Re: [ccp4bb] AIMLESS unmerged MTZ output

[Phil]

 Yes. With the caveat that ISYM refers to the symmetry operators in the order 
they are stored in the MTZ file
Phil

Sent from my iPhone

> On 22 Jun 2017, at 17:04, wtempel  wrote:
> 
> Hello all,
> 
> Are the following statements regarding AIMLESS unmerged mtz output accurate?
> - the H, K, L, M/ISYM columns are sufficient to recover the "original" H K L 
> indices.
> - a combination of "original" H K L and the BATCH value is unique inside the 
> file.
> 
> Thank you in advance.
> Wolfram Tempel

Re: [ccp4bb] AIMLESS unmerged MTZ output

[Eleanor Dodson]

I believe so!
Eleanor

On 22 June 2017 at 17:04, wtempel  wrote:

> Hello all,
>
> Are the following statements regarding AIMLESS unmerged mtz output
> accurate?
> - the H, K, L, M/ISYM columns are sufficient to recover the "original" H K
> L indices.
> - a combination of "original" H K L and the BATCH value is unique inside
> the file.
>
> Thank you in advance.
> Wolfram Tempel
>

[ccp4bb] postdoctoral position at the University of Kentucky, USA

[David Rodgers]

A postdoctoral position is available immediately in the laboratory of Dr. 
Emilia Galperin in the Department of Molecular and Cellular Biochemistry in the 
College of Medicine at the University of Kentucky to work on externally funded, 
multi-year projects. The overall goal of the lab is to understand the molecular 
mechanisms of spatial and temporal regulation of signal transduction. 
Preference will be given to applicant with a strong background in protein 
purification and characterization; experience in other biophysical methods such 
as X-ray crystallography is a plus. The successful applicant will be a highly 
motivated individual with good communication skills, will have a proven 
publication record, and the ability to work independently as well as within a 
group. This work is in collaboration with the protein structural group of Dr. 
David Rodgers.

The applicant must currently have a Ph.D. in Biochemistry or a related field. 
The appointment will be for 1 year with renewal up to 5 years. Interested 
candidates should assemble a i) cover letter, ii) CV, iii) list of references, 
and iv) first-author manuscript that have been published or accepted for 
publication. The cover letter should describe the candidate's research 
experience, scientific interests, and preferred start date.

E-mail the application as a single PDF file to emilia.galpe...@uky.edu.

Re: [ccp4bb] Questions about antibody in crystallization

[Jarrod Mousa]

Yes, the hinge region between Fab and Fc is highly flexible. If you look at
IgG by EM you will see many different conformations. Best to use Fab or
scFv.

Jarrod Mousa

On Thu, Jun 22, 2017 at 1:39 PM, Cheng Zhang  wrote:

> Hi all,
>
> I have a naive question about antibodies. Many people used Fab fragments
> in crystallization. I am wondering if it is possible to use the whole IgG
> molecule with Fc fragment as well. Or it would be too flexible and bad for
> crystallization?
>
> Thanks,
>
> Cheng
>
>
> --
> -
> Cheng Zhang
>

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] Questions about antibody in crystallization

[Gert Vriend]

The fact that the PDB holds hundreds of FABs and a handful whole ABs 
suggests to me that the latter are hard to get crystals for. So, all the 
more reason for us bioinformaticians that you, experimentalists try it :-)



Gert


On 22-6-2017 20:39, Cheng Zhang wrote:

Hi all,

I have a naive question about antibodies. Many people used Fab 
fragments in crystallization. I am wondering if it is possible to use 
the whole IgG molecule with Fc fragment as well. Or it would be too 
flexible and bad for crystallization?


Thanks,

Cheng


--
-
Cheng Zhang

[ccp4bb] Questions about antibody in crystallization

[Cheng Zhang]

Hi all,

I have a naive question about antibodies. Many people used Fab fragments in
crystallization. I am wondering if it is possible to use the whole IgG
molecule with Fc fragment as well. Or it would be too flexible and bad for
crystallization?

Thanks,

Cheng


-- 
-
Cheng Zhang

[ccp4bb] Noncoding RNA Structural Biology trainee position at NIH

[Jinwei Zhang]

A postdoctoral or technician position is immediately available in the 
Structural Biology of Noncoding RNAs and Ribonucleoproteins Section, Laboratory 
of Molecular Biology (LMB), NIDDK, in NIH’s vibrant main campus in Bethesda, MD 
near Washington DC. The lab addresses a widening gap between the accelerated 
discovery and functional description of the noncoding transcriptome, and a 
paucity of 3D structures and mechanistic understanding of complex noncoding 
RNAs. We seek a new member to join our diverse group to study gene-regulatory 
riboswitches, highly structured viral RNAs, circular and other structured long 
noncoding RNAs, and their protein complexes. See 
https://www-mslmb.niddk.nih.gov/zhang/zhanglab.html

The lab is part of the Earl Stadtman Investigator program for high-risk, 
high-impact research at the NIH intramural program consisting of 1100 labs. The 
well-supported lab has dedicated access to complete suites of state-of-the-art 
equipment in structural biology (Mosquito, Dragonfly, Rock Imager, Akta Pures, 
FSEC, etc. for X-ray crystallography; new Titan Krios for single-particle 
Cryo-EM; SAXS, AFM, etc), efficient biochemistry, biophysics (ITC, DSC, SPR, 
BLI, AUC, DLS, SEC-MALS, CD, fluorescence, thermophoresis, etc), fermentation, 
genomics, and proteomics core facilities with hands-on training or service by 
PhD-level staff scientists. The NIH, NIDDK, and LMB are committed to the 
continued education and career development of trainees through numerous courses 
and workshops offered by OITE and FAES. 

We apply innovative technologies to study RNA and RNP structure/function, 
including RNA cryo-EM, time-resolved XFEL, picosecond SAXS/WAXS, etc. Ongoing 
research include structural and mechanistic elucidations of how the T-box 
riboswitches (in bacteria) and Gcn2 kinase (in eukaryotes) recognize the 
structure and aminoacylation state of tRNA, and couple this readout of nutrient 
availability with initiating cellular starvation response. A second project 
addresses how viral and cellular RNA structures differentially manipulate 
immune response protein activities such as dsRNA-binding PKR. We are 
delineating the structural hallmarks of self vs. non-self RNA, which are 
deterministic for activation or suppression of immune protein activity. The lab 
also works closely with the Center for HIV RNA Studies (CRNA) as a core lab. 
https://sites.google.com/a/umich.edu/the-center-for-hiv-rna-studies/faculty-cores.
 Incoming fellows are also encouraged to bring your own ideas that you could 
develop into research programs that you can take to your independent positions. 

Requirements: Postdoctoral candidates must have received (or be expecting) a 
Ph.D. or M.D. within the past five years in molecular or structural biology, 
biochemistry, or biophysics, and be strongly self-motivated to lead innovative 
and rigorous research projects. Technician or Postbac candidates should have a 
BS or MS degree in similar disciplines and extensive laboratory experiences. 
Strong background in protein expression and purification, enzyme kinetics, RNA, 
or structural biology is desirable. 

To apply: Please email a preferred start date, CV, a brief summary of research 
interests, accomplishments, and career goals, and names and contact information 
for at least three references to: Dr. Jinwei Zhang, Email: 
jinwei.zh...@nih.gov. The NIH is dedicated to building a diverse community and 
DHHS/NIH is an Equal Opportunity Employer.

Re: [ccp4bb] What are acceptable Rwork/Rfree for publication

[Randy Read]

Dear Gerard,

I was puzzled by the statement that the UCLA anisotropy server characterises 
anisotropy in terms of a combination of effects restricted to lie along the 
crystallographic axes.  That server is built on the anisotropy correction 
algorithm in Phaser, and from the beginning Phaser has used an anisotropic 
tensor that is constrained only by the Laue symmetry.  I've been in touch with 
Mike Sawaya to clarify whether they were doing anything different.  Perhaps he 
will comment directly, but he said that their server indeed uses the general 
ellipsoid for the anisotropic truncation.  The confusion arises from a 
simplified description on their results page, which describes the principal 
axes in terms of which reciprocal cell edge is closest.

In seeking to clarify exactly what the server is doing, I attempted to track 
down the example underlying the illustration on the STARANISO web page.  I 
believe that this is PDB entry 5j1i, but it is not possible to repeat the 
calculations with the data deposited in the PDB because what has been deposited 
appears to be the anisotropy-corrected, truncated data used in refinement.

This brings up an important point.  I am confident you would agree with me 
that, in cases such as this, the uncorrected, untruncated data should always be 
deposited as well as any massaged data used for refinement.  Once an anisotropy 
correction has been applied, the systematically weak data may not contribute 
any useful information to the refinement, but those data provided essential 
information to the characterisation of anisotropy.  Without access to those 
weak intensities, it becomes impossible to make use of new information (e.g. 
from the atomic model) or to apply any new, improved algorithms for analysing 
anisotropy.  Similar issues are even more important for the treatment of the 
systematically weak intensities arising from translational non-crystallographic 
symmetry (tNCS).

By the way, we have a rather different reflection-by-reflection approach to 
data truncation in Phaser, implemented last year but not yet published.  After 
characterising the anisotropy and (if present) tNCS, we determine how much 
information (measured in bits) each intensity measurement contains, relative to 
what was expected from the Wilson distribution.  By default, reflections that 
contribute less than 0.01 bits of information are omitted from any subsequent 
calculations, though we do not truncate them from the output reflection list.  
In some data sets with severe anisotropy and/or tNCS, over 40% of the 
reflections are omitted.  Importantly, we could not have characterised the 
anisotropy and tNCS without having those extremely weak measurements included 
in the data set!  

We feel that the reflection-by-reflection approach has the advantage that the 
significant number of reflections with some signal that will be found beyond 
any hard limit can still be included, without a large increase in computational 
cost.

It's also worth reminding the community that if the French & Wilson algorithm 
has been applied without accounting for anisotropy and tNCS, the resulting 
amplitudes will significantly over-estimate the systematically weak 
reflections.  That's another reason to strongly encourage everyone to deposit 
their data in terms of intensities and their standard deviations (as discussed 
here: http://scripts.iucr.org/cgi-bin/paper?S2059798315013236 
).

Best wishes,

Randy

> On 18 Jun 2017, at 13:58, Gerard Bricogne  wrote:
> 
> Dear Khoa,
> 
> You are asking a very pertinent question, that will resonate in
> (too) many people's minds.
> 
> Somehow anisotropy is very much "an inconvenient truth" in MX, as
> many things have long been set up and operated on the basis of having
> resolution be a single number and of all statistics being calculated
> in spherical shells, throwing in everything that has an HKL associated
> with it. We are repeatedly told that resolution *has* to be a single
> number, because the title of a Nature paper only has room for one
> number - if that isn't a case of the tail wagging the dog, I don't
> know what is ;-) . 
> 
> There are more serious contexts in which the requirement for a
> single number can be a more complex issue, such as contractual
> arrangements where the achievement of certain milestones and the 
> remuneration attached to them depend on reaching a certain resolution.
> Here, whoever writes and/or accepts resolution criteria of this kind
> will simply have to undergo further education and learn about that
> inconvenient fact called anisotropy. Probably - especially in the case
> of membrane protein structures - it is the highest resolution limit
> that matters; but you can't have good completeness to that highest
> resolution, by the very definition of anisotropy! Therefore, some
> quality criteria that are compatible in the case of 

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.

[ccp4bb] Course: Bridging solution methods: from NMR to X-ray scattering and biophysics"

[Anastassis Perrakis]

Dear all,

Announcing in the mailing list for crystallography and in the era of the single 
particles EM resolution revolution, a course for solution methods? 
NMR, SAXS, calorimetry, fluorescent methods and modeling? Seriously?

Well, yes! For many, many reasons, you should be at least curious, and make 
sure you read through - and apply!

Registrations are open for this iNEXT (http://www.inext-eu.org 
) 4-day training course 

September 18-22, 2017, University of Patras, Greece

The course, organized with lectures in the mornings followed by hands-on 
training in the afternoon, will cover the main approaches for the 
characterization of the structure and dynamics of biomolecules in solution. The 
course is aimed primarily at PhD students and young Post-Docs, but more senior 
researchers interested in structural biology are also welcome. The registration 
fee, including accommodation, is 150 Euro; personal fellowships are also 
available.

For applications and further information please visit

http://www.bionmr.upatras.gr/index.php?option=com_content=article=251=220
 


On the behalf of the organizers Georgios Spyroulias and Giacomo Parigi

Tassos Perrakis

[ccp4bb] AIMLESS unmerged MTZ output

[wtempel]

Hello all,

Are the following statements regarding AIMLESS unmerged mtz output accurate?
- the H, K, L, M/ISYM columns are sufficient to recover the "original" H K
L indices.
- a combination of "original" H K L and the BATCH value is unique inside
the file.

Thank you in advance.
Wolfram Tempel

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  on behalf of Bonsor, Daniel 

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] off-topic:fluorescence polarization displacement assay

[R. Gasper-Schönenbrücher]

Hi Megha,

my explanation would be that you have a 2:2 complex. Meaning in your 
standard assay, you need 2 peptides as dimer to get a dimer of your protein.


Now that you do the competition assay, you probably start with 
concentrations, where this 2:2 complex is not saturated yet. Meaning 
that your unlabelled peptide forms a dimer with a labelled one and this 
induces the 2:2 to form, meaning higher polarisation. At some point, 
where your 2:2 is saturated, you start actually competing and 
polarisation drops.


I would use another method to investigate the actual stoichiometry. 
Also, thinking about your starting concentrations might be important. 
Are you sure that your labelled-peptide - protein starting 
concentrations are high enough above Kd that you have 99% complex formation?


Best,

Raphael


Am 21.06.17 um 21:20 schrieb megha abbey:

Hello All,

This is an off-topic question. I have some issues regarding 
Fluorescence Polarization competitive displacement assay and would 
need some advice.


I have developed an in vitro fluorescence polarization based assay 
using a N-terminus labelled FITC peptide. The peptide is 21 amino 
acids long and the binding protein is 50 Kda in size. The Kd for 
interaction is 1 uM. I am further running a competitive displacement 
assay using the exactly same unlabelled peptide. Here, with increasing 
concentration of the unlabeled peptide, the polarization signal first 
increases and then shows a sharp decline. Attached is the raw data 
file for the above.


I believe that if the unlabelled peptide had been aggregating, the 
polarization signal would increase but not drop. If the binding would 
have been non-specific, then the unlabelled peptide should not 
displace at all, but here I see an increase followed by a decrease in 
the signal. What does this increase and sharp drop in polarization 
signify and how do I fix this? Please help.


I have checked the polarization for titration of the unlabelled 
peptide mixed with fixed conc. of FITC-peptide (no protein added). 
Here, the polarization signals are the same for the entire range of 
unlabeled peptide. I have also tried incubating the unlabelled peptide 
with the protein (for ~15min) first followed by addition of 
FITC-peptide, but the results are the same.


Thank you,
Megha


--
Dr. Raphael Gasper-Schönenbrücher
Manager of X-ray Crystallography and Biophysics Unit
Max-Planck-Institute of Molecular Physiology
Room A2.23
Otto-Hahn-Str. 11
44227 Dortmund, Germany
Tel +49 (0)231-133 2111

[ccp4bb] AW: [ccp4bb] off-topic:fluorescence polarization displacement assay

[Herman . Schreuder]

Dear Megha,

I am puzzled by the results you presented. If you only see the effect in the 
presence of your protein, the protein must have something to do with it. The 
steep decline points to some highly cooperative effect, which might be 
aggregation/precipitation. Did you check that your protein is still in solution 
beyond the point of steep decline? Precipitation of your protein may explain 
what you are seeing.

My 2 cts,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von megha 
abbey
Gesendet: Mittwoch, 21. Juni 2017 21:21
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] off-topic:fluorescence polarization displacement assay

Hello All,

This is an off-topic question. I have some issues regarding Fluorescence 
Polarization competitive displacement assay and would need some advice.

I have developed an in vitro fluorescence polarization based assay using a 
N-terminus labelled FITC peptide. The peptide is 21 amino acids long and the 
binding protein is 50 Kda in size. The Kd for interaction is 1 uM. I am further 
running a competitive displacement assay using the exactly same unlabelled 
peptide. Here, with increasing concentration of the unlabeled peptide, the 
polarization signal first increases and then shows a sharp decline. Attached is 
the raw data file for the above.

I believe that if the unlabelled peptide had been aggregating, the polarization 
signal would increase but not drop. If the binding would have been 
non-specific, then the unlabelled peptide should not displace at all, but here 
I see an increase followed by a decrease in the signal. What does this increase 
and sharp drop in polarization signify and how do I fix this? Please help.

I have checked the polarization for titration of the unlabelled peptide mixed 
with fixed conc. of FITC-peptide (no protein added). Here, the polarization 
signals are the same for the entire range of unlabeled peptide. I have also 
tried incubating the unlabelled peptide with the protein (for ~15min) first 
followed by addition of FITC-peptide, but the results are the same.

Thank you,
Megha