[ccp4bb] bioassay g-csf

[megha goyal]

we perform bioassay of g-csf using m-nfs-60 cell lines but we do not get
reproducible results. some time a sample fails and on repeating the assay
for same sample it passes. we do not get proper gradation too. can someone
please provide me a protocol for g-csf bioassay that has been successfully
used. please help me.

[ccp4bb] detectors on home sources

[Fareed Aboul-Ela]

Thanks to all who generously shared your experiences with Pilatus and CMOS
detectors with me, both on and offline.  I’ve compiled the online responses
here, but given that the vast majority of the responses were offline I’ll
attempt to briefly summarize what I learned.



First no one has had either of these detectors at their home source for a
very long time (> ~1 year).  But, the good news is that there are no
complaints so far. In fact, I only received positive reports from users of
both detectors, especially the Pilatus.  One caution, though, none of the
Pilatus users have yet used the version (200K) which Rigaku are now
offering as standard on their instruments.



There is skepticism about whether advantages in sensitivity and dynamic
range for the Pilatus will be noticeable in a home source. Rather, the beam
intensity may be the limiting factor even for some of the microfocus
rotating anode sources. Another point was raised about the large pixel
size. There is a gap in the detector and one user suggested that a triple
axis goniometer may be necessary to generate the redundancy to compensate.



Agilent have a poster which I’m sure they will be happy to share showing
sensitivity on their ATLAS CCD superior to that of a CMOS detector.
Nonetheless, those with experience using either Photon-100 or Pilatus 200k
detectors at home were very happy with the shutter-less data collection.
Another advantage is that the currently offered versions of these detectors
do not require cooling water. So, with respect to maintenance both seem to
be easier that CCD detectors.



So, overall it’s early days and there is skepticism, but, so far the actual
users seem to have no complaints.



We still haven’t found anyone who has used the Bruker TXS microfocus
rotating anode at home. I think we’re going to have to find forums for
small molecule crystallographers.



Many thanks again.



Fareed



Date:Thu, 2 May 2013 16:04:56 +0200
From:Dworkowski Florian 
Subject: Re: detectors on home sources

I can not really speak for a home-source setup, but here at the SLS MX
group we run two Pilatus6M (one of which is the first Pilatus ever made)
and one Pilatus2M. In nearly four years since we installed the first one
we never had any hardware issue other than users damaging the case. So
I'd say the reliability of the DECTRIS detectors is excellent.

Cheers,
Florian

-
Paul Scherrer Institut
Dr. Florian Dworkowski
Beamline Scientist X10SA

Swiss Light Source
WSLA/219
5232 Villigen PSI
Switzerland

Phone +41 56 310 3584
Fax +41 56 310 5292
florian.dworkow...@psi.ch
http://www.psi.ch/macromolecular-crystallography
http://www.psi.ch/macromolecular-crystallography>



 On Tue, Apr 30, 2013 at 11:44 PM,  wrote:

>  I second this opinion. At the end of next week our Pilatus 200K will be
> delivered. Soon after that I will be able to report on its characterist
> ics.
>
> But really, Boaz nailed it: reliability and service are very important.
> It does not matter how good something is on paper if you cannot keep it
> running. And with this e-mail I think it is clear what my recommendation
> was to our department and I am pleased that the recommendation was
> followed. Exactly as Boaz suggests, it was based in significant part on
consideration
> of reliability and quality of service. It is to be noted that reliability
> of instruments and quality of service could vary from region to region,
> that is, good service in the US may and may not translate to good service
> elsewhere. It would be good to do a "regional poll" for this.
>
> Having said all this, it is my impression that the newer technology has
> fewer moving parts and therefore should be expected to be more reliable.
> But I don't know that for sure, please ask again in 3-5 years. :-)
>
> Mark
>
>
>  -Original Message-
> From: Boaz Shaanan 
> To: CCP4BB 
> Sent: Tue, Apr 30, 2013 2:53 pm
> Subject: Re: [ccp4bb] detectors on home sources
>
>  One of the main things (if not THE main thing) to worry about when
> investing in such expensive equipment is long-time reliability and quality
> of service in your place. Nothing is more frustrating than seeing your
> wonderful and expensive equipment standing idle for long periods because
of
> lack of service. This may mean quite often taking compromises and going
> perhaps not for the front-line state-of-art piece of equipment but rather
> for the sturdy, hard-working equipment. It worked for us very well.
>
>  My 2p advice.
>
>Boaz
>
>
>
> *Boaz Shaanan, Ph.D.
> Dept. of Life Sciences
> Ben-Gurion University of the Negev
> Beer-Sheva 84105
> Israel
>
> E-mail: bshaa...@bgu.ac.il
> Phone: 972-8-647-2220  Skype: boaz.shaanan
> Fax:   972-8-647-2992 or 972-8-646-1710*
> **
> **
> *
>
> *
>   --
> *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Fareed
> Aboul-Ela [faboul...@zewailcity.edu.eg]
> *Sent:* Tue

[ccp4bb] Cauliflower looking crystals

[Browning Christopher]

Hi Everybody,

I was wondering if anybody had a similar case to mine. The protein I'm working 
on is a tough one. Its around 75 KDa in size, and it purifies well, but soon 
after the full length protein starts to autolyse. Consequently, I don't get any 
hits after setting up the screens.

To get around this I digest the protein with trypsin and end up with a stable 
fragment of around 25KDa. When I screen this I get many hits, but none of them 
look like something that can be made into single crystals. All the hits give me 
large lobe-like clusters and they look like cauliflower heads but they are 
optically active. I currently have the protein in 300mM NaCl buffer. Would 
dropping or increasing the salt or even using a different salt make any 
difference? Any ideas?

Cheers,

Chris

Re: [ccp4bb] Cauliflower looking crystals

[Evgeny Osipov]

Hi, Chris,
My coworkers have the same problem with the ubiquitin-like protein. 
Sadly, they still can't overcome this issue.
First, about autolysation: do your protein contain an any metal ions? 
May be you should add EDTA to the protein buffer. Are you sure about 
protein homogeneity? Is it moves as single band on SDS-PAGE?


If you generally prefer to work with 25 kDa protein:
1) try seeding!
2) what is common for all crystallization conditions? Are they PEG or 
salt based? If PEG-based, then consider low salt buffer.
3) Use excessive purification: after gel filtration discard fractions 
below 50-70% of the max UV absorption peak. Of course yield will drop 
considerably, but it may worth it


06.05.2013 14:17, Browning Christopher ?:

Hi Everybody,

I was wondering if anybody had a similar case to mine. The protein I'm 
working on is a tough one. Its around 75 KDa in size, and it purifies 
well, but soon after the full length protein starts to autolyse. 
Consequently, I don't get any hits after setting up the screens.


To get around this I digest the protein with trypsin and end up with a 
stable fragment of around 25KDa. When I screen this I get many hits, 
but none of them look like something that can be made into single 
crystals. All the hits give me large lobe-like clusters and they look 
like cauliflower heads but they are optically active. I currently have 
the protein in 300mM NaCl buffer. Would dropping or increasing the 
salt or even using a different salt make any difference? Any ideas?


Cheers,

Chris



--
Eugene Osipov
Junior Research Scientist
Laboratory of Enzyme Engineering
A.N. Bach Institute of Biochemistry
Russian Academy of Sciences
Leninsky pr. 33, 119071 Moscow, Russia
e-mail: e.m.osi...@gmail.com

Re: [ccp4bb] Cauliflower looking crystals

[David Schuller]

I haven't seen cauliflower but I have seen brussel sprouts. Condtions 
(esp. pH) were changing over the course of crystallization, and the 
stalks and sprouts were different crystal forms.


On 05/06/13 06:17, Browning Christopher wrote:

Hi Everybody,

I was wondering if anybody had a similar case to mine. The protein I'm 
working on is a tough one. Its around 75 KDa in size, and it purifies 
well, but soon after the full length protein starts to autolyse. 
Consequently, I don't get any hits after setting up the screens.


To get around this I digest the protein with trypsin and end up with a 
stable fragment of around 25KDa. When I screen this I get many hits, 
but none of them look like something that can be made into single 
crystals. All the hits give me large lobe-like clusters and they look 
like cauliflower heads but they are optically active. I currently have 
the protein in 300mM NaCl buffer. Would dropping or increasing the 
salt or even using a different salt make any difference? Any ideas?


Cheers,

Chris



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

[ccp4bb] PILATUS data collection

[Theresa Hsu]

Dear crystallographers

Is there a good source/review/software to obtain tips for good data collection 
strategy using PILATUS detectors at synchrotron? Do we need to collect sweeps 
of high and low resolution data separately? For anomalous phasing (MAD), does 
the order of wavelengths used affect structure solution or limit radiation 
damage?

Thank you.

Theresa

Re: [ccp4bb] PILATUS data collection

[jan]

Hi Theresa,
one issue I noticed during -admittedly few- collections on PILATUS detectors, 
are low symmetry space groups and perfectly centered detectors. The many seams 
of the detector left us with <90% complete triclinic data sets. A slightly 
imperfect centering of the detector or collections at different distances 
should remedy that.

Cheers,
Jan

--
Jan Abendroth
Emerald BioStructures
Seattle / Bainbridge Island WA, USA
home: Jan.Abendroth_at_gmail.com
work: JAbendroth_at_embios.com
http://www.emeraldbiostructures.com

On May 6, 2013, at 4:04 PM, Theresa Hsu  wrote:

> Dear crystallographers
> 
> Is there a good source/review/software to obtain tips for good data 
> collection strategy using PILATUS detectors at synchrotron? Do we need to 
> collect sweeps of high and low resolution data separately? For anomalous 
> phasing (MAD), does the order of wavelengths used affect structure solution 
> or limit radiation damage?
> 
> Thank you.
> 
> Theresa

Re: [ccp4bb] PILATUS data collection

[Robert Sweet]

The seminal paper on actually how to collect data from detectors like this 
and others with negligible read-out time is this one, which I strongly 
recommend:


Optimal Fine phi-slicing for Single-Photon-Counting Pixel Detectors Marcus 
Mueller, Meitian Wang, and Clemens Schulze-Briese, Acta Cryst.(2012) D68, 
42-56


And you can pick up a copy of the paper from the RapiData web site: 
http://www.px.nsls.bnl.gov/courses/papers/Mueller_ACD68_2012.pdf



The classic paper on data-collection strategies is this:

Data-Collection Stragegies, Dauter, Z. Acta Cryst. (1999). D55, 1703-1717.

Also available from the RapiData site:

http://www.px.nsls.bnl.gov/courses/papers/dauter_strategy.pdf


Then there are multiple papers on damage and its impact on your data. I 
suggest this one:


Radiation damage in macromolecular crystallography: what is it and why 
should we care?, E.Garman, Acta Cryst. D66, 339-351(2010).


which you can find here:

http://www.px.nsls.bnl.gov/courses/papers/actad-garman-2010.pdf

With this under your belt you'll be able to decide how to collect your 
phasing data.  The bottom line is probably that you should go for SAD 
data.  Employ multiple crystals and average them together in a judicious 
way, keeping only the sweeps from barely damaged x-tals.


Good luck,

Bob Sweet

=
Robert M. Sweet E-Dress: sw...@bnl.gov
Group Leader, PXRR: Macromolecular   ^ (that's L
  Crystallography Research Resource at NSLSnot 1)
  http://px.nsls.bnl.gov/
Photon Sciences and Biosciences Dept
Office and mail, Bldg 745, a.k.a. LOB-5
Brookhaven Nat'l Lab.   Phones:
Upton, NY  11973631 344 3401  (Office)
U.S.A.  631 344 2741  (Facsimile)
=

On Mon, 6 May 2013, Theresa Hsu wrote:


Dear crystallographers

Is there a good source/review/software to obtain tips for good data 
collection strategy using PILATUS detectors at synchrotron? Do we need 
to collect sweeps of high and low resolution data separately? For 
anomalous phasing (MAD), does the order of wavelengths used affect 
structure solution or limit radiation damage?


Thank you.

Theresa

[ccp4bb] A crystallographer on Mars

[Ethan Merritt]

The _New Yorker_ frequently publishes decently written articles on a
huge variety of topics.  Occasionally they come out with one about
science, sometimes with a focus on a public policy issue, sometimes a
biographical piece about a mainstream or not-so-mainstream scientist,
sometimes a serialized first publication of a book by a scientist
written for a wide audience.  So I was not terribly surprised to find
in the 22 April issue an article about the Mars rover Curiosity and
the team that designed it.  

Spread across two pages in the center of the issue was a color image
of the Curiosity rover itself.  Just the thing to inspire creative use 
of one's Lego collection
.

But then it got a bit strange.  The caption reads:

  "... the mission includes a nuclear-powered mobile laboratory,
  equipped with lasers, spectrometers, and an X-ray crystallographer".

Wow!  Who's the lucky Mars-going crystallographer?  Anyone we know?

The article text goes to quote one of the JPL rover team members:

  "Curiosity came equipped with lasers, spectrometers, and a gas
  chromatograph. It had a radiation detector, an X-ray crystallographer,
  and a complete weather station. [...] It was like a Hummer with a
  half-dozen scientists crammed inside".  

OK, so at least our lucky crystallographic colleague has some company
out there on Mars.  Still, I do wonder exactly what it said in the
job ad they responded to. Anyone know what sort of X-ray source they
packed with them?

Ethan

Re: [ccp4bb] A crystallographer on Mars

[Frank von Delft]

A 3rd generation synchrotron, surely. Unless she's happy to use FedEx 
(do you think Customs in space ports are as anal as their terrestrial 
counterparts?)



On 07/05/2013 06:00, Ethan Merritt wrote:

The _New Yorker_ frequently publishes decently written articles on a
huge variety of topics.  Occasionally they come out with one about
science, sometimes with a focus on a public policy issue, sometimes a
biographical piece about a mainstream or not-so-mainstream scientist,
sometimes a serialized first publication of a book by a scientist
written for a wide audience.  So I was not terribly surprised to find
in the 22 April issue an article about the Mars rover Curiosity and
the team that designed it.

Spread across two pages in the center of the issue was a color image
of the Curiosity rover itself.  Just the thing to inspire creative use
of one's Lego collection
.

But then it got a bit strange.  The caption reads:

   "... the mission includes a nuclear-powered mobile laboratory,
   equipped with lasers, spectrometers, and an X-ray crystallographer".

Wow!  Who's the lucky Mars-going crystallographer?  Anyone we know?

The article text goes to quote one of the JPL rover team members:

   "Curiosity came equipped with lasers, spectrometers, and a gas
   chromatograph. It had a radiation detector, an X-ray crystallographer,
   and a complete weather station. [...] It was like a Hummer with a
   half-dozen scientists crammed inside".

OK, so at least our lucky crystallographic colleague has some company
out there on Mars.  Still, I do wonder exactly what it said in the
job ad they responded to. Anyone know what sort of X-ray source they
packed with them?

Ethan