444907035160
>
> The paper offers a plausible explanation of why this effect occurs. The
> evidence is from x-ray spectroscopy rather than x-ray diffraction. Will the
> "subtle yet important changes" be seen with x-ray diffraction? Your plea
> for low temperature capability in conjunction with spectroscopic analysis
> is therefore relevant.
>
> I too would be interested in any updates on this.
>
> Regards
> Colin
>
>
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
> Keller, Jacob
> Sent: 04 April 2017 00:06
> To: ccp4bb
> Subject: [ccp4bb] Helium-Temp Cryo-Cooling
>
> Dear Crystallographers,
>
> It is my recollection from a while ago that cryocooling to Helium
> temperatures has modest if any effects on protein structure or diffraction
> data quality, and I've found a couple of papers just now to that effect.
> Does anyone know differently? Do the b-factors even change?
>
> Further, is anyone aware of a beamline which has really low temperature
> capability, ideally in conjunction with spectroscopic analysis?
>
> All the best,
>
> Jacob Keller
>
>
>
> ***
> Jacob Pearson Keller, PhD
> Research Scientist
> HHMI Janelia Research Campus / Looger lab
> Phone: (571)209-4000 x3159
> Email: kell...@janelia.hhmi.org
> ***
>
>
> --
> This e-mail and any attachments may contain confidential, copyright and or
> privileged material, and are for the use of the intended addressee only. If
> you are not the intended addressee or an authorised recipient of the
> addressee please notify us of receipt by returning the e-mail and do not
> use, copy, retain, distribute or disclose the information in or attached to
> the e-mail.
> Any opinions expressed within this e-mail are those of the individual and
> not necessarily of Diamond Light Source Ltd.
> Diamond Light Source Ltd. cannot guarantee that this e-mail or any
> attachments are free from viruses and we cannot accept liability for any
> damage which you may sustain as a result of software viruses which may be
> transmitted in or with the message.
> Diamond Light Source Limited (company no. 4375679). Registered in England
> and Wales with its registered office at Diamond House, Harwell Science and
> Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
>

Re: [ccp4bb] Using a codon-optimised gene to improve proteinsolubility

[mesters]

Dear Sutapa,

As already pointed out, codon optimization for increasing the solubility 
is not really a viable option but it can be for production /per se/ in 
insect cells.


Among others, slowing down growth, changing the construct or 
coexpression (of for example foldase/chaperone) or witching to a 
homologe can be viable options though.


PLease note that instead of slowing down the growth of /E. coli /by, for 
example, lowering the temperature, add the IPTG at a very late stage of 
the growth, about 1.5 hours before reaching the stationary phase (late 
log phase) when cell density is really high. Big advantage is, lots of 
cells that produce the protein of interest slowly (because of 
methylation processes of ribosomes and elongation factors etc. if I 
recall correctly) and harvest after about 1 hour.


- J. -





Dear all,

We’re trying to express and purify a 1000 residue long protein and 
have run into the problem that it is completely insoluble when 
expressed in E.coli and is not expressed at all in insect cells. The 
usual tricks for improving solubility in E.coli, such as addition of 
GST/MBP tags, optimising expression media and induction conditions 
and use of different cell strains, have not led to any improvement.


We are now looking into ordering a codon-optimised synthetic gene for 
this protein and are trying to decide whether it would be worthwhile 
to codon-optimise for expression in E.coli (given that the protein 
was expressed but not soluble) or if we should attempt baculovirus 
expression again with a gene that has been codon-optimised for insect 
cells.


My question is:
has anyone observed an improvement in the solubility of their target 
protein using a codon optimised gene?


I know of several instances where the use of a codon-optimised gene 
has led to expression where the native gene sequence did not but am 
unable to find any references for improvement in solubility. Since 
codon optimisation significantly alters the translation rate of a 
gene, I believe this should affect solubility as well; but I’d like 
to know what the community thinks/has observed before I order an 
exorbitantly priced gene!


