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

[Kay Diederichs]

Hi vito,

I don't think you need double labelling. Two (if there is a Met at the N-term 
it might be less ordered) or three SeMet should suffice to phase 360 residues. 
It was true in the old days that a Se could phase less than a hundred residues, 
but if your measurement is good (resolution better than 3A, 360° rotation 
range, fine slicing, PAD, little radiation damage, stable beamline) then I see 
no reason why SeMet-SAD (or even better MAD) shouldn't work.

One should mine the literature to find how many residues can be phased with one 
SeMet nowadays - sorry, lost track.

good luck,
Kay


On Wed, 21 Jun 2017 17:46:56 +0200, Vito Calderone  
wrote:

>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] Se-Met and Se-Cys double labelling

[James Holton]

I have only heard of a few cases of successful Se incorporation into 
Cys, and none of them sounded like fun.  Sounds like you have gotten a 
few suggestions already.  There is not much literature on this.


As for alternative solutions to your original question, I can  tell you 
the success or failure of MAD or SAD phasing depends primarily on the 
signal-to-noise ratio of your data and how it compares to your anomalous 
signal.  A good rule of thumb is that you need I/sigma to be better than 
the expected F/delta-F from anomalous scattering. This is the reciprocal 
of your "Bijvoet ratio".  You don't say if one of your Met residues is 
the first one, but if it is you can seldom count on it, so let's say you 
only have two.  You can get a rough guess of what Bijvoet ratio you can 
expect, and what I/sigma you will need using a little web calculator I made:

http://bl831.als.lbl.gov/xtalsize.html

360 residues is roughly 45 kDa, and pessimistically assuming the minimum 
4 electrons from SeMet, with 2 sites per 44 kDa I get a Bijvoet ratio of 
2%.  This isn't all that bad.  You need I/sigma > 50 to solve this 
structure.  Do your crystals typically give you a peak I/sigma of around 
30? That is, in the lowest-angle bin? If so, you only need a 
multiplicity of (50/30)^2 ~ 3x higher than you usually do to get good 
enough signal.  This might not be all that hard to do.  On the other 
hand, if your data collections typically give you I/sigma ~10, then you 
need 25x more multiplicity than the dataset that gave you I/sigma=10.  
That can also be done, but usually involves either very large crystals 
or multiple crystals.  If you can grow isomorphous crystals, then you 
are golden.  Just keep shooting and merging until it solves.  You can 
even do this with native SAD if you have enough time.


If you have a problem with isomorphism, then your are not alone. Your 
best shot with current technologies is to be extra careful with your 
harvesting and cryo-cooling protocols:

Warkentin et al. (2006) http://doi.org/10.1107/S0021889806037484
Farley et al. (2014) http://dx.doi.org/10.1107%2FS1399004714012310

Good luck!

-James Holton
MAD Scientist

On 6/21/2017 8:46 AM, Vito Calderone wrote:

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] Se-Met and Se-Cys double labelling

[Daniel Rigden]

Dear Vito

AMPLE is another option to easily and automatically find conserved cores 
among a set of distant homologues. You point it to a directory 
containing your homologues and use the


-homologs True

flag. You can use the command line or CCP4i. It will then use GESAMT to 
find the multiple structural superposition and then generate a series of 
ensemble search models containing 100, 95, 90...% of the superimposable 
set of residues shared among the structures. The processing uses 
information about structural variance to trim progressively down to 
smaller but more structurally homogeneous cores. Very often this further 
processing, more dramatic than would be attempted manually, is necessary 
for success.


If you only have a single homologous structure we have had some success 
using it as the basis to generate ensembles using distance geometry 
(CONCOORD) and treating the result in the same way as a set of ab initio 
models.


These approaches work best where resolution allows for automated Shelxe 
main chain tracing and phase modification since the resulting statistics 
very clearly indicate success or failure.


These methods will be submitted for publication in Study Weekend papers. 
In the meantime you can hear some explanation here

https://www.youtube.com/watch?v=3B1-Qr00zXk=2s
from about 19:37

There's also a multiple homolog tutorial here
https://amplemr.wordpress.com/programs/ample/ample-tutorials/ample-tutorial-3/

Do feel free to contact us if you need a hand trying this approach.

Best wishes

Daniel Rigden


On 22/06/17 16:37, Randy Read wrote:
Yes, I agree that MR is worth a shot, though depending on the 
resolution your life may be vastly easier with experimental phases! 
 We've had several cases where the following kind of procedure works: 
find related structures with a very sensitive homology search (we like 
HHPRED, though other options probably work), use the corresponding 
sequence alignment to prune the models (with sculptor or chainsaw or 
other tools) and make a trimmed ensemble with ensembler.  The last 
step can be very important, in trimming off any bits of the collection 
of models that do not constitute a conserved core.  Then provide the 
ensemble to Phaser as a molecular replacement model.  The trimmed 
ensemble is usually the best model, in our experience, but one of the 
individual models may be better in some cases, so that's also worth 
testing.


Also, the MR-Rosetta pipeline has had a substantial number of 
successes when the highest sequence identity was in the range of 15-25%.


