Re: [ccp4bb] Difficult Molecular replacement

[Oganesyan, Vaheh]

Thank you!
At the end it appears that Zanuda is not really a "зануда", translated from 
Russian as something being depressing, disagreeable or unsatisfactory.

Vaheh

From: CCP4 bulletin board  on behalf of Randy John Read 

Sent: Wednesday, February 21, 2024 11:55 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Difficult Molecular replacement

Hi,

This is also one of my favourite methods, although I hadn’t realised that you 
could just say “labin F=FC” instead of making fake intensities!

However, at a recent workshop I ran into a case where pointless misled me. This 
was a case with tNCS where one of the translation components was half a unit 
cell edge. As a result, the calculated diffraction data would have alternately 
strong and weak reflections along that axis regardless of whether there was a 
crystallographic 2(1) axis or an NCS translation of 1/2 parallel to that axis. 
Pointless assigned a 2(1) axis where there should have been a pure two-fold. On 
the other hand, Zanuda tried refinement in all the different possibilities and 
only one refined well.

Best wishes,

Randy

> On 21 Feb 2024, at 16:05, Kay Diederichs  
> wrote:
>
> Hi Vaheh,
>
> for this purpose, I use
>
> pointless hklin refmacXY.mtz < labin F=FC
> eof
>
> Thus, pointless determines the space group, including the crystallographic 
> screw axes, from the Fcalc.
>
> Best wishes,
> Kay
>
> On Wed, 21 Feb 2024 15:40:07 +, Oganesyan, Vaheh 
>  wrote:
> ...
>> This might be a silly question, but I do not know the answer: After 
>> refinement in P1 how do I distinguish which axis is crystallographic and 
>> which one in non-crystallographic?
>>
>> Vaheh
>
> 
>
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-
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research Tel: +44 1223 336500
The Keith Peters Building
Hills Road E-mail: rj...@cam.ac.uk
Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk




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Re: [ccp4bb] Difficult Molecular replacement

[Oganesyan, Vaheh]

Hi All,

Interesting discussion as I have a similar case. In my case molecular 
replacement solution can be found easily in P21, P212121, with very similar 
looking electron densities. However, R-factors remain relatively high (mid 
30s). In P1 completeness suffers (75% completeness), maps look decent, R 
factors are in high 20's. Resolution limit is 1.9A with CC1/2 in high res. 
shell ~0.7.
This might be a silly question, but I do not know the answer: After refinement 
in P1 how do I distinguish which axis is crystallographic and which one in 
non-crystallographic?

Vaheh

From: CCP4 bulletin board  on behalf of Randy John Read 

Sent: Wednesday, February 21, 2024 9:18 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Difficult Molecular replacement

Hi,

It’s possible the true space group is P2(1) with the b-axis unique, and that 
subset of true symmetry is found repeatedly with different incorrect 
backgrounds of other copies. But I think the easiest way to resolve this 
unambiguously is to solve in P1, and let that uncover the true symmetry.

Best wishes,

Randy

> On 21 Feb 2024, at 13:56, Pedro Matias  wrote:
>
> But curiously, all the 4 best solutions correspond to a SG with a 21 screw 
> along b.
> And amazingly none of the TF solutions is rejected due to clashes.
> On 21/02/2024 12:20, Eleanor Dodson wrote:
>> Lots of comments, but it would be easier to actually look at your integrated 
>> data!
>> Some of the stats look a bit ropey -
>> 621 reflections labelled as outliers by PHASER?
>> Very anisotropic
>> Moments go mad at the highest resolution..
>>
>> The good news - extremely strong signal from the rotation function means the 
>> model is probably a good one.
>> Bad news - translation function results do not select a definitive solution..
>> Possible reasons
>> Unit Cell: 72.61 73.73 147.23 90.00 90.00 90.00
>> Most likely data problems - a axis ~ = b axis so twinning is possible
>>
>> I could add more comments if either you could share the unmerged data, or at 
>> least a pointless logfile..
>> Cheers Eleanor
>>
>>
>>
>>
>>
>> On Wed, 21 Feb 2024 at 11:06, Randy John Read  wrote:
>> Hi Marco,
>>
>> To add to what Kay has said:
>>
>> The intensity moments from Phaser (between 1.5 and 2 for the second moments 
>> after correcting for anisotropy) are indicative of likely twinning. With the 
>> cell dimensions, it might be possible to have pseudomerohedral twinning in 
>> an orthorhombic space group, but given the lack of distinction among 
>> possible choices of orthorhombic spac group (noted by Kay), it seems much 
>> more likely that the true symmetry is lower and that you have pseudosymmetry 
>> combined with perfect twinning.
>>
>> Judging from the strong and unambiguous rotation peak, your model is clearly 
>> very good, so I think it would be easy to ask Phaser to solve this by 
>> looking for 4 copies in space group P1. You can get P1 data either by 
>> expanding the orthorhombic data to P1 or by re-merging the data in P1. If 
>> the merging statistics were good, that would indicate that any twinning 
>> would be close to perfect, so just expanding the data would be a reasonable 
>> choice. Alternatively, you have reasonable redundancy so merging in P1 would 
>> be a plausible choice. I would probably go with expanding the data, figuring 
>> out from the MR solution what the real symmetry is, and then merging the 
>> data with that symmetry.
>>
>> Unless there are some other pathologies, I think the MR in P1 is very likely 
>> to give a clear answer (or maybe 2 answers related by the twin operator). 
>> It’s formally possible that you could have a different number of copies 
>> (e.g. 6 in the unit cell) if the true symmetry were monoclinic, so keep an 
>> open mind on that question. You could just try finding the largest domain 
>> from splitting the AlphaFold model (presumably the domain for which you sent 
>> the log file), work out the symmetry from that solution, and then run a job 
>> to search for all the domains in the right space group. It’s generally a 
>> good idea, by the way, to ask Phaser to look for everything you expect to 
>> find in one job, because it has built-in logic to predict the best search 
>> order but then update it on the basis of preliminary results.
>>
>> There are different ways to sort out the true symmetry from the MR solution, 
>> but within CCP4 the Zanuda procedure is a very effective choice.
>>
>> Get in touch if you have any difficulties following this procedure, and it 
>> would be great to let the BB know the outcome!
>>
>> Best wishes,
>>
>> Randy Read
>>
>> > On 21 Feb 2024, at 08:42, Kay Diederichs  
>> > wrote:
>> >
>> > Hi Marco:
>> >
>> > short comments (I sent you also a private mail):
>> > - the stats in CORRECT.LP look ok, but I'd like to know what ISa is, and 
>> > what the number of outliers is ("misfits"). Seeing the delta-CC1/2 stats 
>> > as a function of frame number is also useful for judging e.g. 

Re: [ccp4bb] Crystallizing a tough target

[Oganesyan, Vaheh]

For what it is worth, human serum albumin has been crystallized initially from 
250 mg/ml solution back in ‘90s. When I started working on ternary complex 
hSA-FcRn-Fc (PDB Id 4N0U) was afraid that such high concentration couldn’t be 
achieved with amounts of FcRn I can reasonably express. However, complex got 
crystallized at around 6-8 mg/ml, if I remember it correctly. What I’m trying 
to tell is it might be worth trying to find binding partner for your protein 
and then co-crystallize the complex.

Vaheh Oganesyan, Ph.D.
[cid:image001.png@01DA5817.C3AEDD00]
R | Biologics Engineering
One Medimmune Way, Gaithersburg, MD 20878
T:  301-398-5851
vaheh.oganes...@astrazeneca.com



From: CCP4 bulletin board  On Behalf Of kavyashreem
Sent: Monday, February 5, 2024 5:27 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Crystallizing a tough target


Dear All,

Has anyone worked on a protein which is highly soluble even at 80mg/ml?

We have one such candidate, which does not precipitate even at 80mg/ml instead 
forms phase separated globules in crystallization plate, which eventually 
hardens over a period of 1 to 1.5 months (which is florescent under UV 
microscope.)

We tried screening at different pH, but failed to get any hits.

Since we got few conditions in which the phase separated globules solidified, 
we focused on them and expanded with 120mg/ml protein, still there were not 
visible precipitates except for the phase separation. This has been a 
challenging target so far. We have tried with different constructs, which 
unfortunately are not soluble!

Does POMs help in such cases? Or do you have any other suggestion.

Thank you

Regards

Kavya




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Re: [ccp4bb] Error in "Prepare and validate files for deposition" task"

[Oganesyan, Vaheh]

Nick,

What files do you mean when call them CCP4 formatted? Coordinates are accepted 
only in mmCIF. Experimental data in mtz. What else?

Thank you.

Vaheh

From: CCP4 bulletin board  On Behalf Of Nicholas Clark
Sent: Wednesday, January 31, 2024 3:49 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Error in "Prepare and validate files for deposition" task"

Maria,

Apologies for the second email. I forgot that "Prepare files for deposition" 
also merges the CCP4 formatted files into an MTZ. If I recall correctly, I used 
"CONVERT2MTZ" in the command line to complete the merging of the CCP4 files to 
generate the necessary MTZ for deposition. Instructions can be found here: 
https://www.ccp4.ac.uk/html/convert2mtz.html.

Best regards,

Nick Clark

On Tue, Jan 30, 2024 at 12:47 PM mdimarog 
mailto:mdima...@central.ntua.gr>> wrote:
Dear all,

I am trying to deposit 2 structures to the PDBe, solved and refined in
CCP4 (version 7), and checked in PRIVATEER as they have a lot of sugars
in. To this end, I used "Prepare and validate files for deposition"
task, as I had previously (and succesfully) done. However, and in spite
of switching to the latest CCP4 version (8.0.017), I keep getting this
error message "adding_stats_to_mmcif_i2_gui:Error in wrapper Failed to
add 204". I am using macOS Monterey, version 12.6.5.

Does anybody have an idea what is going wrong? If not, does anybody know
a different way to submit the reflection data to the PDBe? For example,
would this be somehow possible just using the output from Refmac and
AIMLESS? I wouldn't want to repeat the scaling/MR/refinement procedure
because there are a lot of sugars there, quite difficult to built.

Many thanks in advance for your help!

Kind regards,

Maria




--
Maria Dimarogona
Assistant Professor
Department of Chemical Engineering
University of Patras
Greece



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Re: [ccp4bb] What could these crystals be?

[Oganesyan, Vaheh]

Run SEC on complex prior to crystallization to remove single entities, provided 
size difference makes sense. Not too many column options though.

Vaheh

From: CCP4 bulletin board  On Behalf Of 
careinaedgo...@yahoo.com
Sent: Friday, November 24, 2023 4:00 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] What could these crystals be?


I wanted to thank everyone for their suggestions and ideas regarding the 
eyeball shaped crystals that I got at the beginning of the month.
I can confirm that these crystals do indeed contain protein and DNA which is 
good news.
I have tried a number of different buffers and salts since then. I have also 
tried seeding but nothing I have tried has changed the morphology of the 
crystals. They remain thin, flat and eyeball/pumpkin seed shaped.
It is not difficult to make the crystals, they form quite easily under quite a 
few conditions. The difficulty is in the diffraction. By changing the buffer 
conditions we can now see some very weak diffraction (previously there was no 
diffraction at all).
Are there any suggestions as to how to improve diffraction of these crystals? I 
did try different cryoprotectants, parabar, glycerol, PEG but no difference. I 
think perhaps the problem is heterogeneity considering my sample contains both 
protein and DNA.
Any suggestions or thoughts are welcome.
Thank you
Careina

On Wednesday, November 8, 2023 at 06:18:46 PM GMT+2, 
careinaedgo...@yahoo.com 
<02531c126adf-dmarc-requ...@jiscmail.ac.uk>
 wrote:



The reservior solution is 0.2 M NaCl2, 0.1 M HEPES pH 7.5, and 25% 3350 PEG

Protein buffer is 300 mM NaCl, 20 mM HEPES pH 7.5, 3mM TCEP

The drop contains 1.5ul protein-DNA complex and 1.5 ul reservior solution


On Wednesday, November 8, 2023 at 05:12:25 PM GMT+2, 
stephen.c...@rc-harwell.ac.uk 
mailto:stephen.c...@rc-harwell.ac.uk>> wrote:


Hi Careina,

Without knowing what's in your protein buffer or crystallisation condition it's 
hard to comment.

Best wishes,
Steve

Dr Stephen Carr
Research Complex at Harwell (RCaH)
Rutherford Appleton Laboratory
Harwell Oxford
Didcot
Oxon OX11 0FA
United Kingdom
Email stephen.c...@rc-harwell.ac.uk
tel 01235 567717

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of careinaedgo...@yahoo.com 
<02531c126adf-dmarc-requ...@jiscmail.ac.uk>
Sent: 08 November 2023 15:00
To: ccp4bb mailto:ccp4bb@jiscmail.ac.uk>>
Subject: [ccp4bb] What could these crystals be?

Hi all
We have been trying with no success to crystalize a protein. Recently we got 
these strange shape "crystals". They are hard and flat but they do not diffract 
at all. Any ideas as to what could cause this?
Careina



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Re: [ccp4bb] Fragile Crystals

[Oganesyan, Vaheh]

Hi Morgan Elizabeth,

In some cases adding 1-2% of cryoprotectant into crystallization drop during 
setting those drops up helps to introduce 25-30% of the same cryoprotectant 
during harvest, provided you still can get those crystals to grow. Worked for 
me in several cases.

Vaheh

From: CCP4 bulletin board  On Behalf Of Blake, Morgan 
Elizabeth
Sent: Wednesday, November 22, 2023 11:44 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Fragile Crystals

Hello!

I am a PhD student working on a crystallography project to wrap up my 
dissertation research. I have purified a complex of two proteins, and I can 
consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane pH 
7.5. These crystals have sharp edges and can grow to a large size (greater than 
0.5 mm), but the crystals seem to be very fragile. When we open the drops to 
harvest the crystals, we have little time to harvest the crystals before they 
crack. When we move the crystals to a cryoprotectant, over time they start 
fracturing. We've tried using different percentages of glycerol, ethylene 
glycol, PEG400, and oil for cryoprotectants with no success. Needless to say, 
the crystals do not diffract well, with spot patterns that look very 
streaky/mosaic, which I presume is due to the defects that we see in 
harvesting/handling. We have screened for alternate crystallization conditions, 
but we seem to get the same morphology in other conditions. Does anyone have 
suggestions for additives we could use post-crystallization to help stabilize 
our crystals?

Thanks for your advice!



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Re: [ccp4bb] RES: [ccp4bb] About model building

[Oganesyan, Vaheh]

You may as well have fun by manually building your molecule B into 1.9A ed map 
when R-factors are already in mid 30s.

Vaheh

From: CCP4 bulletin board  On Behalf Of Sam Tang
Sent: Monday, November 6, 2023 2:02 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] RES: [ccp4bb] About model building

Dear all

Thanks again for more input to the question. And credits to Eleanor and Kay for 
pointing out the high R-factor and possible issue with the space group. Their 
advice prompted me to revisit the MR solution and it happens that another 
solution, in P3121, gave a better map with R-factor after one round of 
refinement being 0.33/0.36, which was a remarkable difference with the P321 
solution (R~0.5).

So the next step I would take would be to re-try Arp/warp and see if things 
work out with poly-A. I shall update the community with the outcome.

Kind regards

Sam



On Tue, 7 Nov 2023 at 01:13, Rafael Marques 
mailto:rafael_mmsi...@hotmail.com>> wrote:
Hi Sam.

If you still have any of your crystals or any protein solution left in the well 
you harvested your crystals, I would run a MS/MS with them. Next step would be 
to run AF with your known chain A and your best Mass Spec hit (s), and use the 
resulting model for MR.

Good luck


Rafael Marques da Silva
PhD Student – Structural Biology
University of Leicester

Mestre em Física Biomolecular
Universidade de São Paulo

Bacharel em Ciências Biológicas
Universidade Federal de São Carlos

phone: +55 16 99766-0021

   "A sorte acompanha uma mente bem treinada"



De: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
em nome de Boaz Shaanan mailto:bshaa...@bgu.ac.il>>
Enviado: Monday, November 6, 2023 2:41:43 PM
Para: CCP4BB@JISCMAIL.AC.UK 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Assunto: Re: [ccp4bb] About model building

Hi,
If you still have crystals left, you could soak crystals with KI3 and collect 
data at Cu wavelength for SAD phasing, which could help you to resolve the 
missing piece. Maybe.
Cheers,
Boaz

Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben Gurion University
Beer Sheva, Israel

On Nov 4, 2023 10:04, Sam Tang 
mailto:samtys0...@gmail.com>> wrote:
Dear community,

I am solving the structure of a complex between proteins A and B, where A is a 
protein with known homologs and B is a novel protein isolated from plant. The 
diffraction data was at 1.9 Ang collected in-house, indexed to P321. Using A as 
the search model, we have got a reasonable solution where, after one round of 
refinement, the A chain fits the map pretty well. What's left was to extend the 
termini and fit a few rotamers.

For protein B (B chain) I have tried the web version of ARP/wARP but the 
outcome was not really good. The model was not successfully built as indicated 
by low model completeness and score. The tricky thing may be that we do not 
have the complete sequence information of this protein B in-hand. (The other 
way round, we more or less wish to rely on the high resolution data to confirm 
its sequence.) What approach would you then recommend to build the B chain in 
this scenario?

Thanks in advance and best regards,

Sam



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Re: [ccp4bb] How to fix ARG planarity outliers?

[Oganesyan, Vaheh]

Whatever it is worth, I agree with Dale. I see those "Major validation issues" 
all the time. Some structures at medium resolution (2-3A), and some are at high 
(1.1-1.4A). Not all Arg residues are "in violation". At low and medium 
resolution cases it is hard to argue. But high resolution electron density maps 
are showing correct fit, yet those Arg are called an "issue". Fixing restrains 
for all Arg is not right solution. Embracing the difference and understanding 
why those are different feels like scientific.

Thank you.

Vaheh Oganesyan, Ph.D.
[cid:image001.png@01D9D10E.4C64C4C0]
R | Biologics Engineering
One Medimmune Way, Gaithersburg, MD 20878
T:  301-398-5851
vaheh.oganes...@astrazeneca.com



From: CCP4 bulletin board  On Behalf Of Vladyslav 
Yadrykhinsky
Sent: Thursday, August 17, 2023 11:44 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] How to fix ARG planarity outliers?

Hello,

Thank you for your feedback.
Yes, I meant those that appear in pdb validation report.

I will try to inspect surrounding residues as well as run with suggested 
keywords and let you know how it went.

Thank you!

Best regards,
Vladyslav

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Keitaro Yamashita 
mailto:kyamash...@mrc-lmb.cam.ac.uk>>
Sent: Thursday, August 17, 2023 5:03:00 PM
To: CCP4BB@JISCMAIL.AC.UK 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: Re: [ccp4bb] How to fix ARG planarity outliers?

Hi,

I assume the planarity you mentioned is what is discussed in Moriarty
et al (2020) 
https://doi.org/10.1107/S2059798320013534

As shown there, it is known to have some deviation. In Refmac (or in
the CCP4 monomer library) it is restrained using a torsion angle with
5 degree sigma instead of the planarity restraint. You can tighten the
restraint using an even smaller sigma. The keyword could be:

restr tors include resi ARG name chi5 sigma 2.0

By the way, did you mean by "outliers" those from the PDB validation
report? I have had a feeling that their criterion is a bit too strict.
Could anyone from the PDB tell how the outlier of the ARG sidechain is
calculated?

Best regards,
Keitaro

On Thu, 17 Aug 2023 at 15:02, Vladyslav Yadrykhinsky
mailto:vladyslav.yadrykhin...@student.uib.no>>
 wrote:
>
> Hello,
>
> I am refining a structure and I have ARG planarity outliers in the sidechain.
> Could someone please tell me how should I set the planarity restraint in 
> REFMAC5 (version 5.8.0403) to correct them?
>
> Should I use: plane [value1] [value2] in the advanced settings? If so, what 
> values 1 and 2 should be? My current weight restraints are 0.128
>
> Please let me know if you need more information and I will appreciate any 
> assistance on the matter.
>
> Best regards,
> Vladyslav
>
> 
>
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Re: [ccp4bb] Ligand superposition!

[Oganesyan, Vaheh]

Hi Rams,

Given the ligand is identical in all structures I'd create and object from just 
the ligand and then write a script to align all 50 structures with that ligand 
object. I've done that on number of cases.

Hope it works in your case as well.

Vaheh

From: CCP4 bulletin board  On Behalf Of Subramanian, 
Ramaswamy
Sent: Saturday, July 15, 2023 8:10 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Ligand superposition!

Thanks Boaz,

The proteins are all completely different in folds and structures!


Rams
subra...@purdue.edu




On Jul 15, 2023, at 8:04 AM, Boaz Shaanan 
mailto:bshaa...@bgu.ac.il>> wrote:

 External Email: Use caution with attachments, links, or sharing data 

Hi,
-You may want to send a query to the UCSF chimerax/chimera people. They may 
suggest how to this using scripts.
- I have not done this on 50 structures, only on few ones. In my experience, if 
the proteins are sufficiently similar there is a good chance that superimposing 
the proteins will also bring the ligands to near superposition.
-Using chimera/chimerax you can select protein residues within certain radius 
from the ligands, write out the pdb's and then perhaps superimpose those 
smaller pdb's. I have not tried this.
Sorry I can't be of more help.
Cheers,
Boaz



Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

E-mail: bshaa...@bgu.ac.il
Phone: 972-8-647-2220
Fax:   972-8-647-2992 or 972-8-646-1710





From: CCP4 bulletin board on behalf of Subramanian, Ramaswamy
Sent: Friday, July 14, 2023 10:49 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Ligand superposition!

Hi All,

I have over 50 pdb files that I have downloaded from PDB that all have the same 
ligand bound.

I wamt to superpose the ligands (and move the protein coordinates using that 
matrix).  The goal is for me to see how difference in ligand environments in 
different complexes.

Is there an easy way to do it?  I am sure it has been done before and I do not 
want to reinvent the wheel.

Thanks.



Rams
subra...@purdue.edu




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Re: [ccp4bb] To Trim or Not to To Trim

[Oganesyan, Vaheh]

Hi Ben,

All copies created by multiplying cell dimensions will act exactly same as the 
original one, mathematically exactly. Nick’s approach is better. Something 
similar to what Nick said was published around 2002-2003. I was reviewing it. I 
did not understand then what the author was trying to achieve and kept thinking 
about it for few months. The author split model into 20 each with 5% occupancy. 
After refinement he got an ensemble that looked like NMR structures. I’m not 
sure, however, that adding that uncertainty will help answering any question.

Vaheh

From: CCP4 bulletin board  On Behalf Of benjamin bax
Sent: Saturday, March 18, 2023 5:07 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] To Trim or Not to To Trim
Hi,
Probably a stupid question.
Could you multiply a, b and c cell dimensions by 2 or 3 (to give 8 or 27 
structures) and restrain well defined parts of structure to be ‘identical’ ? To 
give you a more NMR like chemically sensible ensemble of structures?
Ben


> On 18 Mar 2023, at 12:04, Helen Ginn 
> mailto:ccp...@hginn.co.uk>> wrote:
>
> Models for crystallography have two purposes: refinement and interpretation. 
> Here these two purposes are in conflict. Neither case is handled well by 
> either trim or not trim scenario, but trimming results in a deficit for 
> refinement and not-trimming results in a deficit for interpretation.
>
> Our computational tools are not “fixed” in the same way that the standard 
> amino acids are “fixed” or your government’s bureaucracy pathways are 
> “fixed”. They are open for debate and for adjustments. This is a fine example 
> where it may be more productive to discuss the options for making changes to 
> the model itself or its representation, to better account for awkward 
> situations such as these. Otherwise we are left figuring out the best 
> imperfect way to use an imperfect tool (as all tools are, to varying 
> degrees!), which isn’t satisfying for enough people, enough of the time.
>
> I now appreciate the hypocrisy in the argument “do not trim, but also don’t 
> model disordered regions”, even though I’d be keen to avoid trimming. This 
> discussion has therefore softened my own viewpoint.
>
> My refinement models (as implemented in Vagabond) do away with the concept of 
> B factors precisely for the anguish it causes here, and refines a 
> distribution of protein conformations which is sampled to generate an 
> ensemble. By describing the conformations through the torsion angles that 
> comprise the protein, modelling flexibility of a disordered lysine is 
> comparatively trivial, and indeed modelling all possible conformations of a 
> disordered loop becomes feasible. Lysines end up looking like a frayed end of 
> a rope. Each conformation can produce its own solvent mask, which can be 
> summed together to produce a blurring of density that matches what you would 
> expect to see in the crystal.
>
> In my experience this doesn’t drop the R factors as much as you’d assume, 
> because blurred out protein density does look very much like solvent, but it 
> vastly improves the interpretability of the model. This also better models 
> the boundary between the atoms you would trim and those you’d leave 
> untrimmed, by avoiding such a binary distinction. No fear of trimming and 
> pushing those errors unseen into the rest of the structure. No fear of 
> leaving atoms in with an inadequate B factor model that cannot capture the 
> nature of the disorder.
>
> Vagabond is undergoing a heavy rewrite though, and is not yet ready for human 
> consumption. Its first iteration worked on single-dataset-single-model 
> refinement, which handled disordered side chains well enough, with no need to 
> decide to exclude atoms. The heart of the issue lies in main chain 
> flexibility, and this must be handled correctly, for reasons of 
> interpretability and elucidating the biological impact. This model isn’t 
> perfect either, and necessitates its own compromises - but will provide 
> another tool in the structural biology arsenal.
>
> —-
>
> Dr Helen Ginn
> Group leader, DESY
> Hamburg Advanced Research Centre for Bioorganic Chemistry (HARBOR)
> Luruper Chaussee 149
> 22607 Hamburg
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

[Oganesyan, Vaheh]

Hi Stephan,

You’re mostly correct, however, gradients made on instrument with one pump are 
less reliable.

Thank you.

Vaheh

From: CCP4 bulletin board  On Behalf Of Stephan Rempel
Sent: Friday, September 16, 2022 7:38 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

Hi,

I had some experience for about 2-3 years with the BioRad systems before I 
moved on and they worked just fine like the AKTAs. At the time, the price point 
was a huge plus over the AKTA Pure and in my opinion for protein purification 
there was no reason to fork out the money for an AKTA Pure because you would 
never use the two pumps anyway. We had the NGC quest with multi wavelength 
detection for protein labeling, which worked fine. I liked the NGCs a lot, they 
were reliable, and I would be happy to get one any time.

In the meantime, Cytiva has released the AKTA Go, which is a scaled down 
version of the Pure with only one pump. And I would give this one a thought if 
you only want to purify proteins.

It is a great (!) little system for protein purification and the price is very 
reasonable in my opinion. Our version with the additional inlet sample valve 
(allows for scouting over night), air detector (very convenient when loading 
large lysate volumes on IMAC columns), and column valve was in the region of 
20-25k. You also have the choice between a simpler fraction collector and the 
one from the AKTA Pure. The only caveat is that they only offer a fixed 
wavelength detector. After two years, we had not a single fault (they have been 
on the market for 2.5-3 years). I very much like the Unicorn software.

I hope this helps,
Stephan.





Stephan Rempel, PhD
Senior Scientist, FoRx Therapeutics AG

Lichtstrasse 35, WSJ-350.3.10
4056 Basel, Switzerland
stephan.rem...@forxtherapeutics.com
www.forxtherapeutics.com

  [cid:image001.png@01D8C9B6.8D851690]

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Marko Hyvonen
Sent: Friday, 16 September 2022 13:17
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

Hello Eike,

A couple of comments on the AKTA Pures (while I have not experience with the 
BioRad systems).

We have a couple of AKTA Pures and we (and another lab close to us) had a 
catastrophic failure of the 3-wavelength detector “block”. Price tag for the 
replacement part (apparently a black box  with nothing accessible for repair – 
happy to hear if this was misinformation!) was over £12,000. Someone commented 
that they had even more eye-watering price tag for an upgrade to triple 
wavelength. We switched to a single wavelength detector at £5k and I will get 
these from now on only.

I really dislike the database system used in new Unicorn for storing data etc: 
another black box. I much prefer the way older Unicorns worked, but maybe that 
is just me being old and grumpy and stuck in my ways. That reminds also that we 
tend to source computers separately. Much better systems available for less 
money and with MUCH smaller footprint.

The price tags on Pures (like almost everything else with Cytiva) are in my 
opinion a reflection of the market dominance. I wish there was more competition 
(bring back Biocad!). Service contracts with Cytiva are also ridiculously 
expensive – we have, luckily,  a very knowledgeable company here that does all 
the servicing and maintenance, at less than half the price.

Having just complained, I do like AKTA Pures and there is much to like in them. 
Time will tell if these will be as robust as the original “grey” AKTA 
purifiers. Several of ours Purifiers still running perfectly after over 15 
years. As long as the detector works and flow is accurate, the proteins elute 
just the same from the columns. However, once you have even one of the shinier 
systems around, those old ones start collecting dust very soon.

Hth, Marko


Marko Hyvonen
Department of Biochemistry
University of Cambridge
https://hyvonen.bioc.cam.ac.uk
@HyvonenGroup
mh...@cam.ac.uk




From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Schulz, Eike-Christian
Sent: 16 September 2022 08:40
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

Dear all,

I am considering to purchase a chromatography system for routine protein 
purification. The device is supposed to be used in a multi-user environment, 
hence ease of use, ease of training and ease of maintenance is important. I am 
rather looking for a robust system people like to use than a system that comes 
with many bells and whistles that no one dares to touch.


·   Is there any particularly _bad_ experience with either system?


·   Is there any specific 

Re: [ccp4bb] Coot sporadically crashes

[Oganesyan, Vaheh]

Thank you, Paul!

From: CCP4 bulletin board  On Behalf Of Paul Emsley
Sent: Friday, July 8, 2022 11:50 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Coot sporadically crashes



 Is there an option to save the session automatically every 10-15 minutes or so?





And now to actually answer your question:

yes: Edit -> Settings ->Enable Quick-Save Check-pointing... (the default is 30 
seconds)







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[ccp4bb] Coot sporadically crashes

[Oganesyan, Vaheh]

Hi All,

Sorry for posting on CCP4BB.

Coot installed together with latest CCP4 on Win10 crashes time to time with no 
messages/warnings. Is there an option to save the session automatically every 
10-15 minutes or so?

Thank you.

Vaheh



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Re: [ccp4bb] displaying residues (as a surface perhaps) for one component of a p-p-i

[Oganesyan, Vaheh]

So does PISA with great detailed info on interface residues available.

VO

From: CCP4 bulletin board  On Behalf Of Jan Dohnalek
Sent: Tuesday, May 31, 2022 2:48 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] displaying residues (as a surface perhaps) for one 
component of a p-p-i

Dear Fred,
CCP4MG has this capability of automated interface display.
When opening a structure you can select from many predefined styles of display. 
My favourite is
Interfaces, then "residues", the surface can be displayed when you ask for PISA 
analysis within CCP4MG and then select a particular interface and display it.
I hope it works for you,

Jan




On Tue, May 31, 2022 at 8:12 AM Fred Vellieux 
mailto:frederic.velli...@lf1.cuni.cz>> wrote:
Dear bb members,

I am quite certain that someone must have needed to do this already. I
looked at publications but no details were given concerning how figures
were prepared.

So here is the problem:

Given a protein protein interface (with the 3D structure of the complex
available) I am looking for a method allowing me to identify and display
the interface forming residues of one of the protein components. Plus a
surface representation of that part, for the 3D structure.

Thanks in advance for any tips.

Regards,

Fred. Vellieux

--
MedChem, 1st F. Medicine, Charles University
BIOCEV, Vestec, Czech Republic



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--
Jan Dohnalek, Ph.D
Institute of Biotechnology
Academy of Sciences of the Czech Republic
Biocev
Prumyslova 595
252 50 Vestec near Prague
Czech Republic
Tel. +420 325 873 758



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Re: [ccp4bb] ligand binds to one molecule

[Oganesyan, Vaheh]

Well, there is almost nothing left to suggest, but one. Try co-crystallizing. 
While in solution molecules will have more flexibility to adopt a conformation 
that fits ligand bound state better. When soaking there might be restrictions 
on conformational changes needed for better binding due to already packed 
interactions. What you see in soaked-in crystals could be an incomplete bound 
state.

Just an idea.

Vaheh

From: CCP4 bulletin board  On Behalf Of 
Sent: Saturday, March 5, 2022 3:01 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] ligand binds to one molecule

Hello all,

In homo-dimeric or homo-oligomeric protein crystal structures, what would be 
the reason for having a ligand (chemical compound or fragment) binds to one 
molecule and not all molecules in the asymmetric unit?

I have soaked a fragment that has an affinity of 200 uM to a viral protein but 
I can only see it binds to one molecule (we have eight molecules in the AU). 
This is was also notable as well in some published PDB (dimeric protein).

Any suggestions?

Best wishes,
Shymaa



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Re: [ccp4bb] Glutaraldehyde and gel filtration

[Oganesyan, Vaheh]

It would probably be safe if you run dialysis first. If you still think that 
may harm the expensive SEC column then running PD-10 column first would be 
advised.

Hope this helps.
Vaheh Oganesyan, Ph.D.
[cid:image001.png@01D80EB0.8E5CA9A0]
R | Biologics Engineering
One Medimmune Way, Gaithersburg, MD 20878
T:  301-398-5851
vaheh.oganes...@astrazeneca.com


From: CCP4 bulletin board  On Behalf Of Maria Jose 
Sanchez Barrena
Sent: Friday, January 21, 2022 9:59 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Glutaraldehyde and gel filtration

Dear all:

Can I ask for some help-opinion on possible problems for a superdex200 column 
to separate a glutaraldehyde-crosslinked sample?

We are using 0.1% Glutaraldehyde to crosslink and protein complex and after 
incubation, we stop the reaction by adding 50mM Tris buffer. We have analyzed 
the sample on an SDS-PAGE gel and we do not observe aggregates, only the 
crosslinked complex and some uncrosslinked protein.

We would like to further analyze this sample on a gel filtration Superdex 200 
Increase 3.2/300 column and although I cannot see any incompatibility in the 
column specifications, I would like to have some advice on how you deal with 
this. Since the glutaraldehyde is quenched with Tris, I imagine that passing 
this sample through the column would not make any harm in future purifications 
or will have a bad effect on resin stability. Would I need to wash the column 
with something special? Do you have columns dedicated to 
glutaraldhyde-containing samples? I am a bit concerned on “contaminating” the 
column or losing resolution in the future.

Many thanks in advance and all best wishes for 2022,

María J. Sánchez-Barrena


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Re: [ccp4bb] renaming chains

[Oganesyan, Vaheh]

Dear John Helliwell,

I should have been more clear in addressing my email. It was in response to 
John Berrisford’s answer.

Regards,

Vaheh

From: John R Helliwell 
Sent: Monday, November 1, 2021 3:13 PM
To: Oganesyan, Vaheh 
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] renaming chains

Dear Valeh
Apologies, I was solely replying to the numbering/labelling of waters 
observation/query of Mohamed, ie as a function of in situ parameter (time, 
temperature and pressure).
Best wishes
John
Emeritus Professor John R Helliwell DSc


On 1 Nov 2021, at 18:39, Oganesyan, Vaheh 
mailto:vaheh.oganes...@astrazeneca.com>> wrote:

Hi John,

Thank you for explanation.
The most recent encounter I had on this issue was with 3DMM and 3B9K. You would 
know better what caused chain renaming here, but it doesn’t look like any of 
the two scenarios you describe. Whatever the reason was I’m glad to hear that 
this is not a widespread rule that applies to all structures.

Regards,
Vaheh Oganesyan, Ph.D.
[cid:image001.png@01D7CF33.8D9C2020]
R | Biologics Engineering
One Medimmune Way, Gaithersburg, MD 20878
T:  301-398-5851
vaheh.oganes...@astrazeneca.com<mailto:oganesy...@medimmune.com>



From: John Berrisford mailto:j...@ebi.ac.uk>>
Sent: Monday, November 1, 2021 12:53 PM
To: Oganesyan, Vaheh 
mailto:vaheh.oganes...@astrazeneca.com>>
Cc: CCP4BB@jiscmail.ac.uk<mailto:CCP4BB@jiscmail.ac.uk>
Subject: Re: [ccp4bb] renaming chains

Dear Vaheh

Usually we do not rename chains as part of the curation procedure.
There are instances when we do, for example when a chain has to be split
into two chains and a new chain has to be defined, but this isn't
typical.

Because of this the wwPDB mmCIF file for each entry will usually contain
the chains as defined by the depositor.
If two letter chain IDs were used by the depositor then this is
incompatible with the PDB format and a best effort PDB file is created.
This will contain remapped chain IDs with a single letter for the
chains.
If you are using this best effort PDB file then I would encourage using
the mmCIF file instead.

If this is not the case, please can you share examples through our
helpdesk (deposit-h...@mail.wwpdb.org<mailto:deposit-h...@mail.wwpdb.org>) 
where this has occurred so we can
investigate.

Many thanks

John

On 2021-11-01 15:00, Oganesyan, Vaheh wrote:
> Hi All,
>
> This question is mostly for RCSB and PDBe: why are you renaming chains
> in the deposited PDB files? Why does it matter what letter is assigned
> to the chain? For 1,2 or 3 chain structures it is manageable, but for
> more chains and/or many complexes per asu this becomes quite a
> challenge. And if chains are similar in shape it is a real pain.
> Reading associated manuscripts and looking at those structures is an
> additive that feels unnecessary.
>
> Thank you in advance for explaining the logic behind.
>
> Vaheh Oganesyan, Ph.D.
>
> R | Biologics Engineering
>
> One Medimmune Way, Gaithersburg, MD 20878
>
> T: 301-398-5851
>
> vaheh.oganes...@astrazeneca.com<mailto:vaheh.oganes...@astrazeneca.com>
>
> -
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1<https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1>

--
John Berrisford
PDBe
European Bioinformatics Institute (EMBL-EBI)
European Molecular Biology Laboratory
Wellcome Trust Genome Campus
Hinxton
Cambridge CB10 1SD UK
Tel: +44 1223 492529



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Re: [ccp4bb] renaming chains

[Oganesyan, Vaheh]

Hi John,

Thank you for explanation.
The most recent encounter I had on this issue was with 3DMM and 3B9K. You would 
know better what caused chain renaming here, but it doesn't look like any of 
the two scenarios you describe. Whatever the reason was I'm glad to hear that 
this is not a widespread rule that applies to all structures.

Regards,
Vaheh Oganesyan, Ph.D.
[cid:image001.png@01D7CF2E.22CE9C50]
R | Biologics Engineering
One Medimmune Way, Gaithersburg, MD 20878
T:  301-398-5851
vaheh.oganes...@astrazeneca.com<mailto:oganesy...@medimmune.com>



From: John Berrisford 
Sent: Monday, November 1, 2021 12:53 PM
To: Oganesyan, Vaheh 
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] renaming chains

Dear Vaheh

Usually we do not rename chains as part of the curation procedure.
There are instances when we do, for example when a chain has to be split
into two chains and a new chain has to be defined, but this isn't
typical.

Because of this the wwPDB mmCIF file for each entry will usually contain
the chains as defined by the depositor.
If two letter chain IDs were used by the depositor then this is
incompatible with the PDB format and a best effort PDB file is created.
This will contain remapped chain IDs with a single letter for the
chains.
If you are using this best effort PDB file then I would encourage using
the mmCIF file instead.

If this is not the case, please can you share examples through our
helpdesk (deposit-h...@mail.wwpdb.org<mailto:deposit-h...@mail.wwpdb.org>) 
where this has occurred so we can
investigate.

Many thanks

John

On 2021-11-01 15:00, Oganesyan, Vaheh wrote:
> Hi All,
>
> This question is mostly for RCSB and PDBe: why are you renaming chains
> in the deposited PDB files? Why does it matter what letter is assigned
> to the chain? For 1,2 or 3 chain structures it is manageable, but for
> more chains and/or many complexes per asu this becomes quite a
> challenge. And if chains are similar in shape it is a real pain.
> Reading associated manuscripts and looking at those structures is an
> additive that feels unnecessary.
>
> Thank you in advance for explaining the logic behind.
>
> Vaheh Oganesyan, Ph.D.
>
> R | Biologics Engineering
>
> One Medimmune Way, Gaithersburg, MD 20878
>
> T: 301-398-5851
>
> vaheh.oganes...@astrazeneca.com<mailto:vaheh.oganes...@astrazeneca.com>
>
> -
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1<https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1>

--
John Berrisford
PDBe
European Bioinformatics Institute (EMBL-EBI)
European Molecular Biology Laboratory
Wellcome Trust Genome Campus
Hinxton
Cambridge CB10 1SD UK
Tel: +44 1223 492529



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[ccp4bb] renaming chains

[Oganesyan, Vaheh]

Hi All,

This question is mostly for RCSB and PDBe: why are you renaming chains in the 
deposited PDB files? Why does it matter what letter is assigned to the chain? 
For 1,2 or 3 chain structures it is manageable, but for more chains and/or many 
complexes per asu this becomes quite a challenge. And if chains are similar in 
shape it is a real pain. Reading associated manuscripts and looking at those 
structures is an additive that feels unnecessary.

Thank you in advance for explaining the logic behind.

Vaheh Oganesyan, Ph.D.
[cid:image001.png@01D7CF0E.5D61A0D0]
R | Biologics Engineering
One Medimmune Way, Gaithersburg, MD 20878
T:  301-398-5851
vaheh.oganes...@astrazeneca.com




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Re: [ccp4bb] AW: [ccp4bb] Antwort: Re: [ccp4bb] chain on 2-fold axis?

[Oganesyan, Vaheh]

How P3221 can be an option if it assumes chain on axis? I guess I'm missing 
something, but per my belief only those sg will be possible for which there is 
no axis going through the extra molecule. P1 sg looks the only correct option 
here in my humble opinion.
Democracy (voting) depends on science. However, the reverse is not, thankfully.

Vaheh

From: CCP4 bulletin board  On Behalf Of Peer Mittl
Sent: Friday, August 27, 2021 6:32 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] AW: [ccp4bb] Antwort: Re: [ccp4bb] chain on 2-fold axis?

Dear Herman,

The answer probably depends on the impact of the "extra" chain on the
sublattice. If there is no impact the "true" space group is P3221 with
one chain on the special position. If the swapping of the extra chain
influences the sublattice P32 (or C2 or P1, as pointed out by Kay)
twinned to P3221 might be the better description.

All the best,
Peer

On 27.08.2021 10:56, Schreuder, Herman /DE wrote:
>
> Dear Peer and Eleanor,
>
> This is indeed what I am suspecting: If the "twinning operator" in P32
> puts 4 out of 5 protein chains on top of symmetry mates, is the "true"
> space group then P32, with 5 twinned chains, or P3221 with 4 normal
> chains and 1 chain on a special position? I would vote for the latter.
>
> Best,
>
> Herman
>
> *Von:* CCP4 bulletin board 
> mailto:CCP4BB@JISCMAIL.AC.UK>> *Im Auftrag von
> *Peer Mittl
> *Gesendet:* Freitag, 27. August 2021 10:17
> *An:* CCP4BB@JISCMAIL.AC.UK
> *Betreff:* Re: [ccp4bb] Antwort: Re: [ccp4bb] chain on 2-fold axis?
>
> Dear Eleanor,
>
> I indeed used r/tefmac for the refinement and it came up with the values
> HKL (a=0.56), KH-L (a=0.44). It would be interesting to see if a
> refinement in P3221 would come up with the same occupancies for the
> alternative conformations for the "extra" chain on the 2-fold axis. It
> seems as if the "well-ordered" chains (2 in P3221, 4 in P32) form a
> sublattice with P3221 symmetry and it's just the "extra" chain, which
> generates the twinning.
>
> All the best,
> Peer
>
> On 26.08.2021 18:09, Eleanor Dodson wrote:
> > Motto =mitti in predictive text!
> >
> > On Thu, 26 Aug 2021 at 16:52, Eleanor Dodson
> > mailto:eleanor.dod...@york.ac.uk
> 
>>
> wrote:
> >
> > Great, motto. I think you have nailed it! Did you use tefmac for
> > twinned refinement? And if so what did it suggest the twin
> >  fraction is?
> >
> > On Thu, 26 Aug 2021 at 16:30, Peer Mittl mailto:mi...@bioc.uzh.ch%0b>> > 
> >> wrote:
> >
> > Yes, the data indeed seems to be twinned and the tNCS has
> > masked the twinning statistics, which is why I haven't
> > considered it so far.
> >
> > I have not tried twinned refinement in C2 and P1 yet, but
> > refining 4 chains in P32 with twinning yields a difference ED
> > map that clearly indicates one (and just on!) orientation for
> > the 5th chain. Thank you all for your suggestions.
> >
> > Have a nice evening,
> > Peer
> >
> > -"CCP4 bulletin board" mailto:CCP4BB@JISCMAIL.AC.UK%0b>> > 
> >> schrieb: 
-
> > An: CCP4BB@JISCMAIL.AC.UK 
> > 
>  >>
> > Von: "Kay Diederichs"
> > Gesendet von: "CCP4 bulletin board"
> > Datum: 26.08.2021 16:41
> > Betreff: Re: [ccp4bb] chain on 2-fold axis?
> >
> > Dear Peer,
> >
> > I suspect that the true spacegroup has lower symmetry than
> > P3221, and that there may be twinning masked by tNCS.
> > Subgroups of P3221 are C2 and P32 (
> >
> https://strucbio.biologie.unikonstanz.de/xdswiki/index.php/Space_group_determination#Subgroup_and_supergroup_relations_of_these_space_groups
> >
> >
> 
> 

Re: [ccp4bb] sugestions on weak diffracting protein crystals

[Oganesyan, Vaheh]

Herman's suggestion is very good. But I'd go one step further and try to grow 
crystals in 30-40% PEG400. Then you definitely do not need any additional 
cryoprotectant. There is a chance that your crystals are of poor quality to 
start with. In that case no cryoprotection will help. You can test that by 
collecting few diffraction images at ambient temps. If that will prove true 
that your crystals are not good enough then either that particular 
crystallization condition is not suitable for your complex or, most likely, 
more attention should be paid on getting more homogeneous protein solution.
For more project oriented advice you may contact off list.

Vaheh Oganesyan, Ph.D.
[cid:image001.png@01D74BC2.18B85070]
R | Antibody Discovery and Protein Engineering
One Medimmune Way, Gaithersburg, MD 20878
T:  301-398-5851
vaheh.oganes...@astrazeneca.com




From: CCP4 bulletin board  On Behalf Of Schreuder, 
Herman /DE
Sent: Tuesday, May 18, 2021 8:33 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] AW: [ccp4bb] sugestions on weak diffracting protein crystals

Dear Deepak,
the cryoprotection may destroy the diffraction. I would also try diffraction at 
room temperature, using special sleeves to prevent dehydration. With 20% 
PEG400, you could also try to freeze the crystals without using additional 
cryoprotectant. If you are lucky, it may work.

Best,
Herman

Von: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
Im Auftrag von Deepak Deepak
Gesendet: Dienstag, 18. Mai 2021 12:08
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] sugestions on weak diffracting protein crystals

Dear all,

I have got multiple crystals (see picture 1) of a protein (8kDa) with a helical 
aromatic oligoamide foldamer (5kDa) but these crystals diffract very poorly 
(see the diffraction pattern in picture 2).

I prepare a 1.3mM:1.3mM complex of protein: foldamer in 20mM Tris, pH 7.5 
buffer. Crystals grew in 3-5 days in sitting and hanging drop at 20 Deg C and 
25 Deg C in the following conditions:

- 20% PEG 400, 0.1M MES pH 6.0
-20% PEG 400, 0.1M Sodium Cacodylate pH 6.0

Multiple cryo used were:
-25%Glycerol in mother solution
 -30% glycerol in water
-30%PEG 400,
-35% PEG 400
-20% PEG 8000 + 40% PEG 400 mix

Kindly suggest some methods/modifications on how can I improve the resolution 
and get better-diffracting crystals. Please let me know if you need more 
information.

Kind regards,
Deepak
Ph.D. Student

PS: The protein is a DNA binding protein and I have crystallized and solved the 
structure of this protein with its DNA partner and now I crystallized it with 
our foldamers but diffraction is not good. There are multiple structures of the 
Protein+DNA complex in literature but no apo-protein structure as the protein 
needs a binding partner to crystallize. We already have solution studies 
showing a good binding.



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Re: [ccp4bb] metal coordination at low resolution - restraints

[Oganesyan, Vaheh]

Hi Jan,

They hold nice because of high occupancy or because you have very high 
resolution and no restraints are necessary at all (even for protein part)?

Thank you

Vaheh

From: CCP4 bulletin board  On Behalf Of Jan Dohnalek
Sent: Tuesday, September 8, 2020 8:01 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] metal coordination at low resolution - restraints

Hi Garib,




On 8 Sep 2020, at 11:39, Jan Dohnalek 
mailto:dohnalek...@gmail.com>> wrote:

These are structural.

Are they tetrahedral or octahedral? From the list of neighbours they do not 
look like tetrahedral. Some of them do look like octahedral.

They are involved in reaction.
Two are ~ octahedral (skewed though, two positions filled by catalysis 
participant), one is ~tetrahedral, but actually can also accept a fifth 
coordinating atom.

But as I said - in all our structures restraining the coordination geometry is 
not necessary, they hold nice.

Jan


Jan


On Tue, Sep 8, 2020 at 12:22 PM Garib Murshudov 
mailto:ga...@mrc-lmb.cam.ac.uk>> wrote:
What are these numbers?

If I understand these numbers correctly: none of your Zn atoms is structural (4 
coordinated tetrahedral). If that is the case then you need specific links or 
restraints. If my reading of your numbers is correct then there could be some 
chemistry change of the surrounding residues.

If it is not structural Zn then it is likely that coordination is 6. But 
without seeing coordinates and maps it is difficult to say what is there.

Regards
Garib



On 8 Sep 2020, at 11:11, Eleanor Dodson 
mailto:eleanor.dod...@york.ac.uk>> wrote:

Hmm - here is my problem - a list of ZN contacts for the two molecules..
residue 602 is a phosphate, and there possibly should be a few more waters ..
No idea how best to tackle it..
E


 Z  401 ZN   A   W   21 NA  2.057 X,Y,Z   1.008.73
 Z  401 ZN   A   W   21 OA  2.220 X,Y,Z   1.008.76
 Z  401 ZN   A   H   26 NE2  A  2.000 X,Y,Z   1.008.39
 Z  401 ZN   A   D  139 OD1  A  2.085 X,Y,Z   1.008.61
 Z  401 ZN   A   Z  601 O2   A  1.927 X,Y,Z   0.60   10.74
 Z  401 ZN   A   O  821 OC  2.006 X,Y,Z   0.407.51

 Z  402 ZN   A   H   80 ND1  A  2.033 X,Y,Z   1.008.94
 Z  402 ZN   A   H  135 NE2  A  2.032 X,Y,Z   1.008.70
 Z  402 ZN   A   D  139 OD2  A  2.024 X,Y,Z   1.008.70
 Z  402 ZN   A   Z  601 O2   A  2.131 X,Y,Z   0.60   11.05
 Z  402 ZN   A   O  821 OC  1.829 X,Y,Z   0.407.81

 Z  403 ZN   A   H  145 NE2  A  2.027 X,Y,Z   1.00   10.50
 Z  403 ZN   A   H  168 NE2  A  2.030 X,Y,Z   1.00   10.19
 Z  403 ZN   A   D  172 OD2  A  2.062 X,Y,Z   1.00   12.66
 Z  403 ZN   A   Z  601 O3   A  1.953 X,Y,Z   0.60   11.54
 Z  403 ZN   A   O  820 OC  2.207 X,Y,Z   0.209.09
 Z  403 ZN   A   O  822 OC  2.059 X,Y,Z   0.40   13.79

 Z  401 ZN   A   Z  402 ZN   A  3.349 X,Y,Z   1.008.73


 Z  401 ZN   B   W   21 NB  2.099 X,Y,Z   1.009.22
 Z  401 ZN   B   W   21 OB  2.184 X,Y,Z   1.008.91
 Z  401 ZN   B   H   26 NE2  B  2.009 X,Y,Z   1.008.79
 Z  401 ZN   B   D  139 OD1  B  2.069 X,Y,Z   1.008.76
 Z  401 ZN   B   Z  601 O3   B  1.981 X,Y,Z   0.709.31

 Z  402 ZN   B   H   80 ND1  B  2.032 X,Y,Z   1.009.49
 Z  402 ZN   B   H  135 NE2  B  2.024 X,Y,Z   1.009.22
 Z  402 ZN   B   D  139 OD2  B  2.032 X,Y,Z   1.009.70
 Z  402 ZN   B   Z  601 O3   B  1.973 X,Y,Z   0.709.58

 Z  403 ZN   B   H  145 NE2  B  2.027 X,Y,Z   1.00   10.80
 Z  403 ZN   B   H  168 NE2  B  2.029 X,Y,Z   1.00   10.65
 Z  403 ZN   B   D  172 OD2  B  2.089 X,Y,Z   1.00   13.12
 Z  403 ZN   B   Z  601 O4   B  1.938 X,Y,Z   0.70   14.10
 Z  403 ZN   B   O  825 OC  2.322 X,Y,Z   0.20   10.61
~

On Tue, 8 Sep 2020 at 10:47, Garib Murshudov 
mailto:ga...@mrc-lmb.cam.ac.uk>> wrote:
Hi Robbie and Eleanor

There are links for Zn-His and Zn-Cys. They meant to be used automatically, 
obviously something is not entirely right.

Link names are:
ZN-CYS

It has a bond between Zn and S as well as an angle:
ZN-CYS   1 ZN  2 SG  2 CB  109.0003.000

This also removes H of Cys to make covalent bond between Zn and Cys.

Similar links are available for Zn and His ND1 and Zn - HIS NE2
Link names are:

ZN-HISND
ZN-HISNE

Again these links have angles between Zn and atoms of His.

Angle centred at Zn is missing. But these distances and angles defined in the 
link it 

Re: [ccp4bb] Accessing full list of programs in CCP4I2

[Oganesyan, Vaheh]

Thank you for explanation. There are many small programs in CCP4 that have not 
been updated for years and they still work despite being in unsupported folder. 
By making them inaccessible (or more difficult to access) nobody gets 
easier/cheaper life. Even if there are newer programs that may do the same it 
is not a good reason to forget the old ones. It is like forgetting the history 
and the culture. Then making new discoveries just to find that it is the same 
old “wheel” just a bit more round. It is probably very personal, but I do not 
throw away old edition of Thermodynamics text book when new one is coming out. 
And I’m not a hoarder, do not have OCD or ADHD.

This is already 4 cents together. Sorry to clutter your mailbox.

Vaheh (CCP4 user since 1992)

From: Christian Roth 
Sent: Tuesday, July 7, 2020 12:42 PM
To: Oganesyan, Vaheh 
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] Accessing full list of programs in CCP4I2

I understand that one is used to the programs one is familiar with and Eleanor 
especially is very fond of these things, but there are several things which can 
be done for example in Coot (symmetry coordinates etc) . It is just another way 
to do it. I had the chance and pleasure to work for a while with the 
programmers of some of these programs in one building and such decisions are 
not taken lightly. But one cannot support all programs and write for every 
program interfaces or even keep two interfaces alive. There are just not enough 
people and money to do that. Also most scientific programmers don't get paid to 
keep the things just running. Would it be better to have also programmers to 
keep legacy up to date and write and update GUI's, maybe? But again someone 
needs to pay for that.
Just my 2 cents.

Cheers
Christian


On Tue, Jul 7, 2020 at 6:30 PM Oganesyan, Vaheh 
mailto:vaheh.oganes...@astrazeneca.com>> wrote:
… and how all these changes being justified?

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Eleanor Dodson
Sent: Tuesday, July 7, 2020 12:25 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] Accessing full list of programs in CCP4I2

Yes - there are things I use all the time which are not part of the CCP4I2 list
pdbset to generate symmetry equivalents  or find the centre of mass etc etc
coordconv to turn orthogonal coordinates to fractional - after all we are 
crystallographers and need to relate models to unit cells..
distang to do a quick check on crystal contacts..
nd there must be more..
Eleanor

On Tue, 7 Jul 2020 at 17:16, Palm, Gottfried 
mailto:p...@uni-greifswald.de>> wrote:
For occasions like Laus, it would be useful to further on have access to the
CCP4 v7.0 Program Documentation,
even if the programs are not updated as Christian explained.
I was for instance looking for translating coordinates as in pdbset, but 
couldn't find a replacement in the ccp4i2 gui.
Greetings
  Gottfried


On Tuesday, 07-07-2020 at 18:00 Lau Kelvin wrote:
Ah I see! Great I can run it that way

And yes, Eleanor, after I realized that list was gone, I was panicking that 
there are some random things I used to do with those programs would no longer 
be possible. Good to know that the command line versions are still there in the 
7.1 distro.

Best,

Kelvin

--
Kelvin Lau
https://people.epfl.ch/kelvin.lau<https://people.epfl.ch/kelvin.lau>

Protein production and structure core facility - PTPSP
EPFL SV PTECH PTPSP
AI 2146 (Bâtiment AI)
Station 19
CH-1015 Lausanne
Switzerland
Email: kelvin@epfl.ch<mailto:kelvin@epfl.ch>
Phone: +41 21 69 30267
If unreachable: +41 21 69 34494


On 07.07.20, 15:51, "CCP4 bulletin board on behalf of Christian Roth" 
mailto:CCP4BB@JISCMAIL.AC.UK> on behalf of 
christianroth...@gmail.com<mailto:christianroth...@gmail.com>> wrote:

yes Eleanor is right. command line still works.[:-)]
fft is also in 7.1 distribution.



On Tue, Jul 7, 2020 at 3:43 PM Eleanor Dodson 
mailto:eleanor.dod...@york.ac.uk>> wrote:
Oh Lau - how I miss that list!
But if you just run fft online it is still distributed..wombat:Downloads 
eleanor$

fft hklin  mapout 

LABIN FP=  and so on..


On Tue, 7 Jul 2020 at 14:22, Christian Roth 
mailto:christianroth...@gmail.com>> wrote:
Hi Kelvin,
well fft as single program is kind of not longer supported as is not ccp4i. In 
i2 internally, as well in communication with mg or coot, everything is done 
using map coefficients.
Their are two options:
First via i2: Use the unusual map coefficients Task and choose not to compare 
maps, but to generate the map coefficients plus a map (button is in Advanced 
tab) the standard grid parameters can be changed, but are actually optimized 
already.
Second just save the map out of Coot (Export map)

To avoid redundancy, the old fft task was discontinued. i2 works with 
coefficients, which generates smaller files and Coot provides all the functions 
to gen

Re: [ccp4bb] Accessing full list of programs in CCP4I2

[Oganesyan, Vaheh]

… and how all these changes being justified?

From: CCP4 bulletin board  On Behalf Of Eleanor Dodson
Sent: Tuesday, July 7, 2020 12:25 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Accessing full list of programs in CCP4I2

Yes - there are things I use all the time which are not part of the CCP4I2 list
pdbset to generate symmetry equivalents  or find the centre of mass etc etc
coordconv to turn orthogonal coordinates to fractional - after all we are 
crystallographers and need to relate models to unit cells..
distang to do a quick check on crystal contacts..
nd there must be more..
Eleanor

On Tue, 7 Jul 2020 at 17:16, Palm, Gottfried 
mailto:p...@uni-greifswald.de>> wrote:
For occasions like Laus, it would be useful to further on have access to the
CCP4 v7.0 Program Documentation,
even if the programs are not updated as Christian explained.
I was for instance looking for translating coordinates as in pdbset, but 
couldn't find a replacement in the ccp4i2 gui.
Greetings
  Gottfried


On Tuesday, 07-07-2020 at 18:00 Lau Kelvin wrote:

Ah I see! Great I can run it that way

And yes, Eleanor, after I realized that list was gone, I was panicking that 
there are some random things I used to do with those programs would no longer 
be possible. Good to know that the command line versions are still there in the 
7.1 distro.

Best,

Kelvin

--
Kelvin Lau
https://people.epfl.ch/kelvin.lau

Protein production and structure core facility - PTPSP
EPFL SV PTECH PTPSP
AI 2146 (Bâtiment AI)
Station 19
CH-1015 Lausanne
Switzerland
Email: kelvin@epfl.ch
Phone: +41 21 69 30267
If unreachable: +41 21 69 34494


On 07.07.20, 15:51, "CCP4 bulletin board on behalf of Christian Roth" 
mailto:CCP4BB@JISCMAIL.AC.UK> on behalf of 
christianroth...@gmail.com> wrote:

yes Eleanor is right. command line still works.[:-)]
fft is also in 7.1 distribution.



On Tue, Jul 7, 2020 at 3:43 PM Eleanor Dodson 
mailto:eleanor.dod...@york.ac.uk>> wrote:
Oh Lau - how I miss that list!
But if you just run fft online it is still distributed..wombat:Downloads 
eleanor$

fft hklin  mapout 

LABIN FP=  and so on..


On Tue, 7 Jul 2020 at 14:22, Christian Roth 
mailto:christianroth...@gmail.com>> wrote:
Hi Kelvin,
well fft as single program is kind of not longer supported as is not ccp4i. In 
i2 internally, as well in communication with mg or coot, everything is done 
using map coefficients.
Their are two options:
First via i2: Use the unusual map coefficients Task and choose not to compare 
maps, but to generate the map coefficients plus a map (button is in Advanced 
tab) the standard grid parameters can be changed, but are actually optimized 
already.
Second just save the map out of Coot (Export map)

To avoid redundancy, the old fft task was discontinued. i2 works with 
coefficients, which generates smaller files and Coot provides all the functions 
to generate the map and is its own gui.

Hope that explains a bit why things are how they are now.

Cheers
Christian

On Tue, Jul 7, 2020 at 1:56 PM Lau Kelvin 
mailto:kelvin@epfl.ch>> wrote:
Hi Christian,

I was in particular looking for the fft program (I couldn’t find that using the 
method you described) just to convert an mtz into a .map. Before in version 7.0 
I could just browse all programs, now it seems like I cannot do that (other 
than using the filter, and some seem to be missing)

At the end I just used mtz2map in phenix.

--
Kelvin Lau
https://people.epfl.ch/kelvin.lau

Protein production and structure core facility - PTPSP
EPFL SV PTECH PTPSP
AI 2146 (Bâtiment AI)
Station 19
CH-1015 Lausanne
Switzerland
Email: kelvin@epfl.ch
Phone: +41 21 69 30267
If unreachable: +41 21 69 34494


On 06.07.20, 13:34, "Christian Roth" 
mailto:christianroth...@gmail.com>> wrote:

Hi Kelvin,
not quite sure if I understand you correctly,  but if you press the Task 
Manager button you get on the right sight a list of topics (import data, 
Molecular Replacemnt etc.) each point can be open up like a file tree to see 
all programs or pipelines available. You can search with the search field 
(Filter) on top for specific program names.
Does that help?

Cheers
Christian

On Mon, Jul 6, 2020 at 1:15 PM Lau Kelvin 
mailto:kelvin@epfl.ch>> wrote:
Hello,

I am looking for a way to find the list of programs accessible using the new 
7.1 CCP4I2 interface? Is this still possible or do I have to revert back to 
version 7.0?

Best regards,

Kelvin

--
Kelvin Lau
https://people.epfl.ch/kelvin.lau

Protein production and structure core facility - PTPSP
EPFL SV PTECH PTPSP
AI 2146 (Bâtiment AI)
Station 19
CH-1015 Lausanne
Switzerland
Email: kelvin@epfl.ch
Phone: +41 21 69 30267
If unreachable: +41 21 69 34494




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Re: [ccp4bb] Flexible C terminus

[Oganesyan, Vaheh]

Hi Chitra Latka,

By far the best approach is to find what protein is interacting with the one 
you have the structure and try co-crystallizing them together. At least there 
will be some more biology (science) involved in what you will be doing. You may 
get lucky and get different packing of your full length protein and get some 
sort of structure for last 20 aa. Then what?

Regards,

From: CCP4 bulletin board  On Behalf Of chitra latka
Sent: Thursday, March 12, 2020 3:54 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Flexible C terminus

Dear All,

I am working on a protein that has flexible C terminus. None of the available 
structures even in homologs have density for C term region (around 20 odd 
residues). All the available pdb entries have missing density for these 20 
residues at C terminus.

I am going to try my luck crystallising the entire protein in hope of getting 
density for C term residues as well (Fingers crossed).

Has anyone faced a similar problem where they have managed to get density for a 
flexible terminus successfully?

Any suggestions would be appreciated.

Cheers !

Chitra Latka




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Re: [ccp4bb] nVidia 3D Vision2 glasses

[Oganesyan, Vaheh]

Colleagues,
While on stereo issues here is problem I’m trying to resolve with community 
help:
my hardware includes nVidia quadro FX1400, Xpand emiter and NewVision glasses. 
Driver taken from nVIDIA site specifically for Quadro FX 1400, long lived. On 
old Linux station these worked together fine for PyMOL and O. On newer machine 
PyMOL doesn’t complain but doesn’t show stereo either. “O” complains:


freeglut (/home/Vaheh/o/bin/170211_lin64_ono): ERROR: Internal error  in function fgOpenWindow

and quits.

Will appreciate help on resolving this issue.


Regards,

Vaheh Oganesyan, Ph.D.
Scientist, Biologic Therapeutics

AstraZeneca
R | Antibody Discovery and Protein Engineering
One Medimmune Way, Gaithersburg, MD 20878
T:  301-398-5851
vaheh.oganes...@astrazeneca.com




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[ccp4bb] "O" executables

[Oganesyan, Vaheh]

Hello Colleagues,

A bit off topic, but could someone point me in the direction of Alwyn’s 
executables for “O”?
It doesn’t reside at xray.bmc.uu.se anymore.

Regards,

Vaheh Oganesyan, Ph.D.
Scientist, Biologic Therapeutics

AstraZeneca
R | Antibody Discovery and Protein Engineering
One Medimmune Way, Gaithersburg, MD 20878
T:  301-398-5851
vaheh.oganes...@astrazeneca.com








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Re: [ccp4bb] Another difficult MR case

[Oganesyan, Vaheh]

Hi Napo,

Just in case nobody suggested this option I want to add it. Very recently I’ve 
solved structure of Fab with 8 molecules per au. It wasn’t easy to do because 
some of tNCS were dropped by programs. After contacting the developer of MolRep 
Alexey Vagin and asking him to help understand why is that happening he found a 
way to improve the program. After that improvement it worked fine. Give it a 
try. If I remember correctly you’ve said that sequence identity is around 50%. 
That is considered (from previous life in Structural Genomics center) extremely 
similar to target. Our cut-off was 30%. Anything above this value was 
considered similar and wasn’t within new fold designation. Let me know 
personally (off the bb) if you need help.


Regards,

Vaheh Oganesyan, Ph.D.
Scientist, Biologic Therapeutics

AstraZeneca
R | Antibody Discovery and Protein Engineering
One Medimmune Way, Gaithersburg, MD 20878
T:  301-398-4640  M:  240-398-0046
vaheh.oganes...@astrazeneca.com



From: CCP4 bulletin board  On Behalf Of Napoleão
Sent: Thursday, September 5, 2019 10:04 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Another difficult MR case


Dear all,
Thank you for your kind expert suggestions, and sorry for the late reply, I'm 
still trying all the suggested approaches.
Some comments:
- I can't use buccaneer yet, since I'm still working to resolve the structure.
- Robyn Stanfield's suggestion of using Shelxe to try to verify the validity of 
the MR solutions was very helpful. I'm trying it a lot, however without 
success. Thank you Robyn and all Shelx developers!
- The maps phased with partial molecular replacement solutions look reasonable, 
but do not show any sign of the rest of the molecules or of the missing side 
chains.
- ContaMiner gave me green lights, so it does not appear to be a contaminant 
(thank you Ivan Shabalin).
- Similarly, SAUC indicates there is no other PDB with cell parameters similar 
to this data set (thank you Jonathan Cooper).
- I am currently using EPMR to try to resolve the structure by MR (thank you 
Roger Rowlett).
- I am not sure if there is translational NCS, as Phil Jeffrey suggested 
despite phenix.xtriage's green lights. I'm not experienced with this issue, so 
I'm sending the log file to Randy Read as he kindly offered to help.

I think one of my problems is that the AU is big (at least to me), apparently 
containing from 9 to 12 copies of my protein, so the MR solutions contain less 
than 10% of the scatters in the AU.
Maps look good when I refine one chain of the MR solutions in Phenix (blue maps 
over most of the protein, with reasonable main chain continuity and very few 
green or red blobs), but the R values are 0.50 and I can't see any feature 
suggesting the solution is good (like a well-defined new side chain).
I will keep trying.

Thank you all again! And please let me know if any ideas cross your wise minds.
Regards,
Napo




Em 2019-08-30 06:29, Randy Read escreveu:
Dear Napoleão,

Yes, I agree with Phil that it looks like a case where there *is* translational 
NCS but it's not being used by Phaser, either because it wasn't automatically 
detected (native Patterson peak just under the default limit? more than one 
off-origin Patterson peak?) or because Phaser was told to ignore tNCS.  You 
should look in detail at the relevant section of the logfile, and manually 
override Phaser's automated decision if you think there's good evidence for 
tNCS.  I would say that, as Phil noted, the fact that two molecules are in 
basically the same orientation separated by a translation is pretty strong 
evidence.  In particular, the fact that placing the second molecule in the same 
orientation gave such a large increase in both LLG and TFZ is strong evidence: 
this tells us that having two molecules in the same orientation (even if 
they're the wrong molecule or in the wrong orientation) explains some feature 
of the data, i.e. the modulation caused by tNCS.

I'd be happy to look at the log file for you if you find it hard to interpret.

Best wishes,

Randy Read


On 29 Aug 2019, at 17:24, Phil Jeffrey 
mailto:pjeff...@princeton.edu>> wrote:

Are you *sure* there's no translational NCS ?

For example your first molecular replacement solution out of Phenix shows

EULER  293.6   27.7  288.7
FRAC -0.02  0.02  0.02
(that's "first molecule at origin in P1")

and

EULER  294.0   27.9  288.8
FRAC -0.37  0.02  0.02

which is essentially the same orientation, and a translation down one 
crystallographic axis (a*)

And this suggests to me that either Xtriage or Phaser is missing something 
here.  Does Phaser find translational NCS in its initial data analysis ?  
Unmodeled translational NCS could cause significant problems with the molecular 
replacement search.

Phil Jeffrey
Princeton




On 8/29/19 11:28 AM, Napoleão wrote:

Deal all,
Sorry for the long post.
I have a data 

Re: [ccp4bb] image

[Oganesyan, Vaheh]

Try seeding at lower concentration.

From: CCP4 bulletin board  On Behalf Of zhangyi19950109
Sent: Friday, March 22, 2019 1:55 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] image

Dear all,
A single plus-stranded RNA virus RDRP monomer grows crystals, precipitants 
(PEG8000)
with low concentration, more nucleuses and poor resolution.Reduce precipitant 
concentration can reduce the crystal nucleus, but worse resolution, precipitant 
concentration increased, many crystal nucleus and crystal is very small.  Try 
to optimize the temperature, pH, protein concentration, dehydration, additives, 
detergent, sit and hanging drop, but were not improved much.  Pease provide 
some advice methods.
This is a picture of this crystal
[cid:image001.png@01D4E091.7C7EEE00]






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[ccp4bb] R Associate II or Associate Scientist I - Protein Engineering

[Oganesyan, Vaheh]

Posting on behalf of colleague (please, do not reply to me):

·
o
§
US - Gaithersburg - MD
· Apply
· Apply with 
LinkedIn
· MedImmune is the worldwide biologics research and development arm of 
AstraZeneca. Here, you’ll have the opportunity to make a difference in people’s 
lives every day. As one of the world’s premier biotechnology companies, our 
mission is centered on delivering life-changing products that advance world 
health, and help fight and cure disease.
We’re constantly pushing the boundaries of science to deliver life-changing 
medicines to patients, with a passion for discovery and a pipeline to show for 
it. We’re pioneering innovative research and exploring novel pathways across 
key therapeutic areas including oncology, respiratory, inflammation and 
autoimmunity, cardiovascular and metabolic disease, and infection and vaccines. 
And we’re industry-leading in immunology, protein engineering and translational 
science. We offer a unique and strong collaborative network as part of the 
AstraZeneca family, as together we explore synergies between small and large 
molecules.

MedImmune has a dynamic environment that fosters collaboration and innovation. 
We attract top minds, and we nurture and build top talent.
Main Duties and Responsibilities
Major responsibilities include planning and executing experiments related to 
developing protein engineering platforms for therapeutic antibody generation 
and optimization. She/he will work with scientists to create research plans, 
execute experiments, analyze and interpret data and present results to the 
group and the department. She/he is expected to have a strong work ethic, 
excellent organizational skills and keep good records. Strong team-based 
communication and presentation skills will be required.
Essential Requirements
· The ideal candidate should have strong hands-on experience in 
Molecular Biology or Biochemistry, particularly PCR and molecular cloning 
techniques.
· Protein expression, purification and characterization experience is 
also required.
· Experience in cell culture, FACS and antibody engineering 
(phage/yeast display-based selections) is not required but is also highly 
desirable.
Education and Experience
· Research Associate II - Bachelor’s Degree in Molecular Biology or 
other related fields with two to five years of relevant experience in industry 
(preferred) or academia, or Master’s Degree with zero to two years of relevant 
experience in industry (preferred) or academia
· Associate Scientist I – Bachelor’s Degree in Molecular Biology or 
other related fields with five to eight years of relevant experience in 
industry (preferred) or academia, or Master’s Degree with two to five years of 
relevant experience in industry (preferred) or academia
Knowledge
· Hands-on experience with advanced PCR and molecular cloning 
techniques, particularly where troubleshooting was required to drive a project 
forward
· Familiarity with cell culture, recombinant protein production and 
purification. Biochemical characterization such as affinity determination and 
immunoassays (ELISA).
· Experience with antibody engineering and FACS is highly desirable.
· The ability to learn and embrace new technologies quickly, work 
independently and communicate well with the team.




Regards,

Vaheh Oganesyan, PhD
www.medimmune.com




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Re: [ccp4bb] suggestions for cryoprotectant

[Oganesyan, Vaheh]

Firdous,

Many crystallographers (if not all) went through case like you describe. So far 
you’ve got great suggestions that are worth trying. I’ll add one more from my 
own experience. In all cases I tried it appeared that addition of 0.5 to 1% v/v 
of glycerol or ethylene glycol to drop (either having it added to protein 
solution or to precipitant mix) doesn’t change “crystallizability” of the 
protein. However, that minor presence of the cryo agent in the solution where 
crystals grew allows increasing the concentration of cryo agent to 20-25% 
during harvesting, which should be enough for cryopreservation.

Good luck.


Regards,

Vaheh Oganesyan, PhD
www.medimmune.com



From: CCP4 bulletin board  On Behalf Of Firdous Tarique
Sent: Friday, October 19, 2018 5:58 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] suggestions for cryoprotectant

Dear members

I have got beautiful crystal hits in SaltRx screens which are not diffracting 
to a good resoultion. All of them are salt based condition and I am not able to 
formulate a good cryoprotectant for these crystals. I also think that in my 
case the poor resolution is due to a poor cryoprotectant selection.

The conditions are as follows:

1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0
2>0.5M KCN 100mM Tris pH8.5
3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0
4>4M Sodium Nitrate 100mM Tris pH8.5
5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6

There are few more conditions but so far not able to see good diffraction with 
using lower peg and glycerol based cryoprotectants.

Can anybody suggest me good cryos conditions for salt based crystallization 
conditions or anything good for SaltRx crystallization hits.

Thanks

Firdous



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Re: [ccp4bb] Sulphate or phosphate?

[Oganesyan, Vaheh]

Not very elegant way of doing what you want but as a last resort I used 
distance criteria. P-O distance is 1.6A, S-O distance is 1.4A. Provided your 
coordinate error is in the range of 0.1A you may cautiously suggest one or the 
other. However, it may be impossible to prove that what you see is not a 
mixture of both.


Regards,

Vaheh Oganesyan, PhD
www.medimmune.com



-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of David 
Schuller
Sent: Tuesday, July 31, 2018 9:39 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Sulphate or phosphate?

How can one distinguish between a sulphate or phosphate in an electron density 
map? Both are present in the mother liquor, and resolution is in the range of 
1.75 - 2.25 A


--
===
All Things Serve the Beam
===
David J. Schuller
modern man in a post-modern world
MacCHESS, Cornell University
schul...@cornell.edu



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[ccp4bb] Arg distorsion during refinement

[Oganesyan, Vaheh]

Dear crystallographers,

Lately when refining a structure (at 2.8A) with Refmac5 I've found that nearly 
all Arg residues get distorted at one angle:  NE-CZ-NH1(2). Starting model has 
120*, final model 123*(117*), which validation server considers a major issue. 
May any of you recognize why is this happening? Don't remember seeing anything 
like this before.
Current CCP4 version 7.0.050; Refmac5 version 5.8.0189.

Last structure deposited in December'17 did not have those issues. CCP4 version 
then was 7.0.047; Refmac5 version was the same.

Thank you for your time.

Regards,

Vaheh Oganesyan
MedImmune, ADPE
www.medimmune.com


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information that is not in the public domain, such information is considered by 
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Re: [ccp4bb] His-6 versus His-10 tag

[Oganesyan, Vaheh]

Hi Herman,

I haven't done His-6 versus His-10 for the same protein, but have done that for 
different ones with success. However, if in His-6 containing protein structure 
the packing or folding is such that you don't see His-6 then it shouldn't 
matter it is 6 or 10. Just an opinion.

Regards,

Vaheh Oganesyan
www.medimmune.com

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
herman.schreu...@sanofi.com
Sent: Tuesday, September 19, 2017 6:11 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] His-6 versus His-10 tag

Dear BB,

We are planning the production of a protein for crystallization. From 
literature, we know that the construct with a 6-histidine tag crystallizes. 
However, for other biophysical measurements, we would prefer to have a 
10-histidine tag.

Does anyone has experience with His-6 versus His-10 tags in terms of 
crystallization success?

Thanks for your help!
Herman


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Re: [ccp4bb] Risk assessment for heavy atom soaking - examples?

[Oganesyan, Vaheh]

James,

What you wrote doesn't look like official risk assessment document. However, 
your essay is very informative and entertaining. Thank you.

Regards,

Vaheh Oganesyan
www.medimmune.com


-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of James 
Holton
Sent: Wednesday, September 06, 2017 2:45 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Risk assessment for heavy atom soaking - examples?

Something that could perhaps be of use here is what I like to call the 
"Anchovie Pizza Equivalent" (APE), which is about 1 microgram of mercury.  
According to the Food and Drug Administration website here in the USA the 
average mercury content of anchovies is 0.34 ppm, which is about 1 microgram 
per ounce of fish.  Tuna can be higher, but varies a lot from fish to fish.  My 
point here is that most institutions regard the amount of mercury you bring 
onsite for purposes of eating for lunch, be it sushi or pizza, as small enough 
to be negligible.  I tend to agree.  So, one could argue that 1 microgram of Hg 
per day is a "safe amount".  Especially if you don't eat it.

In terms of protein crystals, a 100 micron wide cube has a volume of 1 
nanoliter, and if it were soaked to a final concentration of 50 mM Hg that is 
1e-9 L * 50e-3 mol/L *200 g/mol = 10 ng.  So, 100 protein crystals soaked with 
Hg add up to roughly 1 APE.  Please note that I am in no way encouraging you to 
eat your protein crystals, and especially not the solutions you soak them in.  
You should do your own APE calculations for those.  But I do think it important 
to note just how tiny the amount of metal in our crystals really is.

Now, mercury is purportedly the second-most-toxic metal after Plutonium.  But 
Pu derivatives are uncommon.  In fact, until recently
(4zhd) Pu derivatives were unheard of. The authors I'm sure will tell you 4zhd 
involved no small amount of paperwork.  But as long as you are not working with 
Pu, you can regard every other metal as less toxic than Hg.

Another good example is selenium; by far the most common metal derivative.  
Although toxic, Se is also a dietary requirement.  I suppose this is an 
excellent demonstration of what "moderation" really means.  The Recommended 
Daily Allowance (RDA) of selenium in the USA for adult men and pregnant women 
is 55-60 micrograms per day.  In crystals, one Se atom per 100 amino acids at 
50% solvent comes to an overall concentration of 50 mM.  So, a 100 micron 
crystal contains about 4 ng of Se.  It would take 15,000 such crystals to add 
up to the US RDA.  The synchrotrons I work at don't go thought that many 
crystals every day. But even if they did, I'd stick to my commercially 
available multivitamin to get my dietary selenium.

So, although it is never a good idea to be sloppy with chemicals in the lab, I 
think it is also important to do the math and think about not just the toxicity 
of the things we work with on the bench, but the everyday items all around us.  
It is never a good idea to be antagonistic with regulators about such things.  
They are only trying to do their job, and all they are trained to know about 
are LD50s and how to stay as far below them as possible.  A little 
gently-pointed-out insight into non-lethal applications of heavy metals can be 
helpful all around.  The over-the-counter drug Pepto Bismol (bismuth 
subsalicylate) is almost 50% bismuth by weight, a metal that is right next to 
mercury on the periodic table. Brominated vegetable oil contains no bromine, by 
the way.  And dandruff shampoos such as Selsun Blue make an excellent and 
surprisingly radiation-hard reference for the selenium edge.

-James Holton
MAD Scientist

On 9/4/2017 3:13 AM, Dr Stephen Graham wrote:
> Hi all,
>
> (This email is aimed primarily at my UK colleagues, but feel free to
> read on and gloat that you don't have to write safety forms in your
> lab/country!).
>
> I need to sort out written risk assessments for heavy atom soaking of
> crystals in my lab. I wondered whether anyone would be willing to
> share the risk assessments they have in their institute/company so
> that I can seek inspiration and make sure I'm keeping up with best
> practice.
>
> Many thanks,
>
> Stephen
>
To the extent this electronic communication or any of its attachments contain 
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Re: [ccp4bb] NMR or Homology Model as a MR model

[Oganesyan, Vaheh]

Percent of identity/similarity is a number that might be often misleading when 
used as a judgement rule for molecular replacement. Very high or very low 
numbers are almost always indicative of corresponding outcome. The numbers in 
twilight zone of 20 to 35%, however, are not. When aligning sequences of target 
protein with potential MR models pay attention to residues like Cys, Trp, Phe, 
Tyr. Last three could be used in place of each other. Aligned Cys, for example, 
are very good indication of fold similarity. Ding between aligned Cys is 
indicative of difference in loop lengths, etc. When possible use more sequences 
to align, not just two. Also, there might be some info about your protein’s 
either binding partner or an active site, if it is an enzyme. In that case 
check out alignment of residues associated with the function.

Regards,

Vaheh Oganesyan
www.medimmune.com

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nishant 
Varshney
Sent: Tuesday, August 29, 2017 6:51 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] NMR or Homology Model as a MR model

Dear Crystallographers,

I am working to solve an human protein structure which has a domain sequence 
identity of 24% with domain of another protein.  As Phaser as well as Molrep 
failed to give any definite solution (TFZ=3.7 from MR), I want to ask, if 
solution structure of another protein having domain sequence similarity of 25% 
or a homology model can be used as a template for MR?
Many thanks and Regards
Nishant

--
Dr. Nishant Kumar Varshney,
Research Associate,
C/O Dr. Sameena Khan,
Drug Discovery Research Center,
Translational Health Science and Technology Institute (THSTI)
NCR Biotech Science Cluster,
3rd Milestone, Faridabad – Gurgaon Expressway,
Faridabad – 121001 (HARYANA), India
Ph: +91- 0129-2876477
Mob: 8390564690
To the extent this electronic communication or any of its attachments contain 
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Re: [ccp4bb] crystallization optimization

[Oganesyan, Vaheh]

What I’m about to write should be referred as a question rather than an answer. 
However, it might also help to find the answer to crystallization question 
discussed here.
The good old crystallization diagram so far for me was something that I’d look 
after successful crystallization story and find in which direction my 
optimization went. Each condition in every screen is just a point at the 
diagram. Were on the diagram that point is situated you don’t know because the 
scales of X and Y axes are unknown. You can find those scales by deliberately 
setting up similar screens with diluted (or concentrated, or both) of protein 
sample (Y axis scale) and diluted (mostly) crystallization screen. This is the 
way I can make use of the crystallization diagram. Unfortunately, often we 
cannot spare enough protein to do so. In such cases going through different 
screens and looking for similar conditions sometime allows finding horizontal 
line on which your crystallization position should be. After this few 
optimization attempts at different protein concentrations may help finding 
position on the diagram and clues where to go.

I hope what I just wrote makes sense. If there is a better way of using 
crystallization diagram I’d love to hear.

Regards,

Vaheh Oganesyan
www.medimmune.com

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Philippe 
BENAS
Sent: Thursday, July 13, 2017 12:47 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] crystallization optimization

Dear all,

I fully agree with all the suggestions, but it seems that no one has raised the 
issue of the solubility curve changes on the pH. If the dilution of the protein 
or precipating agent can indeed modify starting and the equilbrium points on 
the phase diagram, I would also suggest trying various pH as they can change a 
whole lot of the net protein charge, therefore the corresponding solubility 
curve and nucleation zone and hence the entire corresponding phase diagram (for 
more info PubMed search with "Madeleine Riess-Kautt" as keywords, a great 
scientist who dedidacted her career to understanding of the so-called 
Hofmeister series).


All the best,
Philippe


Philippe BENAS, Ph.D.
Dog in the manger
"Un importun survient qui trouble l'intimité, qui arrête l'expansion, qui glace 
le plaisir, - probablement comme un étranger tombant au milieu d'enfants en 
train de danser une ronde", Alfred Delvau, Dictionnaire de la langue verte 
(1866).

Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS
Faculté de Pharmacie, Université Paris Descartes
Case 48
Av, de l'Observatoire
F-75270 PARIS cedex 06
+33.1.5373.1599
E-mails: 
philippe.be...@parisdescartes.fr, 
philippe_be...@yahoo.fr
URLs: 
http://lcrbw.pharmacie.univ-paris5.fr/
 , 
http://lcrbw.pharmacie.univ-paris5.fr/spip.php?article18




De : Patrick Shaw Stewart >
À : CCP4BB@JISCMAIL.AC.UK
Envoyé le : Mercredi 12 juillet 2017 17h28
Objet : Re: [ccp4bb] crystallization optimization


Alun

I agree Frank's point is very interesting - and he intriguingly refers us to 
the phase diagram.

Is the point that Line A is longer than Line B ?

Best wishes

Patrick




[Inline images 2]






On 12 July 2017 at 14:40, Alun R Coker 
> wrote:
Hi Everyone,
Franks point is really interesting. We routinely reduce the protein 
concentration when we see too many precipitated wells, but we never dilute the 
screen. Has anyone tried this?
All the best,
Alun

On 12/07/17 08:48, Frank von Delft wrote:
The point I was failing to make:  reducing either protein or precipitant 
concentration will indeed reduce 

Re: [ccp4bb] Problem with a cell content

[Oganesyan, Vaheh]

Anna,

Eleanor is raising very important question: if you have NCS then there should 
be another similar entity in the asu. Have you detected off origin peak while 
using your final P6222 or lower sg?


From: Koromyslova, Anna [mailto:a.koromysl...@dkfz-heidelberg.de]
Sent: Tuesday, July 11, 2017 1:38 PM
To: Oganesyan, Vaheh; Phil Jeffrey; CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Problem with a cell content

Dear Vaheh and Phil,

Sorry, I was a bit misleading – antibody fragment I used is a nanobody (VHH, 
15kDa). I was worried about the cell content because when I started the pdb 
deposition, there was a warning that solvent content is expected to be below 
80%, so I thought maybe I missed a correct solution. But if that’s acceptable 
value the problem is solved.
Thank you for the quick answer and the suggestion about the resolution!

Best regards,

Anna

Dr. Anna Koromyslova, Postdoctoral researcher
German Cancer Research Center (DKFZ), F150
Im Neuenheimer Feld 242
D-69120 Heidelberg
Germany


From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of "Oganesyan, Vaheh" 
<oganesy...@medimmune.com<mailto:oganesy...@medimmune.com>>
Reply-To: "Oganesyan, Vaheh" 
<oganesy...@medimmune.com<mailto:oganesy...@medimmune.com>>
Date: Tuesday, July 11, 2017 at 7:05 PM
To: "CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>" 
<CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: Re: [ccp4bb] Problem with a cell content

Похоже, что вы уже решили структуру. Почему вас беспокоит плотность ваших 
кристаллов? Кристаллы бывают разные.

First of all Fab by itself is already almost 50 kDa, so complex with antigen 
should be more than 50 kDa. Because you already solved the structure calculate 
the molecular mass based on your pdb file and rerun Matthews with correct mass. 
New numbers may be quite a bit different. Good indication of relatively low 
crystal density and consequently loose packing is the resolution of your data 
set. If you did not throw away data beyond 2.9A I’d suggest use them all. The 
reflections are too valuable to throw away. If data beyond some resolution is 
weak then they will have low contribution to the structure. Best if you 
calculate electron density maps at different resolutions at the end of 
refinement, compare them and use resolution that makes difference.



Regards,

Vaheh
8-5851

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Koromyslova, Anna
Sent: Tuesday, July 11, 2017 12:32 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] Problem with a cell content

Dear CCP4 members,

I am working on a structure of a protein in complex with an antibody fragment 
(approx. 50kDa together). Molecular replacement with closely related proteins 
always comes up with one complex in the asymmetric unit, although MW of protein 
to which Matthews applies is 125kDa and corresponds to two complexes.
Phaser gives two warnings:
Large non-origin Patterson peak indicates that translational NCS is present.
Solutions with Z-scores greater than 27.2 (the threshold indicating a definite 
solution) were rejected for failing packing test

I couldn’t get a solution with two subunits although I have tried multiple 
combinations including only conserved parts of both proteins and different 
space groups including P1. Phenix Autobuild also yielded only one complex.

So, the question is whether I can use that structure as is despite very high 
solvent content (80%) or should I try smth else. I would be very grateful for 
any suggestions.

When the solution with a single complex is refined the statistics are the 
following:

R-work  0.2129
R-free  0.2459
Matthews Coefficient: 6.22
Percentage Solvent: 80.22
Resolution range (Å)  48.34  - 2.9 (2.98  - 2.9)
Space group P 62 2 2
Unit cell  167.45 167.45 143.538 90 90 120
Multiplicity  19.1 (18.3)
Completeness (%)99.44 (94.39)
Mean I/sigma(I) 24.59 (2.71)
Wilson B-factor64.28
R-merge   0.1256 (1.186)
R-meas   0.1291
CC1/2 0.999 (0.85)
CC*1 (0.959)

Thank you very much for your help,

Anna


Dr. Anna Koromyslova, Postdoctoral researcher
German Cancer Research Center (DKFZ), F150
Im Neuenheimer Feld 242
D-69120 Heidelberg
Germany

To the extent this electronic communication or any of its attachments contain 
information that is not in the public domain, such information is considered by 
MedImmune to be confidential and proprietary. This communication is expected to 
be read and/or used only by the individual(s) for whom it is intended. If you 
have received this electronic communication in error, please reply to the 
sender advising of the error in transmission an

Re: [ccp4bb] Problem with a cell content

[Oganesyan, Vaheh]

Похоже, что вы уже решили структуру. Почему вас беспокоит плотность ваших 
кристаллов? Кристаллы бывают разные.

First of all Fab by itself is already almost 50 kDa, so complex with antigen 
should be more than 50 kDa. Because you already solved the structure calculate 
the molecular mass based on your pdb file and rerun Matthews with correct mass. 
New numbers may be quite a bit different. Good indication of relatively low 
crystal density and consequently loose packing is the resolution of your data 
set. If you did not throw away data beyond 2.9A I’d suggest use them all. The 
reflections are too valuable to throw away. If data beyond some resolution is 
weak then they will have low contribution to the structure. Best if you 
calculate electron density maps at different resolutions at the end of 
refinement, compare them and use resolution that makes difference.



Regards,

Vaheh
8-5851

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Koromyslova, Anna
Sent: Tuesday, July 11, 2017 12:32 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Problem with a cell content

Dear CCP4 members,

I am working on a structure of a protein in complex with an antibody fragment 
(approx. 50kDa together). Molecular replacement with closely related proteins 
always comes up with one complex in the asymmetric unit, although MW of protein 
to which Matthews applies is 125kDa and corresponds to two complexes.
Phaser gives two warnings:
Large non-origin Patterson peak indicates that translational NCS is present.
Solutions with Z-scores greater than 27.2 (the threshold indicating a definite 
solution) were rejected for failing packing test

I couldn’t get a solution with two subunits although I have tried multiple 
combinations including only conserved parts of both proteins and different 
space groups including P1. Phenix Autobuild also yielded only one complex.

So, the question is whether I can use that structure as is despite very high 
solvent content (80%) or should I try smth else. I would be very grateful for 
any suggestions.

When the solution with a single complex is refined the statistics are the 
following:

R-work  0.2129
R-free  0.2459
Matthews Coefficient: 6.22
Percentage Solvent: 80.22
Resolution range (Å)  48.34  - 2.9 (2.98  - 2.9)
Space group P 62 2 2
Unit cell  167.45 167.45 143.538 90 90 120
Multiplicity  19.1 (18.3)
Completeness (%)99.44 (94.39)
Mean I/sigma(I) 24.59 (2.71)
Wilson B-factor64.28
R-merge   0.1256 (1.186)
R-meas   0.1291
CC1/2 0.999 (0.85)
CC*1 (0.959)

Thank you very much for your help,

Anna


Dr. Anna Koromyslova, Postdoctoral researcher
German Cancer Research Center (DKFZ), F150
Im Neuenheimer Feld 242
D-69120 Heidelberg
Germany

To the extent this electronic communication or any of its attachments contain 
information that is not in the public domain, such information is considered by 
MedImmune to be confidential and proprietary. This communication is expected to 
be read and/or used only by the individual(s) for whom it is intended. If you 
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sender advising of the error in transmission and delete the original message 
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cooperation.

Re: [ccp4bb] [phenixbb] Asymmetric unit content

[Oganesyan, Vaheh]

Matthews coef. is not made to give you exact answer about number of molecules 
in the asu. As you rightly say, it is just probability.
I’d have been convinced seeing unbiased (before refinement with fifth molecule) 
diff. electron density map or discontinuous packing in the crystal in the 
absence of that last molecule. It might be that the fifth molecule occupancy is 
not 100%. Have it at lower occupancy and see if this will get reflected in the 
quality of the EDM. Alternatively, refine with and without the fifth molecule. 
Difference in Rfree could be a good measure here. At resolution of 7 A there is 
very little to do except few cycles of rigid body refinement. However, the best 
advice and probably the most hated one is to get better diffracting crystals.


Regards,

Vaheh
8-5851

From: phenixbb-boun...@phenix-online.org 
[mailto:phenixbb-boun...@phenix-online.org] On Behalf Of Sharan Karade
Sent: Monday, December 12, 2016 9:56 AM
To: o...@quantumbioinc.com
Cc: pheni...@phenix-online.org
Subject: Re: [phenixbb] Asymmetric unit content

I have calculated Mathews coefficient also, the probability shows five 
molecules in ASU. But it fails to convince my supervisor.

On Mon, Dec 12, 2016 at 6:52 AM, 
> wrote:
Compute Matthews coefficient.
e.g. using
http://www.ruppweb.org/mattprob/default.html

Oleg



-Original Message-
From: "Sharan Karade" >
Sent: Sunday, December 11, 2016 6:05am
To: pheni...@phenix-online.org
Subject: [phenixbb] Asymmetric unit content

___
phenixbb mailing list
pheni...@phenix-online.org
http://phenix-online.org/mailman/listinfo/phenixbb
Unsubscribe: 
phenixbb-leave@phenix-online.orgDear
 all,

   I have solved the structure in C121 space group, having five
molecules in asymmetric unit, my supervisor have doubt about the fifth
molecule becoz of poor density fit, if i delete the fifth molecule, there
is a space for the molecule and crystal is not continuous, i calculated
difference map from Phenix by deleting fifth molecule and got the Map
showing presence of molecule. My supervisor want me to get SetMet data, but
crystals are diffracting poor (7A). Is there any way to convince my guide.


advance thanks for valuable suggestions.

--
Sharan
C/O Dr. J V Pratap
Senior Research Fellow,
CSIR-Central Drug Research Institute,
Lucknow
Oleg Borbulevych, Ph.D.
Staff Scientist
QuantumBio, Inc.
2790 W. College Ave.
State College, PA 16801
E-mail: oborbulev...@yahoo.com, 
o...@quantumbioinc.com
Linkedin Professional Profile: 
http://www.linkedin.com/pub/oleg-borbulevych/22/454/b80



--
Sharan
C/O Dr. J V Pratap
Senior Research Fellow,
CSIR-Central Drug Research Institute,
Lucknow


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MedImmune to be confidential and proprietary. This communication is expected to 
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cooperation.

Re: [ccp4bb] Nitrate versus Carbonate

[Oganesyan, Vaheh]

Would this figure answer your question? Cells must have way of feeling the 
difference in the bond lengths, and strength and nuances in hybridization. At 
least, I hope they do.

Regards,

Vaheh Oganesyan
www.medimmune.com

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, 
Jacob
Sent: Thursday, November 10, 2016 3:42 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Nitrate versus Carbonate

Dear Crystallographers,

I don't think there is any feasible way crystallographically to distinguish 
between nitrate and carbonate or bicarbonate-correct? But that is not my main 
question.

My main question is: given that nitrate and carbonate are both very important 
and also very different physiologically, and therefore they must be 
distinguished/recognized by cells, how is this done, since the ions are so 
similar in structure? Is there some aspect of these ions that differs 
dramatically of which I am not aware? What kind of "handles" could a protein 
grab onto to distinguish between nitrate and carbonate/bicarbonate?

JPK


***
Jacob Pearson Keller, PhD
Research Scientist
HHMI Janelia Research Campus / Looger lab
Phone: (571)209-4000 x3159
Email: kell...@janelia.hhmi.org
***

To the extent this electronic communication or any of its attachments contain 
information that is not in the public domain, such information is considered by 
MedImmune to be confidential and proprietary. This communication is expected to 
be read and/or used only by the individual(s) for whom it is intended. If you 
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[ccp4bb] alternative side chain locations

[Oganesyan, Vaheh]

Colleagues,

Working at relatively high resolution of 1.5A allows in some cases seeing 
alternative positions of side chains. After placing those in the coordinate 
file with occ adding to 1.0 and running refinement (latest REFMAC on Win) I 
find that occupancies of all atoms in the specific residue with altlocs are 
being modified.
In the refinement through GUI I've failed to find place where occ refinement is 
turned on and assumed that occ refinement will be automatically turned on upon 
presence of altlocs. Apart from being aware of danger associated with some 
assumptions would you be able to advise what I'm missing in refinement with 
altlocs?
Thank you in advance.


Regards,

Vaheh Oganesyan
MedImmune, ADPE
www.medimmune.com
To the extent this electronic communication or any of its attachments contain 
information that is not in the public domain, such information is considered by 
MedImmune to be confidential and proprietary. This communication is expected to 
be read and/or used only by the individual(s) for whom it is intended. If you 
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Re: [ccp4bb] Rfree below Rwork

[Oganesyan, Vaheh]

Hi Herman,

While you're correct regarding increase in number of entities in the asu upon 
lowering the symmetry, you're not correct for specific case of R32. One 
molecule per asu in R32 equals 18 molecules per asu in P1.

Regards,

Vaheh Oganesyan
www.medimmune.com

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
herman.schreu...@sanofi.com
Sent: Wednesday, July 01, 2015 7:34 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] AW: [ccp4bb] Rfree below Rwork

Dear Boaz,

One can equally well describe a R32 crystal with one molecule in the asymmetric 
unit as P1 and 6 molecules in the asymmetric unit. In this case, the NCS in P1 
is identical to the crystallographic symmetry in R32.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Boaz 
Shaanan
Gesendet: Mittwoch, 1. Juli 2015 12:10
An: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Rfree below Rwork

Just wondering about Eleanor's interesting remark: would the Rf  Rw go as low 
as reported by Wolfram (0.22) in case of a wrong space group?

 Boaz


Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

E-mail: bshaa...@bgu.ac.ilmailto:bshaa...@bgu.ac.il
Phone: 972-8-647-2220  Skype: boaz.shaanan
Fax:   972-8-647-2992 or 972-8-646-1710




From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Eleanor Dodson 
[eleanor.dod...@york.ac.uk]
Sent: Tuesday, June 30, 2015 8:55 PM
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Rfree below Rwork
I suppose if I was the referee for this structure and your FreeR is so close to 
the Rfactor I would ask you to ensure you had the right space group - is the 6 
fold NCS actually 2 fold NCS with a crystallographic 3 fold..
Cases occur where R32 is indexed as C2..

Certainly if the Rfree set is assigned randomly to reflections which are 
symmetry equivalents then you see this phenomena of Rfree = Rfactor

Eleanor

On 30 June 2015 at 18:26, Gerard Bricogne 
g...@globalphasing.commailto:g...@globalphasing.com wrote:
Dear Wolfram,

 I have a perhaps optimistic view of the effect of high-order NCS
on Rfree, in the sense that I don't view it as a problem. People
have agonised to extreme degrees over the difficulty of choosing a
free set of reflections that would produce the expected gap between
Rwork and Rfree, and some of the conclusions were that you would need
to hide almost half of your data in some cases!

 I think it is best to remember that the idea of cross-validation
by Rfree is to prevent overfitting, i.e. ending up with a model that
fits the amplitudes too well compared to how well it determines the
phases. In the case of high-order NCS (in your case, the U/V ratio
that the old papers on NCS identified as the key quantity to measure
the phasing power of NCS would be less than 0.1!) the phases and the
amplitudes are so tightly coupled that it is simply impossible to fit
the amplitudes without delivering phases of an equally good quality.
In other words there is no overfitting problem (provided you do have
good and complete data) and the difference between Rfree and Rwork is
simply within the bounds of the statistical spread of Rfree depending
on the free set chosen.

 You are lucky to have 6-fold NCS, so don't let any reviewer
convince you that it is a curse, and make you suffer for it :-) .


 With best wishes,

  Gerard.

--
On Tue, Jun 30, 2015 at 12:58:44PM -0400, wtempel wrote:
 Hello,
 my question concerns refinement of a structure with 6-fold NCS (local
 automatic restraints in REFMAC) against 2.8 A data. The size of my free set
 is 1172 selected in thin resolution shells (SFTOOLS) and corresponding to
 4.3 % of reflections.
 A refmac run of 10 cycles of TLS and 10 cycles of CGMAT starts out at
 Rfree/Rcryst 0.271/0.272. After the 10th TLS cycle I have 0.227/0.224. Yes,
 Rfree  Rcryst. At the end of CGMAT I have 0.2072/0.2071.
 I understand that NCS stresses the independence assumption of the free set.
 Am I correct in believing that Rfree *may* be smaller than Rcryst even in
 the absence of a major mistake? My hope is that the combined wisdom of
 ccp4bb followers can point out my possible mistake,  suggest tests that I
 may perform to avoid them and, possibly, arguments in defense of a
 crystallographic model with Rfree  Rcryst.
 Many thanks,
 Wolfram Tempel
--

 ===
 * *
 * Gerard Bricogne 
g...@globalphasing.commailto:g...@globalphasing.com  *
 * *
 * Global Phasing Ltd. *
 * Sheraton House, Castle Park Tel: 
+44-(0)1223-353033tel:%2B44-%280%291223-353033 *
 * Cambridge CB3 0AX, UK   Fax: 

Re: [ccp4bb] AW: [ccp4bb] Rfree below Rwork

[Oganesyan, Vaheh]

Dirk, you're right. With rhombohedral setting there are only six copies of 
asymmetric units in the unit cell. So, technically, Herman was not wrong.

Regards,

Vaheh Oganesyan
www.medimmune.com

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dirk 
Kostrewa
Sent: Thursday, July 02, 2015 10:03 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] AW: [ccp4bb] Rfree below Rwork

Hi Herman and Boaz,

in the trigonal setting R32 (not in the hexagonal setting H32), the unit cell 
in R32 contains 6 copies. If you take the whole R32 unit cell as a P1 cell, you 
would have 6 copies in the asymmetric unit, as Hermann wrote.

Best regards,

Dirk.
Am 02.07.15 um 15:52 schrieb 
herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.com:
You are right. After I sent the email to the bulletin board, I realized that in 
R32 there must be more then unit cells but did not send a correction.
Next time, I will check the space group before sending an email.
Best regards,
Herman

Von: Oganesyan, Vaheh [mailto:oganesy...@medimmune.com]
Gesendet: Donnerstag, 2. Juli 2015 15:48
An: Schreuder, Herman RD/DE; 
CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Betreff: RE: [ccp4bb] Rfree below Rwork

Hi Herman,

While you're correct regarding increase in number of entities in the asu upon 
lowering the symmetry, you're not correct for specific case of R32. One 
molecule per asu in R32 equals 18 molecules per asu in P1.

Regards,

Vaheh Oganesyan
www.medimmune.com

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.com
Sent: Wednesday, July 01, 2015 7:34 AM
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] AW: [ccp4bb] Rfree below Rwork

Dear Boaz,

One can equally well describe a R32 crystal with one molecule in the asymmetric 
unit as P1 and 6 molecules in the asymmetric unit. In this case, the NCS in P1 
is identical to the crystallographic symmetry in R32.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Boaz 
Shaanan
Gesendet: Mittwoch, 1. Juli 2015 12:10
An: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Rfree below Rwork

Just wondering about Eleanor's interesting remark: would the Rf  Rw go as low 
as reported by Wolfram (0.22) in case of a wrong space group?

 Boaz


Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

E-mail: bshaa...@bgu.ac.ilmailto:bshaa...@bgu.ac.il
Phone: 972-8-647-2220  Skype: boaz.shaanan
Fax:   972-8-647-2992 or 972-8-646-1710




From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] 
on behalf of Eleanor Dodson 
[eleanor.dod...@york.ac.ukmailto:eleanor.dod...@york.ac.uk]
Sent: Tuesday, June 30, 2015 8:55 PM
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Rfree below Rwork
I suppose if I was the referee for this structure and your FreeR is so close to 
the Rfactor I would ask you to ensure you had the right space group - is the 6 
fold NCS actually 2 fold NCS with a crystallographic 3 fold..
Cases occur where R32 is indexed as C2..

Certainly if the Rfree set is assigned randomly to reflections which are 
symmetry equivalents then you see this phenomena of Rfree = Rfactor

Eleanor

On 30 June 2015 at 18:26, Gerard Bricogne 
g...@globalphasing.commailto:g...@globalphasing.com wrote:
Dear Wolfram,

 I have a perhaps optimistic view of the effect of high-order NCS
on Rfree, in the sense that I don't view it as a problem. People
have agonised to extreme degrees over the difficulty of choosing a
free set of reflections that would produce the expected gap between
Rwork and Rfree, and some of the conclusions were that you would need
to hide almost half of your data in some cases!

 I think it is best to remember that the idea of cross-validation
by Rfree is to prevent overfitting, i.e. ending up with a model that
fits the amplitudes too well compared to how well it determines the
phases. In the case of high-order NCS (in your case, the U/V ratio
that the old papers on NCS identified as the key quantity to measure
the phasing power of NCS would be less than 0.1!) the phases and the
amplitudes are so tightly coupled that it is simply impossible to fit
the amplitudes without delivering phases of an equally good quality.
In other words there is no overfitting problem (provided you do have
good and complete data) and the difference between Rfree and Rwork is
simply within the bounds of the statistical spread of Rfree depending
on the free set chosen.

 You are lucky to have 6-fold NCS, so don't let any reviewer
convince you that it is a curse, and make you suffer for it :-) .


 With best wishes,

  Gerard.

--
On Tue, Jun 30, 2015 at 12:58:44PM -0400, wtempel wrote:
 Hello,
 my question concerns refinement of a structure with 6-fold NCS (local
 automatic restraints in REFMAC

Re: [ccp4bb] 3BDN, 16.5% Ramachandran Outliers!!!!!

[Oganesyan, Vaheh]

Hi Robbie and Co,

These things are happening now too. Look at the entry 4x4m. The paper got 
published in January, PDB released coordinates in April. That means reviewers 
did not have a chance to look even at validation report. In my opinion, 
whatever it is worth, every journal dealing with crystal structures should, at 
the very least, request the validation report from PDB including Nature, 
Science and PNAS.
What is also interesting that at the end PDB released the coordinates with 
large number of outliers. I don't think those can be justified with low 
resolution of the data.

Regards,

Vaheh Oganesyan
www.medimmune.com

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Robbie 
Joosten
Sent: Friday, April 24, 2015 2:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] 3BDN, 16.5% Ramachandran Outliers!

The PDB_REDO entry for 3bdn was pretty old, so I replaced it using a newer 
version of PDB_REDO that can use nucleic acid restraints from LibG: 
http://www.cmbi.ru.nl/pdb_redo/bd/3bdn/index.html. Obviously, the new structure 
model is far from brilliant (PDB_REDO doesn't rebuild at this resolution), but 
Molprobity seems to like it quite a bit more.

I agree with the replies so far in that:
- The topic starter was rather blunt and could have been more subtle. He should 
probably go work in the Netherlands ;)
- Building structure models at 3.9A is incredibly difficult.
- The tools we have now are much better than in 2008.

However, we should not act like 2008 were still in the dark ages of 
crystallography. There are a lot of good structures available from that time 
(and also from long before) even at that resolution. That is not surprising 
seeing that we also already had very good building and refinement tools 
available. We also had enough validation tools available to tell us that this 
particular structure model isn't very good. I really believe that a good 
crystallographer that was not pressed for time (or at least didn't rush) could 
have done better with the data and the tools available.

I'm now going to hide behind an asbestos wall to say this:
The manuscript was submitted in July 29th 2007, the PDB entry was deposited 
November 15th 2007. That means that the referees probably did not have a chance 
to see the finished structure model, at least not in the first pass. This 
implies that the authors didn't want to deposit the model on time. There are a 
whole lot of excuses for this, that are fortunately dealt with now 
(http://onlinelibrary.wiley.com/doi/10.1107/S0907444913029168/abstract), but 
the referees could have been a bit more critical. They should have at least 
seen that the supplemental table 1 did not show any Ramachandran statistics. We 
can only speculate what happened. I'm guessing that the authors didn't finish 
the structure yet and rushed the publication through to avoid being scooped or 
for the general glory of a Nature paper. To bad that came at the expense of the 
crystallography.

Cheers,
Robbie

Date: Thu, 23 Apr 2015 18:43:13 +
From: kell...@janelia.hhmi.orgmailto:kell...@janelia.hhmi.org
Subject: Re: [ccp4bb] 3BDN, 16.5% Ramachandran Outliers!
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Is it in pdb redo? Take a look here: 
http://www.cmbi.ru.nl/pdb_redo/bd/3bdn/index.html

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Misbah ud 
Din Ahmad
Sent: Thursday, April 23, 2015 2:28 PM
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] 3BDN, 16.5% Ramachandran Outliers!

Dear Phoebe A. Rice,
I didn't mean to discredit the work but the statistics of the structure just 
shocked me at the first instance.
I could for example point out to another structure 1ZR2, which has the same 
resolution (the protein Molecular weight is almost the same) and the statistics 
are:
R-work: 0.27
Rfree: 0.319
Ramachandran outliers: 2.8%
The structure was solved by a combination of MIR and MAD three years earlier in 
2005 and definitely they didn't had better softwares.
As per your advice, I tried one cycle of refinement of this structure in Phenix 
and the statistics are:
Start R-work = 0.2816 ; Start R-free = 0.3551
Final R-work = 0.2887 ; Final R-free = 0.3671
Ramachandran outliers: 18.91%
Rotamer Outliers: 25.19%
Being a crystallographic community, isn't it our responsibility that if better 
softwares are available now, we try to re-refine our older structures and 
deposit better models in the PDB. That would help the users immensely.

Misbha







On Thu, Apr 23, 2015 at 7:06 PM, Mark J van Raaij 
mjvanra...@cnb.csic.esmailto:mjvanra...@cnb.csic.es wrote:
The abstract of the papers says they used MIR.

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/~mjvanraaij








 On 23 Apr 2015, at 18:57, Todd Jason Green wrote:

 My guess is 

Re: [ccp4bb] nVidia quadro

[Oganesyan, Vaheh]

Kay and others,

I think Quadro 5000 available @
http://www.amazon.com/PNY-VCQ5000-PB-DisplayPort-Profesional-Graphics/dp/B003X26T7K
is a better option both price wise and need of transforming the output into 
3-pin mini DIN. Having said that I should mention difference: the memory is 
2.5GB for 5000 model while K4200 has 4 GB. I believe the memory shouldn't be an 
issue at that level since my 10 years old FX1400 has only 1 GB and I did not 
encounter problems so far.



Vaheh Oganesyan
www.medimmune.com


-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kay 
Diederichs
Sent: Wednesday, April 01, 2015 3:31 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] nVidia quadro

On Tue, 31 Mar 2015 15:04:31 -0400, Andreas Schenk 
andreas_sch...@hms.harvard.edu wrote:

On 3/25/2015 18:10, Kay Diederichs wrote:
 On Wed, 25 Mar 2015 14:16:55 -0400, David Schuller schul...@cornell.edu 
 wrote:

 You could check the nVidia page of officially supported displays. It
 includes a search tab so you can check for Built-in Emitter.
 http://www.nvidia.com/object/3d-vision-displays.html

 Performing that search brings up 5 contenders. Good luck finding any
 of these products still for sale.
 Unfortunately, this NVidia page has not been updated for years.

 The qualifier 3D-fähig (aktiv) at 
 http://www.heise.de/preisvergleich/?cat=monlcd19widexf=5848_3D-f%E4hig+(aktiv)#xf_top
  should indicate a built-in emitter, but I looked at some of the 
 descriptions of these 11 monitors and was unable to confirm that they indeed 
 have a built-in emitter. So one has to research every specific case.

 I changed the wording on the wiki page.

 Kay

I went through the specs for the monitors on the Heise list, and none
of them seems to have a built in emitter compatible with Nvidia 3D
Vision 2. It looks like it is a bad time to buy a 3D monitor. At this
point it might be easier to just go for a Quadro with 3-pin connector.

Best,
Andreas


I asked the company who runs the price info site to check their assignments, 
and they fixed the categories: http://geizhals.eu/?cat=monlcd19wide now has a 
inkl. 3D-emitter attribute. This currently only returns the Asus 278HR which 
can only be bought in Poland, or through EBay.

The cheapest current Nvidia Quadro with (optional?) 3-pin DIN Stereo connector 
(needed for Linux) is the K4200 
(http://www.nvidia.de/object/quadro-desktop-gpu-specs-de.html) which starts at 
~ €700.

best,

Kay
To the extent this electronic communication or any of its attachments contain 
information that is not in the public domain, such information is considered by 
MedImmune to be confidential and proprietary. This communication is expected to 
be read and/or used only by the individual(s) for whom it is intended. If you 
have received this electronic communication in error, please reply to the 
sender advising of the error in transmission and delete the original message 
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cooperation.

Re: [ccp4bb] nVidia quadro

[Oganesyan, Vaheh]

Colleagues,

I’d like to thank everyone who took time to answer my question regarding Quadro 
cards that support quad buffered stereo. I now hope to build a workstation with 
Quadro 5000.

Regards,

Vaheh Oganesyan
www.medimmune.com

To the extent this electronic communication or any of its attachments contain 
information that is not in the public domain, such information is considered by 
MedImmune to be confidential and proprietary. This communication is expected to 
be read and/or used only by the individual(s) for whom it is intended. If you 
have received this electronic communication in error, please reply to the 
sender advising of the error in transmission and delete the original message 
and any accompanying documents from your system immediately, without copying, 
reviewing or otherwise using them for any purpose. Thank you for your 
cooperation.

[ccp4bb] nVidia quadro

[Oganesyan, Vaheh]

Graphics gurus,

http://www.amazon.com/PNY-DisplayPort-Profesional-Graphics-VCQ6000-PB/dp/B0044XUD1U/ref=sr_1_8?ie=UTF8qid=1427151570sr=8-8keywords=nvidia+quadro

Would this card work for quad buffered stereo on Linux workstation? It does 
have 3-pin mini Din.


Regards,

Vaheh Oganesyan
MedImmune, ADPE
www.medimmune.com

To the extent this electronic communication or any of its attachments contain 
information that is not in the public domain, such information is considered by 
MedImmune to be confidential and proprietary. This communication is expected to 
be read and/or used only by the individual(s) for whom it is intended. If you 
have received this electronic communication in error, please reply to the 
sender advising of the error in transmission and delete the original message 
and any accompanying documents from your system immediately, without copying, 
reviewing or otherwise using them for any purpose. Thank you for your 
cooperation.

Re: [ccp4bb] CCP4 Scalepack2mtz problem: Anisotropy correction failed

[Oganesyan, Vaheh]

I might be completely wrong here but doesn’t it bother that the number of 
rejected reflections shown in log file is ~83000? It shows, at least to me, 
that either indexing step is far from being correct or crystal is not really 
good enough due to defects.



Vaheh Oganesyan
www.medimmune.com

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Xiao Lei
Sent: Saturday, January 10, 2015 7:08 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] CCP4 Scalepack2mtz problem: Anisotropy correction failed

Hi Eleanor,

Thanks for the input, I attach the scale log file here.

On Sat, Jan 10, 2015 at 2:16 PM, Eleanor Dodson 
eleanor.dod...@york.ac.ukmailto:eleanor.dod...@york.ac.uk wrote:
Certainly a negative eigen value is bad - can you attach the data? There may be 
some obvious problem..
Eleanor

On 10 January 2015 at 04:42, Xiao Lei 
xiaolei...@gmail.commailto:xiaolei...@gmail.com wrote:
Dear All,

I tried to convert my x-ray diffraction sca data from HKL200 (.sca file) to mtz 
in CCP4 using Scalepack2mtz and it failed, I do not know what should I supposed 
to do next to correct the problem, any suggestions are appreciated. I pasted 
the message from part of log file below:


ANISOTROPY ANALYSIS (using intensities):


Eigenvalues: -0.5668 0.1479 0.3584

Eigenvalue ratios: -1.5815 0.4128 1.

ctruncate: Anisotropy correction failed - negative eigenvalue.

Times: User: 0.4s System: 0.0s Elapsed: 0:00

***

* Information from CCP4Interface script

***

The program run with command: /Applications/ccp4-6.4.0/bin/ctruncate -hklin 
/LAB/CCP4/TEMP/RelADD_12212014ALS502_3_1_mtz.tmp -hklout 
/LAB/CCP4/TEMP/RelADD_12212014ALS502_3_3_mtz.tmp -colin 
/*/*/\[IMEAN,SIGIMEAN\] -colout P1_12

has failed with error message

ctruncate: Anisotropy correction failed - negative eigenvalue.

ObjectCache: Leaked 0005 refs to

***



#CCP4I TERMINATION STATUS 0  ctruncate:  Anisotropy correction failed - 
negative eigenvalue. ObjectCache: Leaked 0005 refs to



#CCP4I TERMINATION TIME 09 Jan 2015  19:55:50

#CCP4I MESSAGE Task failed






To the extent this electronic communication or any of its attachments contain 
information that is not in the public domain, such information is considered by 
MedImmune to be confidential and proprietary. This communication is expected to 
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Re: [ccp4bb] asymmetric homotrimer in the asu

[Oganesyan, Vaheh]

This fact by itself is unusual to say the least (for me):
 we have NO rotational symmetry (2, 3, or 4-fold) whatsoever between 
interacting monomers in the ASU or relating those built up by the 
crystallographic symmetry
There might be several ways of choosing molecules to represent the asymmetric 
unit. Is it possible to find ones that are related? Say something like 
non-crystallographic translation (or pseudo translation) + non-crystallographic 
rotation.

For long time I was thinking about such a possibility of having more than one 
molecule in au but no rotation or pst. May be I've missed but never found an 
evidence, nor can I explain why would that be impossible.


Vaheh Oganesyan
www.medimmune.com

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hay Dvir
Sent: Thursday, December 11, 2014 12:04 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] asymmetric homotrimer in the asu

Dear Michael,

Thank you very much for the useful comments.

Indeed, we are of course looking at it biochemically, which isn't a clear cut 
so far..
As you pointed out it could be a monomer in solution, but the interface between 
monomers within this asymmetric trimer seems too extensive (compared to those 
responsible for the lattice packing) not to suspect a trimer as a solution 
assembly. PISA suggested this asymmetric trimer as the most likely assembly but 
it falls into the grey region of their criteria (see attached pic.)

[cid:image001.png@01D01549.A63DFAA0]


Since it's rare, we are interested to know of other similar reports, if any, to 
learn how they were resolved/concluded. I believe the case you describe is not 
similar, as we have NO rotational symmetry (2, 3, or 4-fold) whatsoever between 
interacting monomers in the ASU or relating those built up by the 
crystallographic symmetry. Therefore I can't see how the space group 
information may help, but it is p212121 in case it helps boosting your morning 
coffee experience with symmetry pondering ... :).

Cheers,
Hay



On Dec 11, 2014, at 3:47 PM, R. M. Garavito wrote:


Dear Hay,

And your point is?  I am not trying to be snarky (although I am just starting 
my morning coffee), but to bring up the fact that CCP4BB readers need more info 
to comment on your case, like space group, local interactions, and how packed 
is tightly packed.

I have had two cases of trimers, as my students initially called them, that 
were actually a dimer and a half.  The half dimer had its mate in another 
ASU.   Can it be a biological monomer that just happened to crystallize 3 
monomers to an ASU?  Non-symmetric homo-oligomers are rare, but sadly cannot be 
absolutely confirmed by crystallography alone, but by good old biochemistry.  
The PISA website (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html) can give 
you estimations of the strengths of the interfacial interactions, but they are 
mere estimates.  What does gel filtration say or cross linking? Does it fit 
with the biology/biochemistry expected of this protein?

Anyway, have fun with your structure, but use a lot of skepticism in your 
interpretation.  That will help you convince the reviewers.

Cheers,

Michael


R. Michael Garavito, Ph.D.
Professor of Biochemistry  Molecular Biology
603 Wilson Rd., Rm. 513
Michigan State University
East Lansing, MI 48824-1319
Office:  (517) 355-9724 Lab:  (517) 353-9125
FAX:  (517) 353-9334Email:  
rmgarav...@gmail.commailto:garav...@gmail.com




On Dec 11, 2014, at 7:27 AM, Hay Dvir 
hd...@tx.technion.ac.ilmailto:hd...@tx.technion.ac.il wrote:


Dear all,


We have a structure of a rather tightly packed homotrimer protein in the ASU 
with no apparent crystallographic or non-crystallographic rotational symmetry 
between monomers.
Attempting to establish the biological assembly, we are very interested to hear 
about additional similar cases you might know of.

Thanks in advance,
Hay


---
Hay Dvir   Ph. D.
Head   Technion Center for Structural Biology
TechnionHaifa 323, Israel
Tel:   +(972)-77-887-1901
Fax:  +(972)-77-887-1935
E-mail   hd...@technion.ac.ilmailto:hd...@technion.ac.il
Websitehttp://tcsb.technion.ac.ilhttp://tcsb.technion.ac.il/



To the extent this electronic communication or any of its attachments contain 
information that is not in the public domain, such information is considered by 
MedImmune to be confidential and proprietary. This communication is expected to 
be read and/or used only by the individual(s) for whom it is intended. If you 
have received this electronic communication in error, please reply to the 
sender advising of the error in transmission and delete the original message 
and any accompanying documents from your system immediately, without copying, 
reviewing or 

Re: [ccp4bb] Off topic: Precast gels

[Oganesyan, Vaheh]

Theresa,

Try Genscript. They have probably special now going on, so box of 10 SDS PAGE 
costs $39.

I'm not associated with Genscript in any way and I don't use their SDS PAGE.



Vaheh Oganesyan
www.medimmune.com

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Theresa 
Hsu
Sent: Friday, August 29, 2014 2:24 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Off topic: Precast gels

Dear all

Would anyone knows of source of cheap precast SDS-page gels?

Thank you.
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[ccp4bb] migration from monitor to wall mounted screen and projector

[Oganesyan, Vaheh]

Hi all,

I'm planning migration from CRT monitor to wall mounted screen with projector 
that will support stereo. Mid-range emitter like AE125 for small office (4m by 
4m) like mine is sufficient. I'm looking for advise on projector and screen. It 
looks like ViewSonic PJD7820HD ($699) with native (at least 1280 x 1024 at 120 
Hz) support should work.
Have any of you tried this? What are other options for projector and are there 
preferences for wall mount screens?

Thank you.

Regards,

Vaheh Oganesyan
MedImmune, ADPE
www.medimmune.com

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Re: [ccp4bb] Tenure track junior Group Leader Positions in Milan, Italy

[Oganesyan, Vaheh]

It sounds very interesting: experimental and computational dance biology. Any 
type of computational dance or there are style limitations?

Regards,

Vaheh Oganesyan
www.medimmune.com
[MedI Logo Sig]

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Sebastiano Pasqualato
Sent: Friday, April 04, 2014 12:01 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Tenure track junior Group Leader Positions in Milan, Italy


Dear all,
there are open positions for junior Group Leaders in the field of experimental 
and computational dance biology at the European Institute of Oncology in milan, 
Italy.
Please, find enclosed the details.

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inline: image001.png

[ccp4bb] SO4 geometry

[Oganesyan, Vaheh]

Colleagues,

Sorry to bother for something really minor. The Refmac usually always 
recognizes tetrahedral SO4 groups and there were no problems related to its 
geometry. Two attached files demonstrate SO4 geometry before and after 
refinement. Would you be able to point me to mistake I’m doing?
Thank you.

Regards,

Vaheh Oganesyan, PhD
Antibody discovery and protein engineering
MedImmune, LLC.
www.medimmune.com


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so4-6after.pdb
Description: so4-6after.pdb


so4-6.pdb
Description: so4-6.pdb

Re: [ccp4bb] SO4 geometry

[Oganesyan, Vaheh]

Thank you, Garib, Ethan, Robbie and Matthias,

It was indeed chirality issue. RTFLog file is as important as RTFManual.

Regards,

Vaheh Oganesyan
www.medimmune.com


-Original Message-
From: Ethan A Merritt [mailto:merr...@u.washington.edu]
Sent: Monday, March 31, 2014 3:25 PM
To: Oganesyan, Vaheh
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] SO4 geometry

On Monday, 31 March, 2014 19:01:33 Oganesyan, Vaheh wrote:
 Colleagues,

 Sorry to bother for something really minor. The Refmac usually always 
 recognizes tetrahedral SO4 groups and there were no problems related to its 
 geometry. Two attached files demonstrate SO4 geometry before and after 
 refinement. Would you be able to point me to mistake I’m doing?


Apart from any question of geometry, I conclude from the B factors  100  that 
refmac doesn't think the sulfate belongs there.

As to geometry - the refmac log file should contain mention of whatever target 
was problematic.  I would guess that the chiral volume has the wrong sign.

Ethan


 Thank you.

 Regards,

 Vaheh Oganesyan, PhD

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[ccp4bb] stereo emitter

[Oganesyan, Vaheh]

Colleagues,

I was happily using my NuVision 60GX emitter with Quadro FX1400 graphics card 
for number of years on CRT and recently something went bad and image will flip 
regularly sending front to back and vice versa. First I thought the card went 
bad but after installing new one nothing changed. I'm suspecting the emitter 
now and wondering if now there are solutions better than those emitters. 
Hopefully it is not too off topic.

Thanks in advance for suggestions.

Vaheh Oganesyan, PhD
Antibody development and protein engineering
1 MedImmune Way, Gaithersburg, MD 20878
www.medimmune.comhttp://www.medimmune.com/
[logo1]

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inline: image002.jpg

Re: [ccp4bb] high purity imidazole

[Oganesyan, Vaheh]

Thanks to Debanu Das, Pedro J. B. Pereira and Bernhard Rupp for pointing me in 
the right direction.

https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind1308L=ccp4bbP=R2552751=ccp4bb9=AJ=ond=No+Match%3BMatch%3BMatchesz=4






Regards,



Vaheh Oganesyan

www.medimmune.com



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[ccp4bb] high purity imidazole

[Oganesyan, Vaheh]

Colleagues,

In August of 2013 there was a tread regarding high purity Imidazole and someone 
posted list of products with absorption values at 280 nm and manufacturer.
I can find most of the e-mails from that tread but not the one with list of 
Imidazoles. Has anyone saved that e-mail for future use and willing to share it 
with me?

Vaheh Oganesyan, PhD
Antibody development and protein engineering
1 MedImmune Way, Gaithersburg, MD 20878
www.medimmune.com

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Re: [ccp4bb] Dose anyone see this ligand before?

[Oganesyan, Vaheh]

It actually looks much like pyrophosphate! If your protein is phosphatase and 
the extra density is in vicinity of the active site it might be the remaining 
product of reaction.

 Vaheh




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bosch, 
Juergen
Sent: Tuesday, July 16, 2013 11:53 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Dose anyone see this ligand before?

Special position ?
sulfate-size ?
Jürgen

On Jul 16, 2013, at 11:35 AM, Wei Feng wrote:


Dear all,
I found some redundant density in my structure beween two molecule. (see 
picture 1 and 2)
But I am not sure which ligand will be.
Dose everyone see this ligand before? If so, can you tell the PDB code or send 
me the struture file?
Thank you for your time!
Wei



picture1.jpgpicture2.jpg

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry  Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu



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Re: [ccp4bb] Improve diffraction ...any ideas?

[Oganesyan, Vaheh]

I think this is an advice not to follow.

 Vaheh




-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Rajiv K 
Bedi
Sent: Friday, May 24, 2013 1:44 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Improve diffraction ...any ideas?

Dear Umri,

I think the main problem is co-crystallization.

What I would do is crystallize protein and antibody separately and then soak 
protein crystals into reservoir solution containing antibody or vice versa.
And do try to get crystals from different conditions which may alter the space 
group and thereby improve diffraction quality, hopefully.

All the best,
Rajiv
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Re: [ccp4bb] Etiquette on publishing if there is a crystallization report from someone else.

[Oganesyan, Vaheh]

Herman,

I don't know which early days you refer to, but from late 80s until structural 
genomics era there were relatively few crystallization reports. May be I didn't 
see them, and then I apologize. But crystallization reports in large started in 
late 90s through early 21st century and Acta F has been created to accommodate 
them. As far as my understanding goes, you publish crystallization results only 
if you're sure the structure will be solved or is already solved but not ready 
for different reasons to be published.
Some time ago I was in position similar to Christine's. And I waited and waited 
until I decided to contact the authors of the notes. Sure enough, they intended 
to publish structure but the postdoc left and nobody else was able to do the 
work.
Christine, you have got good advises already. Contact the authors and if they 
are reasonable publish back to back, if they are not - you do not have any 
legal/moral obligations to wait.

My two drams,

 Vaheh




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
herman.schreu...@sanofi.com
Sent: Tuesday, September 25, 2012 11:04 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Etiquette on publishing if there is a crystallization 
report from someone else.

In the very early days, solving a protein structure was an enormous amount of 
work and since hardly any protein structures were solved there was a huge pool 
of unsolved structures. Under these circumstances, it was a waste of resources 
if two groups would work on the same protein.  To prevent this, people would 
publish crystallization notes so other groups could choose another protein to 
work on and this is what usually happened. Also, the purpose of scientific 
publications is that other people can use this information to progress their 
results.

Unless unethical actions were involved (holding up referee reports, making 
shortcuts to publish before the competition) I do not see a reason why you 
could not publish your paper. As Jürgen suggested, you may want to contact the 
other group to see if you could publish back to back.

my two cents,
Herman


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Lukacs, 
Christine
Sent: Tuesday, September 25, 2012 3:33 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Etiquette on publishing if there is a crystallization report 
from someone else.
I'd like to get a community opinion on something.

If a group has published crystallization and diffraction data (Acta Cryst F 
style crystallization report), and you happen to have the same crystal form and 
have solved the structure, is there an unspoken rule that you don't publish, or 
an amount of time that you wait to allow the other group to publish before you 
do?  I am not talking about a high impact structure with a race to publish.

Just looking for a general consensus.

Thanks
Christine

Christine Lukacs, Ph.D.
Principal Scientist
Roche
christine.luk...@roche.com
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[ccp4bb] UV microscope

[Oganesyan, Vaheh]

Hi All,

May I ask opinion of those who currently have standalone UV microscope whether 
or not they are happy with their choice? Few that I know are from Formulatrix, 
JanScientific and Corima. Pros and cons of your instument are greatly 
appreciated.

I'll send the summary to the list.

Regards,

Vaheh Oganesyan, PhD
Antibody Discovery and Protein Engineering
MedImmune, LLC.
1 MedImmune Way, Gaithersburg, MD 20878
oganesy...@medimmune.commailto:oganesy...@medimmune.com

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inline: image001.gif

Re: [ccp4bb] Protein concentration vs Molecular wt...

[Oganesyan, Vaheh]

Number of years ago Jaru Jancarik (the author of Screen I  II sold by HR) 
while in Berkeley Structural Genomics Center (or may be even earlier) made an 
observation regarding protein precipitation in condition A6 in that very 
screen. Based on this observation HR sells now PCT (protein concentration test 
or something similar). In brief, if your protein is concentrated enough and is 
a regular protein, not a stellar, you will see medium to heavy precipitation. 
In the case of proteins that are really of great quality you'll end up having 
crystals in A6. After many years of experience I realized that A9 and A10 from 
the same screen could be added to the list of conditions that are somewhat 
indicative for choosing right concentration same way like Jaru did for A6.

My two Armenian drams worth,

 Vaheh




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger 
Rowlett
Sent: Thursday, July 19, 2012 4:42 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Protein concentration vs Molecular wt...

This is almost exactly our basic approach, too. Before we got a dropsetter, we 
did 24 wells (1/2 screen) to get a feel for the correct protein concentration. 
Some additional rules of thumb we use:

  1.  We usually start at 10 mg/mL protein and go up or down from there 
depending on the results of the initial screen
  2.  If we observe a high frequency of precipitation in a 10 mg/mL protein 
screen, we will usually set a 1/2 concentration screen by diluting the screen 
solutions 1:1 with water. This frequently uncovers additional hits in wells 
that were heavily precipitated in the original screen. Empirically, proteins we 
study seem to crystallize better in the higher protein/lower precipitant zone 
of the phase diagram than the lower protein/higher precipitant zone. 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.edumailto:rrowl...@colgate.edu
On 7/19/2012 4:31 PM, mjvdwo...@netscape.netmailto:mjvdwo...@netscape.net 
wrote:
I don't think there is such a rule, but in the old days, when we only had 
Hampton Screen I and II, the rule was:

- Set up screen 1, look at the drops and you should expect some kind of 
precipitation in 50% of the drops. If much less than that, increase your 
protein concentration. If much more than that, decrease protein concentration.
- Set up screen 2, look and expect 30% precipitation.

I used to cut corners and do the statistics at 1/2 of a screen (one 24-well 
plate). You can probably use this method to get within a factor of 2 of the 
optimal concentration.

There are probably good statistics in the papers for the screens that you may 
use. One of the advantages of structural genomics efforts is that these things 
are known (and hopefully published).

Even older trick is to take a drop of protein and look under a microscope, 
record how much AmSO4 it takes to cause precipitation. Do the same with PEG. 
Keep adding a little at a time and look immediately. This will give you an idea 
if you are near a reasonable concentration. I think that this latter method 
does not tell you much more than physics-information - which is how many 
zeroes there are: whether 1 mg/ml or 10mg/ml or 100mg/ml is reasonable.

Mark

-Original Message-
From: james09 pruza james09x...@gmail.commailto:james09x...@gmail.com
To: CCP4BB CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Sent: Thu, Jul 19, 2012 1:59 pm
Subject: [ccp4bb] Protein concentration vs Molecular wt...
Dear Crystallographers,
Is there any rule of thumb for Protein concentration and molecular weight for 
crystallization trials of a soluble protein? Looking for high molecular wt. 
protein ~50kDa.
James.

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Re: [ccp4bb] Death of Rmerge

[Oganesyan, Vaheh]

It wasn't doing well lately. So, it was expected.


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Jacob Keller 
[j-kell...@fsm.northwestern.edu]
Sent: Thursday, May 31, 2012 2:20 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Death of Rmerge

Dear Crystallographers,

in case you have not heard, it would appear that the Rmerge statistic
has died as of the publication of  PMID: 22628654. Ding Dong...?

JPK

--
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***
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[ccp4bb] water picking

[Oganesyan, Vaheh]

All,

For those who still don't use Coot is there an automatic water picking 
procedure in CCP4?

Many years ago there was a peakmax which is now for Patterson peaks only. Then 
there was routine through Arp/wArp. Now is Coot only. Is this right?

Thanks.

Vaheh Oganesyan, PhD
Antibody Discovery and Protein Engineering
MedImmune, LLC.
1 MedImmune Way, Gaithersburg, MD 20878
oganesy...@medimmune.commailto:oganesy...@medimmune.com

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inline: image001.gif

[ccp4bb] FW: error

[Oganesyan, Vaheh]

BBers,

In my new 6.2.0 installation I'm getting child killed: segmentation violation 
while running Truncate. The /tmp/Vaheh directory does exist and is writable.
Below is the message:

[Vaheh] ===
The program run with command: /usr/bin/ccp4-6.2.0/bin/ctruncate -mtzin 
/home/Vaheh/[Vaheh] /scala_tern_pointless1.mtz -mtzout 
/tmp/Vaheh/11-42.tmp -colin /*/*/\[I,SIGI\] -colout 11_42
has failed with error message
child killed: segmentation violation
***


#CCP4I TERMINATION STATUS 0 child killed: segmentation violation
#CCP4I TERMINATION TIME 23 Apr 2012  11:49:16
#CCP4I MESSAGE Task failed
[Vaheh] ===


Will appreciate a solution to problem.

Thank you.

 Vaheh
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Re: [ccp4bb] To archive or not to archive, that's the question!

[Oganesyan, Vaheh]

I was hesitant to add my opinion so far because I'm used more to listen this 
forum rather than tell others what I think.
Why and what to deposit are absolutely interconnected. Once you decide why 
you want to do it, then you will probably know what will be the best format and 
vice versa.
Whether this deposition of raw images will or will not help in future 
understanding the biology better I'm not sure.
But to store those difficult datasets to help the future software development 
sounds really farfetched. This assumes that in the future crystallographers 
will never grow crystals that will deliver difficult datasets. If that is the 
case and in 10-20-30 years next generation will be growing much better crystals 
then they don't need such a software development.
If that is not the case, and once in a while (or more often) they will be 
getting something out of ordinary then software developers will take them and 
develop whatever they need to develop to consider such cases.

Am I missing a point of discussion here?

Regards,

 Vaheh




-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Robert 
Esnouf
Sent: Monday, October 31, 2011 10:31 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] To archive or not to archive, that's the question!

Dear All,

As someone who recently left crystallography for sequencing, I
should modify Tassos's point...

A full data-set is a few terabytes, but post-processing
reduces it to sub-Gb size.

My experience from HiSeqs is that this full here means the
base calls - equivalent to the unmerged HKLs - hardly raw
data. NGS (short-read) sequencing is an imaging technique and
the images are more like 100TB for a 15-day run on a single
flow cell. The raw base calls are about 5TB. The compressed,
mapped data (BAM file, for a human genome, 30x coverage) is
about 120GB. It is only a variant call file (VCF, difference
from a stated human reference genome) that is sub-Gb and these
files are - unsurprisingly - unsuited to detailed statistical
analysis. Also $1k is a not yet an economic cost...

The DNA information capacity in a single human body dwarfs the
entire world disk capacity, so storing DNA is a no brainer
here. Sequencing groups are making very hard-nosed economic
decisions about what to store - indeed it is a source of
research in itself - but the scale of the problem is very much
bigger.

My tuppence ha'penny is that depositing raw images along
with everything else in the PDB is a nice idea but would have
little impact on science (human/animal/plant health or
understanding of biology).

1) If confined to structures in the PDB, the images would just
be the ones giving the final best data - hence the ones least
likely to have been problematic. I'd be more interested in
SFs/maps for looking at ligand-binding etc...

2) Unless this were done before paper acceptance they would be
of little use to referees seeking to review important
structural papers. I'd like to see PDB validation reports
(which could include automated data processing, perhaps culled
from synchrotron sites, SFs and/or maps) made available to
referees in advance of publication. This would be enabled by
deposition, but could be achieved in other ways.

3) The datasets of interest to methods developers are unlikely
to be the ones deposited. They should be in contact with
synchrotron archives directly. Processing multiple lattices is
a case in point here.

4) Remember the average consumer of a PDB file is not a
crystallographer. More likely to be a graduate student in a
clinical lab. For him/her things like occupancies and B-
factors are far more serious concerns... I'm not trivializing
the issue, but importance is always relative. Are there
outsiders on the panel to keep perspective?

Robert


--

Dr. Robert Esnouf,
University Research Lecturer, ex-crystallographer
and Head of Research Computing,
Wellcome Trust Centre for Human Genetics,
Roosevelt Drive, Oxford OX3 7BN, UK

Emails: rob...@strubi.ox.ac.uk   Tel: (+44) - 1865 - 287783
and rob...@esnouf.comFax: (+44) - 1865 - 287547


 Original message 
Date: Mon, 31 Oct 2011 11:37:47 +0100
From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf
of Anastassis Perrakis a.perra...@nki.nl)
Subject: Re: [ccp4bb] To archive or not to archive, that's
the question!
To: CCP4BB@JISCMAIL.AC.UK

   Dear all,
   The discussion about keeping primary data, and what
   level of data can be considered 'primary', has -
   rather unsurprisingly - come up also in areas other
   than structural biology.
   An example is next generation sequencing. A
   full-dataset is a few tera bytes, but
   post-processing reduces it to sub-Gb size. However,
   the post-processed data, as in our case,
   have suffered the inadequacy of computational
   reduction ... At least out institute has decided
   to create double back-up of the primary data in
   triplicate. For that reason our facility bought
   three -80 freezers, one 

Re: [ccp4bb] Akta Prime / FPLC Options / Off Topic

[Oganesyan, Vaheh]

GE has a policy on Product Obsolescence, which, afaik, means that service 
contracts will not be issued to those instruments that were discontinued 7 
years ago. Among instruments affected by this deadline are AktaPrime INCL, 
AktaPrime EXCL and AktaPrime COMPLETE. You have time to service these 
chromatography systems until April 2012. After that time GE will offer service 
upon the availability of parts. AktaPrime Plus is still good to go.

I know all these because I've got a letter from GE regarding my AktaPrime.

HTH,

 Vaheh




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Paul Smith
Sent: Wednesday, October 12, 2011 9:19 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Akta Prime / FPLC Options / Off Topic

Michael,

Unfortunately, I actually don't know who serves these machines apart from GE.

Because you brought up the subject of GE equipment and service, I thought I 
would ask the community about the best options for routine crystallographic 
scale FPLC.

In my opinion, following the takeover of Pharmacia by GE the the price of GE 
machines, replacement parts, and service has skyrocketed and GE service reps 
seem determined to squeeze and extort every dollar they can.  Personally, I'd 
love to never do business with GE again.

However, in some ways, they are the only game in town.  GE is the de facto 
standard for our line of work and the Akta line are very good machines.  
However, GE's consistent price gouging and outright crooked service practices 
encourage me look elsewhere.

I've used systems from AP-biotech (junk) and have heard some good things about 
Bio-rad.  What does the community at large think?  Are there other good 
options?  Does anyone have some spare millions and manufacturing connections in 
India/China to consider starting a competing company?

Sorry to hijack your thread Michael.  Let me know what you find out.  The less 
money I send to GE the better.


--Paul


From: Michael Colaneri colane...@gmail.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wednesday, October 12, 2011 2:28 PM
Subject: [ccp4bb] Akta Prime


Dear all,

We have an AktaPrime and GE Lifesciences stop servicing these instruments 
because they are getting old.  Does anyone know of a third party company that 
gives contracts to maintain these instruments?  Thank you.

Mike Colaneri

To the extent this electronic communication or any of its attachments contain 
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Re: [ccp4bb] Cadmium sites and co-ordinations in structure

[Oganesyan, Vaheh]

While I believe there is plenty written about metal coordination, the best 
approach, IMHO, is to search PDB for metal of your choice at resolution as high 
as you can get and compare to your case.

 Vaheh




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dhirendra 
K Simanshu
Sent: Friday, August 26, 2011 10:34 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Cadmium sites and co-ordinations in structure

Hello Sandeep,

I have been in this situation many times before but with different metal ions..
I have found papers published by Marjorie M Harding very useful in such 
situations. In fact, there are lots of information on-line on which is 
available here (including all the references for his papers):

METAL COORDINATION SITES IN PROTEINS

http://tanna.bch.ed.ac.uk/
http://tanna.bch.ed.ac.uk/qg3.htm
http://tanna.bch.ed.ac.uk/newtargs_06.html

You will at least find information for Ca and Co here for sure.

All the best
Simanshu

On Fri, Aug 26, 2011 at 10:21 AM, Partha Chakrabarti 
ppc...@gmail.commailto:ppc...@gmail.com wrote:
Hi Sandeep, if someone sends one, kindly share the references.

In general, Ca2+ could have more Asp, Asn kind of coordination and distorted 
pentagonal bipyramidal geometry with waters (about 2.5A), Cd can also have S- 
since it is softer, I guess Co might have N/O/S (i.e all three with paired 
electrons). An inorganic chemistry textbook like Greenwood  Earnshaw or Cotton 
 Wilkinson could be handy.. or a bioinorganic chemistry book.

HTH,
Partha




On Fri, Aug 26, 2011 at 7:24 PM, Sandeep 
s.talapa...@beatson.gla.ac.ukmailto:s.talapa...@beatson.gla.ac.uk wrote:
Hi,

I crystallised a protein in the presence of Calcium, Cobalt, and Cadmium and 
determined its structure. It turns out that I see several metal sites in the 
structure, mostly cadmiums. Is there any information published (preferably a 
review) which summarises data on cadmium sites in proteins such as for example 
the possible coordination numbers of cadmium, distances, type of side chains 
found to coordinate with cadmium, etc.? I could extract all this from the PDB, 
but a nice review would be simpler to start with.

Thank you in advance for your help

Sandeep




--
Dhirendra K Simanshu
Memorial Sloan-Kettering Cancer Center
Structural Biology Program
New York, NY, USA 10065

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MedImmune to be confidential and proprietary. This communication is expected to 
be read and/or used only by the individual(s) for whom it is intended. If you 
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Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

[Oganesyan, Vaheh]

I completely disagree with Filip's assessment. I've been using nanodrop nearly 
5 years and never had inconsistency issues. If you work at reasonable speed (if 
you put a drop there then lower the lever and click measure before you do 
anything else) there will be no issues. At very high concentrations the 
accuracy and therefore consistency may become lower. Concentrations between 5 
and 10 mg/ml should be fine. The instrument is pricey though.

 Vaheh




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Filip Van 
Petegem
Sent: Thursday, June 16, 2011 3:34 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old 
Bradford.

Dear Arnon,

the Bradford method is not recommended for accurate measurements.  The readings 
are strongly dependent on the amino acid composition.  A much better method is 
using the absorption at 280nm under denaturing conditions (6M Guanidine), and 
using calculated extinction coefficients based on the composition of mostly 
Tyrosine and Tryptophan residues (+ disulfide bonds).  This method is also old 
(Edelhoch, 1967), but very reliable.

One thing about the nanodrop: smaller volume = more evaporation.  On the demo 
we've had, I was so unimpressed with the precision (25% variability between 
two consecutive measurement) that we didn't consider this instrument at all.  
So unless you just want a 'rough' estimate, I wouldn't recommend it at all. But 
most respectable spectrophotometers will take cuvettes with 50ul volumes - a 
big step up from 1ml volumes...

Filip Van Petegem



On Thu, Jun 16, 2011 at 12:15 PM, Arnon Lavie 
la...@uic.edumailto:la...@uic.edu wrote:
Dear fellow crystallographers - a question about spectrophotometers for protein 
concentration determination.

We are so last millennium - using Bradford reagent/ 1 ml cuvette for protein 
conc. determination.

We have been considering buying a Nanodrop machine (small volume, no dilution 
needed, fast, easy).
However, while testing our samples using a colleague's machine, we have gotten 
readings up to 100% different to our Bradford assay (all fully purified 
proteins). For example, Bradford says 6 mg/ml, Nanodrop 3 mg/ml. So while it is 
fun/easy to use the Nanodrop, I am not sure how reliable are the measurements 
(your thoughts?).

So QUESTION 1: What are people's experience regarding the correlation between 
Nanodrop and Bradford?

While researching the Nanodrop machine, I heard about the Implen NanoPhotmeter 
Pearl.
So Question 2: Is the Pearl better/worse/same as the Nanodrop for our purpose?

Thank you for helping us to advance to the next millennium, even if it is 
nearly a dozen years late.

Arnon

--
***
Arnon Lavie, Professor
Dept. of Biochemistry and Molecular Genetics
University of Illinois at Chicago
900 S. Ashland Ave.
Molecular Biology Research Building, Room 1108 (M/C 669)
Chicago, IL 60607
U.S.A.
Tel:(312) 355-5029tel:%28312%29%20355-5029
Fax:(312) 355-4535tel:%28312%29%20355-4535
E-mail: la...@uic.edumailto:la...@uic.edu
http://www.uic.edu/labs/lavie/
***



--
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.commailto:filip.vanpete...@gmail.com
http://crg.ubc.ca/VanPetegem/
To the extent this electronic communication or any of its attachments contain 
information that is not in the public domain, such information is considered by 
MedImmune to be confidential and proprietary. This communication is expected to 
be read and/or used only by the individual(s) for whom it is intended. If you 
have received this electronic communication in error, please reply to the 
sender advising of the error in transmission and delete the original message 
and any accompanying documents from your system immediately, without copying, 
reviewing or otherwise using them for any purpose. Thank you for your 
cooperation.

Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

[Oganesyan, Vaheh]

I think that the absolute value of protein concentration is not very important. 
Some proteins get crystallized at 1 mg/ml, others at 50. What is important is 
to be able to reproducibly estimate it from prep to prep. You probably want to 
start at some reasonable value of about 10 mg/ml. If it in reality is 12.5 I 
don't personally care.
If I publish the result and someone repeats and doesn't get crystal at exactly 
same concentration I don't care either because that person is not taking 
sensible approach.

 Vaheh




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Machius, 
Mischa Christian
Sent: Thursday, June 16, 2011 7:23 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old 
Bradford.

With respect to the Edelhoch method and the ProtParam server, I would strongly 
recommend determining extinction coefficients experimentally and not rely on 
the ProtParam values. The reason is that the underlying extinction coefficients 
in the formula used by ProtParam and referenced there are statistical averages. 
They may or may not be valid for a given protein. I have seen differences of 
more than 20% between the theoretical and experimental extinction 
coefficients, particularly for proteins with few Trp and Tyr residues. When 
relying on relative concentrations, this inaccuracy is not detrimental, but 
when absolute concentrations are needed (CD, AUC, ITC, any binding experiment, 
etc.), such a difference would be considered huge. Determining an extinction 
coefficient experimentally takes but a few minutes.

Cheers!
MM


On Jun 16, 2011, at 6:22 PM, Petr Leiman wrote:


Totally support the statements below. We have had several proteins with A280 
absorbance of 0.1 or less (at 1 mg/ml). You _have_ to use Bradford in the 
Nanodrop or whatnot to measure the concentration.

Before purchasing the Nanodrop we used a Hellma TrayCell and a normal UV/Vis 
instrument. Similar to the Nanodrop, the sample volume in TrayCell is  2-3 ul. 
Traycell works well at a fraction of the Nanodrop cost, but Nanodrop is a lot 
more convenient to use for high concentration quick measurements (especially if 
you need to measure several things in succession), so you get what you pay for.

Petr

P.S. Expasy's Protparam tool has been around for ages (10-12+ years?). That 
plus the Nanodrop are two essential and synergetic tools of a protein 
chemist/crystallographer.

On Jun 16, 2011, at 10:31 PM, Edward A. Berry wrote:


Bradford is an assay, Nanodrop is a spectrophotometer.
Both the A280 and Bradford methods are strongly dependent on
amino acid composition, so unless you correct A280 for that
as mentioned by Filip, either one is semiquantitative.
Occasionally you come across a protein with no tryptophan
which will have a much lower extinction coefficient.
Try making a 1 g/l solution of gelatin (collagen?)
and see what its A280 is!  I noticed recently the
protparam tool at http://ca.expasy.org/cgi-bin/protparam
estimates the extinction coefficient given a sequence.



David Briggs wrote:
~~~

I wouldn't touch Bradford with a barge-pole. I've found it to be
wildly inaccurate for certain proteins I've handled, where as the
OD280 measurements have been fine.

One wonders what does fine mean, like same as with Biuret or
Kjeldahl nitrogen, or solution made up by weight?

---
Mischa Machius, PhD
Director, Center for Structural Biology
Assoc. Professor, Dept. of Pharmacology
Member, Lineberger Comprehensive Cancer Center
University of North Carolina
4079 Genetic Medicine
CB#7365
120 Mason Farm Road
Chapel Hill, NC 27599-7365, U.S.A.
tel: +1-919-843-4485
fax: +1-919-966-5640
email: mach...@unc.edumailto:mach...@med.unc.edu

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MedImmune to be confidential and proprietary. This communication is expected to 
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Re: [ccp4bb] Mg2+ or water

[Oganesyan, Vaheh]

Using different web servers on your refined structure is good thing to
do. But for distinguishing metal ion, specifically Mg, from water was
done unambiguously before these programs existed. The simple rule is two
fold: 1. distance; 2. coordination. For Mg ion distances are between 2
and 2.2 A and coordination is almost always nearly perfect octahedron.
Neither of those criteria met in your structure. Hence the verdict - it
is water.

 

 

 Vaheh  



From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
jlliu liu
Sent: Monday, December 20, 2010 4:16 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Mg2+ or water

 

Hi All,

I am refining a structure and encountered a problem of modeling a
difference density as water or Mg2+, and would like to hear opinions
from the community. It has the following coordinations (attached): the
water/Mg2+ forms salt bridge/H-bonding interaction with a carboxylate
group from the ligand, it also forms salt bridge/H-bonding interaction
with a Glu residue from the protein, it is also within hydrogen bonding
distance to the main chain N of another protein residue. In provious
publication, it was modelled as a Mg2+ and the author reasoned the dual
salt-bridge stabilizes the liganding binding, also the Mg2+ is present
in the protein solution for crystallization. For my case, I have no Mg2+
present in the protein buffer, also modelling it with water refines
perfectly with no indication of positive difference density even at 2.0
sigma cut off. Should I modelled this density as water or as Mg2+. Your
opinions are appreciated.

JL
 




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sender advising of the error in transmission and delete the original message 
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cooperation.

[ccp4bb] on the same note

[Oganesyan, Vaheh]

On the same note with Mirek: does anyone know of a source other than GE for 
resin for purification of FLAG-ed proteins?

Thanks.

 Vaheh  

-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Mirek 
Cygler
Sent: Tuesday, November 02, 2010 11:47 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Glutathione sepharose

Hello,
For various reasons we are frequently expressing proteins with a GST
tag. The glutathione sepharose beads that we are using for affinity
purification seem to be difficult to regenerate and we see much lower
capacity when used the second time. We are following the manufacturer's
instructions for regeneration but this not very effective process as opposed
to NiNTA which can regenerate multiple times. Due to this, the purification
of GST-tagged proteins is quite costly. Do you know of a GSH-sepharose that
can be successfully regenerated several times? Any comments will be greatly
appreciated.

    Mirek



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Re: [ccp4bb] pdf to text

[Oganesyan, Vaheh]

Thanks go to all who took their time and answered and in some cases did
file manipulations. Extremely helpful! Not only for this particular case
but many times CCP4BB has proven to be the best.
Below are received answers in chronological order:

Albert Gluskov: I converted it in adobe acrobat pro, so now you can
manipulate it, but check all numbers, because usually software doesn't
recognize everything 100% correctly. (the file was attached)
___
Tiago Botelho: Why don't you try an OCR program to read the text for
you? It should do the trick...

Let me know if it worked!   
___

Jonas Boehringer: I have run OCR text recognition in Adobe Acrobat over
it and attached the .pdb file. You will still have to remove title, page
numbers and some errors in the last column but better than typing it all
by hand.
___

Judit Murray-Rust: Can't convert your pdf to text (tho note that I have
typed up bgger mols than this hand in the past - it's all a question of
how badly you want it!). But here is one way to get set a set of
coordinates reputedly of polymixin B

1. look up in chemspider (my result =
http://www.chemspider.com/Chemical-Structure.8009375.html) - note there
are other polymixin B deriv here, so you probably want to start at the
front page of chemspider, that link is just so rest of explantion makes
sense.
2. select 3D under the black box on the lhs. you should get a jmol image
(rotatable) 
3. use the save button to save it as a mol file.
4. import the mol file into some displa program (I used pymol) and save
the result in pdb format.
5. It is then up to you to check it is the molecule you want!


can get 3D mol file from chemspider, too. And convert to pdb with one of
many display progs. But whether it is exactly the same as what is in the
pdf file is an exercise for the user ;-) J

___

Paul Mcewan: Try using the prodrug server. if you can draw it, it
can make it!
 
http://davapc1.bioch.dundee.ac.uk/prodrg/
___

Paul Emsley: If you don't need the same conformation, how about
downloading the 2D mol file from chemspider?  Prodrg can convert that to
3D in a trice.
(screenshot attached)

___

Luca Jovin: Just ran your PDF file through an OCR program - output
attached. Obviously it will still needs quite a bit of manual editing,
and I would make super-sure it read numbers correctly, but still it's a
start!
(text file attached)
___
Tim Gruene: You could try a text recognition software. In the
Linux-/Unix-world, gocr is probably to most popular one. Since it is a
courier font, your chances should be pretty good that it works.
___

Fred Vellieux: What you could try to do is print out the pdf file, then
locate a scanner with a suitable scanning software. Several scanning
software have the possibility of generating word processing program
output or ASCII format. Since the pdf file is text only (no figures etc)
then it should be OK. You just have to go through the output generated
and check for errors (such software is not perfect and produces errors
here and there).
___
Tomas Malinauskas: PDB file:
http://129.128.185.122/drugbank2/drugs/DB00781/pdb/download
More information:
http://www.drugbank.ca/drugs/DB00781
___
Mark Brooks: For OCR without installing software, Free OCR
http://www.free-ocr.com/ works quite well for me, but beware that you
may need to do corrections afterwards. 
 
Just upload your file to this web site, as long as it isn't secret!
 
The OCR in Adobe Acrobat works better for me though, and is worth the
money, I think.
___

Ed Pozharski: Ran it through Adobe Acrobat OCR text recognition - I
think now it's a selectable text. (proper pdf file attached).


In case anybody needs any of the files sent to me - just let me know.

Regards,


 Vaheh  




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information that is not in the public domain, such information is considered by 
MedImmune to be confidential and proprietary.  This communication is expected 
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Re: [ccp4bb] PEG 1000

[Oganesyan, Vaheh]

Actually it probably would be better ask the supplier of your screen
first how they got PEG 1K diluted to 12.5%. If they did hit it then you
have to do the same. But even better idea is to order that PEG 1K from
the same company that sells the screens. It will insure identical
treatment.

 

My 2 cents.

 

 

 Vaheh  



From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
R.Srinivasan
Sent: Wednesday, June 23, 2010 3:11 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] PEG 1000

 

Dear All,

 I have got initial crystals in a condition with PEG 1000. The
PEG 1000 stock we had in our lab was rock solid and when i heated it to
about 50 degrees for 15 to 20 minutes it became a solution. We thought
the compound has got out dated or something like that and bought a brand
new bottle from Sigma and this is rock solid too.

Is this something characteristic of PEG1000? The hit condition
says its 12.5% w/v PEG 1000 but it apparently seems i could never get a
powder of it.

   So my question is, Can i go ahead using this melted solution form
of PEG1000 for setting up optimizations?

Thank you all in anticiaption,
Vasan

 




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[ccp4bb] Multiple NCS relations

[Oganesyan, Vaheh]

Dear bb contributors,

I would like to refer to your expertise and get advice regarding
multiple NCS relations between two macromolecular complexes in asu. 
Resolution 2.5A, s.g. C2221, R-merge 6%, synchrotron data, 2 complexes
per a.s.u. Each complex consist of three polypeptide chains, let's say
A, B, C and A', B' and C'. A and A' have one NCS operator and B and B'
have another. C molecule follows A. 
When I impose ncs in refinement my R-factors jump to ~30(35)% while
without they are around 21(27)%. 
Does this mean that I can not use two different NCS operators or it
means I'm not applying ncs correctly?

I'm using refmac5 with TLS through GUI.

Thank you.  

 Vaheh 

P.S. I don't remember if such question have ever been discussed on bb. 




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Re: [ccp4bb] Fwd: [ccp4bb] Blue color upon X-ray exposure?

[Oganesyan, Vaheh]

Protein crystals grown in Phosphate-Citrate buffer, pH 4.2 behave exactly the 
way Richard described: they first turn blue then fade through yellow-brown.

Vaheh


-Original Message-
From: CCP4 bulletin board on behalf of Richard Gillilan
Sent: Mon 3/15/2010 2:23 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Fwd: [ccp4bb] Blue color upon X-ray exposure?
 
 
 I have personally noticed that the blue color only appears when the pH of the 
 hyperquenched solution is higher than 7 or so.  I assume this is because 
 solvated electrons react with protons to form their conjugate base: the 
 hydrogen atom.  The latter species is highly reactive as well, but it is not 
 colored. 
...

We see a very strong dark blue in cryoSAXS experiments on lysozyme buffer at pH 
4.5 (acetate) containing high glycerol content. We've also seen that color fade 
to light yellow/brown over time while in the cryostream once irradiation has 
stopped (I don't recall if that particular solution had protein or not). The 
blue color appears long before the Henderson limit and does not seem to affect 
the scattering profile.

I believe a similar effect is at work in bottle glass that has been exposed to 
sunlight for a long time - color centers. Blue ice seen in the far north 
however, appears to be purely a light-scattering phenomenon and not a result of 
trapped electrons.

Richard Gillilan
MacCHESS


 
 -James Holton
 MAD Scientist
 
 Todd Geders wrote:





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[ccp4bb] definition file

[Oganesyan, Vaheh]

Colleagues,

Do any of you have created a def.site file for LRL-CAT 31-ID-D beamline
to be used by HKL2000? I'll appreciate if you can share. 

__
Oganesyan Vaheh, Ph.D 
Antibody Discovery 
MedImmune, Inc.





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Re: [ccp4bb] mammalian cell culture on IMAC

[Oganesyan, Vaheh]

Brad,

 

It looks like the chelator is a reducing agent added to media for
preservation, I guess.

 

The cure is not found yet. I'm just loading ~300 ml per one 5 ml
column and it so far serves me well.

 

The IEX approach did not work well. I've lost more than with IMAC after
3X dilution (with properly chosen resin and buffer pH).

 

Your idea works fine: that's why I limit load to 300 ml.

 

Thank you.

 

___ 
Vaheh 



From: Brad Bennett [mailto:bradbennet...@gmail.com] 
Sent: Thursday, October 08, 2009 4:43 PM
To: Oganesyan, Vaheh
Subject: Re: [ccp4bb] mammalian cell culture on IMAC

 

Hi Vaheh-
Well (gulp) you could dilute your solution say 2-10X with buffer with no
salt. You could try small volumes at first and see how dilute the [salt]
must be before your protein sticks to your IEX column.

So, what is in the media that is stripping off the Ni ions? A chelator
like EDTA? And you're sure it's not something more sinister like
stripping AND reducing the Ni? If the media formulation is proprietary,
you may never definitively know. But I wonder if you could add free
resin to a small portion of your solution, spin it down and run the
beads on a gel and detect your protein by silver stain or immunoblot?
If, whatever it is, is just stripping your resin, then maybe you could
pull down some of your target protein. Surely some Ni+ will get
chelated but maybe it's worth a shot? Maybe as long as you have plenty
of Ni-NTA, Ni-Sepharose or Talon hanging around, you could give this a
try.

You could try salting it out with saturating ammonium sulfate. Not
elegant but it should work and you could resuspend the pellet in
whatever buffer and volume you wanted.

Any other epitopes/affinity handles on this protein? Like Flag or HA?

HTH-
Brad

On Thu, Oct 8, 2009 at 4:15 PM, Oganesyan, Vaheh
oganesy...@medimmune.com wrote:

In case of cell lysates this may be a good idea since you can adjust the
salt concentration in your sample. In case of secreted proteins it is
probably not so good since the media contains ~150 mM NaCl. In my case
this salt prevents protein from bindinq to Q column.

Thank you anyway.



From: CCP4 bulletin board on behalf of Dima Klenchin
Sent: Thu 10/8/2009 3:54 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] mammalian cell culture on IMAC





When mammalian cell culture is being loaded to GE HisTrap resin Ni ions
are being stripped off the resin, at least in my hands. Did any of you
have similar experience and if so what kind of work-around was found?
Volume is fairly large (3L) and concentration/dialysis have proven to
cause loss of desired protein.
Please share your positive experience.

I get this with insect cell lysates. The solution: load onto ion
exchanger
first - not for purification but to get rid of whatever strips Ni2+.
Crude
wash with 50 mM salt (or as high as your protein allows) followed by
step
elution with 0.5M salt (very few proteins do not elute at 0.5M) -- load
directly onto Ni-NTA. Solves the stripping problem 100%. (If you use
step
elution, make sure to NOT use weak exchangers or you will have pH shift
of
2-3 units). If the protein binds to cation-exchangers at pH 7, even
such a
crude step results in significant purification in itself. If, for some
reason, ion exchangers are not an option, load onto hydroxyapatite and
elute with phosphate. The downside to HA is that it has a lot less
capacity
than ion-exchangers and that it needs frequent repacking.

Good luck,

Dima






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electronic communication in error, please reply to the sender advising
of the error in transmission and delete the original message and any
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cooperation.

 




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MedImmune to be confidential and proprietary.  This communication is expected 
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Re: [ccp4bb] mammalian cell culture on IMAC

[Oganesyan, Vaheh]

Dear All,
 
When mammalian cell culture is being loaded to GE HisTrap resin Ni ions are 
being stripped off the resin, at least in my hands. Did any of you have similar 
experience and if so what kind of work-around was found?
Volume is fairly large (3L) and concentration/dialysis have proven to cause 
loss of desired protein.
Please share your positive experience.
 
Thank you for your time.
 
Vaheh






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information that is not in the public domain, such information is considered by 
MedImmune to be confidential and proprietary.  This communication is expected 
to be read and/or used only by the individual(s) for whom it is intended.  If 
you have received this electronic communication in error, please reply to the 
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and any accompanying documents from your system immediately, without copying, 
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Re: [ccp4bb] mammalian cell culture on IMAC

[Oganesyan, Vaheh]

In case of cell lysates this may be a good idea since you can adjust the salt 
concentration in your sample. In case of secreted proteins it is probably not 
so good since the media contains ~150 mM NaCl. In my case this salt prevents 
protein from bindinq to Q column.
 
Thank you anyway.



From: CCP4 bulletin board on behalf of Dima Klenchin
Sent: Thu 10/8/2009 3:54 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] mammalian cell culture on IMAC




When mammalian cell culture is being loaded to GE HisTrap resin Ni ions
are being stripped off the resin, at least in my hands. Did any of you
have similar experience and if so what kind of work-around was found?
Volume is fairly large (3L) and concentration/dialysis have proven to
cause loss of desired protein.
Please share your positive experience.

I get this with insect cell lysates. The solution: load onto ion exchanger
first - not for purification but to get rid of whatever strips Ni2+. Crude
wash with 50 mM salt (or as high as your protein allows) followed by step
elution with 0.5M salt (very few proteins do not elute at 0.5M) -- load
directly onto Ni-NTA. Solves the stripping problem 100%. (If you use step
elution, make sure to NOT use weak exchangers or you will have pH shift of
2-3 units). If the protein binds to cation-exchangers at pH 7, even such a
crude step results in significant purification in itself. If, for some
reason, ion exchangers are not an option, load onto hydroxyapatite and
elute with phosphate. The downside to HA is that it has a lot less capacity
than ion-exchangers and that it needs frequent repacking.

Good luck,

Dima





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information that is not in the public domain, such information is considered by 
MedImmune to be confidential and proprietary.  This communication is expected 
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cooperation.

[ccp4bb] shape complementarity calculations

[Oganesyan, Vaheh]

Colleagues,

Would some one kindly suggest software that calculates shape
complementarity of two interacting proteins based on co-crystal
structure?
I've seen number of reports with sc parameter included but none of
those mention how it was done.
Among non-runnable programs in CCP4 there is the sc program that indeed
does not run.

Thanks in advance.

___
Vaheh




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[ccp4bb] Planar Systems monitor

[Oganesyan, Vaheh]

I came across this LCD monitor with DVI connection. Would experts in the
field recommend this monitor for stereo viewing?

http://www.pcconnection.com/IPA/Shop/Product/Detail.htm?sku=6220485

Thank you.
___
Vaheh



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information that is not in the public domain, such information is considered by 
MedImmune to be confidential and proprietary.  This communication is expected 
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Re: [ccp4bb] cryo-cooling

[Oganesyan, Vaheh]

I apologize for using BB to address a person (Ethan Merritt, U. of Washington). 
His e-mail address mentioned on TLSMD page is not operational.


Good morning Ethan,

Few months ago I asked you if carbohydrates can be recognized by TLSMD and 
appropriate calculations can be included in on-line version of TLSMD. I'm 
writing to you to find out if there was any progress in that direction and how 
to make carbohydrates to be recognized.

Regards,

Vaheh Oganesyan

___
Vaheh

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Kay Diederichs
Sent: Saturday, June 07, 2008 7:26 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] cryo-cooling

Jose,

check out the 4 papers by R. Thorne listed at
http://www.px.nsls.bnl.gov/courses/papers/ZD_EG_papers.html

HTH,

Kay
-- 
Kay Diederichs  http://strucbio.biologie.uni-konstanz.de
email: [EMAIL PROTECTED]  Tel +49 7531 88 4049 Fax 3183
Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz




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[ccp4bb] HEK293S

[Oganesyan, Vaheh]

Please, forgive my partially off-topic question.

I'm looking for commercial or cell bank source for GnTI-deficient
HEK293S cells and will appreciate any suggestions on how to get them.

P.S. It's partially off-topic since the cells will be used to produce
protein for crystallography.

Thanks.

___
Vaheh




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cooperation.

[ccp4bb] Research Associate I/II position

[Oganesyan, Vaheh]

Colleagues,

I'll appreciate if following advertisement will be brought to attention
of qualified researchrs.

An exciting research opportunity is available at MedImmune, Inc. (now
part of AstraZeneca, Plc.) for a self motivated B.S. or M.S. level
Scientist to contribute to ongoing projects that include expression,
purification, and characterization of antibodies, related proteins and
new protein scaffolds.  The candidate should have extensive experience
in general molecular biology techniques including cloning, site-directed
mutagenesis, protein expression (E. coli and mammalian cells), and
protein purification.  As a part of an integrated team, the candidate
will interact with chemists and biophysicists to design new scaffolds
and alter their properties. Knowledge and experience with antibody
expression and purification is highly desirable.  Experience in phage
display (or similar) or protein engineering is a plus, as is experience
with biophysical characterization of proteins. Other requirements are:
Experience:  3+ years
Special Skills/Abilities:  Excellent written and verbal communication
skills.

Please note that experience in any aspect of protein crystallography is
not required.
To apply, please visit http://www.medimmune.appone.com/ and search by
Req. Number 00616.
__
Oganesyan Vaheh, Ph.D 
Antibody Discovery 
MedImmune, Inc.





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information that is not in the public domain, such information is considered by 
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[ccp4bb] negative LLG in Phaser run

[Oganesyan, Vaheh]

Phaser experts,

When running Phaser I see LL gain becoming negative closer to the end.
What would that mean?
Resolution ~3.0 A, s.g. I222, 2 complexes per a.u.
Here is the line from log file:
ANNOTATION:  RFZ=7.6 TFZ=8.9 PAK=0 LLG=142 RFZ=6.6 TFZ=18.0 PAK=0
LLG=150 RFZ=4.6
   TFZ=12.8 PAK=0 LLG=-44 RFZ=3.8 TFZ=5.8 PAK=19 LLG=-908

Thank you in advance.

__
Oganesyan Vaheh, Ph.D 
Antibody Discovery 
MedImmune, Inc.





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Re: [ccp4bb] crystallisation robot

[Oganesyan, Vaheh]

Mark,

 

What was the state of the larger drops when tiny counterparts had
crystals? My guess - they all precipitated.

I'm trying to understand why some proteins or some conditions require
change in protein concentration while others do not when migrating from
smaller drops to larger ones. If it is protein dependent then I'm afraid
there might be no one answer; if it is not then there should be a trend
and explanation of phenomena.

 

 

Vaheh 



From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
[EMAIL PROTECTED]
Sent: Wednesday, January 16, 2008 8:31 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] crystallisation robot

 

Once upon a time I worked in a group that was interested in developing
crystallization in microfluidics. This was before the time that Fluidigm
existed and we had not heard of crystallization with the aid of
microfluidics at the time. We had good reason to try to make a system
that was as small and light as possible - it had something to do with
the cost of shipping proteins and precipitants - less was better. And we
also wanted all protein drops to be fully enclosed, out of safety
considerations.

Like Tassos, we were very worried what would happen if you scaled back
drops along the lines of this discussion - several uL downto tens of
nanoliters. If the stochastic process had a major influence over this
process, we thought that we would never get any crystals. So we set up
side-by-side experiments at larger volumes and smaller volumes -
basically scanning several orders of magnitude - expecting a decrease of
the number of crystals when volumes decrease. To our great surprise the
outcome was that smaller volumes almost always gave MORE (I almost want
to say 'dramatically more') crystals, more nucleation, and indeed in
various cases the crystals grew much faster also. Indeed, it was trivial
to observe that the surface-to-volume ratio was the primary driver for
the nucleation process. We had control over geometry to some extent and
were able to observe surfaces while crystals grow. The crystals would
most commonly nucleate on a surface. 

So although there probably is something to stochastic aspects, it is
clear that other aspects can be more important and overrule the
stochastic considerations.
The somewhat unpleasant consquence is of course that results acquired in
very small volumes (with larger surface-to-volume ratio) cannot
necessarily be repeated in larger volumes (smaller surface-to-volume
ratio).

This is not a flame, even if heat might be a good thing on a night with
temperatures predicted far below 0F.

 :-)

Mark

 

 

-Original Message-
From: Anastassis Perrakis [EMAIL PROTECTED]
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wed, 16 Jan 2008 6:17 am
Subject: Re: [ccp4bb] crystallisation robot

 Oryxnano 50+50 nL 
 
 Demetres 
 
 
Which, indirectly, brings up an interesting (but not relevant to the
Oryx) question. 
 
Nucleation is a process that does have a stochastic aspect. 
 
Thus, one could argue that compromising to 200-300 nl might be better
than either extremes of 50nl (too small volume and less chance for
nucleation) or 1000 nl (too much sample). 
 
any comments ? (let the flames begin). 
 
A. 
 
PS1 
another interesting issue that has has been hardly touched in these
emails is the real sample loss: left in wells and not easy to recover,
lost because of contamination with system liquid, etc ... 
 
PS2 
I see lots of people with new robots. please do have a look at the
www.BIOXHIT.org page and if you have a few minutes to assemble a table
we will be happy to add your specs to our pages. it can be a nice
resource and it has already enough things and already one response to my
last email ;-) To make life easier to potential contributors we can
provide an Excel sheet to fill up with your specs - just ask. 
 
On Jan 16, 2008, at 12:46, Demetres D. Leonidas wrote: 
 
 
 David Briggs wrote: 
 I'll defend the honour of the phoenix... (again) 
 
 Bernhard Rupp 100+100 nl 
 Dave Briggs (and all users at Univ of Manchester, UK) 100+100nl 
 Others.. 
 
 Only time we have ANY problems is when the nano dispensing tip 
gets clogged. Often a good wash whilst still on the machine will 
clear the blockage. 
 
 Dave 
 
 
 
 
 --  
 David C. Briggs PhD 
 Father  Crystallographer 
 http://www.dbriggs.talktalk.net http://www.dbriggs.talktalk.net 
 AIM ID: dbassophile 
  
 
 -- Demetres D. Leonidas, Ph.D. 
 Structural Biology  Chemistry Group 
 Institute of Organic and Pharmaceutical Chemistry 
 The National Hellenic Research Foundation 
 48, Vassileos Constantinou Avenue 
 Athens 116 35, Greece 
 == 
 Tel. +30 210 7273841 (office) 
 +30 210 7273895 (lab) Fax. +30 210 7273831 
 E-mail: [EMAIL PROTECTED] 
 URL: http://athena.eie.gr 
 == 



size=2 width=100% 

Re: [ccp4bb] crystallisation robot

[Oganesyan, Vaheh]

I have been using Phoenix for more than two years and so far there were
no issues with maintenance. Wash the needles and nano-dispenser before
and after the runs and you are good to go.
Electrostatic effects have been seen in a way of drops being positioned
on the side of the flat bottom plate, but the drops do not climb too
high in my case probably because I'm using fairly large drops (200 +
200 nl).
No matter how robust the system is, there will be always a way to break
it.

Once in a while I get large crystal that can be used for data
collection, but in most of the cases optimization in hanging 1+1 uL
drops is required.
What I found quite difficult to do is to choose the appropriate protein
concentration when moving from 200+200nL to 1+1 uL. Sometimes protein
should be diluted 3-4 times, sometimes it shouldn't. How others are
approaching this issue?

Vaheh

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Anastassis Perrakis
Sent: Wednesday, January 16, 2008 8:23 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] crystallisation robot

 More recently, I've looked at all of the crystallization robot  
 vendors.
 For single lab users, all of the systems work well. Systems like  
 the Hydra
 or Mosquito are less automatic, but provide the basic functions for
 crystallization trial setup. For more of a user facility with a large
 number of users from several groups, you want more automation to avoid
 protocols that can damage the components, like alignment or  
 breakage of
 the needles. You also want to consider the total annual cost of
 expendables and maintenance.

There are two major kinds of  a user facility with a large number of  
users from several groups

a. One with an operator: then buy what the operator likes ;-)  
basically all machines do work
b. NO operator: buy something that is hard to break and needs minimal  
maintenance

We are in b. and happy with our Mosquito for 4 years or more now,  
although I am sure the
other choices mentioned are also clearly fine.

A.

Re: [ccp4bb] SUMMARY: PEG MW vs. cryoprotectivity

[Oganesyan, Vaheh]

Phoebe,

In addition to all other comments I like to add that have had several
cases when crystals would not take/like any cryo agent at all. What I
did is added 1% glycerol in the crystallization solution. That did not
change crystallizability of any of the proteins I worked with, but as a
result those crystals would not mind glycerol at all (up to 40% I
tried). One more thing,
the data I get from such grown crystals are better: low mosaicity,
relatively low B-factors. It might be worth trying.

Regards,

Vaheh Oganesyan, Ph.D.
MedImmune, Inc.
Phone: 1-301-398-5851
Facsimile: 1-301-398-8851

To the extent this electronic communication or any of its attachments
contain information that is not in the public domain, such information
is considered by MedImmune to be confidential and proprietary, and
expected to be used only by the individual(s) for whom it is intended.
If you have received this electronic communication in error, please
reply to the sender advising of the error in transmission and delete the
original message and any accompanying documents from your system
immediately, without copying, reviewing or otherwise using them for any
purpose.  Thank you for your cooperation.



-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Behalf Of
[EMAIL PROTECTED]
Sent: Thursday, December 06, 2007 11:10 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] SUMMARY: PEG MW vs. cryoprotectivity


Many thanks to all who replied.  The answers were remarkably varied - 
see below.
My own two bits worth - vitrification of mother liquor doesn't always 
lead to a nice, low-mosaicity, ice-free crystal freeze in our hands, 
although we're not willing to sacrifice a statistically significant 
number of crystals to figure out why.  Most of our crystals are 
annoyingly fragile, which makes it hard to disentangle the effects of 
bad cryoprotection from those of mechanical damage.
 Phoebe

From: [EMAIL PROTECTED]
PEG 4000 has worked for us at high concentration (35-40%)
depending on what else is in there. The same goes for
PEG 3000. You can try increasing the concentration of
the PEG in the reservoir gradually (over the course of many
days) from the % they are grown at up to 25-40%. Hopefully this
will not crack your crystals.


From: Anastassis Perrakis [EMAIL PROTECTED]
in my experience peg 4k reduces the amount of glycerol that you need
but cant act as cryo on its own.

From: Juergen Bosch [EMAIL PROTECTED]
the larger the worse for cryo. But PEG4000 40% freezes well. PEG8000 
needs some addition of smaller PEGs/Glycerol etc.


From: Kevin Jude [EMAIL PROTECTED]
I have used 23% PEG 3350/5% glycerol as a cryoprotectant (JACS 2006 p 
3011).  The PEG on its own didn't work at that concentration.

It would be enough to test this by making up the solutions and 
shooting empty loops, like Elspeth Garman did for glycerol.

From: Ezra Peisach [EMAIL PROTECTED]
I have seen discussions in the past... The easiest thing to do is 
test it yourself. Try freezing a small loop of high concentrations of
PEG.
If it forms a clear glass it is worth pursuing...

From: Jan Abendroth [EMAIL PROTECTED]
20% PEG 2000 just worked fine, 35% PEG 3350 seems ok too.
also depends on the size of the loop.

From: Edwin Pozharski [EMAIL PROTECTED]
I have used pegmme2000 as cryoprotectant in the past (some 45% of 
it), and it worked fine.  Indeed, PEG4K s included in Hampton's kit.

From: Buz Barstow [EMAIL PROTECTED]
In our experience with freezing protein crystals under high pressure,
we've found that mid weight PEGs do help a little to enhance the 
cryo- protective effect of high pressure, although are not terrifically
effective, especially when at a low concentration of around 5%.


From: Li Sheng [EMAIL PROTECTED]
I used 40% w/v PEG 4000 as cryoprotectant.

From: Moody, Dr P.C.E. [EMAIL PROTECTED]
my experience is that anything over PEG 600 is likely not to be 
a  reliable cryoprotectant, and 400 is the maximum safe 
size.can't comment on the commercial kits, my cynical nature 
would suggest that testing may not be an important part of the 
product pipelinePeter

From: Remy Loris [EMAIL PROTECTED]
PEG 4000 is a very good cryoprotectant in the range of 30-35%. If 
your crystallization condition includes PEG4000, it is a good idea 
for a first trial to find a good cryo condition raise the PEG4000 
concentration to 30-35%. Some of the Hampton ctrystal screen 
conditions (and other commercial kits as well) that contain PEG4000 
do even not need further addition of cryoprotectant (even if in the 
corresponding Hampton cryo screen they are diluted with glycerol, one 
of the most horible cryoprotectants in common use)
Lower MW PEGS can be useful as well, but the required concentrations 
will be higher. Please be aware that in quite a number of cases the 
ideal cryo solution can be very far away from the condition in which 
the protein was crystallized!

From: gengxiang zhao [EMAIL PROTECTED]
Before, I crystallized the 

Re: [ccp4bb] Ni-NTA purifications contaminated with E coli Glucosamine-fructose-6-phosphate aminotransferase

[Oganesyan, Vaheh]

In addition to all good recommendations I would add the following: load
your protein to the whatever-Ni column then wash it with urea. 1 to 4 M
urea will not, in most cases, destroy your protein but will disrupt the
hydrophobic interaction between contaminant and your protein in case if
the contaminant is binding not to Ni column but to your protein.
 
My two cents.
 
Vaheh Oganesyan, Ph.D.
MedImmune, Inc.
Phone: 1-301-398-5851
Facsimile: 1-301-398-8851

To the extent this electronic communication or any of its attachments
contain information that is not in the public domain, such information
is considered by MedImmune to be confidential and proprietary, and
expected to be used only by the individual(s) for whom it is intended.
If you have received this electronic communication in error, please
reply to the sender advising of the error in transmission and delete the
original message and any accompanying documents from your system
immediately, without copying, reviewing or otherwise using them for any
purpose.  Thank you for your cooperation.

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Behalf Of
Ailong Ke
Sent: Friday, November 02, 2007 3:53 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Ni-NTA purifications contaminated with E coli
Glucosamine-fructose-6-phosphate aminotransferase


Thanks for the suggestions, JJ.

The supernatant was loaded onto Ni-NTA in the presence of 300 mM NaCl
and 5 mM imidazole, washed with 20 mM imidazole (could test higher
concentration...), then eluted with 300 mM imidazole. So I don't think
non-specific binding is a problem. We got 90% purity off the Ni-NTA. But
for this particular protein , the Glucosamine-fructose-6-phosphate
aminotransferase just wouldn't go away in subsequent purifications
(which may imply physical interactions?).

I guess I was  wondering if other people had similar problems, and if
there exists a trick similar to using ATP to remove GroEL contamination.


Ailong



Provided your proteins don't stick to each other through exposed
hydrophobic surfaces

then sodium chloride could be used to separate the contaminants more
efficiently prior

to NiNTA purification. You should wash with 30-40 () column volumes
in 20-30mM

imidazole to remove non-specific contaminants.  Conslut your manual
(Ni-resin)

to find out limitations due the presence of higher salt concentraitons.


JJ



On Nov 2, 2007, at 2:42 PM, Ailong Ke wrote:


Hello,




We are trying to purify an N-terminal His6-tagged protein from E. coli,
and the prep was contaminated with the E coli
Glucosamine-fructose-6-phosphate aminotransferase, which co-purifies
with my protein of interest in subsequent ion-exchange and sizing
columns. This protein appears to be a common contamination in the Ni-NTA
purifications. Does anyone have tricks to get rid of it? Thanks.


Ailong




--



..

Joachim Jaeger, DPhil

Center for Medical Sciences, Rm.2009

NYS-DOH, Wadsworth Center

Albany, New York 12201-0509

Tel: +1 518 408-2225  Fax: +1 518 402-2633

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Ailong Ke, Ph.D.
Assistant Professor
Department of Molecular Biology and Genetics
Cornell University
251 Biotechnology Building
Ithaca, NY, 14853

tel: 607-255-3945
fax: 607-255-6249
email: [EMAIL PROTECTED]

Re: [ccp4bb] coot in stereo

[Oganesyan, Vaheh]

Similar thing was happening on the dual Xeon 2.8 GHz machines in O as well. 
But it was 2-5 sec long in time.

Vaheh Oganesyan

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Behalf Of Kay Diederichs
Sent: Tuesday, August 14, 2007 3:54 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] coot in stereo

Paul D. Cook schrieb:
 Hello,  I'm running stereocoot on linux (centos) machines with nvidia
 video cards and stereographics emitters and glasses.  The setup seems to
 work fine most of the time, but the stereo will invert every now and then
 (the right eye is shown the left image and vice versa).  This seems to
 happen especially after a computation such as realspace refine is
 performed.  The stereo will usually spontaneously correct itself, but
 sometimes I have to minimize the window and restore it.  This is occuring
 on three machines with the above configuration.  Swapping emitters and
 glasses seems to do nothing.  Has anyone had such a problem?

 Thanks,
 Paul D. Cook

Paul,

I've seen this problem too, on AMD dual-processor machines with powersaving
enabled. I could not find a version of the NVIDIA driver that did not show the
problem. I believe that the switch in CPU frequency disturbs the 
synchronization.
So you might want to experiment with e.g. service cpuspeed stop (as root) and
see what happens. If it does help, you can switch powersaving off permanently
(chkconfig cpuspeed off).
Unfortunately powersaving depends on proper interaction of kernel, hardware and
usermode software. Intel CPUs are quite different from AMD ones in that respect.

HTH,

Kay

--
Kay Diederichs  http://strucbio.biologie.uni-konstanz.de
email: [EMAIL PROTECTED]  Tel +49 7531 88 4049 Fax 3183
Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz

Re: [ccp4bb] CCP4 Wiki

[Oganesyan, Vaheh]

Isn't this e-mail's topic and it's appearance contradict each other?
It comes from William Scott's e-mail account, but signed as coming from Paul 
Emsley.
How do we figure out who actually wrote this e-mail with or without control 
access.
The easiest way is to assume that it is coming from Bill, he just decided to 
sign as a Paul to disprove his own point.
On the other way, it is only 7:30 am in Santa Cruz, then it is unlikely to be 
Bill, which means that e-mail was sent by Paul.
Worth looking at this http://en.wikipedia.org/wiki/Sherlock_Holmes Wiki page, 
isn't it?

Vaheh Oganesyan

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Behalf Of William Scott
Sent: Friday, July 27, 2007 10:12 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] CCP4 Wiki

Most of us register on the CCP4 bb with our own names.  If we say
something stupid, it damages our reputation in front of our peers.  A
similar approach can be used for the Wiki.  Control access, and limit it
only to people who register with their own name and who are registered to
the ccp4 bb.

-- Paul Emsley

Kay Diederichs wrote:
 Anastassis Perrakis schrieb:
 Kay - disagreeing once was enough ... so:

 I share your thoughts about the wiki !

 However, the dynamics of Wikipedia are an interesting issue and relate
 to 'vandalism'.