Re: [ccp4bb] C6 Webtool

[Janet Newman]

CSIRO has taken this resource offline. Hopefully it will return, but hopes are 
often dashed.

janet

From: CCP4 bulletin board  on behalf of Leonhard Kick 

Sent: Thursday, January 12, 2023 7:56 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] C6 Webtool

You don't often get email from leonhard.k...@wuxiapptec.com. Learn why this is 
important<https://aka.ms/LearnAboutSenderIdentification>

Hi all and a belated Happy New Year,



I was wondering if any of you noticed that the C6 Webtool from Janet Newman 
(https://c6.csiro.au/C6.asp) is discontinued and offline.

We used it quite extensively to get a short and meaningful comparison of 
commercial screens (differences and similarities, mostly).

Do you know if the service was conserved somewhere? Or do you have alternatives 
that you preferred anyway? Would be great if you could share your experiences.



Thank you all



Cheers



Leo





Leonhard Kick

Deputy Head Crystallography

Crelux GmbH – A WuXi AppTec Company



Am Klopferspitz 19a

82152 Martinsried

Germany







Save paper. Protect the environment. Print only when necessary.


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Re: [ccp4bb] suggestion of crystallization optimization

[Janet Newman]

Liuqing Chen,

Everything that has been said seems reasonable, but there are always infinite 
possibilities in crystallisation, so it is more a question of priorities. Do 
the easy (or quick) things first. If you have buckets of prepared protein then 
what you will try first might be different than if you have to go and make your 
protein from scratch each time you set up crystal trays.

1. If you have crystals from an additive screen or seeding - try putting them 
in the beam. If you have access to in-plate screening, you can test the 
crystals without disturbing them, which will give the best idea of their native 
diffraction. Perhaps one of the ugly crystals diffracts well enough?

2. Try cross seeding - seed one or more initial screen(s) (rather than an 
optimisation).  Try initial screening with seeding at different temperatures. 
If you are currently using vapour diffusion, try microbatch. Or vice versa.

3. Try in-situ proteolysis. Add a very small amount of protease to your protein 
sample (chymotrypsin is traditional) - say 1:10,000 or 1:1000 compared to your 
protein concentration then set up that mixture in initial screens. 

4. Is there a ligand/inhibitor/other small molecule that binds? Add that to 
your protein before crystallisation. Maybe even try this first!

5. Lysine methylation/cysteine modification/other side chain modifications.

6. Try using DSF or some other technique to look at your protein's stability in 
the formulation it is in. Maybe you can make happier protein by changing the 
pH, buffer or salt.

7. If you want to be a little more rigorous, take your protein, and a number of 
different proteases, and do a time-course experiment with each protease (add 
1:1000 protease to your protein, then take samples at timepoints - say 0.5 
hours, 1 hour, 5 hours and overnight) then run out on a gel (or analyse by MS) 
and see if you come down to a stable fragment. If you do - then use that 
protease, and while you are waiting for the crystallisation trials to do their 
thing, find out what the end points of the proteolysis fragment are, and make 
that construct.

6. Try a different expression system (different tag, different position of the 
tag, cleave/don't cleave the tag). If the protein is produced in a eukaryotic 
system (and is glycosylated) try a different one to get different glycosylation 
pattern. Try kifunensine treated cells if you are in a mammalian expression 
system.

8. Try the same protein from other species

Janet Newman
Principal Scientist / Director, Collaborative Crystallisation Centre (C3)
CSIRO Material Science and Engineering
343 Royal Parade
Parkville.  VIC. 3052
Australia
Tel +613 9662 7326
Email janet.new...@csiro.au


From: CCP4 bulletin board  on behalf of Liuqing Chen 
<519198...@163.com>
Sent: 04 June 2018 20:57
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] suggestion of crystallization optimization

Hello everyone!

I get a crystal several months ago, but the crystals diffraction very low 
resolution (around 8A)  or no diffraction.
  My sample buffer is 20mm Hepes ph7.0,  50mm NaCl,   my protein pI is 5.
  the codition grow crytal  is : 30% PEG400, 100mm hepes 7.5,  200mm MgCl2.

I also tried  additive screen,  all the crystals appear the same apparence,
even i seeding optimization,  have no improve.
the  attach is  my crystals.

what should   i  do next?

thanks in advance.
sincerely
Liuqing chen



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Re: [ccp4bb] Removing tartrate ions from the active site

[Janet Newman]

Given that your tartrate crystals diffract so much better than your 
non-tartrate crystals, and tartrate is found in the active site, you might 
think that tartrate binding in the active site is related to the good 
diffraction of your crystals. Soaking out a bound tartrate is going to be hard, 
when you have 1.1 M of it around.

You might like to try a gentle cross linking of the tartrate crystals, then 
slowly transferring them to another solution (Perhaps malonate? Test to see 
first if Malonate binds the protein).

Once you are out of 1.1 M tartrate you might have a better chance of displacing 
the tartrate in the active site with your ligand.


Janet

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nemanja 
Vuksanovic
Sent: Sunday, 22 April 2018 12:39 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Removing tartrate ions from the active site

Dear All,
I've recently crystallized a protein under a condition containing 1.1 M 
ammonium tartrate. (I was able to get a few other hits but only these crystals 
diffract below 2.5 A). Upon refinement, it appears that the electron density in 
the active site shows a mixture of tartrate and the native ligand. This occurs 
even after overnight soaks with 50 mM of the ligand ( the Km is about 1 mM). 
Would you recommend dehydrating crystals with PEG when soaking them with ligand 
or some different method to minimize the presence of tartrate?
Thanks in advance,
Nemanja

--
Graduate Student
Department of Chemistry and Biochemistry
University of Wisconsin-Milwaukee

Re: [ccp4bb] Help needed finding hit condition

[Janet Newman]

?Hi Jonathan,


Hopefully you know about the trick of making any precious condition last longer 
- use the 'magic' solution only in the drop itself, and use the best 
approximation you can make in the reservoir of the experiment (if you are doing 
vapour diffusion)


Regards, Janet


Janet Newman
Principal Scientist / Director, Collaborative Crystallisation Centre (C3)
CSIRO Material Science and Engineering
343 Royal Parade
Parkville.  VIC. 3052
Australia
Tel +613 9662 7326
Email janet.new...@csiro.au

From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Jonathan Bailey 
<baile...@tcd.ie>
Sent: 31 July 2017 22:34
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Help needed finding hit condition

Dear CCP4bb community

I apologies for the slightly off topic post.

We have recently had success crystallizing a membrane protein (diffraction > 3 
Å at a synchrotron source) using the in meso method, the hit condition was from 
the Jena Bioscience screen Pi-minimal condition number #57.

Hit condition - 47.1 % w/v PEG1000, 150 mM Tris pH 8.0, 80 mM Potassium bromide

The screen is old and expired 12/20/2013 (lot # JBS00013133), we have tried to 
reproduce the crystals using homemade optimization screens around the hit 
condition but have not had any success. We have tried reproducing the hit using 
a new (not expired) Pi-minimal screen but had no success. We are only able to 
reproduce the crystals using the expired screen and we do not have much of it 
left.

We went back and tested the pH of the condition that had given crystals, the 
expected pH was 7.9 but we found it to be 6 - 6.5 using a pH indicator strip. 
We believe the drop in pH is caused by oxidative degradation of the PEG1000 
resulting in the formation of carboxylic acid species.

We have contacted Jena Bioscience to try and get some of the old screen stock 
but unfortunately they do not have any.

My question is does anyone out there happen to have any expired screen stocks 
of this Pi-minimal condition (#57), ideally from the same lot (lot # 
JB200013133), that they would be willing to send us.

Does anyone have any advice as to how to reproduce the condition? We've 
considered bubbling oxygen through and heating the sample to accelerate the 
oxidation process.

King Regards

Jonathan Bailey (PhD student)

Professor Martin Caffrey Lab MS group Trinity College Dublin

Re: [ccp4bb] preparing DL-Malic acid stock solution

[Janet Newman]

The way I have done it (and it was sort of fun in a mad scientist way) was to 
mix up solid DL-malic acid and sodium hydroxide in the right amounts and add 
water as needed.  This generates  of heat, but gets around the 
solubility minimum which is impossible to get out of:

I mixed 40.23 g of DL-malic acid powder (MW=134.1) with 24g of NaOH pellets 
(MW=40) in a large beaker with a stirrer bar.  Put the beaker in a larger 
container of ice, and put that on a stirrer in the cold room. Add 80 mL H20 to 
the beaker containing the chemicals, stand back - lots of heat evolved.
when everything has quietened down, make up to 100 mL with water (we filtered 
through a 0.22 um filter).

Cheers, Janet

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Edward A. 
Berry
Sent: Friday, 9 June 2017 2:53 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] preparing DL-Malic acid stock solution

(di)Sodium D,L-malate is available from Sigma-Aldrich (# CDS023113) and
probably dissolves to give a 3M solution which is slightly alkaline.
If pK2 is 5.1, then an insignificant amt of malic acid should bring the
pH down to 7 (If you have to add a significant amount, just add more
water to dilute to a final conc of 3M Malate + Malic acid)


On 06/08/2017 12:32 PM, Diana Tomchick wrote:
> As the pKa1 of malic acid is 3.4, it may be that the partially neutralized 
> malic acid salt is less soluble than the acid or the fully neutralized salt.
>
> The pKa2 of malic acid is 5.1.
>
> Contact the people at Hampton Research
>
> https://www.hamptonresearch.com/contact_us.aspx
>
> and ask them. They sell it as a 3.0 M solution, pH neutralized to 7.0.
>
> It is possible that you need to fully neutralize it before it turns clear, or 
> that you may also need to gently heat it.
>
> Diana
>
> **
> Diana R. Tomchick
> Professor
> Departments of Biophysics and Biochemistry
> University of Texas Southwestern Medical Center
> 5323 Harry Hines Blvd.
> Rm. ND10.214A
> Dallas, TX 75390-8816
> diana.tomch...@utsouthwestern.edu 
> (214) 645-6383 (phone)
> (214) 645-6353 (fax)
>
> On Jun 8, 2017, at 9:54 AM, Sebastiano Pasqualato 
> > 
> wrote:
>
>
> Dear all,
> we’ve recently having trouble preparing a 3 M stock solution of DL-Malic 
> Acid, pH 7 (which we had, so it’s doable!).
> When we reach pH 3 - 4 the solution turns milky white and does not goes back 
> to a clear solution even when the pH is raised.
> Does anybody have any advice on how to get a clear solution? Has anyone gone 
> through the same?
> Thanks in advance,
> ciao,
> Sebastiano
>
>
> --
> *Sebastiano Pasqualato, PhD*
> Crystallography Unit
> Department of Experimental Oncology
> European Institute of Oncology
> IFOM-IEO Campus
> via Adamello, 16
> 20139 - Milano
> Italy
>
> tel +39 02 9437 5167
> fax +39 02 9437 5990
> web http://is.gd/IEOXtalUnit
>
>
>
> --
>
> UTSouthwestern
>
> Medical Center
>
> The future of medicine, today.
>

Re: [ccp4bb] [ccp4bb] protein precipitation reg

[Janet Newman]

Just to point out that whatever buffer you purify your protein into is possibly 
not the one that will keep your protein happiest.  We had the opportunity of 
testing about 250 proteins in DSF against 26 different buffer / salt 
combinations (in triplicate, with lots of controls) and found out that about 
1/3 of the time the protein was significantly (4C or more) stable in some other 
buffer system than the one it was in.
Ristic, M., Rosa, N., Seabrook, S.A., Newman, J., 2015. Formulation screening 
by differential scanning fluorimetry: how often does it work? Acta 
Crystallographica Section F Structural Biology Communications 71, 1359–1364. 
doi:10.1107/S2053230X15012662

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 ?

Cheers, Janet

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of CRAIG A 
BINGMAN
Sent: Thursday, 30 March 2017 8:54 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] [ccp4bb] protein precipitation reg


> On Mar 29, 2017, at 4:15 PM, Chun Luo  wrote:
> 
> In addition to price, the prevalence of Ni purification may be another reason 
> for Tris popularity. Some His-tagged constructs don't bind to Ni well in 
> HEPES. I wonder if anyone has similar experience or comments. —Chun

No, I have not specifically noted that before.

Additionally, why would you use a positively charged buffer on a weak cation 
exchange resin?  The Ni affinity resins, in addition to their noteworthy 
affinity for various metals, are also weak cation exchange resins.  Binding a 
positively charged buffer to a negatively charged column material can cause pH 
effects that you probably weren’t expecting.

Tris may have some uses.  But using it in a mobile phase with Ni affinity 
columns or as a final sample buffer aren’t the best cases for choosing Tris.  I 
understand it is widely used in aquaculture applications where they are 
treating tens of thousands of gallons of water, and price of the buffer 
substance is an actual consideration.

Craig

[ccp4bb] App that might be useful - CINDER

[Janet Newman]

Hi All,

We have become quite interested in autoscoring of images of crystallization 
droplets (I think everyone goes through this stage, most grow out of it 
eventually) and as part of our work we wanted to develop a well-scored set of 
images to use as a training set for machine learning approaches.  We decided 
that we would try to classify into four classes

Clear 
Precipitate
Crystal
Other

This very limited scoring would hopefully be more robust than more granular 
scoring, but would give enough information to both help find crystals and to 
help find phase boundaries.  We have developed a mobile app - CINDER - which 
uses swipe technology to assign a score to an image.  Every swipe (score) is 
then recorded in a database, and we would use the consensus score for each 
image as our training set.  Four scores also mean that we can assign a 
direction to each swipe (swiping right for crystal, up for precipitate etc).

To make the app useful to novices, we have included a training set in the 
CINDER app - this training presents the user with an image, then shows the user 
how we would have scored it, and gives an explanation of why we scored it the 
way we did.  Currently our training set ("Cinder Kinder") consists of 75 
images.  We believe that the Cinder Kinder feature might be quite useful to 
train students how to look at crystallization experiments in general.

If you have students or relatives or colleagues who might be interested in a 
'citizen science' project, we would be delighted if you could tell them about 
CINDER (currently available through Googleplay for android devices, iOS version 
coming soon).  If you think Cinder Kinder might be useful, and want to include 
some of your images in the training set, then we would be happy to do so - what 
we would need is the image, and a score (one of the four above) and a reason as 
to why you would assign that score.  Send any images for inclusion to 
janet.new...@csiro.au.  Bugs, improvements and comments all welcome!

Cheers, Janet

Re: [ccp4bb] Formulation of my own mother liquor

[Janet Newman]

Generally one will make this up from stock solutions:

Make up
1 M Tris chloride pH 8
1 M magnesium chloride
50 % (weight/volume) PEG 6K

Then mix these together in the right ratios

(for 1 mL)

100 uL 1 M tris solution
200 uL 1 M MgCl2 solution
400 uL 50% PEG solution
300 uL water.




Janet Newman
Principal Scientist / Director, Collaborative Crystallisation Centre
CSIRO Material Science and Engineering
343 Royal Parade
Parkville.  VIC. 3052
Australia
Tel +613 9662 7326
Email janet.new...@csiro.au

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of amro selem 
[03391c09f749-dmarc-requ...@jiscmail.ac.uk]
Sent: 22 November 2014 03:48
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Formulation of my own mother liquor

Dear All,
first i wish you nice weekend, then i wanna ask about formulation of my own 
mother liquor, i want to optimize a condition containing
 0.2m MgCl2, 0.1m Tris , PH 8; and 20% PEG6K as pricipitant. (PACT)
the question is , should i make a stock solution from every ingredient by 
dissolving it in miliQ water. then mix all stuff  together  and add water till 
final concentration with out adjusting final pH. OR dissolve the PEG in pH 
adjusted buffer and then mix all ingredients , so the final pH will be 8.
any suggest any idea or any one knows what the company do.
CHEERS
Amr

[ccp4bb] Australasian Course in Macromolecular Crystallisation

[Janet Newman]

Dear All,

Expressions of Interest are open for the upcoming Australasian Course in 
Macromolecular Crystallisation.  This course, (the 4th of this type) is an 
intensive, week-long course in macromolecular crystallisation, with lectures 
and lab sessions for the first four days, and the fifth day being spent at 3 
beam lines of the Australian Synchrotron.  The course will be held from the 9th 
to the 13th of December, 2013 in Melbourne, Australia.

20 students will be selected from those who have submitted an EOI by September 
27, 2013.  This course is aimed at everyone interested in protein 
crystallisation - from beginners onwards.  (Admittedly, when Alex McPherson 
attended, he was a lecturer!)

More details, including  links to the program and an expression of interest 
form are available from http://crystal.csiro.au/Events/ACMC-2013.aspx

Please forward this to anybody you think might be interested.

Regards, Janet

Dr Janet Newman
Principal Scientist | C3 Facility Manager
Materials Science and Engineering
CSIRO
Phone: +61 3 9662 7326 | Fax: +61 3 9662 7101 |
janet.new...@csiro.au | www.csiro.au | www.csiro.au/c3
Address: 343 Royal Parade, Parkville VIC 3052  AUSTRALIA

PLEASE NOTE
The information contained in this email may be confidential or privileged. Any 
unauthorised use or disclosure is prohibited. If you have received this email 
in error, please delete it immediately and notify the sender by return email. 
Thank you. To the extent permitted by law, CSIRO does not represent, warrant 
and/or guarantee that the integrity of this communication has been maintained 
or that the communication is free of errors, virus, interception or 
interference.
Please consider the environment before printing this email.

Re: [ccp4bb] database of crystallization condition

[Janet Newman]

Dear Hena,

The BMCD might be a resource worth investigating:

http://xpdb.nist.gov:8060/BMCD4/index.faces

Although there are claims that structurally similar proteins may crystallise 
under similar conditions (the existence of directed screens  -such as the Jena 
Biosciences 'Kinase' screen, you have to wonder (as Enrico points out) how 
these can work, as the bits that change the most in any protein family are the 
outside bits (ie, away from the active site) and thus are generally the parts 
going to affect crystallisation the most.

Janet

Janet Newman
Principal Scientist / Director, Collaborative Crystallisation Centre
CSIRO Material Science and Engineering
343 Royal Parade
Parkville.  VIC. 3052
Australia
Tel +613 9662 7326
Email janet.new...@csiro.au

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Hena Dutta 
[hdutt...@gmail.com]
Sent: 25 July 2013 00:36
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] database of crystallization condition

Hi,
Can anyone tell, if there is any database containing the crystallization 
conditions of published structures? I want to see the conditions people have 
used for those proteins having some structural similarity. Any suggestion would 
be appreciated.
Regards...
Hena

Re: [ccp4bb] cryo condition

[Janet Newman]

I would try 2.5M or 3M sodium malonate pH 7

Janet Newman
Principal Scientist / Director, Collaborative Crystallisation Centre
CSIRO Material Science and Engineering
343 Royal Parade
Parkville.  VIC. 3052
Australia
Tel +613 9662 7326
Email janet.new...@csiro.au

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Roger Rowlett 
[rrowl...@colgate.edu]
Sent: 23 May 2013 21:21
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] cryo condition


30% glycerol or 25% glucose should be sufficient for 1-2 M ammonium sulfate.

Roger Rowlett

On May 23, 2013 5:42 AM, Faisal Tarique 
faisaltari...@gmail.commailto:faisaltari...@gmail.com wrote:

Dear all

Can anybody tell me the appropriate cryo condition for the crystals obtained in 
2M Ammonium sulphate and tris pH8.5..I tried to add 10% glycerol to it but 
still the ice ring is forming..

Thanx in advance
--
Regards

Faisal
School of Life Sciences
JNU

Re: [ccp4bb] Off-topic: Fungal growth in robot trays

[Janet Newman]

We add 0.02% Azide to all the water used for washing in our Phoenix, and that 
seems to help.


Janet Newman
Principal Scientist / Director, Collaborative Crystallisation Centre
CSIRO Material Science and Engineering
343 Royal Parade
Parkville.  VIC. 3052
Australia
Tel +613 9662 7326
Email janet.new...@csiro.au

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Viswanathan 
Chandrasekaran [v...@biochem.utah.edu]
Sent: 09 March 2013 10:24
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Off-topic: Fungal growth in robot trays

Dear All:
I would like some advice on getting rid of persistent fungal growth in 96-well 
sitting drop crystal plates that were set up using a Phoenix robot.
24-well sitting drop trays prepared by hand don’t have this problem. Washing 
the robot with 0.5% bleach followed by plenty of water had no effect. Is adding 
sodium azide directly to commercial screen hotels (or the protein sample) a 
good idea? If so, how much should I add? Other suggestions are welcome.
I will post a summary of all replies.
Thank you.
Best,
Vish

Re: [ccp4bb] Your suggestions needed: Difficulties in reproducing HT crystallization conditions. Thanks!

[Janet Newman]

Hi Joe,

I really want to re-iterate both Annie and Patrick's suggestion of trying 
different drop ratios and protein amounts, however you might also try adding 
some fresh reducing agent, particularly if you are trying the scale-up with 
protein that has been hanging around (or sitting around) since the screening.

Regards, Janet
Dr Janet Newman
Principal Scientist | C3 Facility Manager
Materials Science and Engineering
CSIRO
Phone: +61 3 9662 7326 | Fax: +61 3 9662 7101 |
janet.new...@csiro.au | www.csiro.au | www.csiro.au/c3
Address: 343 Royal Parade, Parkville VIC 3052  AUSTRALIA

PLEASE NOTE
The information contained in this email may be confidential or privileged. Any 
unauthorised use or disclosure is prohibited. If you have received this email 
in error, please delete it immediately and notify the sender by return email. 
Thank you. To the extent permitted by law, CSIRO does not represent, warrant 
and/or guarantee that the integrity of this communication has been maintained 
or that the communication is free of errors, virus, interception or 
interference.
Please consider the environment before printing this email.
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Joe
Sent: Wednesday, 24 November 2010 4:03 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Your suggestions needed: Difficulties in reproducing HT 
crystallization conditions. Thanks!

Hi all,

I recently have problems reproducing some conditions identified from high 
throughput screenings.

The initial screening (10 mg/ml protein, 0.5 ul well+ 0.6 ul protein, 23 C) 
gave rise to at least three hits from different screen kits.  The follow-up 
grid optimization (1.5 ul well + 1.5 ul protein) varying precipitant 
concentrations and pHs did not produce any crystals for all three conditions. 
Instead, the drops have heavier precipitation background.  The following 
experiments have been done in order to get crystals back.

1. Seeding, with various precipitant concentrations
2. Varying volume ratios between well solution and protein (from 2: 1 to 1: 2 
v/v).
3. Using original solutions from screen kits.
4. Hanging drops and sitting drops,
5. Dispensing protein first or well solutions first.
6. Using robot to set up drops on 96-well plate to see if I can reproduce the 
original hits.

But none of them has worked so far.  This is the first time I encountered such 
a scale-up issue.  I am running out of ideas, so hope you could give me some 
suggestions.  Thank you in advance.

--
Best regards,

Joe

Re: [ccp4bb] Mysterious density

[Janet Newman]

Hi Jin,

Could it be the wrong amino acid in the sequence?   Have you sequenced the gene 
construct that you used?  The codon usage for HIS is CAC or CAU, and for TYR is 
UAC or UAU. - 

Cheers, Janet



From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Jin H. Park 
[jinhp...@mail.med.upenn.edu]
Sent: 04 September 2010 09:53
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Mysterious density

Dear CCP4 community,



I have solved the structure. But, there seems oddly strong density above 
histidine residue.
What could this density be?
The protein was in Tris buffer, with NaCl and DTT, and crystallized in NaSCN 
with PEG.
(electron density in the pictures is at 1.5 sigma)

Thanks.

Re: [ccp4bb] Help with improving these crystals

[Janet Newman]

Hi

One of the easiest things to try is in-situ proteolysis - chymotrypsin (1:1000 
or so, we add 1ul of a 1mg/ml solution of chymotrypsin in H20 to about 100ul of 
protein solution) is the classic - mix the protease with the protein and then 
set up as normal.  Of course you can try other proteases as well.  I would 
generally not recommend subtilisin or proteinase K - these are almost 
guarenteed to turn your protein into amino acid soup.

Matrix seeding (Allan D'Arcy is the lead author on the reference for this)  
with the crystals you have is another relatively painless thing to try (ie, use 
your crystals to seed into a screening experiment) - given that your condition 
is ammonium sulfate based, be aware that you will get lots of lovely crystals 
in any screening condition containing calcium.

may the force be with you

Janet



From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Narayanan 
Ramasubbu [ramas...@umdnj.edu]
Sent: 10 September 2009 01:36
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Help with improving these crystals

Dear All:
We have been struggling to improve the crystals shown in the attachment.
These crystals form from a protein solution 8 mg/mL (before drop), drop
sizes are 2 microL of protein + 2 microL of well solution (0.1 MES
buffer, pH 6.5, 1.8 M Ammonium sulfate with 12 mM CoCl2).

The protein was dissolved initially in 20 mM Tris.HCl, pH 8.0 containing
1 mM EDTA.

Low cobalt also gives smaller crystals. These diffract up to 8 A.

Any suggestions to improve these crystals would be most welcome.

Subbu

PS: We have used many additives (Hampton Research - including detergent
additives) to alter the morphology but with little success.

Re: [ccp4bb] Crystal Imaging Systems - possibilities and recommendations

[Janet Newman]

Do you want an imager, or a more sophisticated system that will store plates 
and image them according to a schedule?

With imaging, it is important to think about what you want out of the system, 
as it is easy to be disappointed with them.  The images you get will not be as 
good as what you see down a microscope (imagine if you set the microscope up to 
have drop A1 in focus and well lit, and after that you only translated the 
plate).

Do you want to be able to find an xtal in an image, and then immediately know 
what was in the drop, reservoir, temperature of setup etc?  In which case, you 
should look at the crystallisation database which is behind the imager - all 
software requires that you put information into it before you can retrieve it, 
so how easy is it to get information into your database?  

Janet
-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Pedro M. Matias
Sent: Tuesday, 22 January 2008 12:38 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Crystal Imaging Systems - possibilities and recommendations

Dear Colleagues,

We are currently contemplating the acquisition of 
an automated imaging system for crystallization screen plates (96-well).
I am aware that Molecular Dimensions sells these 
systems ranging in price between 47 and 71 k€ and 
I am inviting your opinions, comments and advice 
on these and other commercially available 
systems, in particular regarding price, reliability and ease of use.

Thanks in advance,

Pedro Matias


Industry and Medicine Applied Crystallography
Macromolecular Crystallography Unit
___
Phones : (351-21) 446-9100 Ext. 1669
   (351-21) 446-9669 (direct)
 Fax   : (351-21) 441-1277 or 443-3644

email : [EMAIL PROTECTED]

Mailing address :
Instituto de Tecnologia Quimica e Biologica
Apartado 127
2781-901 OEIRAS
Portugal

Re: [ccp4bb] crystallization robot

[Janet Newman]

I bought a replacement Cartesian Tip (in Australia)  on the 8th of
october, 2007 and got charged $660 AUD (of which 60 bucks was the GST
tax) - on the 8th of october, the AUD-USD exchange rate was 0.8956, so
that would be $537 USD, which is quite a long way from both '$700' and
'under $300'.

The tips are quite fragile, and you could guess 10-20% breakage each
year of use, gauging from the stories told around here.

Janet

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Boutin, Ray
Sent: Thursday, 10 January 2008 11:42 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] crystallization robot

Not to inject a commercial tone, but there seems to be a bias in the
posting below. The ceramic tips are breakable, but not all find them
incredibly so. And the price is less then $300, not $700. 

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Lisa A Nagy
Sent: Wednesday, January 09, 2008 11:21 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] crystallization robot

We chose the Phoenix crystallization robot because:

It has no expensive consumables (tips) intrinsic to the machine. This
was also a big item for us because we worry about being able to run the
machine for more than 3 years. Would the tips for our 2008 machine be
available in 2014? 

It is easy to program (BIG ITEM) for different tray configurations, and
various dispensing methods- even on the same tray. Right now you may not
think you'll have to vary drop sizes or add additional components
(ligand? detergent?) to your drops, but you probably will.

It can draw from 2ml block plates. Reformatting from block plates to
your trays is a pain.

The nitinol tips won't break (Compared to ~$700 apiece for the
incredibly breakable ceramic tips on some other machines).

It has cooling blocks for your samples. This is more important than you
think.

It's fast.

It is easily integrated into a fully automated lab system. Right now,
though, our humans (including me) are cheaper than rails and robots.

It's incredibly accurate, even with 30% PEG 4000 (we tested this
ourselves). 

You can use it for other low volume dispensing applications.


--
Lisa Nagy
University of Alabama-Birmingham
[EMAIL PROTECTED]  

Re: [ccp4bb] crystallization robot

[Janet Newman]

The Gilson crystallisation robots (horrible machines: no longer
available) were specific for hanging drops - other robots can set them
up (using the Mosquito sheets or equivalent), but the flipping will have
to be done by hand.  The Douglas Instrument machines can basically
pipette onto anything you want, as it is a one-by-one dispense.  You
still have to flip by hand.  The Mosquito has the ability to do a
'mirror' transfer - which makes the setup of hanging drops easy, as it
compensates for the inversion that you necessarily get when you flip the
piece of plastic (it's a good idea to flip the plastic around the same
axis as the mirror function, BTW).

 

If you want to use this arrangement on the phoenix then you will need to
make up a plate or block with the crystallisation solutions mirrored,
and set up a protocol where the normal array is transferred from the
storage block into the experimental plate reservoirs, and then the
mirrored array of crystallisation solutions is used to make up the
crystallisation droplets.  Certainly do-able, but it means that you need
TWO source blocks for each screen that you want to set up this way.
Best of luck keeping that working in a multi-user lab!

 

We have both a Mosquito and a Phoenix (and an older Cartesian 16+1), and
I could not recommend one over the other of the first two machines -
they are both easy to use, but they do different things well.  The
Mosquito can deal with difficult samples (protein which is crashing out
of solution, for example).  We have been able to set up protein which
gelled to the consistency of hair-gel with our gnat.  The Phoenix does
not dispense viscous samples well from the nano-tip (anything over 10%
glycerol is really iffy, for example), but doesn't require the protein
to be aliquoted out into 8 separate puddles.  The Cartesian was always a
bitch to get running, so given the other two machines, it sits and
collects dust.  Anybody want to start negotiating for a (slightly) used
Cartesian?

 

Janet

 

Janet Newman

CSIRO Molecular and Health Technologies

343 Royal Parade

Parkville  VIC  3052

Australia

tel:  +61 (0)3 9662 7326

fax: +61 (0)3 9662 7101

email: [EMAIL PROTECTED]

 

 

  _  

From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Lisa A Nagy
Sent: Thursday, 10 January 2008 4:49 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] crystallization robot

 

The phoenix can dispense multiple drops per well. You can easily
program dispensing on the deck wherever, whatever and whenever, as long
as it fits in the plate holder . It can handle at least the 1536 pitch
(4 x 384), so if you specify the quadrant of the 384 well cell it will
dispense there. Your drops may merge, though. It has a separate
dispensing head for proteins or additives. You can dispense from up to
16 protein tubes (chilled) and 2 ambient tubes, plus whatever is on
the deck, in whatever order you want. Since the setup programming is an
easy gui, it's not a big deal.

 

I am certain that the mosquito sheets would work on any robot. 

 

From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Van Den Berg, Bert
Sent: Wednesday, January 09, 2008 11:00 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] crystallization robot

 

The mosquito has special (albeit fairly pricey at $13 each) plastic
sheets that allow setup of hanging drops in a 96-well format. It can
also do multiple drops per well. As far as I know this is a capability
unique to the Mosquito but I may be wrong.

 

Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: [EMAIL PROTECTED]
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm

 

  _  

From: CCP4 bulletin board on behalf of Lisa A Nagy
Sent: Wed 1/9/2008 11:53 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] crystallization robot

Looking at the mosquito, it doesn't have any cover-slip handling
robotics, either. So it's the same thing- rearrange the dispense
location and flip the cover- which is either a glass plate or mylar or
a tape seal.

--
Lisa Nagy
University of Alabama-Birmingham
[EMAIL PROTECTED] 

Re: [ccp4bb] [Fwd: Re: [ccp4bb] Expired Crystallization Screening? - what about to freeze ?]

[Janet Newman]

The problem with old solutions is not their age as much as they might be
very hard to reproduce, as you won't exactly know what you have.  (for
all the reasons that have been mentioned: evapouration, degradation, pH
shifts, mould growth, contamination from people using the wrong pipette
tip in a different tube  etc)

 

I would use the old solutions, but be aware that you may have to use up
the rest of that very same tube if you want to repeat any hit that you
get.  There was a suggestion on the Hampton website where one uses the
screening solution in the droplet, but make up your own version of the
condition for the reservoir.  This means that you don't piss off the
other members of your lab who might want to use the same screen later.

 

Janet

  _  

From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Artem Evdokimov
Sent: Friday, 28 December 2007 12:22 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] [Fwd: Re: [ccp4bb] Expired Crystallization
Screening? - what about to freeze ?]

 

We used to freeze screens in 96-well deep blocks and plates - with no
apparent issues :-)

 

Some stuff crystallizes out but usually goes right back into solution.

 

Artem

 

  _  

From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
iulek
Sent: Thursday, December 27, 2007 7:39 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] [Fwd: Re: [ccp4bb] Expired Crystallization Screening?
- what about to freeze ?]

 

Dear all,

I was about to write something quite related, but as the topic has
just started ... I had better add my comments.
I am just establishing a crystallization facility, but currently in
my lab proteins to crystallize will be a rare opportunity (hopefully to
increase the number during the years to go ...). Even in labs where
crystallization is a throughput I see many of the screens to expire
date (I must say ones which have an o'ring to tightly close the cover
use to have volumes more equal at the different tubes -  maybe a
suggestion for Hampton or others which do not have so). I have learned
much from the comments below, but what about to freeze the screens ? Any
known  problem (except, of course, to heat again before usage - not
tough for a several month run between each usage) ? Reagent degradation
(I suppose the tubes might resist the small volume expansion at
freezing) ?

Jorge 

 Original Message  

Subject: 

{Spam?} Re: [ccp4bb] Expired Crystallization Screening?

Date: 

Thu, 27 Dec 2007 16:31:46 -0500

From: 

Artem Evdokimov [EMAIL PROTECTED] mailto:[EMAIL PROTECTED] 

Reply-To: 

Artem Evdokimov [EMAIL PROTECTED] mailto:[EMAIL PROTECTED] 

To: 

CCP4BB@JISCMAIL.AC.UK

References: 

[EMAIL PROTECTED]
mailto:[EMAIL PROTECTED] 

 

This is a tough question. Certainly, in an ideal world (and also in the
world that e.g. Hampton or Emerald salespeople would like us to live in)
you
should replace expired reagents. The reality of course is that it's
sometimes quite costly to do so. It is also expensive (in terms of time
and
effort) to not be able to reproduce your results - so there's always a
balance between price in dollars and price in human work.
 
Here are my two cents on this subject:
 
Any screens containing MES should be carefully checked. MES is notorious
for
turning 'scary yellow' with time, and this yellowing is accompanied by a
pH
shift. This is especially prominent if the solution was exposed to
light.
 
Any screens containing PEGs should be checked, and the stock PEG
solutions
should also be tested for pH change. Fresh PEG solutions have almost no
buffering power (they should have none, but there are always small
amounts
of impurities) and their pH should be around 6 (or even better, 7). With
age
PEGs tend to go acidic, see an earlier post on this subject.
 
Anything that contains organic material and not too much salt should
be
looked at - bacterial or fungal contamination is always possible. At 4C
it's
more likely to grow yeasts or molds, whereas at r.t. bacterial and
fungal
growths are equally possible. Swirl the tubes - if you see something
float
up - discard the tube.
 
Relatively few screening solutions are colored (iron, Ni, Cu, jeffamine,
and
several other ingredients confer color). If a colorless solution went
colored - discard it.
 
On the other hand, stock solutions of inorganic, and some of the organic
salts are pretty stable, even at 5-year mark. With those, you have to
watch
out for slow evaporation of water, because water can slowly migrate
through
plastic. If your solutions have crystals in them, it's likely that they
have
evaporated somewhat. What you want to do with these is your choice -
you
can attempt to dilute with ddH2O to the original volume, or you can
relabel
as saturated (a problem, since at different temperatures the
concentration
of stuff in solution changes).
 
Finally, it's good to remember that acetic acid and ammonia are volatile
and
therefore ammonium salts and acetates can change with time in