Re: [ccp4bb] microseeding SeMet drops with native xtal seeds

[Artem Evdokimov]

Yes, on both counts. SeMet proteins can be frustratingly* close to
their native forms - with little quirks like twinning or lack of
useful diffraction etc. It's common practice to micro (or macro) seed
with native crystals and it often works quite well. It's fun and
sometimes useful to add a few ul of micro-seed suspension to the
*reservoir* of the crystallization experiment (this conveniently
dilutes the stock), mix well, and then set up drops the usual way. If
you're not too much off in your initial seed count, this way every
condition has (potentially) been seeded - of course in some cases
seeds don't survive the conditions in the well, but it is generally
safer than adding seeds to protein prior to set up since they are
likely to dissolve in the absence of precipitants/buffers/etc.

Artem

* is it even a word? who cares...

On Wed, Sep 1, 2010 at 10:37 PM, amit sharma <3112a...@gmail.com> wrote:
> Dear All,
> My SeMet protein crystals have a needle-like morphology, worse than that of
> the native xtals. Also, although the cell dimensions of both forms is very
> much similar, the SeMet xtals are twinned(as indicated by Ctruncate plots).
> I was wondering if such cases were commonly seen, also is it alright to
> microseed my SeMet protein drops with seeds from the native xtal. I would be
> grateful if someone could shed light on this.
>
> thanks in advance,
> --
> Amit Sharma,
> Postdoctoral Fellow,
> Department of Biophysics,
> Johns Hopkins University,
> Baltimore,
> MD21218
>

[ccp4bb] microseeding SeMet drops with native xtal seeds

[amit sharma]

Dear All,

My SeMet protein crystals have a needle-like morphology, worse than that of
the native xtals. Also, although the cell dimensions of both forms is very
much similar, the SeMet xtals are twinned(as indicated by Ctruncate plots).
I was wondering if such cases were commonly seen, also is it alright to
microseed my SeMet protein drops with seeds from the native xtal. I would be
grateful if someone could shed light on this.

thanks in advance,
-- 
Amit Sharma,
Postdoctoral Fellow,
Department of Biophysics,
Johns Hopkins University,
Baltimore,
MD21218

Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

[Ho Leung Ng]

Some other things to try:

1. Co-crystallize with a ligand
2. crystallization with lipid cubic phase or bicelles
3. limited proteolysis to define a rigid core



ho

-
Hi all,

   I got a crystal of one membrane protein (~60kD) from Na/K phosphate
condition (see getit_4), then I got the improved crystal like getit_5 after
trying seeding, different detergents, lipids and additives. But this crystal
does not diffract at all, I already tried Izit staining which shows it is
protein crystal (detergent crystal?). Does anyone have any good suggestions
for the optimization of this membrane protein crystallization? Thank you in
advance.

Best regards

Qiangmin Zhang

Biomedical Science Tower 3
Room1034
3501 Fifth Avenue
Pittsburgh, PA 15260





--
张强敏

Re: [ccp4bb] How to optimize protein-DNA complex conditions?

[Phoebe Rice]

Dear Yang,
  You might try acrylamide instead of agarose, since the caging effect of the 
tighter matrix might keep the complex together better.
  I'm not sure why MgATP? If your protein only binds in the presence of ATP you 
might need a non-hydrolyzable analog?
  Your concentrations are also vastly higher than those usually used for gel 
shift assays.  You might think that would gaurentee binding, but many DNA 
binding proteins need quite a bit of salt to be soluble at high concentrations, 
so your protein might all be stuck in the well. Actually, you can coomassie 
stain an agarose gel with that much protein on it - that should tell you 
exactly where the protein went. 
  Good luck!
   Phoebe

 Original message 
>Date: Wed, 1 Sep 2010 14:55:07 -0700
>From: CCP4 bulletin board  (on behalf of yang li 
>)
>Subject: [ccp4bb] How to optimize protein-DNA complex conditions?  
>To: CCP4BB@JISCMAIL.AC.UK
>
>   Dear All,
>          Recently I am working on a protein-DNA
>   complex, and from running agarose gel, there is a
>   weak delayed band after the band of pure DNA which 
>   indicates some DNA has bind to the protein though
>   the binding efficiency is low. Then I tried to
>   optimize the condition to increase the binding, but
>    it
>   did not work since the intensity of the delayed band
>   didnot grow. The condition I used is list below:
>   1. the concentration of protein is about 1.5mg/ml,
>   buffer in Tris-Cl, PH 8 and NaCl.
>   2. the running buffer for agarose gel is 0.5x TBE,
>   PH 8.3.   
>   3. different ratio of protein: DNA has tried, from
>   2:1 to 1:2.
>   4. different concentration of NaCl, MgCl2 and ATP in
>   reaction system have been added, but no significant
>   change.
>   I wonder if there is any way to increase the binding
>   efficiency? Is it possible to set up crystal plates
>   in this situation, with protein and complex
>   together?
>   Any suggestion would be appreciated!
>   Best
>   Yang 

=
Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123
http://www.rsc.org/shop/books/2008/9780854042722.asp

[ccp4bb] How to optimize protein-DNA complex conditions?

