[ccp4bb] Re： [ccp4bb] Fwd: Regarding Sel-Met containing proteing crystallisation

[tat cheung cheng]

Or maybe a problem of oxidation? 
I had a Se-met crystal that could be crystallized and diffracted just like the 
native one, but only in the presence of the reducing agent (TCEP), no crystal 
otherwise.

Tc





寄件人﹕ Frederic VELLIEUX 
收件人﹕ CCP4BB@JISCMAIL.AC.UK
傳送日期﹕ 2011/6/13 (一) 2:37:10 PM
主題： Re: [ccp4bb] Fwd: Regarding Sel-Met containing proteing crystallisation

I've had exactly the same with an enzyme from Trypanosoma brucei, native enzyme 
gives ca. 2 A data, a search around the crystallization conditions for the 
Se-Met 

enzyme returns absolutely nothing. What I should do is to search for new 
crystallisation conditions (which I won't do: trypanosome means no-one will 
provide any 

funding for the project nowadays).

So if you do not get any crystals from your derivatized protein starting from 
the crystallisation conditions of your native protein, then you must start a 
new 
crystallisation 

screening process right from the start. Remember that introducing Se-Mets can 
change the surface of your protein; it can also introduce an equilibrium 
between 
several 

forms of the protein, whereas you only had one form for the native enzyme - so 
you may need to try to purify (rapidly) further. If this de novo 
crystallisation 
fails, then 

you need to try alternative phasing methods (such as heavy atom soaks or 
co-crystallisation with heavy atoms).

Fred.

> Message du 13/06/11 07:53
> De : "atul kumar" 
> A : CCP4BB@JISCMAIL.AC.UK
> Copie à : 
> Objet : [ccp4bb] Fwd: Regarding Sel-Met containing proteing crystallisation
> 
> -- Forwarded message --
> From: Dilip Kumar 
> Date: Mon, Jun 13, 2011 at 11:21 AM
> Subject: Fwd: Regarding Sel-Met containing proteing crystallisation
> To: atulsingh21...@gmail.com
> 
> 
> 
> 
> -- Forwarded message --
> From: Dilip Kumar 
> Date: Sat, Jun 11, 2011 at 6:09 PM
> Subject: Regarding Sel-Met containing proteing crystallisation
> To: CCP4BB@jiscmail.ac.uk
> 
> 
> Dear all
> 
> I have got protein crystals,crystallisation condition (LiCl, PEG and
> HEPES) .Crystals of native protein have been successesfully reproduced but
> when i tried to reproduce these crystals with protein having Met replaced by
> Sel-Met, i could not get any crystal.I tried crystallisation trials by
> varying pH and PEG concentration and diffferent drop ratio but i could not
> get any hit.Please suggest me what could be the possible reasons behind it?
> And also suggest the other variables that i can try ?
> thanks
> With Regards
> 
> -- 
> Dilip Tiwari
> Graduate Student
> Structural Biology Unit
> Institute of Genomics & Integrative Biology
> Delhi-07
> 
> 
> 
> -- 
> Dilip Tiwari
> Graduate Student
> Structural Biology Unit
> Institute of Genomics & Integrative Biology
> Delhi-07
> 

Re: [ccp4bb] Fwd: Regarding Sel-Met containing proteing crystallisation

[Frank von Delft]

Don't forget to do seeding with the native crystals: it's often more about 
nucleation than growth. Phx


Sent from tiny silly touch screen

- Reply message -
From: "Frederic VELLIEUX" 
Date: Mon, Jun 13, 2011 07:37
Subject: [ccp4bb] Fwd: Regarding Sel-Met containing proteing crystallisation
To: "CCP4BB@JISCMAIL.AC.UK" 

I've had exactly the same with an enzyme from Trypanosoma brucei, native enzyme 
gives ca. 2 A data, a search around the crystallization conditions for the 
Se-Met 
enzyme returns absolutely nothing. What I should do is to search for new 
crystallisation conditions (which I won't do: trypanosome means no-one will 
provide any 
funding for the project nowadays).

