Re: [ccp4bb] Message from the Uppsala EDS: "Morituri te salutant"

2016-12-14 Thread Daniel Picot

Veni vidi
I wil have to change my advices and lectures: go and look at the Uppsala 
Electron Density Server EDS

It has been extremely useful.
Even sometimes vici

Le 13/12/2016 à 19:52, Patrick Loll a écrit :

Ave atque vale.

The EDS was hugely useful (and will continue to be so in its new manifestation, 
we hope)—thanks to everyone who made it happen!



On 13 Dec 2016, at 12:51 PM, Gerard DVD Kleywegt  wrote:

Hi all,

After tirelessly serving the scientific community with (mostly) beautiful maps for two 
decades, the Uppsala Electron Density Server (EDS; is now reaching 
the end of its life (in fact, it has been living on borrowed time for several years 
already). Some time in 2017 it will therefore be "phased" out and join the 
choir invisible (despite its beautiful plumage).

The good news is that much of the EDS functionality (and in particular the 
delivery of map and mtz files, as well as a much better 3D viewer) is now 
provided by the Protein Data Bank in Europe (PDBe;

There is a short write-up that explains what this means for users who just want 
to look at maps, for users who want to download files, for users of software 
that retrieves data from EDS, and for developers of such software (incl. URLs 
for map, mtz and other relevant files on the PDBe website) at:

Toodle pip!


   Gerard J. Kleywegt
   The opinions in this message are fictional.  Any similarity
   to actual opinions, living or dead, is purely coincidental.
   Little known gastromathematical curiosity: let "z" be the
   radius and "a" the thickness of a pizza. Then the volume
of that pizza is equal to pi*z*z*a !

Re: [ccp4bb] In search of an article

2014-11-03 Thread Daniel Picot

Hi Phhilippe,

T. Y. Teng,
J. Appl. Crystallogr.
J. Appl. Crystallography 23:387–391 (1990)
Mounting of crystals for macromolecular crystallography in a 
free-standing thin film

A method for mounting single crystals in macromolecular crystallographic 
studies is described in which the crystal is suspended in a thin film. 
The film is formed from a mixture of the crystallization buffer and a 
hydrophilic viscous material, confined within a thin-wire loop by 
surface tension. Compared with conventional crystal mounting methods, 
this method greatly simplifies and speeds the mounting procedure, is 
well suited to shock freezing and to optical monitoring of the crystals, 
deforms fragile crystals less and gives a lower and more uniform 
background in the X-ray diffraction patterns.

I do not have a copy, but if I remember well it was done with a copper 
wire, hair and nylon loops came later.


Le 03/11/2014 18:23, DUMAS Philippe (VIE) a écrit :

I am looking for the reference of this little paper (in J. Appl. Cryst ?) 
describing how to make a loop and fish crystals...
You know, this kind of humble method used by all crystallographers (apart FEL 
Thank you in advance

Philippe Dumas

Re: [ccp4bb] rigaku micromax 007 cooling

2014-07-24 Thread Daniel Picot

Dear Andreas,
 We have a Hyfra water cooled system (VWK50) on our MicroMax 007, 
but  it is probably over-dimensioned
because it is also used by a Xstream equipment, a smaller dedicated 
cooler to the micromax could be better.


Le 24/07/2014 17:53, Andreas Förster a écrit :

Dear all,

I have a Rigaku MicroMax 007 HF X-ray generator that is cooled by 
chilled water supplied rather unreliably through the building 
infrastructure.  I was wondering what alternatives exist.

Could other MicroMax 007 users share their experiences with 
alternative cooling solutions with me?

Thank you.


Re: [ccp4bb] Invisible atoms in ligands

2014-06-16 Thread Daniel Picot

Dear Ian,
  This has been discussed in a review and related articles by Brian 
Matthews and Liljun Liu:

Matthews BW, Liu L. A review about nothing: are apolar cavities in proteins
really empty? Protein Sci. 2009 Mar;18(3):494-502. doi: 10.1002/pro.61. Review.
PubMed PMID: 19241368; PubMed Central PMCID: PMC2760356.