[yang li]

Dear All,

   Recently I am working on a protein-DNA complex, and from running
agarose gel, there is a weak delayed band after the band of pure DNA which
indicates some DNA has bind to the protein though the binding efficiency is
low. Then I tried to optimize the condition to increase the binding, but  it
did not work since the intensity of the delayed band didnot grow. The
condition I used is list below:
1. the concentration of protein is about 1.5mg/ml, buffer in Tris-Cl, PH 8
and NaCl.
2. the running buffer for agarose gel is 0.5x TBE, PH 8.3.
3. different ratio of protein: DNA has tried, from 2:1 to 1:2.
4. different concentration of NaCl, MgCl2 and ATP in reaction system have
been added, but no significant change.

I wonder if there is any way to increase the binding efficiency? Is it
possible to set up crystal plates in this situation, with protein and
complex together?
Any suggestion would be appreciated!

Best
Yang

Re: [ccp4bb] Thank you so much how to optimize crystallization of a membrane protein

[qiangm zhang]

Thank you for all of your suggestions. I think Pascal Egea has already
summarized the points, and of course Edwin Pozharshi etc. Now I am trying to
confirm if this crystal is detergent or not either using uv microscope or
mass-spec. I also ordered Molecular dimensions stuff. Yes, I noticed that
there are very good suggestions in Emerald biosystem webpage for
crystallization of membrane proteins. Hope this topic is also working for
every people. Again, thanks


With all my best wishes to you



2010/9/1 Pascal Egea 

> Hi Qiangmin,
>
> All the comments and references that were already mentioned to you are
> excellent,
>
> I would stress 3 points.
> 1- The detergent.
> A clear distinction should be made between the detergent used for
> extraction/solubilization and the detergent (or cocktail of
> detergents/lipids) used for crystallization. These are two very different
> things.
> If you are lucky you may not need to change, but once you have extracted
> your rmembrane protein in one detergent, you should try to characterize its
> homogeneity by size exclusion chromatography in different detergents ( with
> shorter or longer chains and/or belonging to a different class (change from
> a choline or a phospho-glycero-lipid to an alkyloside or from a charged to
> an uncharged detergent etc). This scouting is tedious but is extremely
> informative and it can be done on analytical scales (so it does not require
> too much protein).
>
> If you like statistics about detergent use you can look there.
> *http://www.mpdb.tcd.ie/*
>
> depending on the class of membrane protein beta-barrel versus all-alpha
> helical etc etc you can initially concentrate your efforts on a subset of
> detergents.
>
> 2- The diffraction.
> As mentioned, starting with very poorly diffracting crystals is not
> uncommon (as it is for RNA crystals). My own personal experience is that you
> can get from 40 A resolution to the dreadful 6-4.0 A resolution barrier by
> tweaking the purification/extraction conditions (1/ changing detergent
> (shorter chain) and 2/ carefully controlling the amount of detergent present
> in the sample used for crystallization (to avoid or at least minimize the
> phase separation problem)).
>
> 3- The cryo conditions.
> Crystallization drops in presence of detergent are actually not as
> homogenous at it seems. Within the same drop you may have crystals of
> identical size and morphology and freeze them in the same condition and
> still get very different diffraction limits. When you freeze your
> crystals matching the detergent concentration in your cryo-condition with
> the 'expected' concentration in the drop  can be extremely important
> especially with alkylosides (personal experience).
>
> 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
>
>


-- 
张强敏

[ccp4bb] Post-doctoral position available at IBS - please do not reply to me !

[Vellieux Frederic]

POST-DOCTORAL POSITION

DATE OF AVAILABILITY : starting as soon as possible, for 2 years (ANR 
contract).


TITLE : purification and crystallization of ABC transporters

POSITION: the position is opened at the Institute for Structural Biology 
(IBS) in Grenoble in the team of Jean-Michel Jault and will be carried 
out in the framework of a collaborative project between chemists and 
biologists, both in Lyon (IBCP and UCB) and in Grenoble (IBS and UJF) 
with funding from an ANR grant.


JOB DESCRIPTION: Multi-Drug Resistance (MDR) is an increasingly 
challenging problem in the biomedical field and is widespread from 
tumour cells to microbes. In bacteria, resistance to multiple 
antibiotics is often associated with the presence of MDR pumps and 
several of these belong to the largest superfamily of membrane proteins, 
the ABC (“ATP-Binding Cassette”) transporters, which include some human 
members whose dysfunction is involved in severe pathologies (cancer, 
cystic fibrosis, adrenoleukodystrophy…). This project will focus on two 
bacterial ABC transporters that we routinely purify in high yields in 
their functional states, with the goal to crystallize them and solve 
their 3-D structure, using both classical detergents and newly 
synthesized ones.


JOB REQUIREMENTS: the applicant should hold a PhD in Biochemistry and 
have a strong background in protein biochemistry (purification and 
biochemical/biophysical characterisation) and in protein 
crystallography. Additional knowledge in "membrane proteins" will be 
appreciated.


SCIENTIFIC ENVIRONMENT: the Institut de Biologie Structurale (Institute 
for Structural Biology) is located in the Grenoble “Polygone 
Scientifique” area, in the close vicinity of the EMBL outstation, ESRF 
and ILL, i.e. it is one of the most renowned scientific area in France 
for biological research. At the IBS, the post-doc will benefit from a 
technical environment devoted to purification, characterization and 
crystallization of membrane proteins (MP3, PAOL and high throughput 
membrane protein crystallization platforms), as well as the scientific 
expertise associated with these. More information available from 
http://www.psb-grenoble.eu/ and http://www.ibs.fr/?lang=en


TO APPLY: please send a CV and the names of two referees to Jean-Michel 
Jault : jean-michel.ja...@ibs.fr

Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

[Pascal Egea]

Hi Qiangmin,

All the comments and references that were already mentioned to you are
excellent,

I would stress 3 points.
1- The detergent.
A clear distinction should be made between the detergent used for
extraction/solubilization and the detergent (or cocktail of
detergents/lipids) used for crystallization. These are two very different
things.
If you are lucky you may not need to change, but once you have extracted
your rmembrane protein in one detergent, you should try to characterize its
homogeneity by size exclusion chromatography in different detergents ( with
shorter or longer chains and/or belonging to a different class (change from
a choline or a phospho-glycero-lipid to an alkyloside or from a charged to
an uncharged detergent etc). This scouting is tedious but is extremely
informative and it can be done on analytical scales (so it does not require
too much protein).

If you like statistics about detergent use you can look there.
*http://www.mpdb.tcd.ie/*

depending on the class of membrane protein beta-barrel versus all-alpha
helical etc etc you can initially concentrate your efforts on a subset of
detergents.

2- The diffraction.
As mentioned, starting with very poorly diffracting crystals is not uncommon
(as it is for RNA crystals). My own personal experience is that you can get
from 40 A resolution to the dreadful 6-4.0 A resolution barrier by tweaking
the purification/extraction conditions (1/ changing detergent (shorter
chain) and 2/ carefully controlling the amount of detergent present in the
sample used for crystallization (to avoid or at least minimize the phase
separation problem)).

3- The cryo conditions.
Crystallization drops in presence of detergent are actually not as
homogenous at it seems. Within the same drop you may have crystals of
identical size and morphology and freeze them in the same condition and
still get very different diffraction limits. When you freeze your
crystals matching the detergent concentration in your cryo-condition with
the 'expected' concentration in the drop  can be extremely important
especially with alkylosides (personal experience).

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

Re: [ccp4bb] Practical MR advice

[David Briggs]

Hi Roger,

I think your ideas are sound, but I would add some "prime-and-switch"
density modification in resolve plus/minus ncs to try and improve the maps
and cut down on phase bias.

Hth,

Dave

--
Hand delivered by Androids

On 1 Sep 2010 16:12, "Roger Rowlett"  wrote:

 I am trying to find a MR solution for a large unit cell (R3:H, 158x158x196)
with a relatively poor, but I think workable search model (30% identity, 50%
similarity). The data set is decent to 2.4 A. I might be able to get better
if necessary. I submitted the  sequence of the target to the Phyre server,
and it returned a PDB file derived from what I had already identified as the
most likely successful search model. Phaser finds a reasonable solution with
four dimers (Z=9-15 depending on the data set) for which the unit cell
packing looks good. Phaser even assembles what looks like "correct"
biological tetramers in the ASU and across the symmetry interfaces. The main
chain appears to be mostly traceable, but the side chains are not all
well-resolved, and I suspect from the better-defined regions, that the chain
is misregistered by a residue or two throughout most of the structure.

Assuming that an MR solution is possible, what is a good approach from here?
FWIW, Phaser does not correctly place a poly-Ala/Gly model, although it may
place three chains similarly to the MR solution I have with the Phyre model.
My first thought is to:

1. Chainsaw the currrent solution, and attempt to identify and build in the
correct register of the side chains. after refinement.
2. Do a low resolution refinement of the poly-Ala/Gly model to better thread
the main chain?
3. Try EPMR with the Phyre model or the poly-Ala/Gly model
4. Give up and get real phases (but I'm so close now!?)

Thanks in advance.

-- 
 --
Roger S. Rowlett
Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@mail.colgate.edu

Re: [ccp4bb] Practical MR advice

[Dirk Kostrewa]

 Hi Roger,

personally, I would take the four dimers from Phaser, strip off all side 
chains (make a poly-ala/gly), do a ML refinement with tight NCS 
restraints. The resulting map could then be on-the-fly 
real-space-averaged with Coot. At your resolution, I would expect to see 
the real register of at least some side chains either in the 2mfodfc-map 
or in one of the real-space-averaged 2mfodfc-maps.


Good luck,

Dirk.

Am 01.09.10 17:12, schrieb Roger Rowlett:
I am trying to find a MR solution for a large unit cell (R3:H, 
158x158x196) with a relatively poor, but I think workable search model 
(30% identity, 50% similarity). The data set is decent to 2.4 A. I 
might be able to get better if necessary. I submitted the  sequence of 
the target to the Phyre server, and it returned a PDB file derived 
from what I had already identified as the most likely successful 
search model. Phaser finds a reasonable solution with four dimers 
(Z=9-15 depending on the data set) for which the unit cell packing 
looks good. Phaser even assembles what looks like "correct" biological 
tetramers in the ASU and across the symmetry interfaces. The main 
chain appears to be mostly traceable, but the side chains are not all 
well-resolved, and I suspect from the better-defined regions, that the 
chain is misregistered by a residue or two throughout most of the 
structure.


Assuming that an MR solution is possible, what is a good approach from 
here? FWIW, Phaser does not correctly place a poly-Ala/Gly model, 
although it may place three chains similarly to the MR solution I have 
with the Phyre model. My first thought is to:


1. Chainsaw the currrent solution, and attempt to identify and build 
in the correct register of the side chains. after refinement.
2. Do a low resolution refinement of the poly-Ala/Gly model to better 
thread the main chain?

3. Try EPMR with the Phyre model or the poly-Ala/Gly model
4. Give up and get real phases (but I'm so close now!?)

Thanks in advance.

--
Roger S. Rowlett
Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@mail.colgate.edu 


--

***
Dirk Kostrewa
Gene Center Munich, A5.07
Department of Biochemistry
Ludwig-Maximilians-Universität München
Feodor-Lynen-Str. 25
D-81377 Munich
Germany
Phone:  +49-89-2180-76845
Fax:+49-89-2180-76999
E-mail: kostr...@genzentrum.lmu.de
WWW:www.genzentrum.lmu.de
***

[ccp4bb] Practical MR advice

[Roger Rowlett]

I am trying to find a MR solution for a large unit
cell (R3:H, 158x158x196) with a relatively poor, but I think workable
search model (30% identity, 50% similarity). The data set is decent to
2.4 A. I might be able to get better if necessary. I submitted the 
sequence of the target to the Phyre server, and it returned a PDB file
derived from what I had already identified as the most likely
successful search model. Phaser finds a reasonable solution with four
dimers (Z=9-15 depending on the data set) for which the unit cell
packing looks good. Phaser even assembles what looks like "correct"
biological tetramers in the ASU and across the symmetry interfaces. The
main chain appears to be mostly traceable, but the side chains are not
all well-resolved, and I suspect from the better-defined regions, that
the chain is misregistered by a residue or two throughout most of the
structure.

Assuming that an MR solution is possible, what is a good approach from
here? FWIW, Phaser does not correctly place a poly-Ala/Gly model,
although it may place three chains similarly to the MR solution I have
with the Phyre model. My first thought is to:

1. Chainsaw the currrent solution, and attempt to identify and build in
the correct register of the side chains. after refinement.
2. Do a low resolution refinement of the poly-Ala/Gly model to better
thread the main chain?
3. Try EPMR with the Phyre model or the poly-Ala/Gly model
4. Give up and get real phases (but I'm so close now!?)

Thanks in advance.

-- 

Roger S. Rowlett
Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@mail.colgate.edu

Re: [ccp4bb] Sftools can not handle non-standard settings?

[Bart Hazes]

Hi Herman,

As a former biomol user you might have guessed why. SFTOOLS found its 
origin as a transitional program helping the Groningen group move to the 
CCP4 mtz format. Since the Groningen MDF and CCP4 mtz had different 
ideas about space group symmetry and, especially, asymmetric unit 
definitions SFTOOLS needed to handle both. Since the biomol space group 
routine was basically a very large spaghetti of nested if-then-elses to 
accommodate all the peculiar choices I chose to reimplement using a 
simple set of symmetry generators and a matrix to define the asymmetric 
unit.


Since there is no longer need to support MDF, sftools could switch to 
use the ccp4 library but my code is used for many other things, 
determining if a reflection is (a)centric, on a symmetry axis, should be 
systematically absent, expected intensity, convertion to standard 
asymmetric unit etc. So this will be a major undertaking. Alternatively, 
you can create a list of symmetry generators and add space groups as 
Claus has apparently already done.


Bart

On 10-09-01 05:39 AM, herman.schreu...@sanofi-aventis.com wrote:

Dear Claus,

Thank you very much for this patch. We will install it, and I hope CCP4
will install it quickly as well ;-). Still I do not understand why
sftools has all symmetry operations hardcoded, while most other programs
use the CCP4 libraries. In that way, sftools would always be up to date
and would not need to be patched.

Best,
Herman

-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Claus Flensburg
Sent: Wednesday, September 01, 2010 1:18 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Sftools can not handle non-standard settings?

Dear Herman,

please find attached a patch for sftools that will add support for the
following non-standard space group settings:

A2, C21, I21, P2122, P2212, P21221, P22121

Note: the number for I21 follows the upcomming change to
syminfo.lib: 3004 ->  5005.

diffstat -p0<
CCP4-20091104-src-sftools_-sftools.f-Add-some-non-standard-spgrps-v1.pat
ch
  src/sftools_/sftools.f |   52
-
  1 files changed, 35 insertions(+), 17 deletions(-)

The patch applies equally well to series-6_1 and trunk.


Regards,

ClAuS

P.S. After applying the patch and compiling sftools, you can use it in
BUSTER with this command line option:

% refine autoBUSTER_Exe_sftools=/path/to/patched-sftools/sftools ...

On Wed, Sep 01, 2010 at 12:29:55PM +0200,
herman.schreu...@sanofi-aventis.com wrote:
   

Dear CCP4,

In our automated data processing and refinement pipeline, phaser
sometimes comes up with solutions in non-standard settings (e.g. P 21
2 21). These solutions subsequently fail in autobuster and it turned
out that this is because autobuster invokes sftools and sftools
apparently is not able to handle non-standard settings.

I am really puzzled. We upgraded to the latest CCP4 version (6.1.13)
and the symmetry libraries have P 21 2 21 (space group 2018) in them.
Other programs like reindex and coot handle this setting without any
 

problems.
   

Is sftools still supported by ccp4, or should we ask the buster people
 
   

to switch to some other program?

Thank you for your help,
Herman Schreuder
 
   


--



Bart Hazes (Associate Professor)
Dept. of Medical Microbiology&  Immunology
University of Alberta
1-15 Medical Sciences Building
Edmonton, Alberta
Canada, T6G 2H7
phone:  1-780-492-0042
fax:1-780-492-7521

Re: [ccp4bb] Is 2mF-DFc the best map possible? Was: Is the difference between mFo and Fo maps supposed to be very small?

[Ed Pozharski]

Following Ian's excellent comment, do I understand correctly that
2mFo-DFc is the maximum likelihood estimate of the full model map (i.e.
the best map possible) or it's simply modification of 2Fo-Fc map where
plain Fo/Fc are replaced by their maximum likelihood estimates?  Other
words, is k=2 the maximum likelihood estimate of the best approximation
of the true map in the following form

DFc + k*(mFo-DFc)

Ed.

On Wed, 2010-09-01 at 10:49 +0100, Ian Tickle wrote:
> On Wed, Sep 1, 2010 at 4:26 AM, Ed Pozharski  wrote:
> > The
> > reason you see the missing region in (2mFo-DFc) map is because it is
> > effectively the sum of model map (mFo) which shows you the parts of the
> > model you have already placed and difference map (mFo-DFc) which shows
> > you the regions which are still missing.
> 
> This is not true.  The 'model map' (i.e. the map calculated from the
> model) is obviously the one with coefficient DFc.  The mFo map
> represents the model (i.e. the structure already placed) + *half* of
> the missing structure (represented by mFo-DFc), for acentric
> reflections.  To get the 'minimally biased' map you have to make it up
> by adding the other half of the missing structure so we have (for
> acentrics):
> 
> 2mFo-DFc = DFc + (mFo-DFc) + (mFo-DFc)
> = DFc + 2(mFo-DFc)
> 
> For centrics mFo represents the model + *all* of the missing
> structure, so in that case no further contribution is needed,
> 
> We had this discussion a while back: it seems to me that it is
> precisely this confusion that is engendered by thinking in terms of
> 2mFo-DFc = mFo + (mFo-DFc).
> 
> Cheers
> 
> -- Ian

-- 
"I'd jump in myself, if I weren't so good at whistling."
   Julian, King of Lemurs

Re: [ccp4bb] Is the difference between mFo and Fo maps supposed to be very small?

[Ed Pozharski]

Ian is, as always, absolutely right.  The only comment/correction I have
is that Hailang was apparently referring to severely incomplete model,
for which the poor phases will dominate the mFo map.  Under such
circumstances, even 2fo-fc map will not correctly reflect the actual
relative contribution of the missing and already modeled regions into
electron density.

On Wed, 2010-09-01 at 10:49 +0100, Ian Tickle wrote:
> This is not true.  The 'model map' (i.e. the map calculated from the
> model) is obviously the one with coefficient DFc.  The mFo map
> represents the model (i.e. the structure already placed) + *half* of
> the missing structure (represented by mFo-DFc) 
-- 
"I'd jump in myself, if I weren't so good at whistling."
   Julian, King of Lemurs

Re: [ccp4bb] Sftools can not handle non-standard settings?

[Herman . Schreuder]

Dear Claus,

Thank you very much for this patch. We will install it, and I hope CCP4
will install it quickly as well ;-). Still I do not understand why
sftools has all symmetry operations hardcoded, while most other programs
use the CCP4 libraries. In that way, sftools would always be up to date
and would not need to be patched.

Best,
Herman

-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Claus Flensburg
Sent: Wednesday, September 01, 2010 1:18 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Sftools can not handle non-standard settings?

Dear Herman,

please find attached a patch for sftools that will add support for the
following non-standard space group settings:

A2, C21, I21, P2122, P2212, P21221, P22121

Note: the number for I21 follows the upcomming change to
syminfo.lib: 3004 -> 5005.

diffstat -p0 <
CCP4-20091104-src-sftools_-sftools.f-Add-some-non-standard-spgrps-v1.pat
ch
 src/sftools_/sftools.f |   52
-
 1 files changed, 35 insertions(+), 17 deletions(-)

The patch applies equally well to series-6_1 and trunk.


Regards,

ClAuS

P.S. After applying the patch and compiling sftools, you can use it in
BUSTER with this command line option:

% refine autoBUSTER_Exe_sftools=/path/to/patched-sftools/sftools ...

On Wed, Sep 01, 2010 at 12:29:55PM +0200,
herman.schreu...@sanofi-aventis.com wrote:
> Dear CCP4,
> 
> In our automated data processing and refinement pipeline, phaser 
> sometimes comes up with solutions in non-standard settings (e.g. P 21 
> 2 21). These solutions subsequently fail in autobuster and it turned 
> out that this is because autobuster invokes sftools and sftools 
> apparently is not able to handle non-standard settings.
> 
> I am really puzzled. We upgraded to the latest CCP4 version (6.1.13) 
> and the symmetry libraries have P 21 2 21 (space group 2018) in them. 
> Other programs like reindex and coot handle this setting without any
problems.
> Is sftools still supported by ccp4, or should we ask the buster people

> to switch to some other program?
> 
> Thank you for your help,
> Herman Schreuder

Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

[Ezra's gmail]

 I cannot say what you have - running a gel, MS, etc of a washed crystal
could confirm what you have.
What you did not indicate is if your lack of diffraction was of frozen
or unfrozen crystals. I have seen too many cases where it is the cryo
condition killing diffraction. So if you have not tried yet - try a wet
mount.

Ezra

On 8/30/2010 9:24 PM, qiangm zhang wrote:
> Hi all,
> I got a crystal of one membrane protein (~60kD) from Na/K phosphate
> condition (see getit_4), then I got the improved crystal like getit_5
> after trying seeding, different detergents, lipids and additives. But
> this crystal does not diffract at all, I already tried Izit staining
> which shows it is protein crystal (detergent crystal?). Does anyone
> have any good suggestions for the optimization of this membrane
> protein crystallization? Thank you in advance.
>
> Best regards
>
> Qiangmin Zhang
>
> Biomedical Science Tower 3
> Room1034
> 3501 Fifth Avenue
> Pittsburgh, PA 15260
>
>
>
>
>
> -- 
> 张强敏

Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

[Ezra's gmail]

 I cannot say what you have - running a gel, MS, etc of a washed crystal
could confirm what you have.
What you did not indicate is if your lack of diffraction was of frozen
or unfrozen crystals. I have seen too many cases where it is the cryo
condition killing diffraction. So if you have not tried yet - try a wet
mount.

Ezra

On 8/30/2010 9:24 PM, qiangm zhang wrote:
> Hi all,
> I got a crystal of one membrane protein (~60kD) from Na/K phosphate
> condition (see getit_4), then I got the improved crystal like getit_5
> after trying seeding, different detergents, lipids and additives. But
> this crystal does not diffract at all, I already tried Izit staining
> which shows it is protein crystal (detergent crystal?). Does anyone
> have any good suggestions for the optimization of this membrane
> protein crystallization? Thank you in advance.
>
> Best regards
>
> Qiangmin Zhang
>
> Biomedical Science Tower 3
> Room1034
> 3501 Fifth Avenue
> Pittsburgh, PA 15260
>
>
>
>
>
> -- 
> 张强敏

Re: [ccp4bb] Sftools can not handle non-standard settings?

[Claus Flensburg]

Dear Herman,

please find attached a patch for sftools that will add support 
for the following non-standard space group settings:

A2, C21, I21, P2122, P2212, P21221, P22121

Note: the number for I21 follows the upcomming change to 
syminfo.lib: 3004 -> 5005.

diffstat -p0 < 
CCP4-20091104-src-sftools_-sftools.f-Add-some-non-standard-spgrps-v1.patch
 src/sftools_/sftools.f |   52 -
 1 files changed, 35 insertions(+), 17 deletions(-)

The patch applies equally well to series-6_1 and trunk.


Regards,

ClAuS

P.S. After applying the patch and compiling sftools, you can use it in BUSTER 
with this command line option:

% refine autoBUSTER_Exe_sftools=/path/to/patched-sftools/sftools ...

On Wed, Sep 01, 2010 at 12:29:55PM +0200, herman.schreu...@sanofi-aventis.com 
wrote:
> Dear CCP4,
> 
> In our automated data processing and refinement pipeline, phaser
> sometimes comes up with solutions in non-standard settings (e.g. P 21 2
> 21). These solutions subsequently fail in autobuster and it turned out
> that this is because autobuster invokes sftools and sftools apparently
> is not able to handle non-standard settings.
> 
> I am really puzzled. We upgraded to the latest CCP4 version (6.1.13) and
> the symmetry libraries have P 21 2 21 (space group 2018) in them. Other
> programs like reindex and coot handle this setting without any problems.
> Is sftools still supported by ccp4, or should we ask the buster people
> to switch to some other program?
> 
> Thank you for your help,
> Herman Schreuder
--- src/sftools_/sftools.f-orig 2008-09-19 09:12:11.0 +0100
+++ src/sftools_/sftools.f  2009-11-04 08:50:05.0 +
@@ -4169,8 +4169,9 @@
 
   implicit none
 
-  integer maxop, maxgrp
-  parameter (maxop=45, maxgrp=73)
+  integer maxop, maxgrp, maxops, maxcen
+  parameter (maxops=45, maxcen=5)
+  parameter (maxop=maxops+maxcen, maxgrp=80)
 
 c subroutine arguments
   integer spnum,klass,nsym,nprim
@@ -4267,7 +4268,15 @@
   data syminf( 70) /'  214 I4132   40   8  25   0   0'/
   data syminf( 71) /' 1146 R3R 25   0   0   0   0'/
   data syminf( 72) /' 1155 R32R25  26   0   0   0'/
-  data syminf( 73) /' 4005 I2   1   0   0   0   0'/
+  data syminf( 73) /' 2005 A2   1   0   0   0   0'/
+  data syminf( 74) /' 3005 C21 41   0   0   0   0'/
+  data syminf( 75) /' 4005 I2   1   0   0   0   0'/
+  data syminf( 76) /' 5005 I21 42   0   0   0   0'/
+  data syminf( 77) /' 1017 P21221  43   0   0   0'/
+  data syminf( 78) /' 2017 P22122  44   0   0   0'/
+  data syminf( 79) /' 2018 P21221   1   7   0   0   0'/
+  data syminf( 80) /' 3018 P22121   8  45   0   0   0'/
+c 5005 (I21) used to be 3004 in syminfo.lib!
 
 c the matrices for each generator
   data (g(n,  1),n=1,12) /-1, 0, 0, 0, 0, 1, 0, 0, 0, 0,-1, 0/
@@ -4310,12 +4319,17 @@
   data (g(n, 38),n=1,12) / 0,-1, 0,18, 1, 0, 0,18, 0, 0, 1, 6/
   data (g(n, 39),n=1,12) / 0,-1, 0,18, 1, 0, 0, 6, 0, 0, 1,18/
   data (g(n, 40),n=1,12) / 0,-1, 0, 6, 1, 0, 0,18, 0, 0, 1, 6/
+  data (g(n, 41),n=1,12) /-1, 0, 0,12, 0, 1, 0, 0, 0, 0,-1, 0/
+  data (g(n, 42),n=1,12) /-1, 0, 0,12, 0, 1, 0, 0, 0, 0,-1,12/
+  data (g(n, 43),n=1,12) / 1, 0, 0,12, 0,-1, 0, 0, 0, 0,-1, 0/
+  data (g(n, 44),n=1,12) / 1, 0, 0, 0, 0,-1, 0,12, 0, 0,-1, 0/
+  data (g(n, 45),n=1,12) / 1, 0, 0, 0, 0,-1, 0, 0, 0, 0,-1, 0/
 c centering operators
-  data (g(n, 41),n=1,12) / 1, 0, 0, 0, 0, 1, 0,12, 0, 0, 1,12/
-  data (g(n, 42),n=1,12) / 1, 0, 0,12, 0, 1, 0, 0, 0, 0, 1,12/
-  data (g(n, 43),n=1,12) / 1, 0, 0,12, 0, 1, 0,12, 0, 0, 1, 0/
-  data (g(n, 44),n=1,12) / 1, 0, 0,12, 0, 1, 0,12, 0, 0, 1,12/
-  data (g(n, 45),n=1,12) / 1, 0, 0,16, 0, 1, 0, 8, 0, 0, 1, 8/
+  data (g(n, maxops+1),n=1,12) / 1, 0, 0, 0, 0, 1, 0,12, 0, 0, 1,12/
+  data (g(n, maxops+2),n=1,12) / 1, 0, 0,12, 0, 1, 0, 0, 0, 0, 1,12/
+  data (g(n, maxops+3),n=1,12) / 1, 0, 0,12, 0, 1, 0,12, 0, 0, 1, 0/
+  data (g(n, maxops+4),n=1,12) / 1, 0, 0,12, 0, 1, 0,12, 0, 0, 1,12/
+  data (g(n, maxops+5),n=1,12) / 1, 0, 0,16, 0, 1, 0, 8, 0, 0, 1, 8/
 
 c point group names according to CCP4
   data pgnams / 'PG1'  ,'PG2'  ,'PG222', 'PG4'  ,'PG422', 'PG3',
@@ -4407,7 +4421,9 @@
 c monoclinic and rhombohedral use the cell parameters to distinguish settings
   if(spnum2.eq.1)then
 klass2 = 1
-  else if((spnum2.ge.3 .and. spnum2.le.5) .or. spnum2.eq.4005)then
+  else if( (spnum2.ge.3 .and. spnum2.le.5) .or. 
+ $ (spnum2.eq.2005 .or. spnum2.eq.3005 .or.
+ $  spnum2.eq.4005 .or. spnum2.eq.5005) ) then
 if(cell(4).ne.90.0)then
   write(*,*)' !!! Illegal, monoclinic A unique is not valid !!!'
   return
@@ -4424,7 +4440,9 @@
   write(*,*)' to work with C-unique (e.g. -3, -4 or -5)'
 endif
  

[ccp4bb] Sftools can not handle non-standard settings?

[Herman . Schreuder]

Dear CCP4,

In our automated data processing and refinement pipeline, phaser
sometimes comes up with solutions in non-standard settings (e.g. P 21 2
21). These solutions subsequently fail in autobuster and it turned out
that this is because autobuster invokes sftools and sftools apparently
is not able to handle non-standard settings.

I am really puzzled. We upgraded to the latest CCP4 version (6.1.13) and
the symmetry libraries have P 21 2 21 (space group 2018) in them. Other
programs like reindex and coot handle this setting without any problems.
Is sftools still supported by ccp4, or should we ask the buster people
to switch to some other program?

Thank you for your help,
Herman Schreuder

Re: [ccp4bb] Is the difference between mFo and Fo maps supposed to be very small?

[Ian Tickle]

On Wed, Sep 1, 2010 at 4:26 AM, Ed Pozharski  wrote:
> The
> reason you see the missing region in (2mFo-DFc) map is because it is
> effectively the sum of model map (mFo) which shows you the parts of the
> model you have already placed and difference map (mFo-DFc) which shows
> you the regions which are still missing.

This is not true.  The 'model map' (i.e. the map calculated from the
model) is obviously the one with coefficient DFc.  The mFo map
represents the model (i.e. the structure already placed) + *half* of
the missing structure (represented by mFo-DFc), for acentric
reflections.  To get the 'minimally biased' map you have to make it up
by adding the other half of the missing structure so we have (for
acentrics):

2mFo-DFc = DFc + (mFo-DFc) + (mFo-DFc)
= DFc + 2(mFo-DFc)

For centrics mFo represents the model + *all* of the missing
structure, so in that case no further contribution is needed,

We had this discussion a while back: it seems to me that it is
precisely this confusion that is engendered by thinking in terms of
2mFo-DFc = mFo + (mFo-DFc).

Cheers

-- Ian

Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

[Joseph Lyons]

When diffraction fails - one can always look at the crystals with a UV
microscope (assuming you have Trp) - they should light up. Or if its
possible - harvest them, wash and run a gel. (or if its detergent a TLC)

Joe



2010/9/1 wu donghui 

> It does not look like protein crystal and IZIT staining is not reliable in
> determining protein or other. Mostly it is like detergent or PEG crystal or
> quasi-crystal.
>
> Good  luck
>
> Wu
>
> 2010/8/31 qiangm zhang 
>
>  Hi all,
>>
>> I got a crystal of one membrane protein (~60kD) from Na/K phosphate
>> condition (see getit_4), then I got the improved crystal like getit_5 after
>> trying seeding, different detergents, lipids and additives. But this crystal
>> does not diffract at all, I already tried Izit staining which shows it is
>> protein crystal (detergent crystal?). Does anyone have any good suggestions
>> for the optimization of this membrane protein crystallization? Thank you in
>> advance.
>>
>> Best regards
>>
>> Qiangmin Zhang
>>
>> Biomedical Science Tower 3
>> Room1034
>> 3501 Fifth Avenue
>> Pittsburgh, PA 15260
>>
>>
>>
>>
>>
>> --
>> 张强敏
>>
>
>

Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

[wu donghui]

It does not look like protein crystal and IZIT staining is not reliable in
determining protein or other. Mostly it is like detergent or PEG crystal or
quasi-crystal.

Good  luck

Wu

2010/8/31 qiangm zhang 

>  Hi all,
>
> I got a crystal of one membrane protein (~60kD) from Na/K phosphate
> condition (see getit_4), then I got the improved crystal like getit_5 after
> trying seeding, different detergents, lipids and additives. But this crystal
> does not diffract at all, I already tried Izit staining which shows it is
> protein crystal (detergent crystal?). Does anyone have any good suggestions
> for the optimization of this membrane protein crystallization? Thank you in
> advance.
>
> Best regards
>
> Qiangmin Zhang
>
> Biomedical Science Tower 3
> Room1034
> 3501 Fifth Avenue
> Pittsburgh, PA 15260
>
>
>
>
>
> --
> 张强敏
>