Thank you in advance,
Sutapa

--
Sutapa Chakrabarti, Ph.D.
Institute of Chemistry and Biochemistry
Freie Universität Berlin
Takustr. 6
14195 Berlin
Germany
Phone: +49-(0)30-83875094








--
Dr.math. et dis. nat. Jeroen R. Mesters
Deputy, Senior Researcher & Lecturer
Program Coordinator /Infection Biology/ 



Institute of Biochemistry, University of Lübeck
Ratzeburger Allee 160, 23538 Lübeck, Germany
phone: +49-451-31013105 (secretariate -31013101)
fax: +49-451-31013104

http://jobs.zeit.de/image-upload/logo_10564.jpg
http://www.biochem.uni-luebeck.de 
http://www.eine-stadt-sieht-gelb.de 
http://www.uni-luebeck.de/studium/studiengaenge/infection-biology
http://www.iobcr.org 

Visiting Professorship in Biophysics, University of South Bohemia (CZ)
--
If you can look into the seeds of time and tell which grain will grow 
and which will not, speak then to me who neither beg nor fear 
(Shakespeare's Macbeth, Act I, Scene 3)

--
Only two things are infinite, the universe and human stupidity, and I'm 
not sure about the former (Albert Einstein)

--
It is invariably the case that high resolution X-ray structures show 
significantly better agreement with solution observables such as 
coupling constants, 13C chemical shifts, and proton chemical shifts, 
than the corresponding NMR structures, including the very best ones. 
Hence, in most cases, a high-resolution crystal structure (< 2.0 Å)will 
provide a better description of the structure in solution than the 
corresponding NMR structure  (Kuszewski, Gronenborn & Clore, 1996, 
Protein Science 5:1067-80)

--
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[ccp4bb] anomalous difference fourier

[Eleanor Dodson]

I suppose I should have expected this, but I still was led astray..

Ir atom - strong anomalous signal.. Good peak in anom diff fourier phased
from protein.

So I add it to the model with occ = 1.00 and refine..

The B factor is very high and it seems clear that the occupancy should be
<<1.0


And one side effect is that now the anomalous difference fourier has a
large HOLE where I have placed the Ir.

Hmmm.

Eleanor

Re: [ccp4bb] Using a codon-optimised gene to improve protein solubility

[Mark J van Raaij]

Hello Sutapa,

With codon optimisation you would get more and faster expression and likely 
even more insolubility in E. coli (less time for folding). To get the protein 
more soluble perhaps slower production might help. I guess you could try codon 
de-optimisation :-). Cheaper possibilities are growth at lower temperature, 
using a slower-growing E coli strain, using less IPTG or even no IPTG and just 
relying on the leaky expression, but perhaps you have tried all these already.
Or another expression plasmid with tighter control or a different system like 
yeast.
Codon optimisation for insect cells might be worth it if your analysis shows 
that the current sequence is really badly optimised.
Or perhaps your protein needs a specific natural chaperone...it  might be worth 
studying the natural production of your protein more before designing an 
expression strategy.
Without knowing more details about your protein it's hard to give more help - 
you could try to contact other researchers studying similar proteins.

Greetings,

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser .cnb.csic.es/~mjvanraaij 




> On 03 Apr 2017, at 07:49, Sutapa Chakrabarti  
> wrote:
> 
> Dear All,
> 
> We’re trying to express and purify a 1000 residue long protein and have run 
> into the problem that it is completely insoluble when expressed in E.coli and 
> is not expressed at all in insect cells. The usual tricks for improving 
> solubility in E.coli, such as addition of GST/MBP tags, optimising expression 
> media and induction conditions and use of different cell strains, have not 
> led to any improvement. 
> 
> We are now looking into ordering a codon-optimised synthetic gene for this 
> protein and are trying to decide whether it would be worthwhile to 
> codon-optimise for expression in E.coli (given that the protein was expressed 
> but not soluble) or if we should attempt baculovirus expression again with a 
> gene that has been codon-optimised for insect cells. 
> 
> My question is:
> has anyone observed an improvement in the solubility of their target protein 
> using a codon optimised gene? 
> 
> I know of several instances where the use of a codon-optimised gene has led 
> to expression where the native gene sequence did not but am unable to find 
> any references for improvement in solubility. Since codon optimisation 
> significantly alters the translation rate of a gene, I believe this should 
> affect solubility as well; but I’d like to know what the community thinks/has 
> observed before I order an exorbitantly priced gene! 
> 
> Thank you in advance,
> Sutapa
> 
> --
> Sutapa Chakrabarti, Ph.D.
> Institute of Chemistry and Biochemistry
> Freie Universität Berlin
> Takustr. 6 
> 14195 Berlin
> Germany
> Phone: +49-(0)30-83875094
> 
> 
> 
> 
> 
> 

Re: [ccp4bb] Ambiguous crystal diffraction pattern

[Keller, Jacob]

It’s multiple crystals of salt.