So if you do not get any crystals from your derivatized protein starting from 
the crystallisation conditions of your native protein, then you must start a 
new crystallisation 
screening process right from the start. Remember that introducing Se-Mets can 
change the surface of your protein; it can also introduce an equilibrium 
between several 
forms of the protein, whereas you only had one form for the native enzyme - so 
you may need to try to purify (rapidly) further. If this de novo 
crystallisation fails, then 
you need to try alternative phasing methods (such as heavy atom soaks or 
co-crystallisation with heavy atoms).

Fred.

> Message du 13/06/11 07:53
> De : "atul kumar" 
> A : CCP4BB@JISCMAIL.AC.UK
> Copie à : 
> Objet : [ccp4bb] Fwd: Regarding Sel-Met containing proteing crystallisation
> 
> -- Forwarded message --
> From: Dilip Kumar 
> Date: Mon, Jun 13, 2011 at 11:21 AM
> Subject: Fwd: Regarding Sel-Met containing proteing crystallisation
> To: atulsingh21...@gmail.com
> 
> 
> 
> 
> -- Forwarded message --
> From: Dilip Kumar 
> Date: Sat, Jun 11, 2011 at 6:09 PM
> Subject: Regarding Sel-Met containing proteing crystallisation
> To: CCP4BB@jiscmail.ac.uk
> 
> 
> Dear all
> 
> I have got protein crystals,crystallisation condition (LiCl, PEG and
> HEPES) .Crystals of native protein have been successesfully reproduced but
> when i tried to reproduce these crystals with protein having Met replaced by
> Sel-Met, i could not get any crystal.I tried crystallisation trials by
> varying pH and PEG concentration and diffferent drop ratio but i could not
> get any hit.Please suggest me what could be the possible reasons behind it?
> And also suggest the other variables that i can try ?
> thanks
> With Regards
> 
> -- 
> Dilip Tiwari
> Graduate Student
> Structural Biology Unit
> Institute of Genomics & Integrative Biology
> Delhi-07
> 
> 
> 
> -- 
> Dilip Tiwari
> Graduate Student
> Structural Biology Unit
> Institute of Genomics & Integrative Biology
> Delhi-07
> 

[ccp4bb] Solvent channel volume measurement

[Matthew BOWLER]

Dear All,
does anyone know of a program that can measure the volume or 
largest dimension of the solvent channels in crystals?  Cheers, Matt.


--
Matthew Bowler
Structural Biology Group
European Synchrotron Radiation Facility
B.P. 220, 6 rue Jules Horowitz
F-38043 GRENOBLE CEDEX
FRANCE
===
Tel: +33 (0) 4.76.88.29.28
Fax: +33 (0) 4.76.88.29.04

http://go.esrf.eu/MX
===

Re: [ccp4bb] Solvent channel volume measurement [SEC=UNCLASSIFIED]

[DUFF, Anthony]

It may require some tricks, such as the creation of walls of CA atoms, but 
Kleywegt's FLOOD will give an answer in terms of number of water molecules.

Anthony Duff



-Original Message-
From: CCP4 bulletin board on behalf of Matthew BOWLER
Sent: Mon 6/13/2011 5:40 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Solvent channel volume measurement
 
Dear All,
 does anyone know of a program that can measure the volume or 
largest dimension of the solvent channels in crystals?  Cheers, Matt.

-- 
Matthew Bowler
Structural Biology Group
European Synchrotron Radiation Facility
B.P. 220, 6 rue Jules Horowitz
F-38043 GRENOBLE CEDEX
FRANCE
===
Tel: +33 (0) 4.76.88.29.28
Fax: +33 (0) 4.76.88.29.04

http://go.esrf.eu/MX
===

Re: [ccp4bb] Fwd: Regarding Sel-Met containing proteing crystallisation

[Marc Kvansakul]

Dear Atul,

Whilst finding Se-Met conditions you could always just phase with mercury if 
you are lucky enough to have an accessible free Cys residue or try NaI or I3C 
soaks. Always worth trying if you have readily reproducible crystals…

