Re: [ccp4bb] Windows 7 and Xtal Software

[Nian Huang]

There are some windows only software, for example 3ds Max. But you have a
point, you probably don't need linux system in this case.

Nian

On Tue, Aug 30, 2011 at 1:55 AM, Antony Oliver
wrote:

>  Erm, somewhat confused — if you are going to buy a Mac — why would you
> need (or want!)  a triple boot system? It all seems to work just fine on OS
> X.
>
>  Tony.
>
>  On 30 Aug 2011, at 07:38, Nian Huang wrote:
>
> A dual boot laptop is all you need. I always reinstall the windows to get
> rid of bloatware anyway. If you are going to buy a mac, you can also try the
> triple boot, but I don't think anybody is doing it. Although it is very
> convenient, a virtual machine will affect the performance of the software.
> Nowadays booting machine is very fast using a good SSD (under 7 second). So
> it is really not a big trouble comparing before. I am using a sub $400
> laptop, and everything runs really well under Ubuntu including model
> building software.
>
> Nian
>
>
>
> On Sun, Aug 28, 2011 at 9:23 PM, Jacob Keller <
> j-kell...@fsm.northwestern.edu> wrote:
>
>> Dear Crystallographers,
>>
>> are there any additional problems or known issues running ccp4 or
>> other xtal software on windows 7 (beyond those of Vista, etc.?) Your
>> input would be really appreciate before I sink my own personal $$$
>> into a new laptop
>>
>> Jacob Keller
>>
>>
>>
>> --
>> ***
>> Jacob Pearson Keller
>> Northwestern University
>> Medical Scientist Training Program
>> cel: 773.608.9185
>> email: j-kell...@northwestern.edu
>> ***
>>
>
>
>

Re: [ccp4bb] Methods for dehydrating crystals

[Hargreaves, David]

Hi Andrea,

If you haven't already done so it might be worth trying a room temperature 
mount (capillary or in-situ) to get a feeling for how well the crystals 
diffract to start with.

Dave

David Hargreaves
Associate Principal Scientist
_
AstraZeneca
DECS, CP&SS
Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF
Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693
David.Hargreaves @astrazeneca.com
 
Please consider the environment before printing this e-mail


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-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Andrea L 
Edwards
Sent: 26 August 2011 21:53
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Methods for dehydrating crystals

Hi all,

What are the most successful methods you know of for dehydrating a crystal 
prior to freezing it? I am trying to push the resolution of my crystals.

Thanks,
Andrea

Re: [ccp4bb] Crystals with Organic solvents

[Patrick Shaw Stewart]

Hi Eswar

Firstly, I would certainly try crystal seeding into random screens if
you haven't already tried it.  Refs below.

Secondly, it's very convenient to grow the crystals under oil, and to
soak the organic solvents into the drops, through the oil.  This makes
it much easier to harvest the crystals because the oil becomes
saturated with the solvent and stops it from evaporating when you pick
up the crystals. This approach can be used at the screening stage too.

See Mortuza, et al. High-resolution structure of a retroviral capsid
hexameric amino-terminal domain.  Nature 431 (2004), pp 481-485.  Also
see http://www.douglas.co.uk/winner1.htm

Good luck

Patrick



Allan D’Arcy, Frederic Villarda, May Marsh.  'An automated microseed
matrix-screening method for protein crystallization'.  Acta
Crystallographica section D63 (2007) 550–554.  On-line at
http://scripts.iucr.org/cgi-bin/paper?S0907444907007652

A. G. Villaseñor, A. Wong, A. Shao, A. Garg, A. Kuglstatter and S. F.
Harris. 'Acoustic matrix microseeding: improving protein crystal
growth with minimal chemical bias.' Acta Crystallographica Section D66
(2010) 568-576. On-line at
http://scripts.iucr.org/cgi-bin/paper?S0907444910005512

Galina Obmolova,* Thomas J. Malia, Alexey Teplyakov, Raymond Sweet and
Gary L. Gilliland. 'Promoting crystallization of antibody–antigen
complexes via microseed matrix screening.' Acta Crystallographica
Section D66 (2010) 927–933.  Open-access at
http://journals.iucr.org/d/issues/2010/08/00/bw5361/bw5361.pdf

Patrick D. Shaw Stewart, Stefan A. Kolek, Richard A. Briggs, Naomi E.
Chayen and Peter F.M. Baldock. 'Getting the most out of the random
microseed matrix-screening method in protein crystallization'.  Cryst.
Growth Des., 2011, 11 (8), pp 3432–3441. On-line at
http://pubs.acs.org/doi/abs/10.1021/cg2001442

See also http://www.douglas.co.uk/mms.htm


On Fri, Aug 26, 2011 at 11:55 AM, eswar reddy  wrote:
>
> Dear All
>                     I was working on a Human protein and expression and 
> solubility is good in E.coli  and purification is One step (His-Tag), and i 
> need to cleave the Histag before screens, if not 
> the protein will precipitated and Aggregated, but after trying for 1.2 years 
> i have crystals and they are with Organic solvents, (10 conditions), these 
> crystals are inter grown like broccoli  shaped  and i tried seeding, but it 
> is not successful, and even i tried  with additive screen but the result is 
> the same  is there is any idea to increase the size and shape of 
> my protein crystals.
> Any suggestions will be helpful for me
> Thanks in Advance
>
>



--
 patr...@douglas.co.uk    Douglas Instruments Ltd.
 Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
 Directors: Peter Baldock, Patrick Shaw Stewart

 http://www.douglas.co.uk
 Tel: 44 (0) 148-864-9090    US toll-free 1-877-225-2034
 Regd. England 2177994, VAT Reg. GB 480 7371 36

Re: [ccp4bb] Windows 7 and Xtal Software

[Pete Meyer]

Paul Smith wrote:


2) ditch all gui support or, from scratch, develop a gui front-end that uses 
none of the following: Qt, Ruby, Perl, Python, TK/TCl, etc.  This gui must 
compile and run on all mainstream hardware on all major operating systems.  The 
custom gui might also need a custom driver for maximizing the capabilities of 
modern GPU's for 3D work, but shouldn't make use of any existing 
shading/tiling/rendering methods (like openGL).


I know you were joking, but I'm all in favor of dropping gui's for tasks 
that don't involve dealing with graphical data (models, maps or 
diffraction images).  It's usually faster to learn new software with a 
gui; but it often seems to result in users not reading the documentation 
- which may contribute to newcomers to crystallography using software to 
produce structures without understanding what the magical black box is 
doing (aka the clickey-button effect; which seems to pop up every time 
there's a discussion of a retracted paper/problematic structure).


But I don't have a slide rule, so my opinion on these matters may be 
invalid.


Pete


3) scratch all binary formats (mtz,ccp4map,etc.) due to interoperability 
issues/dependencies.  Everything in flat text (if you like, all variables can 
have four letters and can be followed by a flag/switch consisting of an integer 
or two, perhaps negative, to control software behavior).
4) abandon rapid software development afforded by modular, object oriented 
programming.

Sounds good to me.

Seriously however, I do like how well-coded monolithic executables simply work 
once compiled without fuss.  I also like the speed and power afforded by using 
a well thought out toolkit of practical modules, a la PHENIX.  I guess I can't 
have it all.  Personally, if you really need windows, I second the idea of a VM 
running a linux environment.  It's vastly simpler to install mature linux 
binaries within a VM then fight to get all of modern crystallographic software 
to run under windows.  Even better, the other way around -- run linux native 
and windows in a VM.

For the record:
Shelx is awesome
Fortran is a perfectly good programming language
I keep a slide rule in my desk.

--Paul

Paul Smith, Ph.D. -- p...@smithops.net
- Memorial Sloan-Kettering Cancer Center
- New York Institute of Technology
- www.paladinscientific.com



--- On Mon, 8/29/11, George M. Sheldrick  wrote:


From: George M. Sheldrick 
Subject: Re: [ccp4bb] Windows 7 and Xtal Software
To: CCP4BB@JISCMAIL.AC.UK
Date: Monday, August 29, 2011, 3:12 PM
It is simply a result of the 'zero
dependency' philosophy. In other words, the
exact opposite of current trends in crystallographic
computing (e.g. Phenix/CCTBX).
 
George


On Mon, Aug 29, 2011 at 12:39:16PM -0500, Jacob Keller
wrote:

You know, why does your software always seem so clean?

Was it

something about the punch cards?

Jacob

On Mon, Aug 29, 2011 at 12:29 PM, George M. Sheldrick


wrote:

The current SHELX binaries (including the

beta-test multi-CPU SHELXD) all

appear to run fine under Windows 7. There is no

need to use a virtual box etc.

George

On Sun, Aug 28, 2011 at 11:53:05PM -0700, Nat

Echols wrote:

On Sun, Aug 28, 2011 at 7:23 PM, Jacob Keller



wrote:

are there any additional problems or

known issues running ccp4 or

other xtal software on windows 7

(beyond those of Vista, etc.?)


Phenix, ARP/wARP, and HKL2000 do not run on

Windows.  I'm pretty sure none of

Global Phasing's software does either (aside

from web interfaces).

-Nat

--
Prof. George M. Sheldrick FRS
Dept. Structural Chemistry,
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-3021 or -3068
Fax. +49-551-39-22582




--
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: j-kell...@northwestern.edu
***


--
Prof. George M. Sheldrick FRS
Dept. Structural Chemistry, 
University of Goettingen,

Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-3021 or -3068
Fax. +49-551-39-22582

Re: [ccp4bb] Windows 7 and Xtal Software

[Ed Pozharski]

On Tue, 2011-08-30 at 09:55 -0500, Pete Meyer wrote:
> but I'm all in favor of dropping gui's for tasks 
> that don't involve dealing with graphical data 

second that.  I was about to say "while it is not expected that everyone
practicing crystallography should master the use of command line", but
the question is why not?  It's really not that hard, makes a lot of
tasks easier and as Pete correctly points out, forces users to RTDM.
But the "tablet mentality" will, of course, win.

As for my recent addiction to the SSRL's autoxds script - guilty as
charged. :)

-- 
Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
Julian, King of Lemurs

[ccp4bb] Protein preps become a jelly

[aidong]

Dear Buddies,

Sorry for bothering you with an off-ccp4 question.  We recently are  
experiencing a very strange phenomena.  A couple of protein preps with  
reasonably high concentration (10-20mg/ml) become a jelly after  
storages for overnight or a couple of days at 4C.  All of them have  
been purified by gel filtration.  Some of these proteins behave like  
this from very first preps but some of them had been very kind to us  
previously.  We have googled extensively in CCP4BB and www but it  
appears this only happens to us.  It would be highly appreciated that  
you could exchange their experiences or provide your suggestions.


Aidong Han, Ph.D

Department of Biomedical Sciences
School of Life Sciences
Xiamen University
Xiamen, Fujian 361005
China
Phone: 0592-218-8172 (O)
  0592-218-8173 (L)
Web: http://life.xmu.edu.cn/adhanlab/

Re: [ccp4bb] Protein preps become a jelly

[Laurie Betts]

I have had protein crystals (so very high protein concentration) that turn
into gummy bear-like objects, where instead of crumbling they are like,
well, a gummy bear or a piece of rubber.  I attributed it to oxidation or
other chemical ageing processes.  I am sure others will have suggestions for
preventing this.

On Tue, Aug 30, 2011 at 11:31 AM, aidong  wrote:

> Dear Buddies,
>
> Sorry for bothering you with an off-ccp4 question.  We recently are
> experiencing a very strange phenomena.  A couple of protein preps with
> reasonably high concentration (10-20mg/ml) become a jelly after storages for
> overnight or a couple of days at 4C.  All of them have been purified by gel
> filtration.  Some of these proteins behave like this from very first preps
> but some of them had been very kind to us previously.  We have googled
> extensively in CCP4BB and www but it appears this only happens to us.  It
> would be highly appreciated that you could exchange their experiences or
> provide your suggestions.
>
> Aidong Han, Ph.D
>
> Department of Biomedical Sciences
> School of Life Sciences
> Xiamen University
> Xiamen, Fujian 361005
> China
> Phone: 0592-218-8172 (O)
>  0592-218-8173 (L)
> Web: http://life.xmu.edu.cn/**adhanlab/ 
>

Re: [ccp4bb] waters shell analysis

[Eleanor Dodson]

On 08/26/2011 10:18 AM, REX PALMER wrote:

Once waters have been located and refined is there a program that analyses 
their positions
in terms of solvation shells?
Can the results be compared easily with those from related known
protein structures?

Rex Palmer
http://www.bbk.ac.uk/biology/our-staff/emeritus-staff
http://rexpalmer2010.homestead.com

watertidy did this, albeit in a non-standard PDB nomenclature

It matches waters associated with chain A say into an associated CHAIN R 
say with the matching residue number and a code to indicate whether it 
is linked to N O OG ODi NDi ...


ie a water hydrogen bonded to N A 1 would be labelled OW0 R 1
  a water hydrogen bonded to ND1 A 1  would be labelled OW4 R 1

etc.

Then you can link the atoms in CHAIN R to another set of labels in CHAIN S



However the PDB wont accept this!

Re: [ccp4bb] Protein preps become a jelly

[Roger Rowlett]

  
  
Not necessarily uncommon. To minimize
  protein-protein interactions that might be causing gelation, you
  might change the pH of the solution so that is is not close to the
  pI, use a small amount of chelator to sequester metal ions, and
  maintain a modest ionic strength. Other additives that
might stabilize soluble protein would include glycerol or other
polyols, but these may interfere with crystallization. A typical
storage buffer for us is 20 mM Tris-Cl, pH 8.0, 10 uM EDTA, 100 mM
NaCl. You can add 5-50% glycerol to stabilize protein if desired,
and/or omit the NaCl if you protein is tolerant. It is also possible
your protein is crosslinking via disulfide bond formation, in which
case a modest amount of reducing agent may be necessary, e.g. 10-100
mM beta-ME, 1-10 mM DTT, or a small amount of TCEP. Usually, the
less stuff you put in the storage buffer, the easier it is to
crystallize the protein, but as always, YMMV.

Cheers,

___
Roger S. Rowlett
Gordon & Dorothy Kline 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...@colgate.edu

On 8/30/2011 11:31 AM, aidong wrote:
Dear Buddies,
  
  
  Sorry for bothering you with an off-ccp4 question.  We recently
  are experiencing a very strange phenomena.  A couple of protein
  preps with reasonably high concentration (10-20mg/ml) become a
  jelly after storages for overnight or a couple of days at 4C.  All
  of them have been purified by gel filtration.  Some of these
  proteins behave like this from very first preps but some of them
  had been very kind to us previously.  We have googled extensively
  in CCP4BB and www but it appears this only happens to us.  It
  would be highly appreciated that you could exchange their
  experiences or provide your suggestions.
  
  
  Aidong Han, Ph.D
  
  
  Department of Biomedical Sciences
  
  School of Life Sciences
  
  Xiamen University
  
  Xiamen, Fujian 361005
  
  China
  
  Phone: 0592-218-8172 (O)
  
    0592-218-8173 (L)
  
  Web: http://life.xmu.edu.cn/adhanlab/
  

  

Re: [ccp4bb] Protein preps become a jelly

[Patrick Loll]

Certainly not unprecedented, or even that unusual (I remember making gels from 
BSA and IgG solutions during grad school rotations).  Gel formation usually 
requires crosslinking, so consider whether you might be getting adventitious 
disulfide bond formation.
Pat

On 30 Aug 2011, at 11:31 AM, aidong wrote:

> Dear Buddies,
> 
> Sorry for bothering you with an off-ccp4 question.  We recently are 
> experiencing a very strange phenomena.  A couple of protein preps with 
> reasonably high concentration (10-20mg/ml) become a jelly after storages for 
> overnight or a couple of days at 4C.  All of them have been purified by gel 
> filtration.  Some of these proteins behave like this from very first preps 
> but some of them had been very kind to us previously.  We have googled 
> extensively in CCP4BB and www but it appears this only happens to us.  It 
> would be highly appreciated that you could exchange their experiences or 
> provide your suggestions.
> 
> Aidong Han, Ph.D
> 
> Department of Biomedical Sciences
> School of Life Sciences
> Xiamen University
> Xiamen, Fujian 361005
> China
> Phone: 0592-218-8172 (O)
>  0592-218-8173 (L)
> Web: http://life.xmu.edu.cn/adhanlab/

Re: [ccp4bb] Windows 7 and Xtal Software

[Roger Rowlett]

  
  
Personally, I like having the GUI front end for
  training and education, especially for undergraduates. It has made
  protein XRD much more accessible as a tool for many labs that
  would otherwise find the barrier for entry very high. In the "old
  days,"--8 years ago (ha ha)--scripting from the command line was
  pretty much the only way to run most protein XRD software. I think
  scripting is much more powerful, and allows for a nicely pipelined
  and controlled refinement workflow, but it is easier to train
  undergraduate students, and get them productive quickly, with the
  GUI. The emergence of Coot and the CCP4i front-end was a
godsend for the undergraduate research laboratory. The GUI is kind
of like a checklist for protein XRD tasks. But just because you have
a GUI doesn't mean you should ignore the log files...sometimes my
new students think that is optional. ;)

Having said all that, the thought of running protein XRD software in
Windows (except when that is the only option, e.g. CrysalisPro) is
not a cheery one: Windows scripting capability is clumsy, and boy,
is it ever slow! At one point, I think I timed a typical Phaser or
EPMR job we ran at 4X slower in WinXP than in Linux. It was the
difference between about an afternoon and overnight. Computers are
faster now... :)

Cheers,

___
Roger S. Rowlett
Gordon & Dorothy Kline 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...@colgate.edu

On 8/30/2011 11:22 AM, Ed Pozharski wrote:

  On Tue, 2011-08-30 at 09:55 -0500, Pete Meyer wrote:

  
but I'm all in favor of dropping gui's for tasks 
that don't involve dealing with graphical data 

  
  
second that.  I was about to say "while it is not expected that everyone
practicing crystallography should master the use of command line", but
the question is why not?  It's really not that hard, makes a lot of
tasks easier and as Pete correctly points out, forces users to RTDM.
But the "tablet mentality" will, of course, win.

As for my recent addiction to the SSRL's autoxds script - guilty as
charged. :)



  

Re: [ccp4bb] Protein preps become a jelly

[Prince, D Bryan]

I had a similar problem with crystals that were obtained from PEG-based
precipitants over long periods of time. If I harvested the crystals in
about 5 days, they would diffract to ~3 Angstrom. If I let them grow any
time past about 7 days, they became PEG-alated and didn't diffract at
all. I personally have not had that happen in salt-based precipitant
conditions. I would try adding up to 10mM DTT or 5mM TCEP to minimize
proteinaceous skin formation, which I believed was crosslinked by the
PEG. Attempts to change PEG's did not work and I couldn't find a salt
based precipitant condition that would give us crystals where the active
site was not occluded by the salt.



BTW, have you purified the prep over SEC followed by IEX or RP columns?
Have you dissolved the gel and run mass spec to see if there is any
proteolysis going on? If you have an active proteolytic fragment, there
may be one or more hydrophobic patches being exposed and this could
cause the protein to gum up in order to bury those patches.



Anyway, good luck!



Bryan



From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Laurie Betts
Sent: Tuesday, August 30, 2011 11:41 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Protein preps become a jelly



I have had protein crystals (so very high protein concentration) that
turn into gummy bear-like objects, where instead of crumbling they are
like, well, a gummy bear or a piece of rubber.  I attributed it to
oxidation or other chemical ageing processes.  I am sure others will
have suggestions for preventing this.

On Tue, Aug 30, 2011 at 11:31 AM, aidong  wrote:

Dear Buddies,

Sorry for bothering you with an off-ccp4 question.  We recently are
experiencing a very strange phenomena.  A couple of protein preps with
reasonably high concentration (10-20mg/ml) become a jelly after storages
for overnight or a couple of days at 4C.  All of them have been purified
by gel filtration.  Some of these proteins behave like this from very
first preps but some of them had been very kind to us previously.  We
have googled extensively in CCP4BB and www but it appears this only
happens to us.  It would be highly appreciated that you could exchange
their experiences or provide your suggestions.

Aidong Han, Ph.D

Department of Biomedical Sciences
School of Life Sciences
Xiamen University
Xiamen, Fujian 361005
China
Phone: 0592-218-8172 (O)
 0592-218-8173 (L)
Web: http://life.xmu.edu.cn/adhanlab/




--
Confidentiality Notice: This message is private and may contain confidential 
and proprietary information. If you have received this message in error, please 
notify us and remove it from your system and note that you must not copy, 
distribute or take any action in reliance on it. Any unauthorized use or 
disclosure of the contents of this message is not permitted and may be unlawful.
 

Re: [ccp4bb] Protein preps become a jelly

[Michael Thompson]

To add to what Pat has mentioned, I work with a protein that has a number of 
exposed Cys residues, and it turns into a gel at 4 degrees within a week, even 
if I store it in buffer with reducing agents. This happens every time I store 
it at 4 degrees. To test if your "jelly" is due to S-S crosslinking, add a 
really huge excess of DTT or beta-mercaptoethanol to a small sample and see if 
it goes back into solution. I usually add 100mM DTT for this test. If it does 
turn out to be disulfide-related, you can try to add reducing agents with 
longer half-lives (ie TCEP instead of DTT), but unfortunately you may just have 
to work with the protein within a couple days of finishing the prep.

Mike




- Original Message -
From: "Patrick Loll" 
To: CCP4BB@JISCMAIL.AC.UK
Sent: Tuesday, August 30, 2011 9:06:52 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] Protein preps become a jelly

Certainly not unprecedented, or even that unusual (I remember making gels from 
BSA and IgG solutions during grad school rotations).  Gel formation usually 
requires crosslinking, so consider whether you might be getting adventitious 
disulfide bond formation.
Pat

On 30 Aug 2011, at 11:31 AM, aidong wrote:

> Dear Buddies,
> 
> Sorry for bothering you with an off-ccp4 question.  We recently are 
> experiencing a very strange phenomena.  A couple of protein preps with 
> reasonably high concentration (10-20mg/ml) become a jelly after storages for 
> overnight or a couple of days at 4C.  All of them have been purified by gel 
> filtration.  Some of these proteins behave like this from very first preps 
> but some of them had been very kind to us previously.  We have googled 
> extensively in CCP4BB and www but it appears this only happens to us.  It 
> would be highly appreciated that you could exchange their experiences or 
> provide your suggestions.
> 
> Aidong Han, Ph.D
> 
> Department of Biomedical Sciences
> School of Life Sciences
> Xiamen University
> Xiamen, Fujian 361005
> China
> Phone: 0592-218-8172 (O)
>  0592-218-8173 (L)
> Web: http://life.xmu.edu.cn/adhanlab/

-- 
Michael C. Thompson

Graduate Student

Biochemistry & Molecular Biology Division

Department of Chemistry & Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu

Re: [ccp4bb] Protein elution in Size Exclusion

[Allan Pang]

Thanks everyone for your response.

The most likely answer to my problem is protein overloaded onto the  
column. I pushed my protein concentration further down to 0.5ml  
instead of the usual method, which to run multiple times on SEC.


Adding NaCl in the buffer may also help, as it seems that the  
broadness of the elution was affected by addition of NaCl.


I don't think the column is knackered because the previous (and  
succeeding) runs of other protein samples were okay.


Different -mer state is a possibility, but something that I am not  
really leaning towards to, since, I managed to purify the protein  
before in a good elution size.


Science mystery solved.

Thanks again!

Allan

Quoting Filip Van Petegem :


worst-case scenario for crystallization purposes:

9. Your protein runs as a mixture of monomers, dimers, trimers,
whatever-mers...

Filip

On Sun, Aug 28, 2011 at 7:24 AM, David Briggs   
wrote:



Following on from Roger's fine suggestions:

8. Your column is knackered. Can you see fine lines or cracks in the
column? Good packing is v.important for SEC columns.

HTH,

Dave



David C. Briggs PhD
Father, Structural Biologist and Sceptic

University of Manchester E-mail:
david.c.bri...@manchester.ac.uk

http://manchester.academia.edu/DavidBriggs (v.sensible)
http://xtaldave.wordpress.com/ (sensible)
http://xtaldave.posterous.com/ (less sensible)
Twitter: @xtaldave
Skype: DocDCB




On 28 August 2011 10:25, Allan Pang  wrote:
> Hi there everyone,
>
> What does it mean when you have proteins eluting in almost the whole
column
> volume of S200?
>
> I ran a gel with fractions from 8ml to 20ml and saw band for my protein
all
> throughout.
>
> Judging peaks on chromatogram is not useful as it doesn't have any
aromatic
> rings.
>
> Cheers,
>
> Allan
>
> --
> Allan Pang
>
> PhD Student
>
> G35 Joseph Priestley Building
> Queen Mary University of London
> London
> E1 4NS
>
> Phone number: 02078828480
>





--
Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3

phone: +1 604 827 4267
email: filip.vanpete...@gmail.com
http://crg.ubc.ca/VanPetegem/





--
Allan Pang

PhD Student

G35 Joseph Priestley Building
Queen Mary University of London
London
E1 4NS

Phone number: 02078828480

[ccp4bb] twinning in hexagonal system

[john peter]

Hello All,

 This is regarding twinning in a data set.

I collected a native data set  to resolution, 1.8 A.  I used XDS suite
to process and scale the data set. It scaled well in P622 and I found
systematic absence (l=6n present).

Hence thought the space group may be P6122/P6522. SFCHECK  did not
show any twinning and also it did not detect pseudo-translation.  Twin
test in http://nihserver.mbi.ucla.edu/pystats/ ( Merohedral Twin
Detector: Padilla-Yeates Algorithm ) showed  perfect twinning.

Scaled in P61/65 and  SFCHECK reported  twinning fraction 0.272  & no
pseudo-translation.
http://nihserver.mbi.ucla.edu/pystats/ ( Merohedral Twin Detector:
Padilla-Yeates Algorithm ) showed  perfect twinning.
Another server from ucla  http://nihserver.mbi.ucla.edu/Twinning/
showed partial twinning  with twin fraction 0.23 as follows.

2 along a, b, a*, b*

No. Twin Law Related Reflections = 18033 (pairs)
No. Twin Law Pairs Considered = 9016

 = 0.266149
 = 0.095303

Twin Fraction = 0.233249 +/- 0.000602

(SHELXL Commands: TWIN 1 0 0 -1 -1 0 0 0 -1 and BASF 0.233249)


In P61/65, I got the following matthews-coeffs

mol/asym  Matthews Coeff  %solvent   P(1.73) P(tot)
_
  19.7587.39  .00  .00
  24.8874.79  .00  .01
  33.2562.18  .06  .14
  4 2.4449.57  .57  .63
  5 1.9536.97  .36  .21
  6 1.6324.36  .00  .00
  7 1.3911.75  .00  .00
_


May I ask what could be the real twin fraction and what is the
likelihood of solving the structure by molecular replacement by models
with 25 % sequence identity and 30 % sequence similarity.

Thank you so much for reading this mail during your  busy hours and
all suggestions, comments would be gratefully welcome &  appreciated.

thank you ccp4 mailing list.

John

Re: [ccp4bb] twinning in hexagonal system

[Garib N Murshudov]

Hello

Space groups with point groups 622 and 432 merohedral twinning is not possible 
(they are the highest groups possible for proteins). 
If you could merge in 622 it means that Rmerge was very small. It is very 
likely that point group is either 622 or 6 with very strong rotational symmetry 
that is perpendicular to 6 fold axis. In these cases H test (sfcheck based on 
this) and N(z) test (truncate is based on this) will overestimate (H) or 
underestimate (N(z)) if twin is present. At the moment better test for these 
cases seems to be L-test (that is in ctruncate I think).

Solving structure using molecular replacement in the presence of twinning does 
not seem to be a problem (unless data are very noisy and/or model is too far). 
Pointless gives very good idea about "true" space group. 
I would solve in P6_{x} space groups and then run zanuda (from ysbl server) to 
correct space group.

I hope it helps

regards
Garib


On 30 Aug 2011, at 20:31, john peter wrote:

> Hello All,
> 
> This is regarding twinning in a data set.
> 
> I collected a native data set  to resolution, 1.8 A.  I used XDS suite
> to process and scale the data set. It scaled well in P622 and I found
> systematic absence (l=6n present).
> 
> Hence thought the space group may be P6122/P6522. SFCHECK  did not
> show any twinning and also it did not detect pseudo-translation.  Twin
> test in http://nihserver.mbi.ucla.edu/pystats/ ( Merohedral Twin
> Detector: Padilla-Yeates Algorithm ) showed  perfect twinning.
> 
> Scaled in P61/65 and  SFCHECK reported  twinning fraction 0.272  & no
> pseudo-translation.
> http://nihserver.mbi.ucla.edu/pystats/ ( Merohedral Twin Detector:
> Padilla-Yeates Algorithm ) showed  perfect twinning.
> Another server from ucla  http://nihserver.mbi.ucla.edu/Twinning/
> showed partial twinning  with twin fraction 0.23 as follows.
> 
> 2 along a, b, a*, b*
> 
> No. Twin Law Related Reflections = 18033 (pairs)
> No. Twin Law Pairs Considered = 9016
> 
>  = 0.266149
>  = 0.095303
> 
> Twin Fraction = 0.233249 +/- 0.000602
> 
> (SHELXL Commands: TWIN 1 0 0 -1 -1 0 0 0 -1 and BASF 0.233249)
> 
> 
> In P61/65, I got the following matthews-coeffs
> 
> mol/asym  Matthews Coeff  %solvent   P(1.73) P(tot)
> _
>  19.7587.39  .00  .00
>  24.8874.79  .00  .01
>  33.2562.18  .06  .14
>  4 2.4449.57  .57  .63
>  5 1.9536.97  .36  .21
>  6 1.6324.36  .00  .00
>  7 1.3911.75  .00  .00
> _
> 
> 
> May I ask what could be the real twin fraction and what is the
> likelihood of solving the structure by molecular replacement by models
> with 25 % sequence identity and 30 % sequence similarity.
> 
> Thank you so much for reading this mail during your  busy hours and
> all suggestions, comments would be gratefully welcome &  appreciated.
> 
> thank you ccp4 mailing list.
> 
> John

Garib N Murshudov 
Structural Studies Division
MRC Laboratory of Molecular Biology
Hills Road 
Cambridge 
CB2 0QH UK
Email: ga...@mrc-lmb.cam.ac.uk 
Web http://www.mrc-lmb.cam.ac.uk

Re: [ccp4bb] Protein elution in Size Exclusion

[Roger Rowlett]

  
  
I'm glad this worked out well for you. For
  conducting molecular sizing determinations with a calibrated GEC
  column, I typically use 100-500 uL injections of approximately 1
  mg/mL or so of purified protein on a 1.6 x 60 cm column. The
  limited protein quantity will prevent saturation of the internal
  space of the GEC media, and the limited concentration minimizes
  vicosity effects which hinder mass transfer between the stationary
  and mobile phases. At least 100 mM NaCl is important to prevent
  residual ion exchange interactions with the gel medium. I once
  worked with a protein that completely "disappeared" on a
Sephadex column--lots of protein in, no protein out--at low ionic
strength. When the column was washed with 500 mM NaCl, the protein
magically re-appeared. Usually 100 mM NaCl is sufficient to prevent
this occurrence. 

Interestingly, I had a very similar conversation about GEC with a
colleague and research collaborator just a couple of weeks ago. The
apparent MW of our protein sample was all over the map until the
sample size, concentration, vicosity, and eluant ionic strength were
properly controlled, then the apparent measured MW was consistently
within 10% of the calculated mass of the holoenzyme suggested by
crystallography. This is typical. Keep in mind GEC measure molecular
VOLUME and not MW, but most of the time we make certain assumptions
that make apparent MW measurements "close enough for government
work." Pay attention to the exclusion limits of the medium as well.
Proteins that elute close to the void and total volumes will have
poor precision in either the calibration curve or unknown
determination.

Cheers,

___
Roger S. Rowlett
Gordon & Dorothy Kline 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...@colgate.edu

On 8/30/2011 2:50 PM, Allan Pang wrote:
Thanks everyone for your response.
  
  
  The most likely answer to my problem is protein overloaded onto
  the column. I pushed my protein concentration further down to
  0.5ml instead of the usual method, which to run multiple times on
  SEC.
  
  
  Adding NaCl in the buffer may also help, as it seems that the
  broadness of the elution was affected by addition of NaCl.
  
  
  I don't think the column is knackered because the previous (and
  succeeding) runs of other protein samples were okay.
  
  
  Different -mer state is a possibility, but something that I am not
  really leaning towards to, since, I managed to purify the protein
  before in a good elution size.
  
  
  Science mystery solved.
  
  
  Thanks again!
  
  
  Allan
  
  
  Quoting Filip Van Petegem :
  
  
  worst-case scenario for crystallization
purposes:


9. Your protein runs as a mixture of monomers, dimers, trimers,

whatever-mers...


Filip


On Sun, Aug 28, 2011 at 7:24 AM, David Briggs 
wrote:


Following on from Roger's fine
  suggestions:
  
  
  8. Your column is knackered. Can you see fine lines or cracks
  in the
  
  column? Good packing is v.important for SEC columns.
  
  
  HTH,
  
  
  Dave
  
  
  
  
  
  David C. Briggs PhD
  
  Father, Structural Biologist and Sceptic
  
  
  
  University of Manchester E-mail:
  
  david.c.bri...@manchester.ac.uk
  
  
  
  http://manchester.academia.edu/DavidBriggs (v.sensible)
  
  http://xtaldave.wordpress.com/ (sensible)
  
  http://xtaldave.posterous.com/ (less sensible)
  
  Twitter: @xtaldave
  
  Skype: DocDCB
  
  
  
  
  
  
  On 28 August 2011 10:25, Allan Pang 
  wrote:
  
  > Hi there everyone,
  
  >
  
  > What does it mean when you have proteins eluting in
  almost the whole
  
  column
  
  > volume of S200?
  
  >
  
  > I ran a gel with fractions from 8ml to 20ml and saw band
  for my protein
  
  all
  
  > throughout.
  
  >
 

Re: [ccp4bb] Protein elution in Size Exclusion

[Nian Huang]

Thanks, David. I believe the leaked column was probably my case,
although I didn't see any visible leakage I re-tightened connections
anyway. But the weird thing was that after I equilibrized the column
for a while, the dry patches and cracks disappeared. Everything
returned to normal even a standard ran fine. That's why I suspected
the mixing of reagents in the column has something to do with it in
the beginning.

Nian Huang, Ph.D.
UT Southwestern Medical Center

On Sun, Aug 28, 2011 at 5:14 PM, David Briggs  wrote:
> Hi Nian,
>
> It can be a number of things - but typically, air getting into the
> column, or a leaky seal somewhere letting it dry out.
>
> Switching from 150mM NaCl to 20% EtOH directly shouldn't cause a problem.
>
> The reason I suggest it is that I have had the same situation as
> described in the original post (protein comes off across entire volume
> of SEC column elution) and it turned out the column had a leak and had
> dried out in places.
>
> Repacking the column solved the problem.
>
> Cheers,
>
> Dave
> 
> David C. Briggs PhD
> Father, Structural Biologist and Sceptic
> 
> University of Manchester E-mail:
> david.c.bri...@manchester.ac.uk
> 
> http://manchester.academia.edu/DavidBriggs (v.sensible)
> http://xtaldave.wordpress.com/ (sensible)
> http://xtaldave.posterous.com/ (less sensible)
> Twitter: @xtaldave
> Skype: DocDCB
> 
>
>
>
> On 28 August 2011 22:35, Nian Huang  wrote:
>> Hi, David,
>> What is the common cause of knackered SEC column? Will equilibrizing a
>> buffer containing 150 mM NaCl directly into a 20% EtOH or vise versa cause
>> the problem. There was no problem just after packing the column.
>>
>> Nian
>>
>> On Sun, Aug 28, 2011 at 9:24 AM, David Briggs 
>> wrote:
>>>
>>> Following on from Roger's fine suggestions:
>>>
>>> 8. Your column is knackered. Can you see fine lines or cracks in the
>>> column? Good packing is v.important for SEC columns.
>>>
>>> HTH,
>>>
>>> Dave
>>>
>>>
>>> 
>>> David C. Briggs PhD
>>> Father, Structural Biologist and Sceptic
>>> 
>>> University of Manchester E-mail:
>>> david.c.bri...@manchester.ac.uk
>>> 
>>> http://manchester.academia.edu/DavidBriggs (v.sensible)
>>> http://xtaldave.wordpress.com/ (sensible)
>>> http://xtaldave.posterous.com/ (less sensible)
>>> Twitter: @xtaldave
>>> Skype: DocDCB
>>> 
>>>
>>>
>>>
>>> On 28 August 2011 10:25, Allan Pang  wrote:
>>> > Hi there everyone,
>>> >
>>> > What does it mean when you have proteins eluting in almost the whole
>>> > column
>>> > volume of S200?
>>> >
>>> > I ran a gel with fractions from 8ml to 20ml and saw band for my protein
>>> > all
>>> > throughout.
>>> >
>>> > Judging peaks on chromatogram is not useful as it doesn't have any
>>> > aromatic
>>> > rings.
>>> >
>>> > Cheers,
>>> >
>>> > Allan
>>> >
>>> > --
>>> > Allan Pang
>>> >
>>> > PhD Student
>>> >
>>> > G35 Joseph Priestley Building
>>> > Queen Mary University of London
>>> > London
>>> > E1 4NS
>>> >
>>> > Phone number: 02078828480
>>> >
>>
>>
>

[ccp4bb] Temperature Factor statistics

[Yuri Pompeu]

Quick newbie question, 
After i get my output file from baverage containing the average b-factor and 
rms by residues,
How can I calculate and display the average (and or mean) B-factors?
Is there a way of calculating it by protein, ligands and solvent separately?
thank you

Re: [ccp4bb] Temperature Factor statistics

[Pavel Afonine]

Hi Yuri,

a possible option:

phenix.model_vs_data model.pdb data.mtz will do it. Look for lines like this
in the output:

  ADP (min,max,mean):
all   (136 atoms): 4.497.6   25.3
side chains   (48 atoms): 4.996.8   21.0
main chains   (64 atoms): 4.497.6   28.3
macromolecule (112 atoms): 4.497.6   25.2
ligands   (1 atoms): 6.66.66.6
solvent   (23 atoms): 8.844.1   26.8
  mean bonded (Bi-Bj) : 27.91
  number_of_anisotropic: 0
  number_of_non_positive_definite  : 0

Pavel


On Tue, Aug 30, 2011 at 5:32 PM, Yuri Pompeu  wrote:

> Quick newbie question,
> After i get my output file from baverage containing the average b-factor
> and rms by residues,
> How can I calculate and display the average (and or mean) B-factors?
> Is there a way of calculating it by protein, ligands and solvent
> separately?
> thank you
>

Re: [ccp4bb] Do manufacturers change their crystallogenesis screens?

[Jan Dohnalek]

I have witnessed a change in the Hampton additive screen some years ago - on
purpose - the formulation simply did not work OK. So I guess there are
changes occasionally.

Jan


On Wed, Aug 24, 2011 at 5:21 PM, Chris Morris wrote:

> HI,
>
> I've recently seen two examples where the description of a screen in a
> local database was different to the current one on the manufacturer's web
> site. This happened in two different labs, using different software, and
> with different screen manufacturers.
>
> This could potentially lead to an optimisation screen that finds no hits,
> because the wrong condition is being optimised. Does anyone have experience
> of this? Am I just looking at a few one-off errors, or is there a general
> problem here?
>
> The ideal solution is for screen manufacturers to give version numbers to
> their screens. Failing that, a good fix at the laboratory is to download the
> screen description every time a deep-well plate is received, and second best
> would be to download it every time a trial plate is set up. If there is a
> real concern here, we will implement one of these in xtalPiMS.
>
> Regards,
> Chris
>
> 
> Chris Morris
> chris.mor...@stfc.ac.uk
> Tel: +44 1925 603689  Fax: +44 1925 603825
> Mobile: 07921-717915
> http://pims.instruct-fp7.eu/
> STFC, Daresbury Lab,  Daresbury,  Warrington,  UK,  WA4 4AD
>
>


-- 
Jan Dohnalek, Ph.D
Institute of Macromolecular Chemistry
Academy of Sciences of the Czech Republic
Heyrovskeho nam. 2
16206 Praha 6
Czech Republic

Tel: +420 296 809 390
Fax: +420 296 809 410