Le 16/06/2014 11:32, Ian Tickle a écrit :

Dear James

You seem to be discounting the possibility of a true vacuum inside a 
structure, which is obviously not the same thing as 'something' (bulk 
solvent or whatever).  I accept that this is unlikely in the case of 
ligand binding sites exposed to solvent, or indeed any site on the 
outer surface of the molecule, since any vacuum in that situation 
would be unstable against the ingress of water molecules, but it is 
possible in the case of fully-enclosed cavities (i.e. 'inner surface') 
that are normally inaccessible to water.  I don't know if anyone has 
done a systematic survey of this, i.e. looking for cavities where the 
density appears to be actually zero (taking into account F000 of 
course), or at least significantly lower compared with the bulk 
solvent density (where the assumed value of F000 wouldn't affect the 


-- Ian

On 16 June 2014 07:37, James Holton wrote:

Thank you Pavel for the clarification!

What I was really trying to point out is that a missing atom,
occ=0.00 and occ=0.01 are not as similar as one might naiively
think.  Also, if you put a ligand into a pocket and the occupancy
refines to  0, that does not necessarily mean the ligand is
partially occupied.  If the pocket is actually filled with flat
bulk solvent, then you expect the ligand occupancy to be non-zero,
simply because something is better than nothing.  However, if the
bulk solvent mask were somehow smarter and filled the pocket of
a, say, 60% occupied ligand with flat bulk density at 40% the
level of bulk density used far away from any atoms, then one might
actually see the occupancy of a bogus ligand refine to zero.  That
is, a ligand built into a pocket that is truly empty (filled
with flat bulk solvent) and then occupancy refined would actually
be a competition between two alternative hypotheses: 1) ligand
in the pocket, 2) nothing but solvent in the pocket.  If the
occupancy of the ligand refines to zero in this context, then you
can be quite confident that it didn't bind, at least not in the
given orientation.

I fully realize that the implementation of this is easier said
than done, but perhaps it would be worth the effort?

-James Holton
MAD Scientist

On 6/16/2014 3:04 PM, Pavel Afonine wrote:

Hi James,

a remark: different programs may treat occ=0 differently. In
phenix.refine (phenix.maps, etc) atoms with zero occupancy will
be ignored for bulk-solvent mask calculation, unless you ask to
do otherwise. For example, this means that if you want to
calculate a ligand OMIT map both options
- removing the ligand from PDB file;
- setting its occupancy to zero and making sure mask does not
ignore occ=0 atoms)
are a) not equivalent and b) both not good.
In first case (removing atoms from file) bulk-solvent will
flatten residual map (as you pointed out). In second case
bulk-solvent will be excluded in a very specific area, so that
residual (green) density you see there may be either just
bulk-solvent or ligand in question or a mixture; obviously not a
very useful information! This highlights the fundamental problem
of flat bulk-solvent model the way it's currently used.


On Sun, Jun 15, 2014 at 3:01 PM, James Holton wrote:

The principle difference between occ=0 and omitting the atom
entirely is that occ=0 atoms exclude bulk solvent.  Or at
least they do for typical operation of contemporary
refinement programs.  So, by defining occ=0 you are forcing
all map voxels within ~0.6A or so of your invisible atom to
be vacuum.  If you omit it, then the bulk solvent may flood
in, perhaps enough to pull the fo-fc peak down below 3x
rms.  How much the bulk solvent floods in depends on how
nearby atoms exclude the bulk solvent, and this, in turn,
depends on which refinement program you are using. Different
bulk solvent implementations use different radii, shrink
parameters, etc.  In addition, bulk solvent always bleeds a
bit into surrounding areas because the solvent B factor is
never zero.

The real problem, I think, is that for any voxel of the map
there is ALWAYS something there.  The only question is:
what is it?  Is there a 100% occupied ligand?  100% occupied
solvent?  Two conformers of the ligand? Or is it some mixture

Re: [ccp4bb] Se-Met and disulfides

2014-02-27 Thread Daniel Picot

Hi Reza,
You have here an example of MAD data collected fromoxidized  and 
reducedselenomethionine-containing protein:
It states :phasing power of theoxidized  data is doubled for the dispersive 
signal and is 20% stronger
for theanomalous  signal at the peak wavelength

Thomazeau K, Curien G, Thompson A, Dumas R, Biou V.
MAD on threonine synthase: the phasing power of oxidized selenomethionine.
Acta Crystallogr D Biol Crystallogr. 2001 Sep;57(Pt 9):1337-40. Epub 2001 Aug 
PubMed PMID: 11526338.


Le 27/02/2014 17:05, Reza Khayat a écrit :


I'm sure this question has been addressed before and I
apologize for asking it again. I'm working on a protein
predicted to have multiple disulfide bonds. The protein is
expressed in an E. coli strain with an oxidizing cytoplasm,
and is purified under nonreducing conditions (no DTT, b-
ME...). I'd like to produce some Se-Met incorporated protein
for phasing. Given that Se-Met can oxidize, its anomalous
scattering is dependent on its oxidation state, and a mixture
of oxidized and reduced Se-Met makes phasing difficult, what
is the suggested protocol to follow in my situation? Here's
hoping that the solution is not dumping a bunch of Se-Met into
all the purification buffers...

Best wishes,

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070

 Original message 

Date: Thu, 27 Feb 2014 11:22:10 +
From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf

of Vicky Tsirkoni

Subject: [ccp4bb]

   Hi Prerana,
   You could try 4°C as well (dont forget to use
   lower protein concentration) in my case this worked
   Vicky G. Tsirkone, MSc
   Laboratory for Biocrystallography
   Department of Pharmaceutical and Pharmacological
   KU Leuven
   ON II Herestraat 49 - box 822
   3000 Leuven | Belgium
   Tel.: +32 16 3 23419


   From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on
   behalf of Prerana G. []
   Sent: Monday, February 24, 2014 5:23 PM
   Subject: [ccp4bb]
   Hi Vicky,
   I tried to change the ratio of paraffin:silicon oil
   to 1:2, but still the protein precipitated. So I
   tried to do it other way round, I kept
   paraffin:silicon oil ratio as 2:1 and so far it has
   not precipitated. Apart from that, I separately used
   the two oils.In silicon oil there was immediate
   precipitation, but none in parafin oil.

[ccp4bb] Meeting announcement Dynamo Paris 2014

2014-02-04 Thread Daniel Picot

Dear colleagues,
On behalf of the organizing committee, I would like to welcome you at 
the Dynamo International Symposium on

Evolution, Biogenesis and Dynamics of Energy Transducing Membranes
in Paris, April 9th-12th 2014.

The meeting cover a wide range of integrated biology,  including 
structural biology, modelling, regulation of gene expression, organelle 
biology in a framework of bioenergetic-related issues.

Program and leaflet and further information are available at
Registration is free but mandatory before February 15th on the  web site.

Daniel Picot

Re: [ccp4bb] preparation of citrate buffer pH3-6.5

2014-01-30 Thread Daniel Picot
But you have to be aware that pH depends on the concentration  of the 
buffer. This is especially the case for phosphate and citrate buffer.


Le 30/01/2014 15:51, Schnicker, Nicholas J a écrit :
It’s a pain but I usually just make each pH of whatever buffer I’m 
using (if you make it concentrated then you’ll only have to do it 
once).  Also, if you haven’t already found it, Hampton has a nice link 
to calculate volume of components while designing a tray as long as 
you tell it the concentrations.


From: Roger Rowlett
Reply-To: Roger Rowlett

Date: Thursday, January 30, 2014 at 7:23 AM

Subject: Re: [ccp4bb] preparation of citrate buffer pH3-6.5

The easiest way to produce repeatable conditions is to titrate a stock 
solution (say 1M) of citric acid with NaOH to the desired pH and use 
that to mix your screen. That's what Hampton does anyway.

If fine sampling pH, you can mix various ratios of pH 3 and 6.5 
buffers. The pH won't be linear with mixing ratio, but will be easily 
repeatable. The actual pH of the final, magic solution can be directly 
measured if desired. Calculations will never be exactly right; pKa 
values are ionic strength dependent. Better to measure.

Roger Rowlett

On Jan 30, 2014 2:37 AM, sreetama das wrote:

Dear All,
   We have obtained many tiny protein crystals in a
condition containing 0.1M citric acid pH 3.5, 2M ammonium sulfate.
The crystals are too small for mounting in loops.

   We intend to vary the salt concentration  pH to obtain
larger crystals.

   Could anyone direct us to some links, or provide us
with a method (with calculations) to calculate the amounts of
citric acid  trisodium citrate required to obtain buffers in a
range of pH 3 - 6.5?
   I have come across online buffer calculators and links
where the amounts of the components required are mentioned in
grams, but none explaining how those values were arrived at.

Thanks  regards,

Re: [ccp4bb] membrane protein optimization

2013-10-23 Thread Daniel Picot

Hi Frank,
Do not forget that membrane protein crystals are often fragile and 
difficult to manipulate.
Finding good cryo condition can be difficult and small temperature 
variation can destroy crystals
within minutes, this makes room temperature diffraction tests not always 
obvious. The time of harvest may also be critical. In addition to the 
previous suggestions, it is often usefull to check if there is  a large 
amount of lipids in the sample. This can often easily be done by thin 
layer chromatography.


Le 23/10/2013 18:22, crystalboy a écrit :

Hi CCP4BB Forks,

In recently I got a membrane protein crystal in the quite normal
membrane protein crystallization conditions as other persons reported,
like PEG400 16-19%, pH 6.0-7.5, with 50 mM MgCl2 (in my case) by using
sitting drop method. These crystals are around 50-100 uM. They look
like trapezoid crystal. My problem is all of my crystals have not
diffraction in home source X-ray and just poor diffraction at
Synchrotron (lower than 20 A). My crystals like to appear on the
surface of the drop. Look like my crystals are quite light.  I had
tried to use a needle to touch them. Unlike other protein crystal, my
crystal looks like quite soft. When I touch it, it didn't crack, but
was bend or mashed.  I had tried to do additive screen and detergent
screen. It seems they are not useful.

Do anyone have good ideas to optimization these crystals? Thanks for
your suggestions.


Re: [ccp4bb] DDM

2012-03-27 Thread Daniel Picot
It is important to distinguish between the solubilisation and the 
purification steps:
1) During the solubisation step you need to care about the 
lipid/detergent ratio. The amount (not the concentration) of detergent 
(i.e. in non monomeric form, the detergent above the cmc) is important. 
You may need a high amount of detergent.
2) During the purification step, you need to keep your protein soluble. 
Here, the concentration is important and you may  keep the concentration 
of detergent around the cmc. But you have to be aware that in the 
initial (and also the not so initial ones!) steps you may have a lot of 
lipids, you need then keep the concentration of detergent fairly high in 
order to keep everything soluble. I like to decrease the detergent 
concentration at each purification steps in order to avoid protein 
denaturation. The protein may sustain a fairly high detergent 
concentration of detergent during the early steps since the 
lipid/detergent mixed micelles will be less denaturing than the pure 
detergent micelles in the later steps. Thin layer chromatography is a 
quick and easy method to check if you have a large amount of lipids in 
your preparation.


Le 26/03/2012 19:17, Katarzyna Rudzka a écrit :

Hi All,
Has anyone had any luck purifying membrane proteins with DDM
(n-dodecyl-D-maltoside) using concentration of detergent ~1-2 x CMC ?
(Its CMC is very low: 0.009%). I would like to keep it as low as
possible, so I don't have too much DDM around when I get to the
crystallization step. I wonder If the amount of detergent sufficient for
the protein extraction has to be determined experimentally for each
protein or maybe there are some good rules of thumb. I appreciate your
help. Thanks.

Katarzyna Rudzka, Postdoctoral Fellow
Department of Biophysics and Biophysical Chemistry
Johns Hopkins University, School of Medicine
Baltimore, Maryland 21205 USA

Re: [ccp4bb] nmr blog

2012-03-23 Thread Daniel Picot

You can find here several links concerning NMR

with a discussion list:


Le 22/03/2012 19:35, Luthra,Amit a écrit :

Is any NMR blog available for discussion?

Amit Luthra, Ph.D.
Post-Doctoral Fellow
The Radolf Laboratory
Department of Medicine
University of Connecticut Health Center

[p]   860/ 679 - 8390

Re: [ccp4bb] off topic: GPCR membane insertion/orientation

2011-03-04 Thread Daniel Picot
In addition to Bert remarks, you can read this paper from the positive 
inside rule instigators.

Seppälä S, Slusky JS, Lloris-Garcerá P, Rapp M, von Heijne G. Control of
membrane protein topology by a single C-terminal residue. Science. 2010 
Jun 25;328(5986):1698-700. Epub 2010 May 27. PubMed PMID: 20508091.

with the cited literature


Le 04/03/2011 17:31, Justin Hall a écrit :

Dear Community,

In trying to trouble shoot an experiment I have become interested in the
cellular process that regulates the insertion and proper orientation of
membrane proteins. I am looking for references for how a GPCR is
correctly oriented during expression (i.e. the extra cellular domain
ends up extra cellularly oriented instead of a 50/50 mix in and out), my
intuition is that there must be an N-terminal sequence that directs this
process, but I am having no luck finding information on what this
sequence is for GPCRs, what players are involved or how orientation is
thought to be controlled. Any suggestions?

This is all spurred by my wanting to use phage display with a protein
that binds to the intracellular side of a GPCR, but of course that is
the hard side to present to the outside of a cell so I need to figure
out how to flip these guys around. I have thought about adding a new TM
helix before TM1 (or removing TM1) to flip these guys, but was hoping
there might be another way around that doesn't involve such massive
architectural rearrangement such as simply clipping the N-terminal
sequence responsible for proper orientation (if such a thing exists).


Re: [ccp4bb] hydrohyapatite column

2010-11-30 Thread Daniel Picot
Hydroxyapatite can be miraculous, but is often very capricious: among 
others, it is  temperature sensitive and two different corners of a 
somewhat inhomogeneous cold room may provide different results.


Le 29/11/2010 16:08, Sebastiano Pasqualato a écrit :

Hi all,
I read/heard that hydroxyapatite column can be used to purify proteins, getting 
separation results orthogonal to ion exchange and size exclusion chromatography.
I was wondering if any of you would be kind enough to share her/his experience 
with me, and would suggest vendors and models for such columns.
Thanks in advance,

Re: [ccp4bb] difficult P1 crystal

2010-09-30 Thread Daniel Picot

Here is an example that is is not cryocongelation but spacegroup change
upon cooling :

Garavito RM, Jenkins J, Jansonius JN, Karlsson R, Rosenbusch JP. X-ray
diffraction analysis of matrix porin, an integral membrane protein from
Escherichia coli outer membranes. J Mol Biol. 1983 Feb 25;164(2):313-27. 
PubMed PMID: 6302273.


Le 30/09/2010 16:56, Daniel Bonsor a écrit :

There are a couple of papers...

Acta Cryst. (2010). F66, 346-351
Crystallization and X-ray diffraction studies of cellobiose phosphorylase from 
Cellulomonas uda

The space group was originally P21. During collection the crystal moved out of 
the beam (and possibly the cyrostream). Upon recentering,  the space group was 
found to be P212121

Acta Cryst. (1998). D54, 448-450
Crystallization and preliminary X-ray analysis of thiaminase I from Bacillus 
thiaminolyticus: space group change upon freezing of crystals

At room temperature the space group was P212121 but upon freezing the space 
group changes to P21212

Hope this helps.

Re: [ccp4bb] heterologous seeding

2010-09-22 Thread Daniel Picot

An old one:

Eichele G, Ford GC, Jansonius JN. Crystallization of pig mitochondrial
aspartate aminotransferase by seeding with crystals of the chicken 

isoenzyme. J Mol Biol. 1979 Dec 5;135(2):513-6. PubMed PMID: 537086.


Le 21/09/2010 22:24, Christopher Rife a écrit :


I seem to recall a paper from a few years back that used heterologous
proteins for macro/micro seeding, and of course now I can't find it. Seems
likely that lysozyme would be involved...  I've found plenty on using
non-protein compounds as seeds, but nothing for protein. Does anyone know
if such a reference is out there?


Re: [ccp4bb] Why appear the grid on the low resolution areas

2010-09-14 Thread Daniel Picot

Dear Xingliang,
 This is vaguely reminiscent to a pattern that was observed by Zora 
Markovic-Housley on precession picture of crystals of  ornithine 
aminotransferase: the even layer (l=2n) had a nice pattern diffracting 
to high resolution while the odd layer (l=2n+1) had a honeycomb stucture 
with the hole at the centre positioned were a diffraction spot was expected.

Marković-Housley Z, Kania M, Lustig A, Vincent MG, Jansonius JN, John RA.
Eur J Biochem. 1987 Jan 15;162(2):345-50.
Quaternary structure of ornithine aminotransferase in solution and 
preliminary crystallographic data.


Le 14/09/2010 02:31, xingliang zhang a écrit :

Dear everyone,

Recently,we collected data of a native protein crystal on synchrotron
in Shanghai. When we did with the original data with HKL2000, we found
an unconversant phenomenon ,just as the picture in the enclosure, there
were some grid indicated by arrows appearing on the low resolution
areas.The crytal grew in 20%PEG3000,100mMTris-Cl,200mM Ca(OAc)2
,and the cryo-protectant is 20% sucrose mixed with well buffer。Who can
tell me the reasons it appear the grid ?because of the sucrose? I’ll
appreciated for any explains and suggestion.

Best wishes



Best wishes!

Protein crystallography Lab ,College of biological sciences ,China
agricultural university.

No. 2 yuanmingyuan west road HaidianDistrict, Beijing, 100193


Re: [ccp4bb] Fwd: [ccp4bb] XSCALE

2010-08-05 Thread Daniel Picot

Another very nice script to run and collect statistics from XDS is

xdsme from Pierre Legrand


Le 05/08/2010 16:55, Jovine Luca a écrit :

WRT the redundancy, I am afraid you have to recompute an approximate value 
yourself using the number of observations and number of unique reflections 
(this is what I do all the time). I suppose one could always write a jiffy 
program to compute the correct values using both files  INTEGRATE.HKL and 
XDS_ASCII.HKL, but I haven't done it myself... Yet ?

No need to do that - you can use the xdspub command within XDSi:

which gives this kind of output:

  Final data processing statistics
   UNIT_CELL_CONSTANTS=78.0978.0978.09  90.000  90.000  90.000
  Unique reflections   11084.
  High res. shell 1.65-1.50
   Redundancy  4.3(  1.9)
   R(sym)  3.1( 53.1)
  R(meas)  3.5( 70.7)
   R(mergd-F)  7.8(106.0)
   I/s(I) 25.1(  1.5)
 completeness 86.2( 57.3)

Best, Luca

Luca Jovine, Ph.D.
Group Leader  EMBO Young Investigator
Karolinska Institutet
Department of Biosciences and Nutrition  Center for Biosciences
Hälsovägen 7, SE-141 57 Huddinge, Sweden
Voice: +46.(0)8.6083-301  FAX: +46.(0)8.6081-501

Re: [ccp4bb] metal-chelating affinity chromatography and FosCholine detergents

2010-07-13 Thread Daniel Picot
A crude purification prior loading the metal-chelating column (like ion 
exchange chromatography) can help and speed up the binding to the column 
(remark not specific to Fos-cholin). Fos-choline is, contrary  to 
popular belief, a quite harsh detergent and this may affect the 
purification yield and integriry of the protein.


Le 13/07/2010 00:59, Pascal Egea a écrit :

Dear All,

I apologize for the not strictly crystallography-related query.
I am currently purifying several membrane proteins solubilized in
fos-cholines detergents and I consistently observe a significant loss of
protein at the binding step (done in absence of imidazole). Has anyone
else experience the same quite systematic (so far in my hands) problem
with this class of detergents.
I would appreciate any comments or advices from biochemists that face(d)
the same situation.

Thanks in advance

Pascal F. Egea, PhD

Re: [ccp4bb] Pictures of DDM crystals?

2009-08-11 Thread Daniel Picot

Hi Joe,
It is often useful to prepare in parallel drop without protein but with 
a large excess of detergent and protein buffer: a typical membrane 
protein of ca 100 kDa will bind something like 100-200 molecules of DDM, 
If you want to match the quantity of detergent that is bound to a 10 
mg/ml protein solution, this amounts to ca 10-20 mM DDM and this does 
not take into account free micelles and micelle concentration that often 
occurs during ultrafiltration. In addition, even if beta-DDM crystals 
could be difficult to obtain, cristalloid formation can be promoted by 
lipids that copurify with protein, this can be checked by thin layer 
chromatography. One also should not forget lessons from soluble protein: 
 spurious amount of phosphate, etc. is often easier to crystallise than 
DDM or membrane protein even with crystallisation conditions supposedly 
designed for membrane protein.


Joe a écrit :

Hi all,

I am doing initial crystallization screening for a membrane protein 
purified in DDM.  The actual concentration of DDM in the protein 
solution is  2 CMC after the protein concentration step (Amicon MWCO 
50 kD used).  I just found over 50 conditions out of 1000 give me 
crystals.  Having worked on soluble proteins, frankly they all look ugly 
to me to different degrees.  I have selected a few conditions to go 
after, hoping that they will grow big enough for me to verify the 
crystal identity by gels.  I wonder if there is a quick way to tell if 
they are detergent crystals.  I know a microscope with UV capacity would 
do the job, but we probably will not get one very soon.  I have read 
through all threads on CCP4BBS regarding detergent crystals.  I have an 
impression that it is actually not easy to crystallize 
detergents--please correct me if I am wrong.  Your thoughts and comments 
are helpful. 

Thank you for your attention.

Best regards,


Re: [ccp4bb] Color of heme containing Xtals

2008-03-13 Thread Daniel Picot

   Crystals containing haem protein are not necessarily red, the colour 
depends on the oxidation state of the haem and on the haem/protein 
ratio. A FeIII haem will look yellowish, gold or brown depending on

the haem/protein ratio, while an FeII haem will look more pink or red.

Ian Ollmann a écrit :

On Mar 12, 2008, at 12:48 PM, Jan Schoepe wrote:

Hello everybody,

I wonder if anybody has experience with heme (or to be more precise: 
heme b) containing proteins which Xtals do not look red under the 
microscope. How might the technique for crystallization (e.g. sitting 
drop, hanging drop) influence the intensity of the color? Many thanks!

If there is no metal in the heme, its color will be very different.  If 
your crystallization buffer contains EDTA or other strong chelating 
agents, I imagine this might happen, though protein crystallization is 
not my area.


Re: [ccp4bb] His tag on membrane protein

2007-11-23 Thread Daniel Picot

Hi Deliang,
We have crystallized the complex cytochrome b6f with an extension of 
His6 (no linker) at the c-terminal of cytochrome f (Stroebel, 2003). The 
His extension takes part to the crystal contacts. The electron density 
is visible but not very well defined. We have not tried to remove the 
His6. Many other structure of membrane protein have used His-tag, but 
still not enough to  make sound statistics.


deliang a écrit :

Hi there,
I purified a membrane protein with traditional His-tag on the C 
terminal. Before crystallization, I wonder how this tag may affect the 
result. Does anyone have  experience that the removel of this tag may 
improve the result or not? or can provide some references which may give 
some statistic, like how many membrane proteins have been crystallized 
with or without His-tag?
Thanks so much.

Re: [ccp4bb] water soluble protein that needs detergent to be stable

2007-08-29 Thread Daniel Picot
I have not dealt myself with this type of protein, but Alex McPherson
tested the detergent beta-octylglucoside (at a concentration of 1.5 %,
i.e. above the cmc) for the crystallisation of soluble protein and tRNA
(1986, J.Biol.Chem 261:1969-75), the detergent did not hampered the
crystallisation, and even sometimes improved the crystal quality. It is
possible to somewhat control the detergent concentration: if the cmc is
high enough (something above 1 mM) you can dialyse the detergent,
otherwise you can bind the protein to a small affinity column and djust
the required concentration of detergent. I stop here because I find
always very difficult to extrapolate the effect of detergent  from
membrane protein to soluble protein.

Daniel Jin a écrit :
 I am working on a 60 kDa C. elegans protein that is predicted to be
 mostly alpha-helix. It is over-expressed in E.coli and the yield is
 about 1 mg/L of cell culture. The CD spec at 4 degree showed the
 presence of dominant alpha-helix. However, we don’t have any functional
 assay to confirm that it is folded correctly.
 It is over-expressed as a GST-fusion. We noticed that after cleavage of
 GST, it will easily precipitate if moved to room temperature (solution
 turns cloudy). Otherwise, it is OK at 4 degree. The CD temperature
 melting experiment showed a gradual change of signal, no sharp
 transition was observed. We later found out that including some
 detergent in the buffer will make it stay soluble at room temperature
 and showed as a dimer on SEC (4C or RT). Glycerol at 10% will help too
 but not as good as detergent.
 My concerns are, first this protein might not folded correctly, second,
 the presence of probably high concentration of detergent in the final
 sample will harm crystallization since the detergent will be
 co-concentrated with the protein. I am wondering whether anyone has deal
 with proteins like this before and their experience on improvement of
 the biochemical behavior and of course crystallization. Many thanks.
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Re: [ccp4bb] phaser oprn error

2007-03-26 Thread Daniel Picot

You may have called the version of phaser installed in the phenix path. Type

wich phaser

in a terminal to check it.

yang li a écrit :

   I am using CCP4 6.0.2, it has phaser contained in this package. When 
I use it

to do mr, error occured:
*** Phaser Module: READ DATA FROM MTZ 
FILE2.0 ***


FILE OPENING ERROR: /home/work/MR/mader/mad_peak.mtz


I donnot know what is wrong with the mtz file, it is simply converted 
from a sca file
with scalepack2mtz, and now I am using molrep, it didnot give such wrong 

What is the problem? Thanks!

Li Yang