Best of luck

Marc


From: atul kumar mailto:atulsingh21...@gmail.com>>
Reply-To: atul kumar mailto:atulsingh21...@gmail.com>>
Date: Mon, 13 Jun 2011 15:53:12 +1000
To: "CCP4BB@JISCMAIL.AC.UK" 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: [ccp4bb] Fwd: Regarding Sel-Met containing proteing crystallisation



-- Forwarded message --
From: Dilip Kumar mailto:dku...@igib.in>>
Date: Mon, Jun 13, 2011 at 11:21 AM
Subject: Fwd: Regarding Sel-Met containing proteing crystallisation
To: atulsingh21...@gmail.com




-- Forwarded message --
From: Dilip Kumar mailto:dku...@igib.in>>
Date: Sat, Jun 11, 2011 at 6:09 PM
Subject: Regarding Sel-Met containing proteing crystallisation
To: CCP4BB@jiscmail.ac.uk


Dear all

I have got  protein crystals,crystallisation condition (LiCl, PEG and HEPES) 
.Crystals of native protein have been successesfully reproduced but when i 
tried to reproduce these crystals with protein having Met replaced by Sel-Met, 
i could not get any crystal.I tried crystallisation trials by varying pH  and 
PEG  concentration and diffferent drop ratio but i could not get any hit.Please 
suggest me what could be the possible reasons behind it? And also suggest the 
other variables that i can try ?
thanks
With Regards

--
Dilip Tiwari
Graduate Student
Structural Biology Unit
Institute of Genomics & Integrative Biology
Delhi-07



--
Dilip Tiwari
Graduate Student
Structural Biology Unit
Institute of Genomics & Integrative Biology
Delhi-07

Re: [ccp4bb] Solvent channel volume measurement [SEC=UNCLASSIFIED]

[Francois Berenger]

On 06/13/2011 04:55 PM, DUFF, Anthony wrote:

It may require some tricks, such as the creation of walls of CA atoms,
but Kleywegt's FLOOD will give an answer in terms of number of water
molecules.


Some more tools are referenced here:
http://hollow.sourceforge.net/

However, I did not tried them.
I don't know how they delimit the limits of channels,
which may be an interesting question.

Regards,
F.


Anthony Duff



-Original Message-
From: CCP4 bulletin board on behalf of Matthew BOWLER
Sent: Mon 6/13/2011 5:40 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Solvent channel volume measurement

Dear All,
does anyone know of a program that can measure the volume or
largest dimension of the solvent channels in crystals? Cheers, Matt.

--
Matthew Bowler
Structural Biology Group
European Synchrotron Radiation Facility
B.P. 220, 6 rue Jules Horowitz
F-38043 GRENOBLE CEDEX
FRANCE
===
Tel: +33 (0) 4.76.88.29.28
Fax: +33 (0) 4.76.88.29.04

http://go.esrf.eu/MX
===

[ccp4bb] 3-year postdoc position at MRC Laboratory of Molecular Biology, Cambridge, UK

[Murray Stewart]

Postdoc opening at the MRC Laboratory of Molecular Biology, Cambridge, UK.

Murray Stewart's Structural Cell Biology group at the MRC Laboratory of 
Molecular Biology in Cambridge is seeking a postdoctoral scientist 
(3-year Career Development Fellow position) to work on the structural 
basis of the nuclear export of mRNA.


The successful applicant will use a combination of structural, 
biochemical and molecular approaches to establish the molecular 
mechanism of mRNA nuclear export and to determine the molecular basis 
for the formation and disassembly of export-competent mRNP particles. 
Applicants should have a PhD, together with practical experience in 
X-ray crystallography, Molecular Biology and Biochemistry, especially 
with respect to determining the structure of proteins, growing crystals, 
determining binding constants, expressing proteins in bacteria, and 
site-directed engineering of mutants. Experience in nuclear transport or 
yeast Cell Biology and having a strong publication record, with at least 
one first-author paper in a peer-reviewed journal, would also be 
advantageous.Further information can be obtained from Dr Stewart 
(m...@mrc-lmb.cam.ac.uk ) or from 
http://www.jobs.ac.uk/job/ACT935/career-development-fellow/


The successful applicant will be awarded an MRC Career Development 
Fellowship, which is a three year training and development position for 
a postdoctoral scientist who has recently completed their doctoral 
studies or is moving into a new research discipline.A starting salary of 
£26,022 to £28,178 per annum will be supported by a flexible pay and 
reward policy, 30 days annual leave entitlement, an optional MRC final 
salary pension scheme and excellent on-site sports and social facilities.


Applications are handled by the RCUK Shared Services Centre; to apply 
please visit the job board at https://ext.ssc.rcuk.ac.uk 
 and complete an online application form.  
Applicants who would like to receive this advert in an alternative 
format (e.g. large print, Braille, audio or hard copy), or who are 
unable to apply online should contact RCUK by telephone on 01793 867003, 
Please quote reference number IRC23438.


Closing date: 27 June 2011Interview date 4 July 2011.

Re: [ccp4bb] Solvent channel volume measurement

[Tim Gruene]

Hallo Matthew,

you can also mark the diameters with some atoms (e.g. water molecules in coot),
cut them out and use moleman2 ("xyz align" to align the object and "stats" to
get the radii of gyration) as a rough estimate.
Tim

On Mon, Jun 13, 2011 at 09:40:47AM +0200, Matthew BOWLER wrote:
> Dear All,
> does anyone know of a program that can measure the volume or
> largest dimension of the solvent channels in crystals?  Cheers,
> Matt.
> 
> -- 
> Matthew Bowler
> Structural Biology Group
> European Synchrotron Radiation Facility
> B.P. 220, 6 rue Jules Horowitz
> F-38043 GRENOBLE CEDEX
> FRANCE
> ===
> Tel: +33 (0) 4.76.88.29.28
> Fax: +33 (0) 4.76.88.29.04
> 
> http://go.esrf.eu/MX
> ===
> 

-- 
--
Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A



pgpJk6ZQBLYPW.pgp
Description: PGP signature

Re: [ccp4bb] XDS question

[Marco Lolicato]

Thank you to all!

@Frederic
> 
> I have a problem with the following sentence:
> "if I collect all spots I get good map, but it is impossible to solve the 
> structure by molecular replacement" - if you have a good map (I assume 
> electron density map) then the structure is solved... for me a good map is a 
> map I can interpret.

You're right, I said "good map" instead of "good output values".


@Konstantin
> 
> It is possible to process diffraction spots from both crystals using XDS. The 
> procedure is described here (under 'Index and integrate multiple-crystal 
> diffraction'): 
> http://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/Tips_and_Tricks

I tried but with no success! :(


@Kay and all the others

The following links are the images:

http://www.facebook.com/photo.php?fbid=2127189780277&set=o.136323896385679&type=1&theater

http://www.facebook.com/photo.php?fbid=2127189180262&set=o.136323896385679&type=1&theater

http://www.facebook.com/photo.php?fbid=2127188820253&set=o.136323896385679&type=1&theater




...then a little bit more details...
so, if I process only the strong spots I have those cell parameters:
 a=b=96.66  c=112.26alpha=beta=gamma= 90

f I process all the spots I have those cell parameters:
a=b=216.4   c=112.4 alpha=beta=gamma= 90

In both cases the space group is I422.


Thank you again to all, do you have any other suggestion?


Marco

Re: [ccp4bb] XDS question

[Vellieux Frederic]

"the space group is I422" "do you have any other suggestion?"

Yes, how certain are you of the space group? For myself, I'm never 
entirely certain of the space group until I have solved the structure... 
I always keep in mind the other possibilities for space group 
assignment, if need be. And sometimes the "obvious space group" is not 
the space group of the "final" structure. The computer programs we use 
only give hints of the solution, but these are only hints. Remember that 
crystals "behave as they want", the fact for example that I(equiv.1) is 
approximately equal to I(equiv.2) is approximately equal to I(equiv.3) 
etc does not mean that the relationship between intensities is in fact 
an equality, it can be just an approximation... With crystals I have 
learned, everything is possible.


It might be an idea to enclose parts of relevant XDS output files for 
our perusal. Using "standard" input parameters (for spot selection) as 
well as your spot selection input parameters.


Fred.

Marco Lolicato wrote:

Thank you to all!

@Frederic
  

I have a problem with the following sentence:
"if I collect all spots I get good map, but it is impossible to solve the structure 
by molecular replacement" - if you have a good map (I assume electron density map) 
then the structure is solved... for me a good map is a map I can interpret.



You're right, I said "good map" instead of "good output values".


@Konstantin
  

It is possible to process diffraction spots from both crystals using XDS. The 
procedure is described here (under 'Index and integrate multiple-crystal 
diffraction'): 
http://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/Tips_and_Tricks



I tried but with no success! :(


@Kay and all the others

The following links are the images:

http://www.facebook.com/photo.php?fbid=2127189780277&set=o.136323896385679&type=1&theater

http://www.facebook.com/photo.php?fbid=2127189180262&set=o.136323896385679&type=1&theater

http://www.facebook.com/photo.php?fbid=2127188820253&set=o.136323896385679&type=1&theater




...then a little bit more details...
so, if I process only the strong spots I have those cell parameters:
 a=b=96.66  c=112.26alpha=beta=gamma= 90

f I process all the spots I have those cell parameters:
a=b=216.4   c=112.4 alpha=beta=gamma= 90

In both cases the space group is I422.


Thank you again to all, do you have any other suggestion?


Marco
  

Re: [ccp4bb] About heavy atom split

[Nat Echols]

On Mon, Jun 13, 2011 at 9:48 AM, Dhanasekaran Varudharasu <
dhana...@gmail.com> wrote:

> We have solved one protein structure with barium as
> a heavy atom using SAD technique. But in the final refinement one barium
> site shows split. But the distance between the two positions is 3.8 Å.
> Please give your valuable comments and suggestions to conclude whether it is
> due to split or due to the occurrence of two individual barium atoms.
> Herewith I have enclosed the image files containing the electron density map
> at 1.00 sigma level and the anomalous map at 4.00 sigma level for your
> reference.
>

Looks like two sites to me, but why not try refining the occupancies?

-Nat

Re: [ccp4bb] About heavy atom split

[Bernhard Rupp (Hofkristallrat a.D.)]

Given that the water and the barium seem to have the same electron density
on image electron…png I’d look careful at relative density levels,
occupancies, B-factors etc. Often the situation becomes clearer during later
stages of refinement. 

 

Peripherally, I also do not exactly understand the difference between ‘split
barium’ and ‘individual barium’. Are two half occupied bariums necessary
non-individuals? The ‘split’ term in crystallography colloquially relates to
multiple conformations of the same entity, which in case of side chains does
require constrained occupancies. This is not necessarily so for isolated
atoms, where the term imho looses its meaning. However, constrained
occupancies can be imposed on individual atoms by split side chain
conformations. I have some examples in the figure library

 

http://www.ruppweb.org/garland/gallery/Ch12/pages/Biomolecular_Crystallograp
hy_Fig_12-33_AandB.htm

Similar but somewhat different situation of correlated occupancies

http://www.ruppweb.org/garland/gallery/Ch13/pages/Biomolecular_Crystallograp
hy_Fig_13-04.htm

 

 

BR 

 

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Dhanasekaran Varudharasu
Sent: Monday, June 13, 2011 9:48 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] About heavy atom split

 

Dear experts,

We have solved one protein structure with barium as
a heavy atom using SAD technique. But in the final refinement one barium
site shows split. But the distance between the two positions is 3.8 Å.
Please give your valuable comments and suggestions to conclude whether it is
due to split or due to the occurrence of two individual barium atoms.
Herewith I have enclosed the image files containing the electron density map
at 1.00 sigma level and the anomalous map at 4.00 sigma level for your
reference.

with regards
Dhana

[ccp4bb] GTP Agarose Resin

[Matthew Bratkowski]

Hi.

I was considering using GTP Agarose Resin for the final clean up step of the
purification of a GTPase and was wondering if anyone has had experience
using this resin.  My main concerns are whether it actually has a decent
binding capacity for GTP binding proteins, considering that endogenous
GTP/GDP may remain tightly bound during purification, and if an active
GTPase would bind without cleaving the GTP off of the resin itself.

