[ccp4bb] AW: [ccp4bb] Off topic - French Press Accessories

[Hughes, Jon]

hi naza,
i wouldn't touch machines like that from the USA – their prices are too high 
and  their tolerances are miserable. we bought a made-to-measure French press 
(including various stainless-steel cells) from a REAL hydraulics manufacturer 
near here
https://watzhydraulik.de/en/
about 20 years ago, and everything's still working as perfectly as it did on 
the first day.
best,
j

Von: CCP4 bulletin board  Im Auftrag von Nazahiyah Rodzli
Gesendet: Mittwoch, 24. April 2019 03:55
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Off topic - French Press Accessories

Dear All,

Our lab is using the good old Thermo French press (FA-078A-picture attached) 
with a mini sample cell which has only 3.5 ml maximum sample capacity and 
planning to use for as long as it lasts. However, we are now in need of a 
bigger sample cell for our membrane protein work. Unfortunately, I have been 
made aware that Thermo has long since discontinued its support and delivery for 
such robust piece of equipment and for the obvious reason I couldn’t find a 
company locally that can provide this. Can anyone suggest if there’s a way I 
can get the larger sample cell (FA-032 40K Standard - second picture attached); 
or any lab that happens to have an extra piece of the accessory or no longer in 
use and willing to let go at a reasonable price?



Best regards,
Naza


___

Nazahiyah Rodzli, PhD (The University of Manchester)
Scientist
Structural and Applied Genomics
Malaysia Genome Institute
National Institutes of Biotechnology Malaysia
Jalan Bangi,
43000 Kajang,
Selangor, MALAYSIA


[X][Das Bild wurde vom Absender entfernt. Image result for thermo french press 
standard cell]



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[ccp4bb] ABP1 has no known function

[Hughes, Jon]

Dear all,
Many people know much more about the plant hormone auxin than I do, but I 
believe I am right in saying that, despite Romana Gáborová's current PDBe 
highlight on auxin binding protein 1 (ABP1) at 
https://www.ebi.ac.uk/pdbe/about/news/growth, there is currently no valid 
evidence that ABP1 has any physiological function whatsoever (see in particular 
Gao et al. 2015, doi: 10.1073/pnas.1500365112). The structure of the known 
auxin receptor TIR1 (Tan et al. 2007, DOI: 
10.1038/nature05731) is much more 
interesting anyhow!
Best,
jon

--
Professor Jon Hughes, BSc, PhD
Institute for Plant Physiology
Justus Liebig University, Giessen
Zeughaus, Rm. 341
Senckenbergstr. 3
D35390 Giessen, Germany.
work phone: (+49/0)6419935430
fax:  (+49/0)6419935429
mobile:   (+49/0)1757929098
email:  jon.hug...@uni-giessen.de
homepage:
http://www.uni-giessen.de/fbz/fb08/Inst/pflphys
Sent without the use of Apple products





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[ccp4bb] PhD position: 3D structure/function of phytochrome photoreceptors

[Hughes, Jon]

I have a position for a PhD student to work in my lab


PhD position

Phytochrome structure/function


A PhD position at the Institute for Plant Physiology of the Justus Liebig 
University is available immediately. The position (E13 65%) is funded for three 
years as part of the project "Phytochrome from photochemistry to signalling" 
(DFG Hu702/12) in collaboration with Chen Song in Jörg Matysik's lab in Leipzig.
The aim of the project is to further our understanding of the functional 
mechanism underlying photoconversion and signal activation in the 
cyanobacterial phytochrome Cph1. To this end we will study the structures of 
the Pr and Pfr parent states as well as those of photocycle intermediates with 
the help of crystallographic and solid-state NMR methods, work that has already 
lead to numerous prominent publications. The successful candidate will work 
closely with Soshi Nagano in the Giessen lab to create appropriate derivatives 
of Cph1 for crystallisation and highly-accurate atomic distance determinations. 
The work will include visits to various international research facilities and 
extensive personal interactions both in the co-operating labs as well as with 
members of Sfb 1078 in Berlin.

The candidate should have graduated in biochemistry, molecular biology or a 
related field. Experience in protein biochemistry and/or physicochemical 
analysis of protein structure/function would be an advantage. Proficiency in 
written and spoken English and ability to work constructively in a 
multinational, interdisciplinary team are important. Applications from women 
and disabled persons are especially welcome.

If you are interested, please apply by e-mail including a tabular CV:  
Professor Jon Hughes, Plant Physiology, JLU Giessen, Germany 
(jon.hug...@uni-giessen.de).

For further details, see
www.uni-giessen.de/fbz/fb08/Inst/pflphys/pflaphygroups/ag-hughes



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[ccp4bb] AW: [ccp4bb] Is there any alternative to siliconized glass coverslips for crystallization?

[Hughes, Jon]

in my experience using silane with PAGE plates, rain-x is much better (at least 
in that case).
jon

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Goldman, 
Adrian
Gesendet: Donnerstag, 31. Januar 2019 17:43
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Is there any alternative to siliconized glass coverslips 
for crystallization?

When I was a graduate student, about 150,000 years ago, we took regular 
coverslips and doused them in ?silane to make siliconised ones.  You then let 
them sit in a rack to dry.  It was a bit tedious but not horrendously so.  
After a while, I stopped doing it altogether, because IMHO it didn’t make a 
massive amount of difference to the behaviour of the solution on the cover 
slip.  It would bead up, or not, depending on what it was.

So my advice would be: 1) siliconise yourself; 2) compare siliconised versus 
non and decide if you can be bothered.

Adrian Goldman



On 31 Jan 2019, at 16:02, Holton, James M 
<270165b9f4cf-dmarc-requ...@jiscmail.ac.uk>
 wrote:

plastic.

Plastic cover slips are no good for UV or polarization, but they are way better 
than glass if you happen to want to try in-situ diffraction. 
(https://doi.org/10.1107/S002188981254)

If you can't afford commercial ones, then you can always cut up some inkjet 
transparency film sheets like McPherson did in the above reference.  Then after 
you've made a few hundred you can ask yourself how much you'd be willing to pay 
somebody else to do it for you.  There is no wrong answer to that question, but 
it will determine which route you take.

-James Holton
MAD Scientist
On 1/31/2019 12:17 AM, Rajnandani Kashyap wrote:
Dear All
I am a PhD student who requires lots of coverslips (!!) for setting up hanging 
drop crystallization. The company sells it for a huge amount. Also there is a 
wide monetary difference between a normal siliconized coverslip and a 22mm 
siliconized circle coverslips. We tried to search for an alternative companies 
but couldn't get any one who sells coverslips with the same dimensions 
(0.19-0.22mm glass thickness and 22 mm glass diameter). Is there any 
alternative company (distribution in India) from where we can buy them for a 
reasonable price?
Thanks in advance for sparing your valuable time and efforts.

Regards
Rajnandani Kashyap
India


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[ccp4bb] AW: [ccp4bb] AW: [EXTERNAL] [ccp4bb] Is there any alternative to siliconized glass coverslips for crystallization?

[Hughes, Jon]

yes, rain-x is excellent (also for PAGE gel plates). a little bottle costs 
almost nothing and will last you a lifetime.
best
jon

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
Nagarajan V
Gesendet: Donnerstag, 31. Januar 2019 16:14
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] AW: [EXTERNAL] [ccp4bb] Is there any alternative to 
siliconized glass coverslips for crystallization?

I know of Rain-X being used.
V. Nagarajan

On Thu, Jan 31, 2019 at 1:17 AM 
mailto:herman.schreu...@sanofi.com>> wrote:
A long time ago, before siliconized coverslips became commercially available, 
we used to siliconize coverslips ourselves. It is not really that much work and 
unsiliconized cover slips should be very cheap. If you wish, I could try to 
find back the protocol.
Best,
Herman

Von: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
Rajnandani Kashyap
Gesendet: Donnerstag, 31. Januar 2019 09:17
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Is there any alternative to siliconized glass 
coverslips for crystallization?

Dear All
I am a PhD student who requires lots of coverslips (!!) for setting up hanging 
drop crystallization. The company sells it for a huge amount. Also there is a 
wide monetary difference between a normal siliconized coverslip and a 22mm 
siliconized circle coverslips. We tried to search for an alternative companies 
but couldn't get any one who sells coverslips with the same dimensions 
(0.19-0.22mm glass thickness and 22 mm glass diameter). Is there any 
alternative company (distribution in India) from where we can buy them for a 
reasonable price?
Thanks in advance for sparing your valuable time and efforts.

Regards
Rajnandani Kashyap
India



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[ccp4bb] AW: [ccp4bb] 3D stereo and pymol

[Hughes, Jon]

...just open pymol, go to display - stereo mode - cross-eye then click stereo. 
it might need a little practice and it makes one look even sillier than one 
does usually, but it works, costs nothing, needs no updates and i'm told that 
it's even good for your eye muscles!
best,
jon

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Jan 
Stransky
Gesendet: Mittwoch, 30. Januar 2019 11:03
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] 3D stereo and pymol


Dear all,

I started looking into the never ending story of stereo in crystallography... 
As with our standardized linux setup we probably are not willing to move to 
X.org-world, if was wondering, if anybody was succesful to make stereo working 
in Windows 10. I did read some NVIDIA forum, and it seems that 3d vision is not 
very supported by NVIDIA, even for gamers... Was anybody able to mke it work 
with Geforce cards, to save few bucks?

Best regards,

Jan
Dne 03.01.2018 v 10:42 Wim Burmeister napsal(a):
I answer a bit late, but I repost a message on 3D graphics from Mai 2017 :
Hello,
we just wanted to share our experience in finding a configuration which allows 
to use 3D graphics under linux using Nvidia GeForce 3D glasses.
We had quite a hard time to find a configurations which works correctly.
We finally used Debian linux with a xfce desktop. Other recent desktops use a 
tiling which is not compatible with 3D graphics.
The hardware consists of

  *   a DELL Precision T5810  desktop computer with an Nvidia Quadro M4000 (8 
Gbyte memory, 4 DP) graphics card
  *   Nvidia GeForce 3D Vision 2 (NVIDIA GEF 3D VISION 2 GLASSES KIT) active 
stereo glasses
  *   a stereo connector PNY Quadro 4000 3D for the synchronization of graphics 
card and glasses
  *   an ASUS 24" LED 3D - VG248QE display
  *   a DisplayPort-DisplayPort cable
The Nvidia linux drivers from version 367.57 can handle the current version of 
the Nvidia glasses.
For an obscure reason a direct DP-DP connection between graphics card and 
display is absolutely required in order to obtain fully working stereo. If a 
DP-DVI dual link adapter is used, the stereo does not work on the top and the 
bottom part of the screen. This is true for a native DELL active adaptor or 
generic models. The exact reason remains unresolved, but the solution is to use 
a direct DP-DP connection. This limits the available choice of displays which 
require 120 Hz for 1080*1980 screen resolution and a DP input. We have been 
choosing a “Nvidia 3D ready” model.
There has been a considerate about of exchange about this problem on
https://devtalk.nvidia.com/default/topic/992892/linux/partially-working-stereoscopic-effect-with-3d-vision-under-debian-linux/
The setup comes with a price tag of about 1600 € free of taxes.
coot, pymol and chimera work straight without problems in hardware stereo mode. 
The experience is absolutely great.
Best
Wim
--

Wim Burmeister
Professeur
Institut de Biologie Structurale (IBS) CIBB
71 avenue des Martyrs
CS 20192
38044 Grenoble Cedex 9, FRANCE
E-mail: wim.burmeis...@ibs.fr
Tel:+33 (0) 457 42 87 41   Fax: +33 (0) 476 20 94 00
website





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[ccp4bb] honeybee repair?

[Hughes, Jon]

hi everyone,
we use a honeybee pipetting robot for 96-well sitting-drop screening – or 
rather we would like to do so. several of the pins are defective, replacements 
seem not to be available, our workshop has not been able to fabricate them 
successfully. the only possibility would seem to be that we fly in an engineer 
from the usa (US$11k) to replace the entire head (US$24k). these numbers are 
ridiculous of course, but they are the formal quotes from "digilab". does 
anyone have an alternative suggestion?
best,
jon




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[ccp4bb] AW: [ccp4bb] [Offtopic] Protein stain in Coomassie Blue dyes

[Hughes, Jon]

hi,
as a comment to zhijie's question, the biggest problem with marion bradford's 
"protein" assay is that it measures the SDS concentration too! therefore, where 
it would be most useful – that is in quantifying the total protein in an 
SDS-treated gel sample – it cannot be used! the amido-black method is far 
better, at least as far as i know. does anyone know why it's seldom used? our 
protocol is below if anyone's interested.
best
jon

Amido-Black assay.
Dilute 10µl of the clarified protein extract (also in SDS sample buffer) to 
200µl with water, add 600µl Amido-Black reagent, mix and incubate at RT for 
5min.  Centrifuge for 5min at 13krcf.  Discard the supernatant and wash the 
pellet twice in 500µl Acid-MeOH [10%(v/v) HAc in MeOH].  The final supernatant 
should be colourless.  Dissolve the pellet in 1ml 1M NaOH (warm if necessary) 
and assay at 615nm.

Amido-Black reagent
Stock = 0.13g Amido-Black 10B (Merck) + 1ml HAc + 9ml MeOH.  Stir well then 
filter.  Stable at 4°C indefinitely.  To use, dilute 1ml of this stock in 50ml 
Acid-MeOH. Stable upto 1 week at 4°C. Calibrate with a dilution series of a 
"standard" protein such as BSA.


Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Alex Lee
Gesendet: Freitag, 5. Oktober 2018 05:37
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] [Offtopic] Protein stain in Coomassie Blue dyes

Hi All,

Thanks for all of your inputs!

Alex

On Thu, Oct 4, 2018 at 7:31 PM Zhijie Li 
mailto:zhijie...@utoronto.ca>> wrote:
Hi,

At high concentration (1-2%) the published saturating SDS:protein binding ratio 
is about 1.4:1 by weight, that is roughly one SDS molecule per two aa on 
average. It is dense but  not that dense to prevent any further interaction.  
More importantly, as a quite hydrophilic small molecule SDS should have no 
trouble dissociating from the peptide when its in-solution concentration drops 
(therefore you can use SDS gel bands for MS). With common procedure, during 
staining the SDS should partially fall off( especially if the gel is heated), 
and partially remain with the protein in the gel, depending on: how hydrophobic 
the protein is, how low the environmental SDS concentration becomes, how much 
organic solvent there is in the solution, etc.. The coomassie should simply 
find whatever hydrophobic/positively charged patch to bind and aggregate. 
Besides, coomassie-R is probably slightly more hydrophobic than SDS so it is 
capable of competing SDS off if necessary. (The even less hydrophobic Coomassie 
G250 definitely binds protein in the presence of detergents - that how Blue 
Native gel works for membrane proteins) Finally, since the only thing you are 
looking for is some deeper blue to indicate the presence of protein, even if 
SDS did prevent dye binding to some extent, your gel still will work. This is 
different from when you want to use the dye to do some quantitative work such 
as the Bradford assay. It would be interesting to know the effect of detergents 
on Bradford.

Zhijie



On Oct 4, 2018, at 9:26 PM, Alex Lee 
mailto:alexlee198...@gmail.com>> wrote:
Dear All,

I am thinking that in an SDS-PAGE experiment, if protein samples are boiled in 
SDS containing loading dye, and supposedly SDS interacts with proteins, why the 
Coomassie Blue dyes could still interact with and stain the proteins?  I am 
thinking SDS is covering the proteins, making no room for the Coomassie Blue 
dyes interaction.  I'd appreciate it if any input from this forum.

Alex



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[ccp4bb] AW: [ccp4bb] Electron density maps for Cryo-EM structures.

[Hughes, Jon]

yes, but irrespective of how much one knows or thinks one knows, one should 
avoid being gratuitously offensive.
cheers
jon

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Ian 
Tickle
Gesendet: Montag, 10. September 2018 12:58
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Electron density maps for Cryo-EM structures.


Hi Marin

I was about to comment on that too but then I realised that Pavel is referring 
to the map _contours_  (which is what most people using a map visualisation 
program like Coot actually see).  So the contoured map does represent an 
iso-potential surface.  I'm sure Pavel is aware that the original cryo-EM maps 
are 3-dimensional objects.

Cheers

-- Ian

On Mon, 10 Sep 2018 at 10:49, Marin van Heel 
<057a89ab08a1-dmarc-requ...@jiscmail.ac.uk>
 wrote:

Unfortunately,

The problem here lies primarily in the answer given,  not so much in the 
question asked by a newcomer:

"1) In cryo-EM maps are not electron density maps but surfaces representing 
electric potential. "

The answer appears to reflect the widespread misunderstanding that EM images 
(and hence cryo-EM maps)  only show the surfaces not the internal density of 
the complexes we study.
In my Imperial College/Leiden University  lecture notes, I have always used the 
below slide to illustrate this point.

Cheers,

Marin

[cid:part1.5DA80E33.F02E5B7D@googlemail.com]



On 10/09/2018 01:38, Pavel Afonine wrote:
Hi,

Is there any sever available to create electron density maps for cryo-em 
structures?

The questions are nonsensical. Here is why:

1) In cryo-EM maps are not electron density maps but surfaces representing 
electric potential.

2) Creating such a map is essentially carrying on from cryo-EM experiment and 
obtaining the 3D reconstruction.

Are you really sure about what you are asking for?

Or, we should create the maps from mmCIF.

mmCIF is a file format. It may contain representations of rabbits, 
boysenberries or some diffraction data. So.. how you think it may be related to 
cryo-EM, in your particular case?

I am particularly interested in those cryo-em structures with high resolution, 
like 2.6~2.8A.

Sure, all are excited about high-res cryo-EM!!!

Please give me an education.

Sure. One of available universities can do this.

Cheers,
Pavel




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--

==



Prof Dr Ir Marin van Heel



Laboratório Nacional de Nanotecnologia - LNNano

CNPEM/LNNano, Campinas, Brazil



Skype:  Marin.van.Heel

email:  marin.vanheel(A_T)gmail.com

marin.vanheel(A_T)lnnano.cnpem.br

and:mvh.office(A_T)gmail.com



--

Emeritus Professor of Cryo-EM Data Processing

Leiden University

--

Emeritus Professor of Structural Biology

Imperial College London

Faculty of Natural Sciences

email: m.vanheel(A_T)imperial.ac.uk

--



I receive many emails per day and, although I try,

there is no guarantee that I will actually read each incoming email.



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[ccp4bb] AW: Re: [ccp4bb] crystals that dont diffract :( :(

[Hughes, Jon]

Yes indeed!
Jon

--
Jon Hughes
(+49/0)1757929098
Sent without the use of Apple products.

 Daniel M. Himmel, Ph. D. schrieb 

Dear JL,

Years ago, this was a common problem when I was crystallizing myosin
constructs for my doctoral work.  Some of the most beautiful crystals I
got showed little or no diffraction.  Often this occurs when there is a very
large water content in the asymmetric unit and in proteins that have a
great deal of intrinsic disorder.  Jon's suggestion to flash-cool them in
oil could work.  Your high PEG concentration in the flash-cooling solution
might work, too, but you probably cannot just plunge your crystals
suddenly into a much higher PEG solution.  A few suggestions:

1) Whichever cryoprotectant you use, introduce it to your crystal GRADUALLY,
such as in steps (e.g., 10% ==> 15% ==> 20% ==>25%) or something like that.
For some proteins, drastic rapid changes of concentrations of anything in
the solution can damage the crystal and introduce enough disorder so that
the protein crystal will not diffract well.  Sometimes you can tell when there's
damage if the crystal cracks (or melts away), but not always.

2) You may have to experiment with different cryoprotectants.  Different
cryoprotectants make different protein crystals happy.  For example, try PEG 
200,
PEG 400, PEG 600, glycerol, sucrose, trehalose, other disaccharide sugars, MPD, 
butanediols.

3) I have found that, as a "rule of thumb", 25% of any cryoprotectant is enough
to protect against ice formation.  If your protein crystal can tolerate it, 
higher
concentrations could be better (because they can shrink the unit cell and reduce
some of the water content).  HOWEVER, many protein crystals will not tolerate
much higher concentrations without taking on damage.

I hope this helps.

-Daniel

On Tue, Aug 14, 2018 at 9:45 AM, ferrer 
mailto:jean-luc.fer...@ibs.fr>> wrote:
Hi

Did you try them at room temp, in situ (straight in the plate). We observe that 
time to time on our beamline, when just harvesting, not mentioning cryo 
protection, is enough to loose all diffraction. It-s rare but happens.

Regards

JL

On 14/08/2018 11:58, Careina Edgooms wrote:
I got the most beautiful crystals I have ever seen and they don't diffract at 
all. Not poor diffraction, NO diffraction. Anyone know why this could be and 
how I can go about fixing it? I had three beautiful crystals and not one 
diffracted. I did leave them in the drop for about 3 weeks before harvesting 
and in liquid nitrogen for about a month before diffracting. Could that be a 
factor? If I regrew more beautiful crystals and diffracted straight away could 
that help?
Careina



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--

Jean-Luc Ferrer
Institut de Biologie Structurale
71 Avenue des 
Martyrs
CS 10090
38044 Grenoble Cedex 9 - FRANCE

Ph.:  +33 (0)4 57 42 85 22
Cell: +33 (0)6 89 45 13 57
email: jean-luc.fer...@ibs.fr





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[ccp4bb] AW: [ccp4bb] crystals that dont diffract :( :(

[Hughes, Jon]

maybe it's the cryobuffer that's the problem (you didn't mention it). you could 
try to fish the crystals with minimal liquid attached by mounting them in oil 
rather than a cryobuffer. or you could test the native diffraction "in situ" 
(at room temperature in the drop): quite a few beamlines offer this possibility 
these days.
best
jon

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Careina 
Edgooms
Gesendet: Dienstag, 14. August 2018 11:59
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] crystals that dont diffract :( :(

I got the most beautiful crystals I have ever seen and they don't diffract at 
all. Not poor diffraction, NO diffraction. Anyone know why this could be and 
how I can go about fixing it? I had three beautiful crystals and not one 
diffracted. I did leave them in the drop for about 3 weeks before harvesting 
and in liquid nitrogen for about a month before diffracting. Could that be a 
factor? If I regrew more beautiful crystals and diffracted straight away could 
that help?
Careina



To unsubscribe from the CCP4BB list, click the following link:
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[ccp4bb] AW: [ccp4bb] AW: [ccp4bb] [ccp4bb] Oxford University Press

[Hughes, Jon]

...that some papers get waived through isn't all that uncommon already, 
especially with Nature.
j

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Vellieux 
Frédéric
Gesendet: Samstag, 30. Juni 2018 12:23
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] AW: [ccp4bb] [ccp4bb] Oxford University Press

"how to get grants without papers in Nature. anyone have a solution to that 
one?"
Simple: Once all of us have reviewed enough papers (with the money placed in a 
common pot) we simply buy the Nature Publishing Group. All those having taken 
part get a Nature paper every 4 years to ensure grant money continues to flow 
in.
F.

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Hughes, Jon 
mailto:jon.hug...@bot3.bio.uni-giessen.de>>
Sent: Saturday, June 30, 2018 12:12:50 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] AW: [ccp4bb] [ccp4bb] Oxford University Press

great idea! 2 hours at €200 per hour makes €1000 - sounds like an eminently 
reasonable starting point for negotiations. if the publishers don't like our 
price, they can do the reviewing themselves - and after a while no one will 
bother to buy the resulting rubbish anyhow! in the mean time we put our stuff 
online directly (without wasting our time, for example, still formatting 
reference lists in the 21st century!). we have them over a barrel. the only 
problem i see is how to get grants without papers in Nature. anyone have a 
solution to that one?
one final aspect is: who gets the money? surely the universities etc. should 
get it, not us: the taxpayer pays us already.
best,
jon

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Keller, 
Jacob
Gesendet: Samstag, 30. Juni 2018 00:00
An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Betreff: Re: [ccp4bb] [ccp4bb] Oxford University Press

The one I don't get is why not pay reviewers? $1000 per review? If you look at 
publishers' profit margins, you will see that they can afford it. I actually 
think the scientific community should go on a "review strike" until reviewers 
get paid.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Petr 
Leiman
Sent: Friday, June 29, 2018 4:47 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] [ccp4bb] Oxford University Press

Indeed! Scientists in the Soviet Bloc got paid for publishing their scientific 
papers (and maybe for citations as well - not sure about that one)! We need to 
change the current system! Although these changes could be accompanied by many 
other pleasant virtues of the Soviet regime.

Petr


> On Jun 29, 2018, at 8:11 AM, Hughes, Jon 
> mailto:jon.hug...@bot3.bio.uni-giessen.de>>
>  wrote:
>
> whose paper? our universities pay subscriptions for these journals and we 
> even pay on top of that for the pages of our publications (even when they're 
> not actually printed!), whilst we review papers for free! sounds like a 
> well-validated way to use taxpayers' money to keep the expensive company cars 
> etc. nice and shiny. why don't universities just require reimbursement for 
> the time we invest to insure that the merchandise is up to standard? €100 per 
> hour would be cheap. seems to me as though some capitalists need to add a few 
> lines to their balance sheets
> best, jon
>
> -Ursprüngliche Nachricht-
> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von
> Robbie Joosten
> Gesendet: Freitag, 29. Juni 2018 13:42
> An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
> Betreff: Re: [ccp4bb] Oxford University Press
>
> Yes, but think of all the money they miss due to your pirating of their paper 
> ;) It's the typical discussion about whether piracy of copyrighted material 
> leads to loss or gain of revenue. There are a lot of models here, but not 
> necessarily well-validated.
>
> Anyway, if people want to read your papers and cannot get them from
> ResearchGate, I'm sure they can find them on another online
> collection, a hub 

[ccp4bb] AW: [ccp4bb] [ccp4bb] Oxford University Press

[Hughes, Jon]

sorry about the typing error. 2 x 200 = 400, i know.
j

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Hughes, 
Jon
Gesendet: Samstag, 30. Juni 2018 12:13
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] AW: [ccp4bb] [ccp4bb] Oxford University Press

great idea! 2 hours at €200 per hour makes €1000 - sounds like an eminently 
reasonable starting point for negotiations. if the publishers don't like our 
price, they can do the reviewing themselves - and after a while no one will 
bother to buy the resulting rubbish anyhow! in the mean time we put our stuff 
online directly (without wasting our time, for example, still formatting 
reference lists in the 21st century!). we have them over a barrel. the only 
problem i see is how to get grants without papers in Nature. anyone have a 
solution to that one?
one final aspect is: who gets the money? surely the universities etc. should 
get it, not us: the taxpayer pays us already. 
best,
jon

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Keller, 
Jacob
Gesendet: Samstag, 30. Juni 2018 00:00
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] [ccp4bb] Oxford University Press

The one I don't get is why not pay reviewers? $1000 per review? If you look at 
publishers' profit margins, you will see that they can afford it. I actually 
think the scientific community should go on a "review strike" until reviewers 
get paid.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Petr 
Leiman
Sent: Friday, June 29, 2018 4:47 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] [ccp4bb] Oxford University Press

Indeed! Scientists in the Soviet Bloc got paid for publishing their scientific 
papers (and maybe for citations as well - not sure about that one)! We need to 
change the current system! Although these changes could be accompanied by many 
other pleasant virtues of the Soviet regime. 

Petr


> On Jun 29, 2018, at 8:11 AM, Hughes, Jon  
> wrote:
> 
> whose paper? our universities pay subscriptions for these journals and we 
> even pay on top of that for the pages of our publications (even when they're 
> not actually printed!), whilst we review papers for free! sounds like a 
> well-validated way to use taxpayers' money to keep the expensive company cars 
> etc. nice and shiny. why don't universities just require reimbursement for 
> the time we invest to insure that the merchandise is up to standard? €100 per 
> hour would be cheap. seems to me as though some capitalists need to add a few 
> lines to their balance sheets
> best, jon
> 
> -Ursprüngliche Nachricht-
> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
> Robbie Joosten
> Gesendet: Freitag, 29. Juni 2018 13:42
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: Re: [ccp4bb] Oxford University Press
> 
> Yes, but think of all the money they miss due to your pirating of their paper 
> ;) It's the typical discussion about whether piracy of copyrighted material 
> leads to loss or gain of revenue. There are a lot of models here, but not 
> necessarily well-validated.
> 
> Anyway, if people want to read your papers and cannot get them from 
> ResearchGate, I'm sure they can find them on another online 
> collection, a hub of some sort ;)
> 
> Cheers,
> Robbie
> 
>> -Original Message-
>> From: Bernhard Rupp [mailto:hofkristall...@gmail.com]
>> Sent: Friday, June 29, 2018 13:23
>> To: 'Robbie Joosten'; CCP4BB@JISCMAIL.AC.UK
>> Subject: RE: [ccp4bb] Oxford University Press
>> 
>> Agreed, but for 10 years old papers this seems a bit of overkill
>> 
>> 
>> 
>> From: CCP4 bulletin board  On Behalf Of Robbie 
>> Joosten
>> Sent: Friday, June 29, 2018 12:11
>> To: CCP4BB@JISCMAIL.AC.UK
>> Subject: Re: [ccp4bb] Oxford University Press
>> 
>> 
>> 
>> Were they open access papers? If they were, than OUP is being too 
>> aggressive (IMO), but otherwise it makes sense. I also find the 
>> ResearchGate is rath

[ccp4bb] AW: [ccp4bb] [ccp4bb] Oxford University Press

[Hughes, Jon]

great idea! 2 hours at €200 per hour makes €1000 - sounds like an eminently 
reasonable starting point for negotiations. if the publishers don't like our 
price, they can do the reviewing themselves - and after a while no one will 
bother to buy the resulting rubbish anyhow! in the mean time we put our stuff 
online directly (without wasting our time, for example, still formatting 
reference lists in the 21st century!). we have them over a barrel. the only 
problem i see is how to get grants without papers in Nature. anyone have a 
solution to that one?
one final aspect is: who gets the money? surely the universities etc. should 
get it, not us: the taxpayer pays us already. 
best,
jon

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Keller, 
Jacob
Gesendet: Samstag, 30. Juni 2018 00:00
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] [ccp4bb] Oxford University Press

The one I don't get is why not pay reviewers? $1000 per review? If you look at 
publishers' profit margins, you will see that they can afford it. I actually 
think the scientific community should go on a "review strike" until reviewers 
get paid.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Petr 
Leiman
Sent: Friday, June 29, 2018 4:47 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] [ccp4bb] Oxford University Press

Indeed! Scientists in the Soviet Bloc got paid for publishing their scientific 
papers (and maybe for citations as well - not sure about that one)! We need to 
change the current system! Although these changes could be accompanied by many 
other pleasant virtues of the Soviet regime. 

Petr


> On Jun 29, 2018, at 8:11 AM, Hughes, Jon  
> wrote:
> 
> whose paper? our universities pay subscriptions for these journals and we 
> even pay on top of that for the pages of our publications (even when they're 
> not actually printed!), whilst we review papers for free! sounds like a 
> well-validated way to use taxpayers' money to keep the expensive company cars 
> etc. nice and shiny. why don't universities just require reimbursement for 
> the time we invest to insure that the merchandise is up to standard? €100 per 
> hour would be cheap. seems to me as though some capitalists need to add a few 
> lines to their balance sheets
> best, jon
> 
> -Ursprüngliche Nachricht-
> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
> Robbie Joosten
> Gesendet: Freitag, 29. Juni 2018 13:42
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: Re: [ccp4bb] Oxford University Press
> 
> Yes, but think of all the money they miss due to your pirating of their paper 
> ;) It's the typical discussion about whether piracy of copyrighted material 
> leads to loss or gain of revenue. There are a lot of models here, but not 
> necessarily well-validated.
> 
> Anyway, if people want to read your papers and cannot get them from 
> ResearchGate, I'm sure they can find them on another online 
> collection, a hub of some sort ;)
> 
> Cheers,
> Robbie
> 
>> -Original Message-
>> From: Bernhard Rupp [mailto:hofkristall...@gmail.com]
>> Sent: Friday, June 29, 2018 13:23
>> To: 'Robbie Joosten'; CCP4BB@JISCMAIL.AC.UK
>> Subject: RE: [ccp4bb] Oxford University Press
>> 
>> Agreed, but for 10 years old papers this seems a bit of overkill
>> 
>> 
>> 
>> From: CCP4 bulletin board  On Behalf Of Robbie 
>> Joosten
>> Sent: Friday, June 29, 2018 12:11
>> To: CCP4BB@JISCMAIL.AC.UK
>> Subject: Re: [ccp4bb] Oxford University Press
>> 
>> 
>> 
>> Were they open access papers? If they were, than OUP is being too 
>> aggressive (IMO), but otherwise it makes sense. I also find the 
>> ResearchGate is rather aggressive in bugging you to upload papers 
>> that are readily available from the publisher. The whole business bit 
>> in scientific publishing is a necessary (?) evil, but I guess if 
>> given the choice one should publish somewhere where you as an author retain 
>> copyright.
>>

[ccp4bb] AW: [ccp4bb] Oxford University Press

[Hughes, Jon]

whose paper? our universities pay subscriptions for these journals and we even 
pay on top of that for the pages of our publications (even when they're not 
actually printed!), whilst we review papers for free! sounds like a 
well-validated way to use taxpayers' money to keep the expensive company cars 
etc. nice and shiny. why don't universities just require reimbursement for the 
time we invest to insure that the merchandise is up to standard? €100 per hour 
would be cheap. seems to me as though some capitalists need to add a few lines 
to their balance sheets
best, jon

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Robbie 
Joosten
Gesendet: Freitag, 29. Juni 2018 13:42
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Oxford University Press

Yes, but think of all the money they miss due to your pirating of their paper 
;) It's the typical discussion about whether piracy of copyrighted material 
leads to loss or gain of revenue. There are a lot of models here, but not 
necessarily well-validated.

Anyway, if people want to read your papers and cannot get them from 
ResearchGate, I'm sure they can find them on another online collection, a hub 
of some sort ;)

Cheers,
Robbie 

> -Original Message-
> From: Bernhard Rupp [mailto:hofkristall...@gmail.com]
> Sent: Friday, June 29, 2018 13:23
> To: 'Robbie Joosten'; CCP4BB@JISCMAIL.AC.UK
> Subject: RE: [ccp4bb] Oxford University Press
> 
> Agreed, but for 10 years old papers this seems a bit of overkill
> 
> 
> 
> From: CCP4 bulletin board  On Behalf Of Robbie 
> Joosten
> Sent: Friday, June 29, 2018 12:11
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Oxford University Press
> 
> 
> 
> Were they open access papers? If they were, than OUP is being too 
> aggressive (IMO), but otherwise it makes sense. I also find the 
> ResearchGate is rather aggressive in bugging you to upload papers that 
> are readily available from the publisher. The whole business bit in 
> scientific publishing is a necessary (?) evil, but I guess if given 
> the choice one should publish somewhere where you as an author retain 
> copyright.
> 
> 
> 
> Cheers,
> 
> Robbie
> 
> 
> 
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
> Bernhard Rupp
> Sent: Friday, June 29, 2018 11:42
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] Oxford University Press
> 
> 
> 
> Hi Fellows,
> 
> 
> 
> just an advisory that Oxford University Press is pretty aggressive in
> 
> enforcing copyright - I had to remove 2 Bioinformatics papers
> 
> from ResearchGate.
> 
> 
> 
> Fortunately, authors have choices, too
> 
> 
> 
> Cheers, BR
> 
> --
> 
> Bernhard Rupp
> 
> http://www.hofkristallamt.org/
> 
> b...@hofkristallamt.org
> 
> +1 925 209 7429
> 
> +43 676 571 0536
> 
> --
> 
> Many plausible ideas vanish
> 
> at the presence of thought
> 
> --
> 
> 
> 
> 
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
> 
> 
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
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[ccp4bb] AW: [ccp4bb] Inquiry - active and non-active state in alternatives

[Hughes, Jon]

dear petr,
several phytochrome structures seem to represent mixed-state crystals.
best,
jon

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Petr 
Kolenko
Gesendet: Dienstag, 11. Juli 2017 16:00
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Inquiry - active and non-active state in alternatives

Dear colleagues,

We are working on a paper, where we want to discuss our crystal structure. We 
have determined structure of non-active state first (much easier). Than we 
tried to convert the protein into active state by soaking (direct 
crystallization not possible). We have observed more than a half of a protein 
chain in shifted location after soaking with respect to the original 
conformation as a 50% alternative. We believe that the alternative conformation 
corresponds to the active state and we are looking for any other possible 
support to our statement.

My questions are:
Has anyone else observed active and non-active state of a protein in single 
chain together as alternatives?
Has anyone an idea how to search through the PDB for such cases? Simple going 
through all structures with alternatives does not seem to be reasonable.

By the way, we are sure with the space group. Soaking may in some cases cause 
symmetry reduction, but if we process the data in lower symmetry, we see two 
chains with alternatives instead of one and this was not the only reason for 
our decision about the space group. We have also performed much more soaking 
experiments, but because of overall difficulty and instability of the 
chemicals, this was the best observation we ever had.

Thanks for any suggestion!
Best regards,
Petr

[ccp4bb] PhD position - Phytochrome structure/function

[Hughes, Jon]

Dear collegues, I would be grateful if you were to draw the attention of 
appropriate persons to the following flyer (if the fancy html formatting 
doesn't work, here's the flyer in PDF:
http://www.uni-giessen.de/fbz/fb08/Inst/pflphys/pflaphygroups/ag-hughes/PositionsAvailable/resolveuid/402bc6a30e8e41bca6e50d12d745062b
With many thanks in advance and best wishes, Jon.



[jlu_logo_homepage] 
[cid:image004.png@01D2E5D7.699138E0]

PhD position: Phytochrome structure/function

A PhD position at the Institute for Plant Physiology of the Justus Liebig 
University is available immediately. The position (E13 66%) is funded for three 
years by the DFG as part of Sfb1078 (Protonation Dynamics in Protein Function; 
see www.sfb1078.de) in Berlin.
The aim of the project is to derive an understanding of the functional 
mechanism underlying photoconversion and signal activation in the phytochrome 
family of photoreceptors. To this end we will use both X-ray and neutron 
crystallography to study structural changes associated with photoconversion in 
various phytochromes. The work is augmented by long-standing co-operations with 
vibrational spectroscopy and MAS NMR groups in Berlin and Leipzig and has 
already lead to numerous prominent publications. The successful candidate will 
work with Dr. Soshi Nagano to create appropriate derivatives of both 
prokaryotic as well as plant phytochromes for crystallisation and structural 
determination at the atomic level. The work will include visits to various 
international research facilities and extensive personal interactions with 
other members of the Berlin Sfb. We have access to advanced robotic facilities 
to assist us in screening for appropriate crystallisation conditions.

The candidate should have graduated in biochemistry, molecular biology or a 
related field. Experience in protein biochemistry and/or physicochemical 
analysis of protein structure/function or would be an advantage. Proficiency in 
written and spoken English and ability to work constructively in a 
multinational, interdisciplinary team is important. Applications from women and 
disabled persons are especially welcome.

If you are interested, please apply immediately by e-mail including a tabular 
CV:  Professor Jon Hughes, Plant Physiology, JLU Giessen, Germany 
(jon.hug...@uni-giessen.de).

For further details see 
www.uni-giessen.de/fbz/fb08/Inst/pflphys/pflaphygroups/ag-hughes

[ccp4bb] AW: [ccp4bb] Sliconizing of cover-slips

[Hughes, Jon]

anyone tried rainex (if it's still on sale)? for us it works 1000x better than 
anything else: rugged, extremely water repellent, and cheap (one bottle lasts a 
lifetime). in the '90s at least you could get it in auto stores in the USA.
cheers
jon

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Tristan 
Croll
Gesendet: Freitag, 28. April 2017 13:36
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Sliconizing of cover-slips

Ahh, this brings back memories of a former life, preparing hydrophobic 
coverslips for my surface chemistry experiments. I used chlorotrimethylsilane, 
and what I remember best is that the secret to a good coating is making sure 
your coverslips are utterly clean and dry. I used to clean them by boiling in 
base piranha (concentrated ammonia and hydrogen peroxide - need I say this 
stuff should be treated with extreme respect?) before rinsing thoroughly in 
milli-Q water and baking under vacuum. Then essentially as David said: dump 
them in the silane solution (again, it helps to make sure to keep your solvent 
dry), then fish them out one-by-one, rinse and dump them into a beaker of fresh 
solvent (acetone, I think I used). Then once they're all done, take them out of 
that, dry in a nitrogen stream and store. Painful & fiddly, but you can easily 
do a hundred or so in an afternoon.


Tristan Croll
Research Fellow
Cambridge Institute for Medical Research
University of Cambridge CB2 0XY




On 27 Apr 2017, at 10:26, Praveen Kumar Tripathi 
mailto:tripathipraveen.i...@gmail.com>> wrote:
Dear all,

sorry for off topic question.

May i know if anybody uses homemade silinization of coverslips for protein 
crystallization purposes?

I have purchased Sigmacote SL2-100 ml for silanizing coverslips for hanging 
drop protein crystallization setup.

Please share your methods to siliconize coverslip using Sigmacote SL2-100 ml if 
anybody uses.

Thanks in advance

regards
Praveen



--
Praveen Kumar Tripathi
PhD Research Scholar
Kusuma School of Biological Sciences
Indian Institute of Technology Delhi-110016
+91-9873625228

[ccp4bb] AW: [ccp4bb] UVEX UV Fluorescence

[Hughes, Jon]

ah! ok, that's a different matter
j

Von: Keller, Jacob [mailto:kell...@janelia.hhmi.org]
Gesendet: Mittwoch, 5. April 2017 14:29
An: Hughes, Jon ; CCP4BB@JISCMAIL.AC.UK
Betreff: RE: [ccp4bb] UVEX UV Fluorescence

But I think he was asking about imaging of intrinsic fluorescence of protein 
crystals…

I like your idea about gel imaging, though.

JPK

From: Hughes, Jon [mailto:jon.hug...@bot3.bio.uni-giessen.de]
Sent: Wednesday, April 05, 2017 8:23 AM
To: Keller, Jacob mailto:kell...@janelia.hhmi.org>>; 
CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: AW: [ccp4bb] UVEX UV Fluorescence

for ethidium bromide you need a 590nm (50-100nm FWHH) bandpass interference 
filter. ccd chips are sensitive to infrared and transilluminators produce a lot 
of it!
best
j

Von: Keller, Jacob [mailto:kell...@janelia.hhmi.org]
Gesendet: Mittwoch, 5. April 2017 14:16
An: Hughes, Jon 
mailto:jon.hug...@bot3.bio.uni-giessen.de>>;
 CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Betreff: RE: [ccp4bb] UVEX UV Fluorescence

Why 590 nm? BP, SP, LP? I would have thought 390 nm LP or similar--was it a 
typo?

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hughes, 
Jon
Sent: Wednesday, April 05, 2017 6:43 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] AW: [ccp4bb] UVEX UV Fluorescence

you can save a fortune by just fitting a 590nm interference filter to the front 
of a ccd camera attached to a PC + a UV/B transilluminator. i set this up in my 
lab for about €1000 total 15 years ago and we've used it everyday since.
cheers
jon

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Cyprian 
Cukier
Gesendet: Mittwoch, 5. April 2017 12:26
An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Betreff: [ccp4bb] UVEX UV Fluorescence

Dear All,

We are about to buy a new imaging system for our laboratories and we consider 
UVEX UV Fluorescence Imaging from MD. Can anyone provide some comments about 
this equipment (eg. is it reliable, robust, no technical issues, 
low-maintenance etc.)? All comments will be appreciated.

Thanks,
Cyprian

[ccp4bb] AW: [ccp4bb] UVEX UV Fluorescence

[Hughes, Jon]

for ethidium bromide you need a 590nm (50-100nm FWHH) bandpass interference 
filter. ccd chips are sensitive to infrared and transilluminators produce a lot 
of it!
best
j

Von: Keller, Jacob [mailto:kell...@janelia.hhmi.org]
Gesendet: Mittwoch, 5. April 2017 14:16
An: Hughes, Jon ; CCP4BB@JISCMAIL.AC.UK
Betreff: RE: [ccp4bb] UVEX UV Fluorescence

Why 590 nm? BP, SP, LP? I would have thought 390 nm LP or similar--was it a 
typo?

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hughes, 
Jon
Sent: Wednesday, April 05, 2017 6:43 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] AW: [ccp4bb] UVEX UV Fluorescence

you can save a fortune by just fitting a 590nm interference filter to the front 
of a ccd camera attached to a PC + a UV/B transilluminator. i set this up in my 
lab for about €1000 total 15 years ago and we've used it everyday since.
cheers
jon

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Cyprian 
Cukier
Gesendet: Mittwoch, 5. April 2017 12:26
An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Betreff: [ccp4bb] UVEX UV Fluorescence

Dear All,

We are about to buy a new imaging system for our laboratories and we consider 
UVEX UV Fluorescence Imaging from MD. Can anyone provide some comments about 
this equipment (eg. is it reliable, robust, no technical issues, 
low-maintenance etc.)? All comments will be appreciated.

Thanks,
Cyprian

[ccp4bb] AW: [ccp4bb] UVEX UV Fluorescence

[Hughes, Jon]

you can save a fortune by just fitting a 590nm interference filter to the front 
of a ccd camera attached to a PC + a UV/B transilluminator. i set this up in my 
lab for about €1000 total 15 years ago and we've used it everyday since.
cheers
jon

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Cyprian 
Cukier
Gesendet: Mittwoch, 5. April 2017 12:26
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] UVEX UV Fluorescence

Dear All,

We are about to buy a new imaging system for our laboratories and we consider 
UVEX UV Fluorescence Imaging from MD. Can anyone provide some comments about 
this equipment (eg. is it reliable, robust, no technical issues, 
low-maintenance etc.)? All comments will be appreciated.

Thanks,
Cyprian

[ccp4bb] AW: [ccp4bb] [ccp4bb] protein precipitation reg

[Hughes, Jon]

yes - really, tris should be the buffer of last resort rather than the 
standard. its only general advantages would seem to be that it's cheap and not 
very toxic. 
j

--
Professor Jon Hughes, BSc, PhD
Institute for Plant Physiology
Justus Liebig University, Giessen
Zeughaus, Rm. 341
Senckenbergstr. 3
D35390 Giessen, Germany.
work phone: (+49/0)6419935430
fax:  (+49/0)6419935429
mobile:   (+49/0)1757929098
email:  jon.hug...@uni-giessen.de
homepage:
http://www.uni-giessen.de/fbz/fb08/Inst/pflphys/pflaphygroups/ag-hughes
Sent without the use of Apple products



-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Mark 
Wilson
Gesendet: Mittwoch, 29. März 2017 22:12
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] [ccp4bb] protein precipitation reg

I heartily concur with Craig.  Tris can be a dangerous buffer for many reasons, 
including those listed below.  In addition, as a primary amine, it can 
complicate work with metalloproteins and has moderate nucleophilicity.  There 
is almost always a better buffer choice than Tris.
Best regards,
Mark

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 






On 3/29/17 2:53 PM, "CCP4 bulletin board on behalf of CRAIG A BINGMAN"
 wrote:

>
>
>
>There are almost always better choices than Tris buffer.
>
>
>Mo Cleland used to call it “Trash” buffer.  He is no longer with us, 
>but today I will happily carry that flag in his honor.
>
>
>Tris may show up in your crystal structure, especially at carbohydrate 
>binding sites.
>Tris may be a surprisingly strong competitive inhibitor in your enzyme 
>assays, especially as above.
>Tris has an absolutely miserably bad change in pKa vs. temperature.  It 
>is larger than -0.03 pKa/dT(C).  It can be a catastrophically bad 
>choice for flash-freezing protein aliquots.
>
>
>If you taken the time and incurred the expense of preparing a 
>macromolecular sample for crystallization studies, and you are worried 
>about the price difference between Tris and HEPES, in my opinion you 
>are absolutely worried about the wrong things.
>
>
>Why are people substantially concerned about the buffering capacity of 
>a buffer for final sample preparation?  You have a purified protein, 
>presumably without substrate present.  Exactly what do you think is 
>generating or absorbing hydrogen ions  in that solution?  Oxidation of 
>reducing agent should be about the only thing that is taxing the 
>buffer.  From the example below, oxidation of 5 mM BME will put some 
>pressure on the buffer, but unfortunately Tris accelerates the 
>oxidation of BME relative,  to, say, HEPES. And surely you aren’t just 
>letting the protein sit and oxidize in the refrigerator? Oh you might 
>be since when you tried to snap freeze it in Tris, it turned into 
>cooked egg white because the pH went to over 10 before it vitrified.
>(http://www.sciencedirect.com/science/article/pii/S0031942200801429)
>
>
>Isn’t the whole point to use a small amount of buffer so you can easily 
>push the pH around in crystallization screens? (At which point the 
>sample is usually in 100+ mM buffer.)
>
>
>On Mar 29, 2017, at 2:03 PM, Hughes, Jon 
> wrote:
>
>...it's just a wonderful tradition! there's an interesting description 
>of the history of tris in maniatis
>cheers
>jon
> 
>Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im  Auftrag von 
>David Briggs
>Gesendet: Mittwoch, 29. März 2017 17:53
>An: CCP4BB@JISCMAIL.AC.UK
>Betreff: Re: [ccp4bb] protein precipitation reg
> 
>It doesn't cost as much as HEPES, iirc.
>On Wed, 29 Mar 2017, 16:36 Keller, Jacob, 
>wrote:
>
>
>A bit off topic, but I’ve always wondered how TRIS got so popular what 
>with it’s pKa of 8.3—does anyone know?
> 
>JPK
> 
>From: CCP4
> bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger  
>Rowlett
>Sent: Wednesday, March 29, 2017 11:10 AM
>To: CCP4BB@JISCMAIL.AC.UK
>Subject: Re: [ccp4bb] protein precipitation reg
>
>
>
>
> 
>What are you dialyzing against? Your storage solution should typically 
>be buffered away from the pI and contain at least a small amount of 
>kosmotropic salt, e.g. NaCl. Some proteins will require additional 
>stabilizing/solubilizing  agents such as glycerol or reducing agents. 
>FYI, Tris-Cl, pH 7.5 has very little buffer capacity (about 15% of the 
>total concentration in the acid direction). We typically use Tris-Cl pH 
>8.0, which is closer to the Tris pKa and has good buffer capacity for  
>both acid and base. For pH 7.5 we would typically use HEPES as the 

[ccp4bb] AW: [ccp4bb] [ccp4bb] protein precipitation reg

[Hughes, Jon]

yes, "oil of vitriol" is sulphuric acid, "blue vitriol" is copper II sulphate 
as i recall.
j


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von CRAIG A 
BINGMAN
Gesendet: Donnerstag, 30. März 2017 05:52
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] [ccp4bb] protein precipitation reg

Since I’m in full-on cranky old biochemist mode now, I think that vitriol is an 
old name for sulfuric acid.

> On Mar 29, 2017, at 8:40 PM, Keller, Jacob  wrote:
> 
>> And if we are going to pour scorn and vitriol on Tris, why not mention its 
>> large dpKa/dT of 0.03 pH units/deg ?
> 
> Hah! That's what many people are doing when they make buffers: pouring 
> vitriol (HCl) on TRIS! I prefer to pour concentrated HEPES, and get two 
> buffers without adding any extra Cl-.
> 
> JPK

[ccp4bb] AW: [ccp4bb] protein precipitation reg

[Hughes, Jon]

...it's just a wonderful tradition! there's an interesting description of the 
history of tris in maniatis
cheers
jon

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von David 
Briggs
Gesendet: Mittwoch, 29. März 2017 17:53
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] protein precipitation reg

It doesn't cost as much as HEPES, iirc.
On Wed, 29 Mar 2017, 16:36 Keller, Jacob, 
mailto:kell...@janelia.hhmi.org>> wrote:
A bit off topic, but I’ve always wondered how TRIS got so popular what with 
it’s pKa of 8.3—does anyone know?

JPK

From: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger 
Rowlett
Sent: Wednesday, March 29, 2017 11:10 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] protein precipitation reg

What are you dialyzing against? Your storage solution should typically be 
buffered away from the pI and contain at least a small amount of kosmotropic 
salt, e.g. NaCl. Some proteins will require additional stabilizing/solubilizing 
agents such as glycerol or reducing agents. FYI, Tris-Cl, pH 7.5 has very 
little buffer capacity (about 15% of the total concentration in the acid 
direction). We typically use Tris-Cl pH 8.0, which is closer to the Tris pKa 
and has good buffer capacity for both acid and base. For pH 7.5 we would 
typically use HEPES as the storage buffer.

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu
On 3/29/2017 9:38 AM, Akilandeswari Gopalan wrote:
Dear all,
I am a PhD student doing structural studies on a few proteins from 
Mycobacterium tuberculosis. The gene encoding the proteins I work on are cloned 
into pet22b with c terminal His tag. the proteins are expressing well. upon 
purification I am getting good yield of protein but during dialysis, the 
proteins precipitate. Kindly suggest some solutions to avoid aggregation. pI of 
one protein is 9.7 and that of the other is 5.6
I am using 25mM Tris pH 7.5 and 100 mM NaCl buffer with 5mM 
beta-mercaptoethanol and 0.5% triton x 100 for lysis, the same buffer with 
20-30mM imidazole for washing and 300mM imidazole for eluting the proteins.

Thank you
Regards
Akila

--
Akilandeswari G

--


Fehler! Es wurde kein Dateiname angegeben.
[Das Bild wurde vom Absender entfernt.]



David Briggs PhD
Fehler! Es wurde kein Dateiname angegeben.about.me/david_briggs

[ccp4bb] AW: [ccp4bb] How to determine the concentration of biotinylatedpeptide?

[Hughes, Jon]

very good point! you need lots of protein and it has to be pure (meaning also 
minimal buffer, salts and stuff) but it worked pretty well in our hands (when 
we were trying to measure the extinction coefficient of phytochrome). 
incidentally, I think that the notion of quantitative amino acid analysis being 
the gold standard is wrong. we had a sample analysed by different labs in 
different parts of the world – and the results varied by about 50%! maybe we 
were just unlucky, but maybe we won't be the only ones
best
jon

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Nicholas 
Larsen
Gesendet: Montag, 6. Februar 2017 19:01
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] How to determine the concentration of biotinylated 
peptide?

These suggestions are all possible, but why not simply lyophilize it into a 
tared tube and weigh it out?

On Mon, Feb 6, 2017 at 12:28 PM, Alex Lee 
mailto:alexlee198...@gmail.com>> wrote:
Thank you all for your suggestions!

On Mon, Feb 6, 2017 at 5:53 AM, Artem Evdokimov 
mailto:artem.evdoki...@gmail.com>> wrote:
Hi,

In addition to HABA dye assay (which will work great but will also be fooled by 
any biotin that is not conjugated) you can do:

* quantitative MS
* TLC
* HPLC
* elemental analysis
* https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3614710/ biotin catalysis of the 
N3- + I3- reaction (also fooled by free biotin of course)
* UV (but beware, biotin only absorbs strongly below 240nm so you're not super 
well off there

Artem
www.harkerbio.com
"all of our Biotin comes only from free-range gummy vitamin bears..."

- Cosmic Cats approve of this message

On Mon, Feb 6, 2017 at 2:03 AM, Debasish Kumar Ghosh 
mailto:dkgh...@cdfd.org.in>> wrote:
Hi Alex,

In addition to Mirella's suggestion I would like to make an addition which 
might be specifically useful for you. Since your peptide has biotin tag, You 
may use HABA dye assay for the exact quatifiation of biotin (and thus 
biotinylated peptide). As far I recall, Thermo scientific provide a kit for 
this assay. The assay is simple and gives accurate results.

Best !!!



Debasish

CSIR- Senior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group
Centre for DNA Fingerprinting and Diagnostics
Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, 
dgho...@gmail.com
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab)
Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html



- Original Message -
From: Alex Lee mailto:alexlee198...@gmail.com>>
To: CCP4BB@JISCMAIL.AC.UK
Sent: Mon, 06 Feb 2017 03:02:07 +0530 (IST)
Subject: [ccp4bb] How to determine the concentration of biotinylated peptide?

Dear All,

Sorry for the off-topic question, I'd like to do Biacore SPR assay with
N-terminal biotinylated peptide as ligand (to Biacore SA chip) and my
protein as analyte. I have a question of how to determine the concentration
of biotinylated peptide （synthetic peptide), if the peptide has no Tyr and
no Trp residue, I guess amino acid analysis may not work because the
N-terminal of the peptide is biotinylated.

I'd appreciate if anyone share his/her experience on this.




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[ccp4bb] trump

[Hughes, Jon]

hi,
one can be uncomfortable with some of hillary clinton's policies and attitudes 
and be disappointed with obama, but to think that an individual like donald 
trump is in possession of moral character and integrity is simply out of this 
world. god save america (and the rest of us), as they say.
j