Re: [ccp4bb] How to identify unknow heavy atom??

[Frank Murphy]

Dear Haytham,

It should be relatively simple (and quick) to determine the metal in your 
sample employing either crystallized sample or solution using X-ray 
fluorescence (http://en.wikipedia.org/wiki/X-ray_fluorescence) at any beamline 
with a silicon drift detector (http://www.amptek.com/drift.html) set up for 
energy dispersive spectrometry (EDS). 

We perform these analyses regularly at NE-CAT and I am sure there are a number 
of other beamlines set up for the experiment.

Frank Murphy
Beamline Scientist
NE-CAT / Cornell University
frankvmur...@gmail.com

Re: [ccp4bb] How to identify unknow heavy atom??

[Marco Mazzorana]

Dear Haytham,
may I address your points (although some nice hints have already come
from previous replies)

1- if i have anomalous peak of unknown heavy atom, How can i identify
this heavy atom in general. (different methods)

To see anomalous peak I guess you have done the experiment at a
synchrotron (Cu and Zn do not show much anomalous if measured at home
sources). It is always good practice to run some fluorescence scan on
a new sample (say, a novel protein). This can be done at all tunable
beamlines (you might be able to have a broad scan of the entire range
of wavelength, such as an MCA scan first. Once you have found a region
of intrerest you can do a proper energy scan, exactly as if you were
looking for peak remote and inflection wavelengths for a MAD
experiment).
XANES (the proper absorption peak scan) and EXAFS are complementary
techniques. They can be performed in solution at dedicated beamlines
and they can provide precious information on the first and second
coordination spheres of the metal, but for the elemental recognition
it might be sufficient what you measure from your crystal.
Additional information can come from the coordination geometry of your
ion, as mentioned above.

2- in my case, i see anomalous peak in heavy atom binding site
(without any soaking). preliminary i did mass spec. i got Zn++ and Cu,
How can i know which one give the anomalous peak in my protein.

They might both give you anomalous signal... if you are working around
12.5 keV (near the Se edge), both metals have anomalous signal of 2-3
electrons... if you have only 1 heavy atom binding site you might
consider collecting anomalous at both the peak of Zn and Cu (9.7 and
9.0 keV approximately)... however this experiment would not be
necessary if you first find from an energy scan that only one is the
metal binding your protein. If you have a mixture of the two, your
final structure should take this into account (i.e. the appropriate
relative occupancies for the two ions)

3- there is way to know if i have Cu+ or Cu++.

Apart from the coordination geometry for the two oxidation states of
Copper, I know of people doing UV-Vis spectroscopy on the crystals
alongside their data collection. Micro-spectrophotometers are
available on demand at most (if not all) of the synchrotron sources.
If your species is radiation sensitive, a dose-dependent oxidation
could be monitored by collecting multiple spectra.


HTH

Best regards,

Marco

Re: [ccp4bb] How to identify unknow heavy atom??

[Boaz Shaanan]

I'm quite sure that EXAFS is done in solution.


         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 Jacob Keller [j-kell...@fsm.northwestern.edu]
Sent: Tuesday, July 24, 2012 9:57 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] How to identify unknow heavy atom??



I think it's done on a crystal itself, but others who know better than I can comment.


JPK

On Tue, Jul 24, 2012 at 1:02 PM, Theresa Hsu 
 wrote:

Does EXAFS requires same amount of samples as ICP-MS/ICP-AES?

Theresa


On Tue, 24 Jul 2012 12:55:31 -0500, Jacob Keller  wrote:

>Perhaps also exafs should be mentioned--I believe the various ion species,
>redox states, and even binding geometry can be determined.
>
>JPK
>
>
>
>


>--
>***
>Jacob Pearson Keller
>Northwestern University
>Medical Scientist Training Program
>email: j-kell...@northwestern.edu
>***
>










-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] How to identify unknow heavy atom??

[Jacob Keller]

I think it's done on a crystal itself, but others who know better than I
can comment.

JPK

On Tue, Jul 24, 2012 at 1:02 PM, Theresa Hsu  wrote:

> Does EXAFS requires same amount of samples as ICP-MS/ICP-AES?
>
> Theresa
>
> On Tue, 24 Jul 2012 12:55:31 -0500, Jacob Keller <
> j-kell...@fsm.northwestern.edu> wrote:
>
> >Perhaps also exafs should be mentioned--I believe the various ion species,
> >redox states, and even binding geometry can be determined.
> >
> >JPK
> >
> >
> >
> >
> >--
> >***
> >Jacob Pearson Keller
> >Northwestern University
> >Medical Scientist Training Program
> >email: j-kell...@northwestern.edu
> >***
> >
>
>
>


-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] How to identify unknow heavy atom??

[Theresa Hsu]

Does EXAFS requires same amount of samples as ICP-MS/ICP-AES?

Theresa

On Tue, 24 Jul 2012 12:55:31 -0500, Jacob Keller 
 wrote:

>Perhaps also exafs should be mentioned--I believe the various ion species,
>redox states, and even binding geometry can be determined.
>
>JPK
>
>
>
>
>--
>***
>Jacob Pearson Keller
>Northwestern University
>Medical Scientist Training Program
>email: j-kell...@northwestern.edu
>***
>

Re: [ccp4bb] How to identify unknow heavy atom??

[James Austin]

Sent using BlackBerry® from Orange

-Original Message-
From: Jacob Keller 
Sender: CCP4 bulletin board 
Date: Tue, 24 Jul 2012 12:55:31 
To: 
Reply-To: Jacob Keller 
Subject: Re: [ccp4bb] How to identify unknow heavy atom??

Perhaps also exafs should be mentioned--I believe the various ion species,
redox states, and even binding geometry can be determined.

JPK

On Tue, Jul 24, 2012 at 12:37 PM, Roberts, Sue A - (suer) <
s...@email.arizona.edu> wrote:

> Hello
>
> Actually, if the home source uses a copper tube, neither copper nor zinc
> have much of an anomalous signal at that wavelength (the energy is below
> the absorption edge for both).
> The best way is to check the location of the absorption edge at the
> synchrotron.  Cu+ and Cu++ can be distinguished this way, but make sure the
> absorption scan is done before you collect data since copper(II) can be
> photoreduced to copper(I) in the synchrotron x-ray beam.  Whether or not
> you can get a clue from geometry depends upon the resolution of the
> structure.
>
> Sue
>
> On Jul 24, 2012, at 10:22 AM, Nat Echols wrote:
>
> > On Tue, Jul 24, 2012 at 10:14 AM, Haytham Wahba 
> wrote:
> >> 1- if i have anomalous peak of unknown heavy atom, How can i identify
> this
> >> heavy atom in general. (different methods)
> >>
> >> 2- in my case, i see anomalous peak in heavy atom binding site (without
> any
> >> soaking). preliminary i did mass spec. i got Zn++ and Cu, How can i know
> >> which one give the anomalous peak in my protein.
> >>
> >> 3- there is way to know if i have Cu+ or Cu++.
> >
> > You may be able to identify the element based on the coordination
> > geometry - I'm assuming (perhaps incorrectly) that it is actually
> > different for Cu and Zn.  Marjorie Harding has written extensively on
> > the geometry of ion binding:
> >
> > http://tanna.bch.ed.ac.uk/
> >
> > The only way to be certain crystallographically, if you have easy
> > access to a synchrotron, is to collect data above and below the K edge
> > of any candidate element, and compare the difference maps.  (For
> > monovalent ions it is more complicated, since they don't have
> > accessible K edges.)  On a home source, Cu should have a larger
> > anomalous map peak, but I'm not sure if this will be enough to
> > identify it conclusively.
> >
> > -Nat
>
> Dr. Sue A. Roberts
> Dept. of Chemistry and Biochemistry
> University of Arizona
> 1041 E. Lowell St.,  Tucson, AZ 85721
> Phone: 520 621 8171 or 520 621 4168
> s...@email.arizona.edu
> http://www.cbc.arizona.edu/xray or
> http://www.cbc.arizona.edu/facilities/x-ray_diffraction
>



--
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] How to identify unknow heavy atom??

[Jacob Keller]

Perhaps also exafs should be mentioned--I believe the various ion species,
redox states, and even binding geometry can be determined.

JPK

On Tue, Jul 24, 2012 at 12:37 PM, Roberts, Sue A - (suer) <
s...@email.arizona.edu> wrote:

> Hello
>
> Actually, if the home source uses a copper tube, neither copper nor zinc
> have much of an anomalous signal at that wavelength (the energy is below
> the absorption edge for both).
> The best way is to check the location of the absorption edge at the
> synchrotron.  Cu+ and Cu++ can be distinguished this way, but make sure the
> absorption scan is done before you collect data since copper(II) can be
> photoreduced to copper(I) in the synchrotron x-ray beam.  Whether or not
> you can get a clue from geometry depends upon the resolution of the
> structure.
>
> Sue
>
> On Jul 24, 2012, at 10:22 AM, Nat Echols wrote:
>
> > On Tue, Jul 24, 2012 at 10:14 AM, Haytham Wahba 
> wrote:
> >> 1- if i have anomalous peak of unknown heavy atom, How can i identify
> this
> >> heavy atom in general. (different methods)
> >>
> >> 2- in my case, i see anomalous peak in heavy atom binding site (without
> any
> >> soaking). preliminary i did mass spec. i got Zn++ and Cu, How can i know
> >> which one give the anomalous peak in my protein.
> >>
> >> 3- there is way to know if i have Cu+ or Cu++.
> >
> > You may be able to identify the element based on the coordination
> > geometry - I'm assuming (perhaps incorrectly) that it is actually
> > different for Cu and Zn.  Marjorie Harding has written extensively on
> > the geometry of ion binding:
> >
> > http://tanna.bch.ed.ac.uk/
> >
> > The only way to be certain crystallographically, if you have easy
> > access to a synchrotron, is to collect data above and below the K edge
> > of any candidate element, and compare the difference maps.  (For
> > monovalent ions it is more complicated, since they don't have
> > accessible K edges.)  On a home source, Cu should have a larger
> > anomalous map peak, but I'm not sure if this will be enough to
> > identify it conclusively.
> >
> > -Nat
>
> Dr. Sue A. Roberts
> Dept. of Chemistry and Biochemistry
> University of Arizona
> 1041 E. Lowell St.,  Tucson, AZ 85721
> Phone: 520 621 8171 or 520 621 4168
> s...@email.arizona.edu
> http://www.cbc.arizona.edu/xray or
> http://www.cbc.arizona.edu/facilities/x-ray_diffraction
>



-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] How to identify unknow heavy atom??

[Roberts, Sue A - (suer)]

Hello

Actually, if the home source uses a copper tube, neither copper nor zinc have 
much of an anomalous signal at that wavelength (the energy is below the 
absorption edge for both).
The best way is to check the location of the absorption edge at the 
synchrotron.  Cu+ and Cu++ can be distinguished this way, but make sure the 
absorption scan is done before you collect data since copper(II) can be 
photoreduced to copper(I) in the synchrotron x-ray beam.  Whether or not you 
can get a clue from geometry depends upon the resolution of the structure.  

Sue

On Jul 24, 2012, at 10:22 AM, Nat Echols wrote:

> On Tue, Jul 24, 2012 at 10:14 AM, Haytham Wahba  
> wrote:
>> 1- if i have anomalous peak of unknown heavy atom, How can i identify this
>> heavy atom in general. (different methods)
>> 
>> 2- in my case, i see anomalous peak in heavy atom binding site (without any
>> soaking). preliminary i did mass spec. i got Zn++ and Cu, How can i know
>> which one give the anomalous peak in my protein.
>> 
>> 3- there is way to know if i have Cu+ or Cu++.
> 
> You may be able to identify the element based on the coordination
> geometry - I'm assuming (perhaps incorrectly) that it is actually
> different for Cu and Zn.  Marjorie Harding has written extensively on
> the geometry of ion binding:
> 
> http://tanna.bch.ed.ac.uk/
> 
> The only way to be certain crystallographically, if you have easy
> access to a synchrotron, is to collect data above and below the K edge
> of any candidate element, and compare the difference maps.  (For
> monovalent ions it is more complicated, since they don't have
> accessible K edges.)  On a home source, Cu should have a larger
> anomalous map peak, but I'm not sure if this will be enough to
> identify it conclusively.
> 
> -Nat

Dr. Sue A. Roberts
Dept. of Chemistry and Biochemistry
University of Arizona
1041 E. Lowell St.,  Tucson, AZ 85721
Phone: 520 621 8171 or 520 621 4168
s...@email.arizona.edu
http://www.cbc.arizona.edu/xray or 
http://www.cbc.arizona.edu/facilities/x-ray_diffraction

Re: [ccp4bb] How to identify unknow heavy atom??

[Nat Echols]

On Tue, Jul 24, 2012 at 10:33 AM, Ethan Merritt
 wrote:
> As to the home source - no.
> Neither Cu nor Zn has appreciable anomalous signal when excited with a
> Cu K-alpha home source.
>   http://www.bmsc.washington.edu/scatter
>
> An element's emission edge (Cu K-alpha in this case) is about 1 keV below
> the corresponding absorption edge.  This makes sense, because after
> absorbing a photon it can only emit at an equal or lower energy, not a
> higher energy.  So you can't reach the Cu absorption edge, where the
> anomalous signal is, by exciting with Cu K-alpha.

Oops, sorry, I was of course comparing the wrong numbers.

-Nat

Re: [ccp4bb] How to identify unknow heavy atom??

[Ethan Merritt]

On Tuesday, July 24, 2012 10:22:18 am Nat Echols wrote:
> On Tue, Jul 24, 2012 at 10:14 AM, Haytham Wahba  
> wrote:
> > 1- if i have anomalous peak of unknown heavy atom, How can i identify this
> > heavy atom in general. (different methods)
> >
> > 2- in my case, i see anomalous peak in heavy atom binding site (without any
> > soaking). preliminary i did mass spec. i got Zn++ and Cu, How can i know
> > which one give the anomalous peak in my protein.
> >
> > 3- there is way to know if i have Cu+ or Cu++.
> 
> You may be able to identify the element based on the coordination
> geometry - I'm assuming (perhaps incorrectly) that it is actually
> different for Cu and Zn.  Marjorie Harding has written extensively on
> the geometry of ion binding:
> 
> http://tanna.bch.ed.ac.uk/
> 
> The only way to be certain crystallographically, if you have easy
> access to a synchrotron, is to collect data above and below the K edge
> of any candidate element, and compare the difference maps.  (For
> monovalent ions it is more complicated, since they don't have
> accessible K edges.)  On a home source, Cu should have a larger
> anomalous map peak, but I'm not sure if this will be enough to
> identify it conclusively.

As to the SR experiment - yes.

As to the home source - no.  
Neither Cu nor Zn has appreciable anomalous signal when excited with a 
Cu K-alpha home source.
  http://www.bmsc.washington.edu/scatter

An element's emission edge (Cu K-alpha in this case) is about 1 keV below
the corresponding absorption edge.  This makes sense, because after
absorbing a photon it can only emit at an equal or lower energy, not a
higher energy.  So you can't reach the Cu absorption edge, where the
anomalous signal is, by exciting with Cu K-alpha.

Ethan

-- 
Ethan A Merritt
Biomolecular Structure Center,  K-428 Health Sciences Bldg
University of Washington, Seattle 98195-7742

Re: [ccp4bb] How to identify unknow heavy atom??

[Nat Echols]

On Tue, Jul 24, 2012 at 10:14 AM, Haytham Wahba  wrote:
> 1- if i have anomalous peak of unknown heavy atom, How can i identify this
> heavy atom in general. (different methods)
>
> 2- in my case, i see anomalous peak in heavy atom binding site (without any
> soaking). preliminary i did mass spec. i got Zn++ and Cu, How can i know
> which one give the anomalous peak in my protein.
>
> 3- there is way to know if i have Cu+ or Cu++.

You may be able to identify the element based on the coordination
geometry - I'm assuming (perhaps incorrectly) that it is actually
different for Cu and Zn.  Marjorie Harding has written extensively on
the geometry of ion binding:

http://tanna.bch.ed.ac.uk/

The only way to be certain crystallographically, if you have easy
access to a synchrotron, is to collect data above and below the K edge
of any candidate element, and compare the difference maps.  (For
monovalent ions it is more complicated, since they don't have
accessible K edges.)  On a home source, Cu should have a larger
anomalous map peak, but I'm not sure if this will be enough to
identify it conclusively.

-Nat

[ccp4bb] How to identify unknow heavy atom??

[Haytham Wahba]

1- if i have anomalous peak of unknown heavy atom, How can i identify this 
heavy atom in general. (different methods)
 
 
2- in my case, i see anomalous peak in heavy atom binding site (without any 
soaking). preliminary i did mass spec. i got Zn++ and Cu, How can i know which 
one give the anomalous peak in my protein.
 
3- there is way to know if i have Cu+ or Cu++.
 
 
Haytham
UdeM
Biochemistry

[ccp4bb] Refmac: ADP is non-positive

[wtempel]

There are atom records for C, O and CA (B-factors 42, 43, 40A**2,
respectively), but not for N, as density tapers off going to the amino
terminus (well, without the "amino" in this case). Residue 3 is the
lowest-numbered residue in its chain. B-factors of N, CA of residue 4 are
38, 33A**2, respectively. Could refmac just be taking exception to the
missing N atom?

-- Forwarded message --
From: Ethan Merritt 
Date: Tue, Jul 24, 2012 at 11:27 AM
Subject: Re: [ccp4bb] Refmac: ADP is non-positive
To: wtempel 
Cc: CCP4BB@jiscmail.ac.uk


On Tuesday, 24 July 2012, wtempel wrote:
> CCP4ers,
>
> a log file from Refmac_5.7.0027 presents me with this line:
>
> 
> Problem with the ADP of the atom N   A  3 ADP is non-positive
> -1.7740907E+35
> 
>
> I did not explicitely refine ADPs or TLS.
> Should I modify my model when I encounter such a message? If yes, does the
> message refer to a specific atom, such as atom N of residue 3 in chain A?
I
> should note that that atom is omitted from my model due to lack of
electron
> density/disorder.

What do you mean by "omitted from the model"?
Are there no ATOM records for that residue in the PDB file?
What are the B factors for the other atoms in that region?

Ethan

>
> Many thanks in advance,
> Wolfram Tempel
>

[ccp4bb] Fwd: request for reprint

[Jacqueline Vitali]

Dear Colleagues,

Does anyone have access to this article?  It is not in my library and they
cannot find it via interlibrary loan.  I know the limitations and that it
is best not to ask papers via the newsgroup but I have even tried to ask
the author for a reprint and my email was returned to me.

Thank you.

Jackie Vitali
Cleveland State University



Cell Mol Biol 
(Noisy-le-grand).2004
Jun;50(4):347-52.
Cooperativity and high pressure: stabilization of the R conformation of the
allosteric aspartate transcarbamylase under the influence of pressure.
Hervé 
G,
Schmitt 
B,
Serre 
V
.

Re: [ccp4bb] Question about weird diffraction map

[Bernhard Rupp (Hofkristallrat a.D.)]

Hmmm..I just fail to see 'lines' in that diffraction map. I see spots along
something that could be segments of diffraction rings, i.e. a number of
these crystals in some clustered random orientations, similar to ice as you
mention. I also wonder how there could be true diffraction 'lines': If it is
a small molecule or salt, then the diffraction spots are pretty far apart in
reciprocal space, and the chance of seeing diffraction lines of closely
spaced adjacent RL points like we see in the lunes of a (single X)
macromolecular rotation image is quite remote. If for example that spot
cluster down left would be a 'line', then the lattice spacing would have to
be quite large. Why some of the low resolution rings are not exactly rings
either can have various reasons we can discuss off board. 

 

Otherwise no dissent.

 

Best, BR

From: Matthew Franklin [mailto:mfrank...@nysbc.org] 
Sent: Tuesday, July 24, 2012 7:08 AM
To: b...@hofkristallamt.org
Cc: Bernhard Rupp (Hofkristallrat a.D.); CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Question about weird diffraction map

 

Hi Bernhard -

Having the spots in regular lines indicates that this is a single crystal
diffraction pattern (to a first approximation).  I have seen ice rings which
contain a few strong spots, probably from microcrystals of ice, but I
haven't yet seen ice diffraction that shows a lattice of spots.  So, the
presence of the spot lattice, plus the spots below 3.9 A, allow one to say
that this image isn't ice diffraction on top of weak/absent protein
diffraction.  Ergo, this crystal is not a macromolecule.

Plus, I was trying to show Zhao that the spots aren't just scattered at
random.

- Matt


On 7/23/12 5:25 PM, Bernhard Rupp (Hofkristallrat a.D.) wrote:

> you'll see that some of them are arranged in regular lines. 

 

I am not sure I understand what the line argument implies?

 

>indicates a very small unit cell, with dimensions probably < 10 A

 

Very indicative also the few strong and isolated high resolution reflections


Cheers, BR




 






-- 
Matthew Franklin, Ph. D.
Senior Scientist
New York Structural Biology Center
89 Convent Avenue, New York, NY 10027
(212) 939-0660 ext. 9374

Re: [ccp4bb] Refmac: ADP is non-positive

[Ethan Merritt]

On Tuesday, 24 July 2012, wtempel wrote:
> CCP4ers,
> 
> a log file from Refmac_5.7.0027 presents me with this line:
> 
> 
> Problem with the ADP of the atom N   A  3 ADP is non-positive
> -1.7740907E+35
> 
> 
> I did not explicitely refine ADPs or TLS.
> Should I modify my model when I encounter such a message? If yes, does the
> message refer to a specific atom, such as atom N of residue 3 in chain A? I
> should note that that atom is omitted from my model due to lack of electron
> density/disorder.

What do you mean by "omitted from the model"?
Are there no ATOM records for that residue in the PDB file?
What are the B factors for the other atoms in that region?

Ethan

> 
> Many thanks in advance,
> Wolfram Tempel
> 

Re: [ccp4bb] Improvement in crystal quality

[Herman . Schreuder]

Dear Nishant,
 
To me, the diffraction looks like powder diffraction from a salt. In
your case, I would look at the protein buffer to see if there are
components present (phosphate, detergent etc.) which might be prone to
form crystals on their own and see if you can find a minimal buffer
where the protein is still happy. With this buffer, I would do another
full screening to see if you get hits with more protein-like
diffraction.
 
Best,
Herman




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On
Behalf Of Niks
Sent: Tuesday, July 24, 2012 4:02 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Improvement in crystal quality


Dear All, 

I am trying to crystallize a recombinant dehydrogenase protein.
Got five hits in PEG ION Screen from Hamptons (20% PEG 3350 with 0.2M
Sodium Acetate, 0.2M Potassium acetate, 0.2M ammonium acetate, 0.2M
sodium formate and 0.2M Potassium Chloride) after two days.
Crystals looks like needles most of time, sometime broader
needles (Pictures attached). UV crystal scanner says those are protein
crystals, but when we tried to pick up one and shoot at room
temperature, diffraction patterns looks like similar like of  powder
diffraction (picture attached).
I have tried 50 of the 96 additives(whichever I can arrange of)
mentioned in the additive screen from Hamptons . I have tried detergent
screen from Hamptons (this time original screen solutions). I have tried
incubating the plate at 28degrees as well as 10degrees, Though waiting
for 10degree results but one drop  showed needles again after normal two
days of growth period.
I tried to slow down the supersaturation by adding 100ul of 1:1
ratio of silicon oil and paraffin oil over the 1ml of well solution.
This time no crystals but some precipitation.

If anyone spare any word of wisdom to improve these crystal
quality, I will be very grateful.
If seeding is the only obvious thing to try, any reference for
the seeding procedure will be highly appreciated.

Thanks very much
Nishant Varshney
PhD student,
National Chemical Laboratory,Pune,India
-- 
"The most difficult phase of  life is not when No one
understands you;It is when you don't understand yourself"




-- 
"The most difficult phase of  life is not when No one
understands you;It is when you don't understand yourself"

Re: [ccp4bb] Improvement in crystal quality

[Kelly Daughtry]

The diffraction you see is probably not the best diffraction you could
obtain from these crystals. I have found long thin needles are
very susceptible to manipulation.
I would highly recommend seeding (I like the Hampton Seed Bead personally,
http://hamptonresearch.com/product_detail.aspx?cid=18&sid=44&pid=42 ).
You need to try to change the shape of these crystals to beef them up.

Other Recommendations:
Vary protein concentration
Vary pH (if there is not a buffer in the well, determine pH of well and add
buffer to stabilize pH and screen around - I've had success changing needle
clusters with a  few 0.1 pH unit changes)
Dioxane to limit crystal nucleation, try to only add it to the well, not
mother liquor present in drop, 1 - 5 % Dioxane
Different MW PEGs

There are almost limitless possibilities of things to try with protein
crystallography.
Good luck,
Kelly Daughtry

***
Kelly Daughtry, Ph.D.
Post-Doctoral Fellow, Raetz Lab
Biochemistry Department
Duke University
Alex H. Sands, Jr. Building
303 Research Drive
RM 250
Durham, NC 27710
P: 919-684-5178
***


On Tue, Jul 24, 2012 at 9:40 AM, Niks  wrote:

> Dear All,
>
> I am trying to crystallize a recombinant dehydrogenase protein. Got five
> hits in PEG ION Screen from Hamptons (20% PEG 3350 with 0.2M Sodium
> Acetate, 0.2M Potassium acetate, 0.2M ammonium acetate, 0.2M sodium formate
> and 0.2M Potassium Chloride) after two days.
> Crystals looks like needles most of time, sometime broader needles
> (Pictures attached). UV crystal scanner says those are protein crystals,
> but when we tried to pick up one and shoot at room temperature, diffraction
> patterns looks like similar like of  powder diffraction (picture attached).
> I have tried 50 of the 96 additives(whichever I can arrange of) mentioned
> in the additive screen from Hamptons . I have tried detergent screen from
> Hamptons (this time original screen solutions). I have tried incubating the
> plate at 28degrees as well as 10degrees, Though waiting for 10degree
> results but one drop  showed needles again after normal two days of growth
> period.
> I tried to slow down the supersaturation by adding 100ul of 1:1 ratio of
> silicon oil and paraffin oil over the 1ml of well solution. This time no
> crystals but some precipitation.
>
> If anyone spare any word of wisdom to improve these crystal quality, I
> will be very grateful.
> If seeding is the only obvious thing to try, any reference for the seeding
> procedure will be highly appreciated.
>
> Thanks very much
> Nishant Varshney
> PhD student,
> National Chemical Laboratory,Pune,India
> --
> "The most difficult phase of  life is not when No one understands you;It
> is when you don't understand yourself"
>

Re: [ccp4bb] Question on stereo monitor: LG D2342P-PN

[William G. Scott]

Dear Zhijie:

I saw this for under $300 at Amazon's Showroom and wondered the same thing.  I 
can verify that the LG TV will work with coot, etc., as long as you turn OFF 
all stereographic processing and allow the software to do the work.  There is 
an extremely detailed review of this at 
http://www.amazon.com/LG-D2342P-PN-23-Inch-Widescreen-Passive/dp/B004WK3R4U/ref=cm_cr-mr-title
  by Bill Costa (he gives his email address at the end).  He seems to suggest 
that the 3D glasses for the LG TV are not compatible with this, which left me 
scratching my head.

Having said that, I'll bet it will work fine.  If you can get it from Best Buy 
for under $300 and return it within 30 days for a full refund, it might be 
worth it.

Either way, let us know.

Bill




William G. Scott
Professor
Department of Chemistry and Biochemistry
and The Center for the Molecular Biology of RNA
228 Sinsheimer Laboratories
University of California at Santa Cruz
Santa Cruz, California 95064
USA

 

On Jul 23, 2012, at 11:42 AM, Zhijie Li wrote:

> Dear CCP4BBers:
>  
> Sorry to bring up this highly repeated topic again. After some internet 
> research, we are about to make the commitment to buy an LG D2342P-PN for 
> setting up a stereo system. I would like to have a final confirmation from 
> someone with real life experience that this model does work with COOT and 
> Chimera the same way as the Zalman monitors. Thanks in advance.
>  
> Zhijie

[ccp4bb] Refmac: ADP is non-positive

[wtempel]

CCP4ers,

a log file from Refmac_5.7.0027 presents me with this line:


Problem with the ADP of the atom N   A  3 ADP is non-positive
-1.7740907E+35


I did not explicitely refine ADPs or TLS.
Should I modify my model when I encounter such a message? If yes, does the
message refer to a specific atom, such as atom N of residue 3 in chain A? I
should note that that atom is omitted from my model due to lack of electron
density/disorder.

Many thanks in advance,
Wolfram Tempel

[ccp4bb] 2D-bar code scanner to read Hampton pins barcodes

[Pietro Roversi]

Dear all,

I am looking into a 2D-bar code scanner to read Hampton pins barcodes.
Something we can plug directly to any computer running Windows, Mac or Linux,
via a supplied USB cable, and feed the barcodes into Excel.

Does anybody know a cheaper alternative to the FOCUS MS-1690-38?

Grazie, ciao!

Pietro

Re: [ccp4bb] Question about weird diffraction map

[Matthew Franklin]

Hi Bernhard -

Having the spots in regular lines indicates that this is a single 
crystal diffraction pattern (to a first approximation).  I have seen ice 
rings which contain a few strong spots, probably from microcrystals of 
ice, but I haven't yet seen ice diffraction that shows a lattice of 
spots.  So, the presence of the spot lattice, plus the spots below 3.9 
A, allow one to say that this image isn't ice diffraction on top of 
weak/absent protein diffraction.  Ergo, this crystal is not a macromolecule.


Plus, I was trying to show Zhao that the spots aren't just scattered at 
random.


- Matt


On 7/23/12 5:25 PM, Bernhard Rupp (Hofkristallrat a.D.) wrote:


> you'll see that some of them are arranged in regular lines.

I am not sure I understand what the line argument implies?

>indicates a very small unit cell, with dimensions probably < 10 A

Very indicative also the few strong and isolated high resolution 
reflections



Cheers, BR

  



--
Matthew Franklin, Ph. D.
Senior Scientist
New York Structural Biology Center
89 Convent Avenue, New York, NY 10027
(212) 939-0660 ext. 9374

Re: [ccp4bb] Improvement in crystal quality

[Frank von Delft]

4Mb attachments, posted to a mailing list  NOT COOL.

On 24/07/2012 14:40, Niks wrote:

Dear All,

I am trying to crystallize a recombinant dehydrogenase protein. Got 
five hits in PEG ION Screen from Hamptons (20% PEG 3350 with 0.2M 
Sodium Acetate, 0.2M Potassium acetate, 0.2M ammonium acetate, 0.2M 
sodium formate and 0.2M Potassium Chloride) after two days.
Crystals looks like needles most of time, sometime broader needles 
(Pictures attached). UV crystal scanner says those are protein 
crystals, but when we tried to pick up one and shoot at room 
temperature, diffraction patterns looks like similar like of  powder 
diffraction (picture attached).
I have tried 50 of the 96 additives(whichever I can arrange 
of) mentioned in the additive screen from Hamptons . I have tried 
detergent screen from Hamptons (this time original screen solutions). 
I have tried incubating the plate at 28degrees as well 
as 10degrees, Though waiting for 10degree results but one drop  showed 
needles again after normal two days of growth period.
I tried to slow down the supersaturation by adding 100ul of 1:1 ratio 
of silicon oil and paraffin oil over the 1ml of well solution. This 
time no crystals but some precipitation.


If anyone spare any word of wisdom to improve these crystal quality, I 
will be very grateful.
If seeding is the only obvious thing to try, any reference for the 
seeding procedure will be highly appreciated.


Thanks very much
Nishant Varshney
PhD student,
National Chemical Laboratory,Pune,India
--
"The most difficult phase of  life is not when No one understands 
you;It is when you don't understand yourself"

Re: [ccp4bb] Problem with making PDB from Coot

[Joel Tyndall]

You should type Dss into pymol and this will assign more appropriate secondary 
structure

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of meisam 
nosrati
Sent: Tuesday, 24 July 2012 9:47 a.m.
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Problem with making PDB from Coot

Dear CCP4ers

It seems like the text has not showed up, but I have a problem with making pdbs 
from coot. As I refine my structure ( with MR solution ) the beta sheets become 
loops while H-bondings are still there.

I am not sure, if the problem is originating from making PDBs from coot.

I will appriciate your help

Meisam
On Mon, Jul 23, 2012 at 5:31 PM, Meisam Nosrati 
mailto:meisam.nosr...@gmail.com>> wrote:

Re: [ccp4bb] Problem with making PDB from Coot

[Remy Loris]

Seconday structure assignement by Pymol is poor and unreliable to say 
the least. This behavior is very common. Best is to assign secondary 
structure using other software and then tell Pymol explicitely from 
where to where the starnds and helices run


Remy Loris
Vriej universiteit Brussel


On 23/07/12 23:46, meisam nosrati wrote:

Dear CCP4ers

It seems like the text has not showed up, but I have a problem with 
making pdbs from coot. As I refine my structure ( with MR solution ) 
the beta sheets become loops while H-bondings are still there.


I am not sure, if the problem is originating from making PDBs from coot.

I will appriciate your help

Meisam

On Mon, Jul 23, 2012 at 5:31 PM, Meisam Nosrati 
mailto:meisam.nosr...@gmail.com>> wrote: