[ccp4bb] Refmac twin refine works for Amplitude based but not Intensity based

[Xiao Lei]

Dear CCP4bb members,

I have a dataset of 3A, twinned data (twin fraction 0.15) set of space
group P6222, I solved the structure by phaser (TFZ>10, LLG>1000) with space
group P32 and build model to Rfree/R 30% 26%.  I then tried twin refinement
in Refmac, after I tried amplitude based refine the Rfree/R drops to 24%
17% with output files of .mtz map, .pdb and .cif. Everything is normal in
amplitude based refine.

I also tried intensity based twin refine, the log of Refmac shows the
process is successful (I attach below) but only .cif is in output, I could
not find .mtz and .pdb (I even check the path but I still could not find
the files), in addition, there is no report of Rfree/R in the results panel:

**

* Information from CCP4Interface script

***

Writing final coordinates (XYZOUT) to XXX.pdb

***

***

* Information from CCP4Interface script

***

Writing final phases (HKLOUT) to XXX.mtz

***

#CCP4I TERMINATION STATUS 1

#CCP4I TERMINATION TIME 02 Mar 2017  18:10:04

#CCP4I TERMINATION OUTPUT_FILES  XXX.cif

#CCP4I MESSAGE Task completed successfully


Is this a bug? I use CCP4 7.0.030 in ubuntu.

[ccp4bb] Test system for binding energy predictions

[Sampson, Jared]

Dear crystallographers - 

I've been trying without success to find a suitable system to test a method for 
predicting change in binding energy (delta-delta-G) due to mutations of 
protein-protein complexes that result in a change in net charge.  In particular 
I'm looking for a system with the following features for a single mutation:

* crystal structures and experimental binding affinity data available for both 
wild-type and mutant complexes
* net change of charge of ±1 upon mutation (i.e. charged residue to neutral or 
vice versa--I'm not considering charge flipping at the moment)
* non-trivial conformational change in the region around the mutated residue

If you happen to know of any systems that may qualify, I would appreciate any 
information you could send my way.  

Thank you!

Cheers,
Jared

--
Jared Sampson
Ph.D. Candidate
Honig/Shapiro and Friesner Labs
Columbia University
New York, NY

Re: [ccp4bb] Coot and Pymol 3D in Quadro M4000 Graphics Card under Windows 10

[Xiao Lei]

Hi All,

Just share my experience, Windows 10 works for pymol 3D under Quadro M4000
with Nvidia 3D kit (no need to connect the 3 pin MINI DIN, Quadro M4000
does not have 3 pin Mini DIN connections). I use Asus 24 inch 3D monitor,
connect the monitor with display port to display port of Quadro M4000, no
active DP to DVI needed in my case. We have a problem for Coot 3D works
even when Pymol 3D works ok, we are trying to figure out the problem but
Windows 10 does work for pymol 3D under DP to DP port.

On Tue, Jan 31, 2017 at 6:39 AM, Yong Wang  wrote:

> Xiao,
>
>
>
> I had a direct connection from display port to display port that had
> worked for several years (Windows 7).  Since last year I have been losing
> the stereo.  Often the control panel won’t show the 120 Hz like what you
> saw.  Sometimes I was able to forcefully add a 120 Hz resolution (using the
> “Customize …” menu).  But it was not stable, i.e. the stereo would stay for
> some time then disappear and go back to the state of not having 120 Hz.  I
> have not had time to investigate further into this but I suspect a driver
> issue here.  You may want to check the drivers for both the graphics card
> and the monitor.
>
>
>
> Yong
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Xiao
> Lei
> *Sent:* Monday, January 30, 2017 11:50 PM
> *To:* CCP4BB@JISCMAIL.AC.UK
>
> *Subject:* Re: [ccp4bb] Coot and Pymol 3D in Quadro M4000 Graphics Card
> under Windows 10
>
>
>
> Taka,
>
>
>
> I really appreciate this information! I do have Displayport cable and can
> connect the card and Asus VG248QE both via the DP cable, somehow on the
> Nvidia control panel in Windows 10, I do not have an option of choosing
> "120HZ" or "144Hz", I only have option choosing "60Hz" or below, which
> sounds weird to me. Windows 10 should make things easier not harder. If
> CentOS 6 works then Windows 10 should work, I guess I have to go back to
> Windows 7 if needed, but as I already ordered the DP to DVI active cable,
> I'll test this cable first and update CCP4bb later.
>
>
>
> On Mon, Jan 30, 2017 at 7:50 PM,  wrote:
>
> Xiao,
>
>
>
> If you connect the board and monitor by DisplayPort cable directly, it
> should work.
>
> I confirmed with Quadro M4000 and BenQ XL2420Z on CentOS 6, though not
> tested on Windows.
>
>
>
> Taka
>
>
>
> *From:* Xiao Lei [mailto:xiaolei...@gmail.com]
> *Sent:* Tuesday, January 31, 2017 10:08 AM
> *Cc:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] Coot and Pymol 3D in Quadro M4000 Graphics Card
> under Windows 10
>
>
>
> I changed my mind, I should order the usb- powered DP to DVI dual link...
>
>
>
> On Mon, Jan 30, 2017 at 5:02 PM, Christine Gee  wrote:
>
> Hi Xiao,
>
>
>
> I can confirm you need to buy an active adapter if you want to convert the
> display port on the graphics card to DVI. The one that they supply with the
> graphics card is passive and won't work. I was in the same boat about a
> year ago. I bought a USB powered active adapter which allowed 120htz. My
> monitor only had DVI input so I didn't try a direct display port
> connection. I can't comment on why that didn't work.
>
>
>
> Cheers
>
> Christine
>
>
>
> Sent from my iPhone
>
>
> On Jan 30, 2017, at 4:25 PM, Takaaki Fukami 
> wrote:
>
> Dear Xiao,
>
>
>
> You need an active converter from DP to DVI.  You can't get 120Hz if you
> use a passive converter, even with a DVI dual link cable.  If you used an
> adapter come with the board, it's probably passive.
>
>
>
> Taka
>
>
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK
> ] *On Behalf Of *Xiao Lei
> *Sent:* Tuesday, January 31, 2017 9:10 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] Coot and Pymol 3D in Quadro M4000 Graphics Card under
> Windows 10
>
>
>
> Dear All,
>
>
>
> I tried to make Coot and Pymol 3D working for a HP Workstation with Quadro
> M4000 graphics card, the card has four displayport. I also has a Asus 24
> inch 3D monitor. I tried to connect  the graphics card with the monitor
> through displayport to displayport connection and displayport to DVI-D dual
> link connection but failed to set the monitor run on 120Hz. It seems I have
> to follow the old way of using  DVI-D dual link connection for both graphic
> card and monitor, but the problem is that the graphics card does not have a
> DVI-D dual link socket, it only have displayport.
>
>
>
> I appreciate any suggestions.
>
>
>
>
>
>
>
>
>

Re: [ccp4bb] How to refine this needle crystal of membrane complex?

[Keller, Jacob]

I'll add another favorite to that one (below.) I especially enjoy the tracking 
of the intensities of the Bragg *rods* along their lengths, showing how the 2D 
lattice only chops reciprocal space in two dimensions, leaving the third 
dimension continuous.

JPK

Nature. 1975 Sep 4;257(5521):28-32.
Three-dimensional model of purple membrane obtained by electron microscopy.
Henderson R, Unwin PN.
Abstract
A 7-A resolution map of the purple membrane has been obtained by electron 
microscopy of tilted, unstained specimens. The protein in the membrane contains 
seven, closely packed, alpha-helical segments which extend roughly 
perpendicular to the plane of the membrane for most of its width. Lipid bilayer 
regions fill the spaces between the protein molecules.
PMID: 1161000



-Original Message-
From: Phoebe A. Rice [mailto:pr...@uchicago.edu] 
Sent: Wednesday, March 01, 2017 3:16 PM
To: Keller, Jacob ; CCP4BB@JISCMAIL.AC.UK
Subject: RE: [ccp4bb] How to refine this needle crystal of membrane complex?

For historical perspective: 

Molecular structure determination by electron microscopy of unstained 
crystalline specimens PNT Unwin, R Henderson - Journal of molecular biology, 
1975
http://www.sciencedirect.com/science/article/pii/0022283675902120


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Keller, Jacob 
[kell...@janelia.hhmi.org]
Sent: Wednesday, March 01, 2017 1:50 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] How to refine this needle crystal of membrane complex?

There are now quite a few structures solved by ED of 3D crystals, esp by Tamir 
Gonen's group here at Janelia, and a quick count in the pdb yields ~15 
3D-crystal ED structures, some of which are not released. They're getting 
better at it all the time. I guess the news has not spread as much as I 
thought. FYI, last I checked they were looking for good samples to try, 
although you'd have to contact Tamir about that to see whether it's still true, 
or whether they've now got their hands full.

All the best,

Jacob Keller





-Original Message-
From: Tim Gruene [mailto:tim.gru...@psi.ch]
Sent: Wednesday, March 01, 2017 2:22 PM
To: Keller, Jacob 
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] How to refine this needle crystal of membrane complex?

Dear Jacob, dear TO,

(I have wondered what can be 'micro' about diffraction - would you know?)

it would be very exciting to get the structure by electron diffraction - as 
much as I know it would be the first new structure solved from a 3D crystal 
with electron radiation in the PDB. You would have to contact one of the 3-4 
labs that to electron diffraction of protein samples (Abrahams, Gonen, 
Hovmoeller / Zou (possibly), and Yonekura/Toyoshima in alphabetic order). The 
first and the last group are equipped with sensitive hybrid pixel detectors 
that might help.

A good test to get an idea about the resolution you might get from electron 
diffraction is to scratch up as many of your crystals and analyse the powder 
pattern with a strong X-ray source (synchrotron, or very good home source).

Note that when you can see the crystals with a light microscope, they are most 
likely too big for electron diffraction - you might end up seeing a black 
screen with no electron penetrating your samples.

Best regards,
Tim

On Wednesday, March 1, 2017 1:59:44 PM CET Keller, Jacob wrote:
> Although trying to refine the crystals is perhaps the most 
> straightforward thing, you might consider trying micro electron 
> diffraction (micro-ED) with the existing samples-the technique is 
> working increasingly better, and would work well with your tiny 
> crystals. Look at Tamir Gonen's recent papers for more info.

> All the best,
>
> Jacob Keller
>
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ???
> Sent: Wednesday, March 01, 2017 3:53 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] How to refine this needle crystal of membrane complex?
>
> Dear all,
> I am trying to obtain crystals of membrane complex. Now, I have 
> obtained the complex with appropriate stoichiometry after a series of 
> purification in DDM, then I get extremely thin needles (as shown in
> figure) in  some conditions such as 28% MPD, 0.1 M NaAc pH 4.5 or 45%
> MPD,0.1 M Bis-Tris pH 6.5, 0.2 M CaCl2. However, there are not any 
> improvements through changing the concentration of precipitants/salts 
> or adding the additives/detergents to the protein.

> I would be very happy if anybody could give me some advice about this 
> crystal refinement.

> With best regards.
>
>
> [cid:image001.png@01D2926A.28CB0C70]

--
--
Paul Scherrer Institut
Tim Gruene
- persoenlich -
OFLC/104
CH-5232 Villigen PSI
phone: +41 (0)56 310 5297

GPG Key ID = A46BEE1A

Re: [ccp4bb] Dimer in SDS-PAGE

[Bert Van-Den-Berg]

Hi Rich,


I'm not sure but I imagine it would also apply to single span proteins.

I have never encountered a helical membrane protein that could be boiled w/o 
aggregating, even very stable ones. Of course, a more elaborate sample 
preparation involving boiling, sonication and urea as mentioned by Ruud might 
work for some proteins.


Bert



From: CCP4 bulletin board  on behalf of Richard Berry 

Sent: 02 March 2017 05:50
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Dimer in SDS-PAGE

Dear Bert
You made the comment a few weeks ago not to boil helical membrane proteins for 
SDS-PAGE. Could i please ask, does this also apply to type I membrane proteins 
that only have a single a-helix, or is it just membrane proteins that are 
predominantly helical?
Thanks
Rich

On 22 February 2017 at 19:18, Bert Van-Den-Berg 
> 
wrote:

like others I'm not clear why you care where your protein runs on SDS-PAGE. I 
think the band you're seeing is in fact the tetramer, suggesting your protein 
(like KcsA) is very stable. Helical membrane proteins often migrate faster than 
expected (by their Mw) on SDS-PAGE.

