Re: [ccp4bb] Sulphur SAD at home source

[Kay Diederichs]

Hi Manoj,

it depends on crystal and hardware (detector, beam, goniometer) quality as well 
as on the correct strategy whether you will be able to solve the structure with 
sulfur SAD. 6 sulfur in 12 kDa should be doable, if the crystals diffract to 
2.5A or better. Others have given good advice or links on these topics.

My only addition is: you should not assume that there is no radiation damage on 
the home source, rather, it is very noticeable - see e.g. Sarma and Karplus, 
"In-house sulfur SAD phasing: a case study of the effects of data quality and 
resolution cutoffs" Acta Cryst. (2006). D62, 707–716. This means: 
high-multiplicity data from a single crystal are not guaranteed to be 
isomorphous. If you have many crystals, however, you could merge several data 
sets.

best wishes,

Kay

On Tue, 3 Apr 2018 10:26:50 -0400, Manoj Saxena  wrote:

>Hi All,
>
>I am writing to seek advice on doing  sulphur SAD data collection
>at Cu based home source for a protein that is 12 KDa and has 6 S atoms.
>I have seen some links online and some references but would be grateful if
>you can share your know-how for success with this.
>Like what multiplicity of data would be good to aim for and
>data processing tips.
>Inputs from people who have tried and failed would also be highly
>appreciated.
>
>Thank you
>Manoj Saxena
>University of Puerto Rico
>

Re: [ccp4bb] Sulphur SAD at home source

[Joseph Ferrara]

Jim Pflugrath and a couple of high school interns shot a short video on halide 
quick soaks a few years back. You can find it at 
https://www.youtube.com/watch?v=45Qc3jOPaKY.

Cheers,

Joseph D. Ferrara, Ph.D.
CSO
Deputy Director, X-ray Research Laboratory
Vice President, American Crystallographic Association

Rigaku Corporation
9009 New Trails Drive
The Woodlands, TX 77381
Tel: 281-362-2300 x 168
Skype: xrayjoe
url: www.rigaku.com



From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger 
Rowlett
Sent: Tuesday, April 03, 2018 9:57 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Sulphur SAD at home source

Iodine is ideally suited for Cu K-alpha SAD phasing, and iodide ions can 
normally be easily added by soaking crystals in potassium iodide containing 
solutions, which can be done at the time of cryopreservation. A quick lit 
search will turn up the appropriate protocols. For structural genomics work 
where MR was unsuccessful or unusable, iodide soaks were found to work as much 
as 80% of the time.

I've used iodide-soaked lysozyme for an XRD teaching lab and undergraduate 
research student training, and SAD phasing works really well on an overnight 
data collection on our Oxford Diffraction PX-ultra system. It's worth a shot, 
and very easy to do. Many proteins will tolerate soaking, especially if 
crystallized from salts.
___
Roger S. Rowlett
Gordon & Dorothy Kline Professor Emeritus
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<mailto:rrowl...@colgate.edu>
On 4/3/2018 10:46 AM, Eleanor Dodson wrote:
Well - the S f" is only ~ 0.5 at Cu Kalpha so the signal will be very weak..
Very accurate data may get a solution but you first have to position the S 
atoms...
Much easier to try to make a heavy atom derivative!
Eleanor

On 3 April 2018 at 15:26, Manoj Saxena 
<1d16aa30e8a1-dmarc-requ...@jiscmail.ac.uk<mailto:1d16aa30e8a1-dmarc-requ...@jiscmail.ac.uk>>
 wrote:
Hi All,

I am writing to seek advice on doing  sulphur SAD data collection
at Cu based home source for a protein that is 12 KDa and has 6 S atoms.
I have seen some links online and some references but would be grateful if
you can share your know-how for success with this.
Like what multiplicity of data would be good to aim for and
data processing tips.
Inputs from people who have tried and failed would also be highly appreciated.

Thank you
Manoj Saxena
University of Puerto Rico

Re: [ccp4bb] Sulphur SAD at home source

[Andreas Förster]

Dear Manoj,

I'm happy to answer your questions and have a look at your data.  Maybe we
can take this off-list and you report back if and when you solve your
structure.

