Re: [ccp4bb] Recommendations for Robotic Crystal Screening Services

[Edward Snell]

Hi Steven,


In a word, lots :)  In somewhat more detail 


If this is a request to the CCP4 community, then the images and crystallization 
metadata from the Hauptman-Woodward Medical Research Institute High-Throughput 
Crystallization Center are packed in a rar format and users of the Center on 
CCP4bb can provide those directly to you. They are readable with the program 
MacroscopeJ, available from 
http://hwi.buffalo.edu/science/high-throughput-crystallization-center/analyzing-your-results/.
 The software is freely available. The Center itself does not share users 
images or data.


There are many examples of the images given in the publications listed on the 
crystallization research link to the Center, 
http://hwi.buffalo.edu/science/high-throughput-crystallization-center/crystallization-research/.
 The most up to date example is Luft, et al., (2015) Struct Dyn, 2, 041710 
which is open access. Images in that paper were recorded by the Crystallization 
Screening Center and typical of those obtained. Further details and other image 
examples are given in the references cited within. The Center encourages 
potential users to check the publications list at the link above to best 
understand the capabilities, effective use, and optimization strategies.


A complete data set involves crystallization metadata and 1,536 images before 
the sample is added, immediately after, one day after, one week, two weeks, 
three weeks, and four, five, and six weeks after. These images are color video 
microscope images (recently upgraded from B/W) and are supplemented with a set 
of SONICC and UV-TPEF images. I'll ask the Center to add a one of  the QC 
sample sets or one from the many PSI program samples to the website for those 
that are interested. In each set you'll have almost 17,000 images to play with 
but at least it will be a good demonstration of the analysis software!


I don't believe it's appropriate to use the CCP4 board as an advertising medium 
and I apologize for this posting that comes close to that. I'd request that 
questions about the crystallization screening service be directed to the 
contacts on the getacrystal.org page. I'm sure they'd be happy to answer any 
specifics and they would be a appropriate direct contacts to get image examples 
than the bulletin board in general.


Best wishes and many successful crystallizations,


Eddie.

Edward Snell Ph.D.
President and CEO Hauptman-Woodward Medical Research Institute
Assistant Prof. Department of Structural Biology, University at Buffalo
700 Ellicott Street, Buffalo, NY 14203-1102
hwi.buffalo.edu
Phone: (716) 898 8631 Fax: (716) 898 8660
Skype:  eddie.snell Email: 
esn...@hwi.buffalo.edu
[https://mail.google.com/mail/u/0/?ui=2=62aec2015e=fimg=15b6914f7fdf6013=0.1=emb=ANGjdJ9g06QLo-D_EH41Fh-YwWWc3MjxJbO2-5rUPyw9xaU5whcTtrcEMgp6IRAWj-flG5nAubbpuMSCQvS1Drhckx7xSgFjo3hKVAHILDzuOysZW5m7BjsDwfq3SJc=w656-h112=1492181842461=15b6914f7fdf6013=1]
Heisenberg was probably here!





From: Steven Herron 
Sent: Saturday, April 15, 2017 6:46 PM
To: Edward Snell; CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Recommendations for Robotic Crystal Screening Services


Does the Hauptman-Woodward Medical Research Institute have any sample 
data-sets, so we can see what the results look like?



On 4/14/2017 8:58 AM, Edward Snell wrote:

Dear Elizabeth,


The High-Throughput Crystallization Screening Center and the Hauptman-Woodward 
Medical Research Institute has been operating for over a decade with 
considerable success. There are comprehensive details at 
http://getacrystal.org but basically your samples are screened against a 
large range of commercially available conditions plus some more unique ones. 
Video microscope images are provided over a period of six weeks or longer by 
request and the imaging also includes SONICC (detecting very tiny crystals or 
crystals in precipitate) and UV-TPEF to ensure the sample is protein. There are 
a large range of analysis tools that can be used on the data. Much of the 
information is provided on the link above. Success rates are pretty high and 
many entries in the PDB have had their initial crystal hits there.


I hope this helps, the center does not advertise much but works with a lot of 
laboratories.


Cheers,


Eddie.

Edward Snell Ph.D.
President and CEO Hauptman-Woodward Medical Research Institute
Assistant Prof. Department of Structural Biology, University at Buffalo
700 Ellicott Street, Buffalo, NY 14203-1102
hwi.buffalo.edu
Phone: (716) 898 8631 Fax: (716) 898 8660
Skype:  eddie.snell Email: 
esn...@hwi.buffalo.edu

Re: [ccp4bb] in crystallo enzymatic activity

[Steven Herron]

Yes, the pH optimum of a reaction can be changed with a mutation of one 
of the catalytic residue or with a neighboring non-catalytic residue.  A 
classic example is the K166R mutation of mandelate racemase.

 see: Biochemistry. 1995 Mar 7;34(9):2788-97
 https://www.ncbi.nlm.nih.gov/pubmed/7893690
 http://pubs.acs.org/doi/pdf/10.1021/bi9a007

When lysine-166 is changed to arginine, one side of the reaction pH 
profile shifts to the basic.  If I remember correctly, the mutation 
causes a 2 pH unit shift of the basic side of the pH reaction profile.


Steven Herron
sherron_...@yahoo.com


On 4/14/2017 1:35 PM, Petri Kursula wrote:

Hi,

apart from the possibilities of conformational flexibility affected by 
crystal packing, just wondering if this mutation is supposed to 
actually cause an increase or decrease in the corresponding activity 
(outside the context of a crystal)? k(cat) and K(M) measured in 
solution would help here. Is the pH in the crystal far from the 
optimum of the wild-type protein? Can optimal pH change with the mutation?


Petri

Petri Kursula
--
Professor
Department of Biomedicine
University of Bergen, Norway
http://www.uib.no/en/rg/petrikursula 


petri.kurs...@uib.no 
--
Group Leader, Adjunct Professor
Faculty of Biochemistry and Molecular Medicine
University of Oulu, Finland
--



On 13 Apr 2017, at 17:27, Pierre Nioche 
> wrote:


Dear CCP4bb,

We work on an enzyme that we crystallized with two substrates bound 
in the active site (the reaction transform two substrates into two 
products). We have also the structure with the two products. We are 
able to see densities for the substrates when we collect data at 
different time point post-crystallization (days or weeks later). 
There is no change over time and no in crystallo enzymatic reaction 
despite the fact that in solution using the same crystallization 
solution, the reaction occurs readily.
This is not surprising and there are already many examples in the 
literature.
However, when we crystallize a single amino acid variant (mutant 
within the active site) with the same two substrates, we initially 
see the substrates but we then observe in crystallo enzymatic 
activity and formation of the final products over time. This 
structure is identical to the one determined with the two products 
co-crystallized with the enzyme. The crystal packing does not seem to 
be at play here.
I understand that in crystallo activities are well documented in the 
literature and can be induced by addition of ligands, X-rays, change 
in oxidative environment, etc?
Here, the substrates are present from the beginning of the 
crystallization experiments with the same concentration. Nothing is 
added to the crystals later on. Only the time differentiate the two 
type of crystals: after a couple of weeks, one has the substrates in 
the active site (wt) while the other has the products (variant).


Is anyone aware of similar examples where a variant induce in 
crystallo enzymatic activity without perturbation of the crystal?


Thanks,

Pierre
Dept of Pharmacology, Toxicology and cellular signaling
Paris Descartes University

Re: [ccp4bb] Recommendations for Robotic Crystal Screening Services

[Steven Herron]

Does the Hauptman-Woodward Medical Research Institute have any sample 
data-sets, so we can see what the results look like?




On 4/14/2017 8:58 AM, Edward Snell wrote:


Dear Elizabeth,


The High-Throughput Crystallization Screening Center and the 
Hauptman-Woodward Medical Research Institute has been operating for 
over a decade with considerable success. There are comprehensive 
details at http://getacrystal.org but basically your samples are 
screened against a large range of commercially available conditions 
plus some more unique ones. Video microscope images are provided over 
a period of six weeks or longer by request and the imaging also 
includes SONICC (detecting very tiny crystals or crystals in 
precipitate) and UV-TPEF to ensure the sample is protein. There are a 
large range of analysis tools that can be used on the data. Much of 
the information is provided on the link above. Success rates are 
pretty high and many entries in the PDB have had their initial crystal 
hits there.



I hope this helps, the center does not advertise much but works with a 
lot of laboratories.



Cheers,


Eddie.


Edward Snell Ph.D.

President and CEO Hauptman-Woodward Medical Research Institute

Assistant Prof. Department of Structural Biology, University at Buffalo

700 Ellicott Street, Buffalo, NY 14203-1102

hwi.buffalo.edu 

Phone: (716) 898 8631 Fax: (716) 898 8660

Skype: eddie.snell Email: esn...@hwi.buffalo.edu 



Heisenberg was probably here!




*From:* CCP4 bulletin board  on behalf of 
Elizabeth Diaz 

*Sent:* Friday, April 14, 2017 9:06 AM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* [ccp4bb] Recommendations for Robotic Crystal Screening 
Services

All,

I am currently attempting to crystallize a peptide/protein complex and 
am wanting to know about robotic screening services that you would 
recommend. We have done some in house conditions with little luck, and 
want to broaden our search, but that would require going externally 
for screening. Do you have any recommendations for robotic crystal 
screening services, preferably in the United States?


Thank you so much,

Elizabeth Diaz
University of Delaware

Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

[Eleanor Dodson]

Yes - convincing..
Follow Jacob's advice and see how the maps look.
Eleanor

On 15 April 2017 at 06:04, Alex Lee  wrote:

> Thanks Eleanor,
> If I understand right, there is just 1 TFZ. TFZ== means "Translation
> Function Z-score equivalent, only calculated for the top solution after
> refinement (or for the number of top files specified by TOPFILES)" so there
> could be many TFZ==.
>
> Twinning analysis attached below:
>
> TWINNING ANALYSIS:
>
>
> Global twinning statistics.
>
>
> These tests rely on the fact that it is highly improbably that very weak or 
> very strong reflections will coincide, therefore, the tails for the 
> distribution of twinned datasets will be less pronounced
>
>
> Data truncated to  67.93 -   3.50 A resolution
>
> $TABLE: Cumulative intensity distribution:
>
> $GRAPHS: Cumulative intensity distribution (Acentric and 
> centric):N:1,2,3,4,5,6:
>
> $$ Z Acent_theor Acent_twin Acent_obser Cent_theor Cent_obser $$
>
> $$
>
>0.0  0.0  0.0  0.01988  0.0   -
>
>0.04000  0.03921  0.00303  0.03225  0.15852   -
>
>0.08000  0.07688  0.01151  0.04745  0.22270   -
>
>0.12000  0.11308  0.02458  0.06655  0.27097   -
>
>0.16000  0.14786  0.04148  0.09008  0.31084   -
>
>0.2  0.18127  0.06155  0.11562  0.34528   -
>
>0.24000  0.21337  0.08420  0.14170  0.37579   -
>
>0.28000  0.24422  0.10891  0.16750  0.40330   -
>
>0.32000  0.27385  0.13524  0.19450  0.42839   -
>
>0.36000  0.30232  0.16279  0.22295  0.45149   -
>
>0.4  0.32968  0.19121  0.25213  0.47291   -
>
>0.44000  0.35596  0.22021  0.28103  0.49288   -
>
>0.48000  0.38122  0.24953  0.30678  0.51158   -
>
>0.52000  0.40548  0.27895  0.33697  0.52916   -
>
>0.56000  0.42879  0.30829  0.36569  0.54574   -
>
>0.6  0.45119  0.33737  0.39332  0.56142   -
>
>0.64000  0.47271  0.36607  0.41967  0.57629   -
>
>0.68000  0.49338  0.39428  0.44513  0.59041   -
>
>0.72000  0.51325  0.42190  0.47060  0.60386   -
>
>0.76000  0.53233  0.44885  0.49430  0.61667   -
>
>0.8  0.55067  0.47507  0.51550  0.62891   -
>
>0.84000  0.56829  0.50052  0.53736  0.64060   -
>
>0.88000  0.58522  0.52516  0.55780  0.65180   -
>
>0.92000  0.60148  0.54896  0.57822  0.66253   -
>
>0.96000  0.61711  0.57191  0.59779  0.67281   -
>
>1.0  0.63212  0.59399  0.61638  0.68269   -
>
>1.04000  0.64655  0.61521  0.63496  0.69218   -
>
>1.08000  0.66040  0.63557  0.65120  0.70130   -
>
>1.12000  0.67372  0.65507  0.66731  0.71008   -
>
>1.16000  0.68651  0.67373  0.68115  0.71853   -
>
>1.2  0.69881  0.69156  0.69557  0.72668   -
>
>1.24000  0.71062  0.70857  0.70990  0.73453   -
>
>1.28000  0.72196  0.72480  0.72386  0.74210   -
>
>1.32000  0.73286  0.74025  0.73801  0.74941   -
>
>1.36000  0.74334  0.75495  0.74895  0.75646   -
>
>1.4  0.75340  0.76892  0.75913  0.76328   -
>
>1.44000  0.76307  0.78220  0.77024  0.76986   -
>
>1.48000  0.77236  0.79480  0.78058  0.77623   -
>
>1.52000  0.78129  0.80675  0.79061  0.78238   -
>
>1.56000  0.78986  0.81807  0.79977  0.78833   -
>
>1.6  0.79810  0.82880  0.80902  0.79410   -
>
>1.64000  0.80602  0.83895  0.81795  0.79967   -
>
>1.68000  0.81363  0.84855  0.82691  0.80508   -
>
>1.72000  0.82093  0.85763  0.83511  0.81031   -
>
>1.76000  0.82796  0.86621  0.84245  0.81538   -
>
>1.8  0.83470  0.87431  0.85141  0.82029   -
>
>1.84000  0.84118  0.88196  0.85966  0.82505   -
>
>1.88000  0.84741  0.88917  0.86721  0.82967   -
>
>1.92000  0.85339  0.89597  0.87415  0.83414   -
>
>1.96000  0.85914  0.90238  0.87977  0.83849   -
>
>2.0  0.86466  0.90842  0.88629  0.84270   -
>
> $$
>
>
>
> The culmulative intensity, N(Z), plot is diagnostic for both twinning and 
> tNCS.  For twinned data there are fewer weak reflections, therefore, N(Z) is 
> sigmoidal for twinned data.  However, if both twinning and tNCS are present, 
> the effects may cancel each out. Therefore the results of the L-test and 
> patterson test should be consulted
>
>
>
> L test for twinning: (Padilla and Yeates Acta Cryst. D59 1124 (2003))
>
> L statistic =  0.416  (untwinned 0.5 perfect twin 0.375)
>
> Data has used to  67.93 -   3.50 A resolution
>
>Relation between L statistics and twinning fraction:
>
>   Twinning fraction = 0.000  L statistics = 0.500:
>
>   Twinning fraction = 0.100  L statistics = 0.440:
>
>   Twinning fraction = 0.500  L statistics = 0.375:
>
>
> The L test suggests data is twinned
>
> All data regardless of I/sigma(I) has been included in the L test
>
>
>
> $TABLE: L test for twinning:
>
> $GRAPHS: cumulative distribution function for |L|, twin fraction