Also, never boil helical membrane protein samples, they will aggregate.


bert



From: CCP4 bulletin board > 
on behalf of amit gaur >
Sent: 21 February 2017 22:22
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Dimer in SDS-PAGE

Hi all,
  I am trying to purify a potassium ion channel from insect cell using 
baculovirus expression system. I am not seeing monomer of this protein in SDS 
instead a dimer appears.So,I increased DTT in SDS buffer but no change and 
dimer was intact. In size exclusion this protein appeared as a tetramer which 
is common oligomerizaton of potassium channel family with GYG motif. Can any 
body suggest what should I do in this case?

Thanks and regards,


--
Dr. Amit Gaur
Post Doctoral Researcher
PI: Dr. Ji-Fang Zhang
Thomas Jefferson University
1020, Locust Street, Suite 418
Philadelphia, PA 19107





--

--

Dr Richard Berry
NHMRC Career Development Fellow
Department of Biochemistry and Molecular Biology
Monash University
Ground Floor, Building 76, ClaytonCampus
Blackburn Road
Clayton VIC 3800
Australia

T: +61 3 9902 9239
E: richard.be...@monash.edu

Re: [ccp4bb] Crystallization with no precipitant

[Enrico Stura]

Dear ccp4bb,

1. Why would you need a precipitant to crystallize a protein?
The principle of salting-in/salting-out means that as you remove the salt  
that
keeps a protein soluble, the protein will tend to come out of solution and  
given

the right conditions, crystallize.

2. From the solubility curve as you increase protein concentration, you  
will reach a point at which
your protein will become supersaturated and possibly crystallize. You just  
evaporate the water.


3. Protein solubility is temperature dependent:
Huang M., Syed R., Stura E.A., Stone M.J., Stefanko R.S., Ruf W.,  
Edgington T.S. & Wilson I.A. (1998) The mechanism of an inhibitory  
antibody on TF-initiated blood coagulation revealed by the crystal  
structures of human tissue factor, Fab 5G9 and TF.5G9 complex. J Mol Biol.  
275:873-894.

Crystallized in the NMR tube when it was stored in the fridge
 De Halleux S., Stura E.A., VanderElst L., Carlier V., Jacquemin M. &  
Saint-Remy J.-M. (2006) Three-dimensional structure and IgE-binding  
properties of mature fully active Der p 1, a clinically relevant major  
allergen. J. Allergy Clin. Immunol.  11: 571-576.
The unglycosylated protein has low solubility - Concentration to 3.5 mg/ml  
and waiting for it to crystallize in the fridge.
Stura E.A., Visse R., Vera L., Cuniasse P., Dive V. & Nagase H. (2013)  
Crystals structure of full-length human collagenase 3 (MMP-13) with  
peptides in the active site defines exosites in the catalytic domain.  
FASEB J. 27:4395-4405.
A bridging peptide and low temperature induces crystallization - No  
precipitant.


4. A precipitant is an agent that is used by protein crystallogenists to  
avoid using too much protein
in the experiment. If salting-out does not work, PEG is used as a common  
crowding-out agent.

Ideally without a precipitant your protein will be in its most pure state.

5. Maïga A., Vera L., Marchetti C., Lorphelin A., Bellanger L., Mourier  
G., Servent D., Gilles N. & Stura E.A. (2013) Crystallization of  
recombinant Green mamba ρ-Da1a toxin during lyophilization procedure and  
its structure determination. Acta Cryst. F 69: 704-709

What about crystallization during lyophilization?

If you check throughout the literature, you  will finnd many more examples.

Enrico.

--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Proxima-2A, Soleil Synchrotron. Tel: 33 (0)1 69 35 8180 Beamline
http://scholar.google.com/citations?hl=en=Kvm06WIoPAsC=100=pubdate