Best wishes,

Randy Read

On 21 Jun 2017, at 17:08, Mark J van Raaij > wrote:


If your data is good enough, your SeMets alone might well be enough.
Soaking native crystals in Hg compounds may also work, avoiding SeMet 
altogether. We have had a lot of success with methylmercury chloride 
binding to free Cys. You may have to experiment with different 
soaking times and protocols.
Finally, don't give up on MR too early, what matters is the 
structural similarity, not directly the sequence identity. We've had 
success once with 19% identity.
Native protein may be much easier to produce than the SeMet and 
SeMet/SeCys versions (and may differ a lot between proteins).


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 21 Jun 2017, at 17:46, Vito Calderone > wrote:


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匢 suppose MR would be very 
unlikely to

work卻o 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




--
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research  Tel: + 44 1223 336500
Wellcome Trust/MRC Building   Fax: + 44 1223 336827
Hills Road  E-mail: rj...@cam.ac.uk 
Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk 





--
Dr Daniel John Rigden Tel:(+44) 151 795 4467
Institute of Integrative Biology  FAX:(+44) 151 795 4406
Room 101, Biosciences Building
University of Liverpool   http://pcwww.liverpool.ac.uk/~drigden/
Crown St.,
Liverpool L69 7ZB, U.K.

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

[Randy Read]

Yes, I agree that MR is worth a shot, though depending on the resolution your 
life may be vastly easier with experimental phases!  We've had several cases 
where the following kind of procedure works: find related structures with a 
very sensitive homology search (we like HHPRED, though other options probably 
work), use the corresponding sequence alignment to prune the models (with 
sculptor or chainsaw or other tools) and make a trimmed ensemble with 
ensembler.  The last step can be very important, in trimming off any bits of 
the collection of models that do not constitute a conserved core.  Then provide 
the ensemble to Phaser as a molecular replacement model.  The trimmed ensemble 
is usually the best model, in our experience, but one of the individual models 
may be better in some cases, so that's also worth testing.

Also, the MR-Rosetta pipeline has had a substantial number of successes when 
the highest sequence identity was in the range of 15-25%.

Best wishes,

Randy Read

> On 21 Jun 2017, at 17:08, Mark J van Raaij  wrote:
> 
> If your data is good enough, your SeMets alone might well be enough.
> Soaking native crystals in Hg compounds may also work, avoiding SeMet 
> altogether. We have had a lot of success with methylmercury chloride binding 
> to free Cys. You may have to experiment with different soaking times and 
> protocols.
> Finally, don't give up on MR too early, what matters is the structural 
> similarity, not directly the sequence identity. We've had success once with 
> 19% identity.
> Native protein may be much easier to produce than the SeMet and SeMet/SeCys 
> versions (and may differ a lot between proteins).
> 
> 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 21 Jun 2017, at 17:46, Vito Calderone > > wrote:
>> 
>> 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匢 suppose MR would be very unlikely to
>> work卻o 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
> 

--
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research  Tel: + 44 1223 336500
Wellcome Trust/MRC Building   Fax: + 44 1223 336827
Hills RoadE-mail: rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.   www-structmed.cimr.cam.ac.uk

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

[Paula Salgado]

We have successfully used non-auxotrophic strains for incorporation of SeMet 
and SeCys, with an incorporation level of >80% and no effects on yield or 
solubility. Details can be found here:


http://scripts.iucr.org/cgi-bin/paper?S0907444910042022


It's a straight-forward protocol and structure solution was trivial, even 
without 100% incorporation. With your number of Met and Cys, it should give you 
enough signal, as long as expression is not affected by minimal media. You can 
do a small test just to ensure protein expression is OK before doing large 
scale purification.


Using derivatives is another option but it might be time consuming and 
extensive to search for appropriate heavy atoms, so this might be good 
alternative.


Good luck!


Paula


===

Dr Paula S. Salgado
Lecturer in Macromolecular Crystallography
Institute for Cell and Molecular Biosciences
Faculty of Medical Sciences
3rd Floor Cookson Building
Newcastle University
Newcastle upon Tyne, NE2 4HH, UK

Tel: +44 (0)191 208 7432
Fax: +44 (0)191 208 7424
Email: paula.salg...@ncl.ac.uk

From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Bonsor, Daniel 
<dbon...@som.umaryland.edu>
Sent: 21 June 2017 22:03:06
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Se-Met and Se-Cys double labelling

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] Se-Met and Se-Cys double labelling

[Frank Von Delft]

Absolutely. But don't bother with SIRAS:  straight SAD will do the job 9/10 
times these days, thanks to magnificent beamlines and algorithms.

Sent from tiny silly touch screen

From: "Whitley, Matthew J" <mjw...@pitt.edu>
Sent: 22 Jun 2017 1:09 am
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Se-Met and Se-Cys double labelling


I second (or third) the suggestion that others have given to try soaking 
mercury compounds into your crystal.  Mercury absolutely loves free cysteines, 
and if you have 5, you have a great chance of getting binding.  If isomorphism 
is maintained after soaking, the isomorphous differences will be huge, and if 
you can collect data at a synchrotron, then the anomalous signal also opens up 
the possibility of SIRAS phasing.  Everybody seems to have their own favorite 
mercury compound to recommend, and mine is potassium tetraiodomercurate 
(K2HgI4).  Just recently I was able to get mercury binding to three free 
cysteines in my protein with this compound with absolutely no effort 
whatsoever.  I simply dissolved a few grains of compound in an 
acetonitrile/water solution, added a drop to my crystal drop, waited 30 
minutes, and then collected the data.  SHELX was able to locate the sites and 
solve the phases by SIRAS in mere moments.  It couldn't have been easier, and I 
thank the experimental phasing gods for their generosity.  In any event, your 
protein has a large number of free cysteines, so I think you probably have an 
above average chance for some flavor of phasing based on mercury to be 
successful.


Good luck!


Matthew


---
Matthew J. Whitley, Ph.D.
Research Associate
Department of Structural Biology
University of Pittsburgh School of Medicine



From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of CCP4BB automatic 
digest system <lists...@jiscmail.ac.uk>
Sent: Wednesday, June 21, 2017 7:01 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: CCP4BB Digest - 20 Jun 2017 to 21 Jun 2017 (#2017-172)

--

Date:Wed, 21 Jun 2017 17:46:56 +0200
From:Vito Calderone <calder...@cerm.unifi.it>
Subject: 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] Se-Met and Se-Cys double labelling

[Whitley, Matthew J]

I second (or third) the suggestion that others have given to try soaking 
mercury compounds into your crystal.  Mercury absolutely loves free cysteines, 
and if you have 5, you have a great chance of getting binding.  If isomorphism 
is maintained after soaking, the isomorphous differences will be huge, and if 
you can collect data at a synchrotron, then the anomalous signal also opens up 
the possibility of SIRAS phasing.  Everybody seems to have their own favorite 
mercury compound to recommend, and mine is potassium tetraiodomercurate 
(K2HgI4).  Just recently I was able to get mercury binding to three free 
cysteines in my protein with this compound with absolutely no effort 
whatsoever.  I simply dissolved a few grains of compound in an 
acetonitrile/water solution, added a drop to my crystal drop, waited 30 
minutes, and then collected the data.  SHELX was able to locate the sites and 
solve the phases by SIRAS in mere moments.  It couldn't have been easier, and I 
thank the experimental phasing gods for their generosity.  In any event, your 
protein has a large number of free cysteines, so I think you probably have an 
above average chance for some flavor of phasing based on mercury to be 
successful.


Good luck!


Matthew


---
Matthew J. Whitley, Ph.D.
Research Associate
Department of Structural Biology
University of Pittsburgh School of Medicine



From: CCP4 bulletin board  on behalf of CCP4BB automatic 
digest system 
Sent: Wednesday, June 21, 2017 7:01 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: CCP4BB Digest - 20 Jun 2017 to 21 Jun 2017 (#2017-172)

--

Date:Wed, 21 Jun 2017 17:46:56 +0200
From:Vito Calderone 
Subject: 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] 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] Se-Met and Se-Cys double labelling

[Guenter Fritz]

Dear Vito,

for SeMet have a look here:
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_of_SeMet_labeled_proteins
 Worked like a charm for E.coli and also for other expression hosts 
with minor modifications 
(https://www.nature.com/nature/journal/v516/n7529/full/nature14003.html#methods). 


HTH
Guenter

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] Se-Met and Se-Cys double labelling

[Keller, Jacob]

Halide soaks anyone? Cs or NaI?

Jacob

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mark J 
van Raaij
Sent: Wednesday, June 21, 2017 12:08 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Se-Met and Se-Cys double labelling

If your data is good enough, your SeMets alone might well be enough.
Soaking native crystals in Hg compounds may also work, avoiding SeMet 
altogether. We have had a lot of success with methylmercury chloride binding to 
free Cys. You may have to experiment with different soaking times and protocols.
Finally, don't give up on MR too early, what matters is the structural 
similarity, not directly the sequence identity. We've had success once with 19% 
identity.
Native protein may be much easier to produce than the SeMet and SeMet/SeCys 
versions (and may differ a lot between proteins).

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 21 Jun 2017, at 17:46, Vito Calderone 
<calder...@cerm.unifi.it<mailto:calder...@cerm.unifi.it>> wrote:

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匢 suppose MR would be very unlikely to
work卻o 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] Se-Met and Se-Cys double labelling

[Mark J van Raaij]

If your data is good enough, your SeMets alone might well be enough.
Soaking native crystals in Hg compounds may also work, avoiding SeMet 
altogether. We have had a lot of success with methylmercury chloride binding to 
free Cys. You may have to experiment with different soaking times and protocols.
Finally, don't give up on MR too early, what matters is the structural 
similarity, not directly the sequence identity. We've had success once with 19% 
identity.
Native protein may be much easier to produce than the SeMet and SeMet/SeCys 
versions (and may differ a lot between proteins).

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 21 Jun 2017, at 17:46, Vito Calderone  wrote:
> 
> 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匢 suppose MR would be very unlikely to
> work卻o 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