By the way, I'm processing native data collected with a MetalJet at the
moment.  The anomalous signal is even weaker at 1.35 Å but can be useful.

All best.


Andreas



On Tue, Apr 3, 2018 at 5:36 PM, Manoj Saxena  wrote:

> Thank you!, Andreas and all others who replied.
>  I have seen your tutorial and that's what I was referring to.
> Maybe I can consult you later when I screen some samples and have an idea
> of signal strength?
>
> Regards
> Manoj
>
>
> 2018-04-03 10:55 GMT-04:00 Andreas Förster :
>
>> Dear Manoj,
>>
>> providing your crystals diffract to at least 2.5 Å and the sulfurs are
>> ordered, you should be able to solve your structure without too many
>> problems.  As a first experiment, I would recommend collecting 360 degrees
>> of data.  Process these with XDS and the FRIEDEL'S_LAW= FALSE option and
>> see how much anomalous signal CORRECT.LP reports, if you've got stars in
>> the values of the Anomal Corr column, you're on the right track.  Try to
>> phase with SHELXD, following the advice on http://shelx.uni-goettingen
>> .de/tutorials.php.
>>
>> If you don't succeed, you might need higher multiplicity.  If you have a
>> kappa or chi goniometer, you can reorient the crystal and collect another
>> 360 degrees and another 360 degrees, and so on.  Merge all data in XSCALE
>> until you can solve the structure.
>>
>> I once wrote a little tutorial for doing S-SAD on the home source that
>> you might want to follow.  http://www.imperial.ac.uk/x-ra
>> y-crystallography/learning-more/sulphur-sad/  With an HPC detector (or
>> even an image plate) you'd getter better data than with a CCD detector
>> and be able to solve your structure quicker.  (Disclaimer: I work for a
>> company that makes HPC detectors.)  I consider the home source a great
>> place to solve structures by native SAD.
>>
>> All best.
>>
>>
>> Andreas
>>
>>
>>
>> On Tue, Apr 3, 2018 at 4:26 PM, Manoj Saxena <
>> 1d16aa30e8a1-dmarc-requ...@jiscmail.ac.uk> wrote:
>>
>>> Hi All,
>>>
>>> I am writing to seek advice on doing  sulphur SAD data collection
>>> at Cu based home source for a protein that is 12 KDa and has 6 S atoms.
>>> I have seen some links online and some references but would be grateful
>>> if
>>> you can share your know-how for success with this.
>>> Like what multiplicity of data would be good to aim for and
>>> data processing tips.
>>> Inputs from people who have tried and failed would also be highly
>>> appreciated.
>>>
>>> Thank you
>>> Manoj Saxena
>>> University of Puerto Rico
>>>
>>>
>>>
>>
>>
>> --
>> 
>> Andreas Förster, Ph.D.
>> MX Application Scientist, Scientific Sales
>> Phone: +41 56 500 21 00 <+41%2056%20500%2021%2000> | Direct: +41 56 500
>> 21 76 <+41%2056%20500%2021%2076> | Email: andreas.foers...@dectris.com
>> DECTRIS Ltd. | Taefernweg 1
>> 
>>
>> |
>> 5405 Baden-Daettwil
>> 
>>
>> 
>> | Switz
>> 
>> erland
>> 
>>
>> 
>> |
>> 
>> www.dectris.com
>>
>> [image: LinkedIn] 
>>  [image: facebook]
>> 
>> 
>>
>>
>>
>>
>>
>> Confidentiality Note: This message is intended only for the use of the
>> named
>> recipient(s) and may contain confidential and/or privileged information. If
>> you
>> are not the intended recipient, please contact the sender and delete the
>> message.
>> Any unauthorized use of the information contained in this message is
>> prohibited.
>>
>>
>>
>


-- 

Andreas Förster, Ph.D.
MX Application Scientist, Scientific Sales
Phone: +41 56 500 21 00 | Direct: +41 56 500 21 76 | Email:
andreas.foers...@dectris.com
DECTRIS Ltd. | Taefernweg 1 | 5405 Baden-Daettwil | Switzerland |
www.dectris.com

[image: LinkedIn] 
 [image: facebook]

Re: [ccp4bb] Sulphur SAD at home source

[Raghurama P Hegde]

Hi Manoj,

Like Eleanor has pointed out a very accurate data may get you a solution. If 
you have crystals that diffract well and give you data with high I/sigma(I) you 
can attempt to find the positions of the S atoms and use that to arrive at a 
structure. Using lysozyme as a test case we have shown that structure can be 
solved from a routine data set collected at 1 Å (with 10 S but a f” of ~0.28), 
with anomalous multiplicity ~12. Details are published in this paper (yes it’s 
a shameless plug for our paper! ):

Hegde et al. (2017), The hidden treasure in your data: phasing with unexpected 
weak anomalous scatterers from routine data sets, Acta Crystallogr F Struct 
Biol Commun, 73, 184-195.
http://scripts.iucr.org/cgi-bin/paper?S2053230X17002680.

However the data was not collected at a home source but at a synchrotron 
beamline. There could also be some weak anomalous scatterers acquired during 
crystallization, from the crystallization condition, so you could collect a 
data set to check for the presence of anomalous signal in it and attempt 
experimental phasing. That could give you pointers on a future course of action 
like going to a synchrotron beamline to collect stronger, more accurate data or 
prepare heavy atom derivates, like Eleanor has pointed out.

As for data processing you’d have to process the data with Friedel mates pairs 
kept separate so that the anomalous differences can be used to find positions 
of the S atoms.

Hope that helps,
Raghu

From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> On Behalf Of Eleanor Dodson
Sent: Tuesday, April 3, 2018 20:17
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Sulphur SAD at home source

Well - the S f" is only ~ 0.5 at Cu Kalpha so the signal will be very weak..
Very accurate data may get a solution but you first have to position the S 
atoms...
Much easier to try to make a heavy atom derivative!
Eleanor

On 3 April 2018 at 15:26, Manoj Saxena 
<1d16aa30e8a1-dmarc-requ...@jiscmail.ac.uk<mailto:1d16aa30e8a1-dmarc-requ...@jiscmail.ac.uk>>
 wrote:
Hi All,

I am writing to seek advice on doing  sulphur SAD data collection
at Cu based home source for a protein that is 12 KDa and has 6 S atoms.
I have seen some links online and some references but would be grateful if
you can share your know-how for success with this.
Like what multiplicity of data would be good to aim for and
data processing tips.
Inputs from people who have tried and failed would also be highly appreciated.

Thank you
Manoj Saxena
University of Puerto Rico

Re: [ccp4bb] Sulphur SAD at home source

[Roger Rowlett]

Iodine is ideally suited for Cu K-alpha SAD phasing, and iodide ions can 
normally be easily added by soaking crystals in potassium iodide 
containing solutions, which can be done at the time of cryopreservation. 
A quick lit search will turn up the appropriate protocols. For 
structural genomics work where MR was unsuccessful or unusable, iodide 
soaks were found to work as much as 80% of the time.


I've used iodide-soaked lysozyme for an XRD teaching lab and 
undergraduate research student training, and SAD phasing works really 
well on an overnight data collection on our Oxford Diffraction PX-ultra 
system. It's worth a shot, and very easy to do. Many proteins will 
tolerate soaking, especially if crystallized from salts.

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor Emeritus
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 4/3/2018 10:46 AM, Eleanor Dodson wrote:
Well - the S f" is only ~ 0.5 at Cu Kalpha so the signal will be very 
weak..
Very accurate data may get a solution but you first have to position 
the S atoms...


Much easier to try to make a heavy atom derivative!

Eleanor

On 3 April 2018 at 15:26, Manoj Saxena 
<1d16aa30e8a1-dmarc-requ...@jiscmail.ac.uk 
> wrote:


Hi All,

I am writing to seek advice on doing  sulphur SAD data collection
at Cu based home source for a protein that is 12 KDa and has 6 S
atoms.
I have seen some links online and some references but would be
grateful if
you can share your know-how for success with this.
Like what multiplicity of data would be good to aim for and
data processing tips.
Inputs from people who have tried and failed would also be highly
appreciated.

Thank you
Manoj Saxena
University of Puerto Rico

Re: [ccp4bb] Sulphur SAD at home source

[Andreas Förster]

Dear Manoj,

providing your crystals diffract to at least 2.5 Å and the sulfurs are
ordered, you should be able to solve your structure without too many
problems.  As a first experiment, I would recommend collecting 360 degrees
of data.  Process these with XDS and the FRIEDEL'S_LAW= FALSE option and
see how much anomalous signal CORRECT.LP reports, if you've got stars in
the values of the Anomal Corr column, you're on the right track.  Try to
phase with SHELXD, following the advice on http://shelx.uni-goettingen
.de/tutorials.php.

If you don't succeed, you might need higher multiplicity.  If you have a
kappa or chi goniometer, you can reorient the crystal and collect another
360 degrees and another 360 degrees, and so on.  Merge all data in XSCALE
until you can solve the structure.

I once wrote a little tutorial for doing S-SAD on the home source that you
might want to follow.  http://www.imperial.ac.uk/x-
ray-crystallography/learning-more/sulphur-sad/  With an HPC detector (or
even an image plate) you'd getter better data than with a CCD detector and
be able to solve your structure quicker.  (Disclaimer: I work for a company
that makes HPC detectors.)  I consider the home source a great place to
solve structures by native SAD.

All best.


Andreas



On Tue, Apr 3, 2018 at 4:26 PM, Manoj Saxena <1d16aa30e8a1-dmarc-reques
t...@jiscmail.ac.uk> wrote:

> Hi All,
>
> I am writing to seek advice on doing  sulphur SAD data collection
> at Cu based home source for a protein that is 12 KDa and has 6 S atoms.
> I have seen some links online and some references but would be grateful if
> you can share your know-how for success with this.
> Like what multiplicity of data would be good to aim for and
> data processing tips.
> Inputs from people who have tried and failed would also be highly
> appreciated.
>
> Thank you
> Manoj Saxena
> University of Puerto Rico
>
>
>


-- 

Andreas Förster, Ph.D.
MX Application Scientist, Scientific Sales
Phone: +41 56 500 21 00 <+41%2056%20500%2021%2000> | Direct: +41 56 500 21
76 <+41%2056%20500%2021%2076> | Email: andreas.foers...@dectris.com
DECTRIS Ltd. | Taefernweg 1 | 5405 Baden-Daettwil | Switzerland |
www.dectris.com

[image: LinkedIn] 
 [image: facebook]







Confidentiality Note: This message is intended only for the use of the
named
recipient(s) and may contain confidential and/or privileged information. If
you
are not the intended recipient, please contact the sender and delete the
message.
Any unauthorized use of the information contained in this message is
prohibited.

Re: [ccp4bb] Sulphur SAD at home source

[Eleanor Dodson]

Well - the S f" is only ~ 0.5 at Cu Kalpha so the signal will be very
weak..
Very accurate data may get a solution but you first have to position the S
atoms...

Much easier to try to make a heavy atom derivative!

Eleanor

On 3 April 2018 at 15:26, Manoj Saxena <
1d16aa30e8a1-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hi All,
>
> I am writing to seek advice on doing  sulphur SAD data collection
> at Cu based home source for a protein that is 12 KDa and has 6 S atoms.
> I have seen some links online and some references but would be grateful if
> you can share your know-how for success with this.
> Like what multiplicity of data would be good to aim for and
> data processing tips.
> Inputs from people who have tried and failed would also be highly
> appreciated.
>
> Thank you
> Manoj Saxena
> University of Puerto Rico
>
>
>

[ccp4bb] Sulphur SAD at home source

[Manoj Saxena]

Hi All,

I am writing to seek advice on doing  sulphur SAD data collection
at Cu based home source for a protein that is 12 KDa and has 6 S atoms.
I have seen some links online and some references but would be grateful if
you can share your know-how for success with this.
Like what multiplicity of data would be good to aim for and
data processing tips.
Inputs from people who have tried and failed would also be highly
appreciated.

Thank you
Manoj Saxena
University of Puerto Rico