I found only a few companies that still carry the resin, but the price for
each is very different.  The resin from Sigma is fairly cheap and is linked
to the resin via ribose hydroxyls, while the resin from Innova Biosciences
is more than four times as much but is linked to the resin via the gamma
phosphate, which supposively prevents cleavage by contaminating
phophatases.  Considering that my protein should be relatively pure during
this purification step, I was wondering whether or not GTP cleavage of the
resin by the GTPase and loss of binding to the column would be a problem if
using the Sigma resin.

If anyone has any other information about purification using this resin,
such as resin binding capacity, an effective protocol with relevant buffers,
and the lifetime of the resin after regeneration, I would be happy to hear
it.

Thanks,
Matt

Re: [ccp4bb] GTP Agarose Resin

[Pascal Egea]

Hi Matthew,

Most GTPases required Magnesium to hydrolyze so you maybe able to reduce
this by working in presence of EDTA and absence of magnesium. This may
promote removal of traces of GTP or GDP ( from previous experience with SRP
GTPases I would be more worried about residual GDP). Having EDTA and no
magnesium during purification helps.
If your GTPase has a very low basal GTPase activity ( and some do as they
require a cognate GAP to really get in the mood to hydrolyze) this might be
enough to minimize hydrolysis on this resin.

Good luck,


-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu

On Mon, Jun 13, 2011 at 12:21 PM, Matthew Bratkowski 
 wrote:

> Hi.


>
> I was considering using GTP Agarose Resin for the final clean up step of
> the purification of a GTPase and was wondering if anyone has had experience
> using this resin.  My main concerns are whether it actually has a decent
> binding capacity for GTP binding proteins, considering that endogenous
> GTP/GDP may remain tightly bound during purification, and if an active
> GTPase would bind without cleaving the GTP off of the resin itself.
>
> I found only a few companies that still carry the resin, but the price for
> each is very different.  The resin from Sigma is fairly cheap and is linked
> to the resin via ribose hydroxyls, while the resin from Innova Biosciences
> is more than four times as much but is linked to the resin via the gamma
> phosphate, which supposively prevents cleavage by contaminating
> phophatases.  Considering that my protein should be relatively pure during
> this purification step, I was wondering whether or not GTP cleavage of the
> resin by the GTPase and loss of binding to the column would be a problem if
> using the Sigma resin.
>
> If anyone has any other information about purification using this resin,
> such as resin binding capacity, an effective protocol with relevant buffers,
> and the lifetime of the resin after regeneration, I would be happy to hear
> it.
>
> Thanks,
> Matt
>

[ccp4bb] "Small Angle X-ray Scattering (SAXS) Techniques for Macromolecules" Webinar

[Angela Criswell]

Dear colleagues,

I would like to draw your attention to an upcoming free, educational webinar to 
be presented by Angela Criswell, Ph. D. titled "Small Angle X-ray Scattering 
(SAXS) Techniques for Macromolecules". Small angle X-ray scattering is a 
technique used to study dilute solutions of macromolecules. SAXS measurements 
can give valuable information about the low resolution structural 
characteristics and in some cases a model of the protein shape. Specifically, 
SAXS is particularly useful for giving immediate feedback whether your protein 
is monodispersed or aggregated and whether your protein is folded or unfolded 
in solution. In addition, several examples in the literature demonstrate the 
use of SAXS to identify the correct multimeric states or proteins, thus 
eliminating the ambiguity associated with crystal-packing induced oligomers. 
This webinar will summarize various applications of SAXS for macromolecules and 
explore some practical considerations for collecting and processing SAXS data.

This webinar is scheduled to occur on Thursday, June 23rd 10:00 AM CDT (8:00 AM 
PDT / 4:00 PM GMT). You can find more information, including a registration 
link at: http://www.rigaku.com/protein/webinars.html.

Best regards,
Angela Criswell

-- 
Angela R. Criswell, Ph. D.
VP, Life Sciences
Rigaku Americas Corporation
9009 New Trails Drive
The Woodlands, TX 77381 USA
Ph: +1 281 362 2300  ext. 216
Fax: +1 281 364 3628
Email: angela.crisw...@rigaku.com
URL: http://www.rigaku.com