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Prashant 
Deshmukh
Sent: Tuesday, April 04, 2017 8:40 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Ambiguous crystal diffraction pattern

Dear Crystallographers,
Please help us in figuring out whether the attached crystal diffraction 
screenshot belongs to a protein crystal or that of a salt crystal. The 
cryoprotection condition was not optimized for the crystal. The crystallization 
conditions for the crystal is :
Ammonium sulphate , lithium sulphate, Sodium Citrate buffer pH = 5.6.

Thanks.
Prashant Deshmukh
Dept. of Biophysics,
NIMHANS,
Bangalore 560 029,
E-mail:prashantbiophys...@gmail.com
Mob.No.: +919620986525

[ccp4bb] rapper

[Kajander, Tommi A]

Dear all,

Has anyone tested RAPPER recently - doenst seem to work with me from the ccp4i 
7.0.015 with a map (from buster-TNT). (either ca-trace option - with partial 
region defined or the "rebuild badly fitting residues"

- complains and generates nothing. Is there something obvious I might be 
missing? the PDB is OK by other programs - i can’t quite see what would be 
problem with it, error looks like this:



ERROR [General Error]:
  in file 
/Users/buildbot/Buildslaves/ccp4-slave/release-7_0-mac10_6/build/devtools/checkout/rapper_ccp4-1.0a/LOOP/analyze.cpp
 at line 529
  No models could be generated under the provided restraints.  This means that 
either (1) the restraints are too specific or there is something wrong with the 
input PDB file.
***


#CCP4I TERMINATION STATUS 0 " 
--- 
ERROR [General Error]:   in file 
/Users/buildbot/Buildslaves/ccp4-slave/release-7_0-mac10_6/build/devtools/checkout/rapper_ccp4-1.0a/LOOP/analyze.cpp
 at line 529   No models could be generated under the provided restraints.  
This means that either (1) the restraints are too specific or there is 
something wrong with the input PDB file."
#CCP4I TERMINATION TIME 03 Apr 2017  14:19:03
#CCP4I MESSAGE Task failed

Thanks,
Tommi

Re: [ccp4bb] Helium-Temp Cryo-Cooling

[Colin Nave]

Jacob
Good question.

One case  I know is for a metalloprotein where the photoreduction of the metal 
centre is decreased significantly at 40K compared with 110K.
Acta Cryst. (2007). 
D63, 951-960
http://scripts.iucr.org/cgi-bin/paper?S0907444907035160

The paper offers a plausible explanation of why this effect occurs. The 
evidence is from x-ray spectroscopy rather than x-ray diffraction. Will the 
"subtle yet important changes" be seen with x-ray diffraction? Your plea for 
low temperature capability in conjunction with spectroscopic analysis is 
therefore relevant.

I too would be interested in any updates on this.

Regards
Colin


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, 
Jacob
Sent: 04 April 2017 00:06
To: ccp4bb
Subject: [ccp4bb] Helium-Temp Cryo-Cooling

Dear Crystallographers,

It is my recollection from a while ago that cryocooling to Helium temperatures 
has modest if any effects on protein structure or diffraction data quality, and 
I've found a couple of papers just now to that effect. Does anyone know 
differently? Do the b-factors even change?

Further, is anyone aware of a beamline which has really low temperature 
capability, ideally in conjunction with spectroscopic analysis?

All the best,

Jacob Keller



***
Jacob Pearson Keller, PhD
Research Scientist
HHMI Janelia Research Campus / Looger lab
Phone: (571)209-4000 x3159
Email: kell...@janelia.hhmi.org
***


-- 
This e-mail and any attachments may contain confidential, copyright and or 
privileged material, and are for the use of the intended addressee only. If you 
are not the intended addressee or an authorised recipient of the addressee 
please notify us of receipt by returning the e-mail and do not use, copy, 
retain, distribute or disclose the information in or attached to the e-mail.
Any opinions expressed within this e-mail are those of the individual and not 
necessarily of Diamond Light Source Ltd. 
Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments 
are free from viruses and we cannot accept liability for any damage which you 
may sustain as a result of software viruses which may be transmitted in or with 
the message.
Diamond Light Source Limited (company no. 4375679). Registered in England and 
Wales with its registered office at Diamond House, Harwell Science and 
Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom