Re: [ccp4bb] Unknown positive electron density

[Shailesh Tripathi]

Ionic Ba2+ has smaller radius 2/3 of metalic Ba. So it will have increased
e/A3 and modelling Ba atom might give positive density centered at Ba site.
This discussion might help:

http://ccp4bb.blogspot.in/2011/11/atomic-scattering-factors-in-refmac.html


Shailesh Kumar Tripathi,
Phone: 9686289668


On Tue, Aug 22, 2017 at 5:32 AM, Keller, Jacob 
wrote:

> Fourier truncation ripples?
>
>
>
> JPK
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
> *CRAIG
> A BINGMAN
> *Sent:* Monday, August 21, 2017 6:20 PM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] Unknown positive electron density
>
>
>
> Betty,
>
>
>
> I think that f’’ for Ba at this wavelength is around 3.5 electrons, and
> f'' for As is expected to be about 3 electrons. Is there a nearby
> crystallographic symmetry axis?
>
>
>
> Craig
>
>
>
> On Aug 21, 2017, at 5:05 PM, Betty Chu  wrote:
>
>
>
> Hi Craig,
>
> The data collection wavelength was 0.92 Angstroms. Since we observe
> anomalous signal for Ba at this wavelength, we would expect greater
> anomalous signal if As were present. There is a possibility for weak
> anomalous signal in this positive density, but the weak anomalous signal
> only shows up if I try to model a Ba in the density. Without modelling
> anything, there is no anomalous signal.
>
> This is what the map looks like after one round of refinement with the Ba
> in the density. But since there are waters that are 1.6 Angstroms, 1.9
> Angstroms, and 2.2 Angstroms away from the Ba, which is smaller than the
> coordination distance between Ba and water, we are skeptical of the Ba
> being there.
>
> https://cbsostorage.chem.umd.edu/owncloud/index.php/s/O4lI2iKvRsQUoRo
>
>
> Thank you,
>
> Betty
>
>
>
> On Mon, Aug 21, 2017 at 5:21 PM, CRAIG A BINGMAN 
> wrote:
>
> What is the data collection wavelength/energy? Would you expect
> significant anomalous diffraction from As at this wavelength?
>
>
>
> On Aug 21, 2017, at 11:37 AM, Betty Chu  wrote:
>
>
>
> Hi Shailesh,
>
> When I modelled in the Barium ion with octahedrally coordinated waters and
> ran the refinement, the distances from the barium to some of the waters
> ended up being too close (<2.2 Angstroms). Also, the positive electron
> density is connected. If the density indicated barium with coordinated
> waters, would that mean there are multiple ones present in the positive
> density?
>
> Here are more views of the connected positive density.
>
>
> https://cbsostorage.chem.umd.edu/owncloud/index.php/s/j5CgZIJe6NKgUlZ
>
> https://cbsostorage.chem.umd.edu/owncloud/index.php/s/YbiNFbT8AW1iHUQ
>
>
>
> On Mon, Aug 21, 2017 at 12:00 PM, Shailesh Tripathi 
> wrote:
>
> Looks like Ba2+. Since it exist with coordination number 6 or above check
> what geometry water is following there (trigonal bipiramidal or so on).
> Water might also be shared by symmetry related Ba cation.
>
>
>
>
>
>
> Shailesh Kumar Tripathi,
>
> Phone: 9686289668
>
>
>
>
>
> On Mon, Aug 21, 2017 at 9:17 PM, Betty Chu  wrote:
>
> Yes, I have. The cacodylate ion does not fit well into the density.
>
>
>
> On Mon, Aug 21, 2017 at 11:44 AM, Pradeep Pallan 
> wrote:
>
> Did you try modelling in a cacodylate ion (CH3)2AsO2-?
>
>
>
> On Mon, Aug 21, 2017 at 10:19 AM, Betty Chu  wrote:
>
> Dear ccp4bb,
>
> I am refining a 1.40 Angstrom data set for a DNA oligonucleotide. While
> the model for the DNA fits very well into the density, there is a patch of
> positive electron density in the solvent space that we are having trouble
> with.
>
> The screenshot can be viewed through this link:
> https://cbsostorage.chem.umd.edu/owncloud/index.php/s/J5cKnOpCC4vb1VC
>
> In the screenshot, the yellow color is the anomalous map and a barium ion
> is fitted into density near the positive green electron density.
>
>
> The oligonucleotide was purchased from IDT. The crystallization condition
> is 15% MPD, 120 mM BaCl2, and 30 mM NaCaC pH 6.4. I have tried modelling
> Ba2+ with coordinated waters, MPD, and sodium cacodylate into the electron
> density, but none of those fit well.
>
> Any suggestions regarding the identity of this electron density is much
> appreciated. Thank you!
>
>
>
> Sincerely,
>
> Betty Chu
>
> Paukstelis Research Group
>
> Department of Chemistry and Biochemistry
>
> University of Maryland, College Park
>
>
>
>
>
> --
>
>
> ---
> Pradeep Pallan
>
>
>
>
>
>
>
>
>
>
>
>
>

Re: [ccp4bb] Unknown positive electron density

[Keller, Jacob]

Fourier truncation ripples?

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of CRAIG A 
BINGMAN
Sent: Monday, August 21, 2017 6:20 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Unknown positive electron density

Betty,

I think that f’’ for Ba at this wavelength is around 3.5 electrons, and f'' for 
As is expected to be about 3 electrons. Is there a nearby crystallographic 
symmetry axis?

Craig

On Aug 21, 2017, at 5:05 PM, Betty Chu 
> wrote:

Hi Craig,
The data collection wavelength was 0.92 Angstroms. Since we observe anomalous 
signal for Ba at this wavelength, we would expect greater anomalous signal if 
As were present. There is a possibility for weak anomalous signal in this 
positive density, but the weak anomalous signal only shows up if I try to model 
a Ba in the density. Without modelling anything, there is no anomalous signal.

This is what the map looks like after one round of refinement with the Ba in 
the density. But since there are waters that are 1.6 Angstroms, 1.9 Angstroms, 
and 2.2 Angstroms away from the Ba, which is smaller than the coordination 
distance between Ba and water, we are skeptical of the Ba being there.

https://cbsostorage.chem.umd.edu/owncloud/index.php/s/O4lI2iKvRsQUoRo

Thank you,
Betty

On Mon, Aug 21, 2017 at 5:21 PM, CRAIG A BINGMAN 
> wrote:
What is the data collection wavelength/energy? Would you expect significant 
anomalous diffraction from As at this wavelength?

On Aug 21, 2017, at 11:37 AM, Betty Chu 
> wrote:

Hi Shailesh,
When I modelled in the Barium ion with octahedrally coordinated waters and ran 
the refinement, the distances from the barium to some of the waters ended up 
being too close (<2.2 Angstroms). Also, the positive electron density is 
connected. If the density indicated barium with coordinated waters, would that 
mean there are multiple ones present in the positive density?
Here are more views of the connected positive density.

https://cbsostorage.chem.umd.edu/owncloud/index.php/s/j5CgZIJe6NKgUlZ

https://cbsostorage.chem.umd.edu/owncloud/index.php/s/YbiNFbT8AW1iHUQ

On Mon, Aug 21, 2017 at 12:00 PM, Shailesh Tripathi 
> wrote:
Looks like Ba2+. Since it exist with coordination number 6 or above check what 
geometry water is following there (trigonal bipiramidal or so on). Water might 
also be shared by symmetry related Ba cation.



Shailesh Kumar Tripathi,
Phone: 9686289668


On Mon, Aug 21, 2017 at 9:17 PM, Betty Chu 
> wrote:
Yes, I have. The cacodylate ion does not fit well into the density.

On Mon, Aug 21, 2017 at 11:44 AM, Pradeep Pallan 
> wrote:
Did you try modelling in a cacodylate ion (CH3)2AsO2-?

On Mon, Aug 21, 2017 at 10:19 AM, Betty Chu 
> wrote:
Dear ccp4bb,
I am refining a 1.40 Angstrom data set for a DNA oligonucleotide. While the 
model for the DNA fits very well into the density, there is a patch of positive 
electron density in the solvent space that we are having trouble with.

The screenshot can be viewed through this link:
https://cbsostorage.chem.umd.edu/owncloud/index.php/s/J5cKnOpCC4vb1VC

In the screenshot, the yellow color is the anomalous map and a barium ion is 
fitted into density near the positive green electron density.

The oligonucleotide was purchased from IDT. The crystallization condition is 
15% MPD, 120 mM BaCl2, and 30 mM NaCaC pH 6.4. I have tried modelling Ba2+ with 
coordinated waters, MPD, and sodium cacodylate into the electron density, but 
none of those fit well.
Any suggestions regarding the identity of this electron density is much 
appreciated. Thank you!

Sincerely,
Betty Chu
Paukstelis Research Group
Department of Chemistry and Biochemistry
University of Maryland, College Park



--

---
Pradeep Pallan

Re: [ccp4bb] Unknown positive electron density

[CRAIG A BINGMAN]

Betty,

I think that f’’ for Ba at this wavelength is around 3.5 electrons, and f'' for 
As is expected to be about 3 electrons. Is there a nearby crystallographic 
symmetry axis?

Craig

On Aug 21, 2017, at 5:05 PM, Betty Chu 
> wrote:

Hi Craig,

The data collection wavelength was 0.92 Angstroms. Since we observe anomalous 
signal for Ba at this wavelength, we would expect greater anomalous signal if 
As were present. There is a possibility for weak anomalous signal in this 
positive density, but the weak anomalous signal only shows up if I try to model 
a Ba in the density. Without modelling anything, there is no anomalous signal.

This is what the map looks like after one round of refinement with the Ba in 
the density. But since there are waters that are 1.6 Angstroms, 1.9 Angstroms, 
and 2.2 Angstroms away from the Ba, which is smaller than the coordination 
distance between Ba and water, we are skeptical of the Ba being there.

https://cbsostorage.chem.umd.edu/owncloud/index.php/s/O4lI2iKvRsQUoRo

Thank you,
Betty

On Mon, Aug 21, 2017 at 5:21 PM, CRAIG A BINGMAN 
> wrote:
What is the data collection wavelength/energy? Would you expect significant 
anomalous diffraction from As at this wavelength?

On Aug 21, 2017, at 11:37 AM, Betty Chu 
> wrote:

Hi Shailesh,

When I modelled in the Barium ion with octahedrally coordinated waters and ran 
the refinement, the distances from the barium to some of the waters ended up 
being too close (<2.2 Angstroms). Also, the positive electron density is 
connected. If the density indicated barium with coordinated waters, would that 
mean there are multiple ones present in the positive density?

Here are more views of the connected positive density.

https://cbsostorage.chem.umd.edu/owncloud/index.php/s/j5CgZIJe6NKgUlZ

https://cbsostorage.chem.umd.edu/owncloud/index.php/s/YbiNFbT8AW1iHUQ

On Mon, Aug 21, 2017 at 12:00 PM, Shailesh Tripathi 
> wrote:
Looks like Ba2+. Since it exist with coordination number 6 or above check what 
geometry water is following there (trigonal bipiramidal or so on). Water might 
also be shared by symmetry related Ba cation.



Shailesh Kumar Tripathi,
Phone: 9686289668


On Mon, Aug 21, 2017 at 9:17 PM, Betty Chu 
> wrote:
Yes, I have. The cacodylate ion does not fit well into the density.

On Mon, Aug 21, 2017 at 11:44 AM, Pradeep Pallan 
> wrote:
Did you try modelling in a cacodylate ion (CH3)2AsO2-?

On Mon, Aug 21, 2017 at 10:19 AM, Betty Chu 
> wrote:
Dear ccp4bb,

I am refining a 1.40 Angstrom data set for a DNA oligonucleotide. While the 
model for the DNA fits very well into the density, there is a patch of positive 
electron density in the solvent space that we are having trouble with.

The screenshot can be viewed through this link:
https://cbsostorage.chem.umd.edu/owncloud/index.php/s/J5cKnOpCC4vb1VC

In the screenshot, the yellow color is the anomalous map and a barium ion is 
fitted into density near the positive green electron density.

The oligonucleotide was purchased from IDT. The crystallization condition is 
15% MPD, 120 mM BaCl2, and 30 mM NaCaC pH 6.4. I have tried modelling Ba2+ with 
coordinated waters, MPD, and sodium cacodylate into the electron density, but 
none of those fit well.

Any suggestions regarding the identity of this electron density is much 
appreciated. Thank you!

Sincerely,

Betty Chu
Paukstelis Research Group
Department of Chemistry and Biochemistry
University of Maryland, College Park



--

---
Pradeep Pallan

Re: [ccp4bb] Unknown positive electron density

[Betty Chu]

Hi Craig,

The data collection wavelength was 0.92 Angstroms. Since we observe
anomalous signal for Ba at this wavelength, we would expect greater
anomalous signal if As were present. There is a possibility for weak
anomalous signal in this positive density, but the weak anomalous signal
only shows up if I try to model a Ba in the density. Without modelling
anything, there is no anomalous signal.

This is what the map looks like after one round of refinement with the Ba
in the density. But since there are waters that are 1.6 Angstroms, 1.9
Angstroms, and 2.2 Angstroms away from the Ba, which is smaller than the
coordination distance between Ba and water, we are skeptical of the Ba
being there.

https://cbsostorage.chem.umd.edu/owncloud/index.php/s/O4lI2iKvRsQUoRo

Thank you,
Betty

On Mon, Aug 21, 2017 at 5:21 PM, CRAIG A BINGMAN  wrote:

> What is the data collection wavelength/energy? Would you expect
> significant anomalous diffraction from As at this wavelength?
>
> On Aug 21, 2017, at 11:37 AM, Betty Chu  wrote:
>
> Hi Shailesh,
>
> When I modelled in the Barium ion with octahedrally coordinated waters and
> ran the refinement, the distances from the barium to some of the waters
> ended up being too close (<2.2 Angstroms). Also, the positive electron
> density is connected. If the density indicated barium with coordinated
> waters, would that mean there are multiple ones present in the positive
> density?
>
> Here are more views of the connected positive density.
>
> https://cbsostorage.chem.umd.edu/owncloud/index.php/s/j5CgZIJe6NKgUlZ
>
> https://cbsostorage.chem.umd.edu/owncloud/index.php/s/YbiNFbT8AW1iHUQ
>
> On Mon, Aug 21, 2017 at 12:00 PM, Shailesh Tripathi 
> wrote:
>
>> Looks like Ba2+. Since it exist with coordination number 6 or above check
>> what geometry water is following there (trigonal bipiramidal or so on).
>> Water might also be shared by symmetry related Ba cation.
>>
>>
>>
>> Shailesh Kumar Tripathi,
>> Phone: 9686289668
>>
>>
>> On Mon, Aug 21, 2017 at 9:17 PM, Betty Chu  wrote:
>>
>>> Yes, I have. The cacodylate ion does not fit well into the density.
>>>
>>> On Mon, Aug 21, 2017 at 11:44 AM, Pradeep Pallan <
>>> pradeeppal...@gmail.com> wrote:
>>>
 Did you try modelling in a cacodylate ion (CH3)2AsO2-?

 On Mon, Aug 21, 2017 at 10:19 AM, Betty Chu  wrote:

> Dear ccp4bb,
>
> I am refining a 1.40 Angstrom data set for a DNA oligonucleotide.
> While the model for the DNA fits very well into the density, there is a
> patch of positive electron density in the solvent space that we are having
> trouble with.
>
> The screenshot can be viewed through this link:
> https://cbsostorage.chem.umd.edu/owncloud/index.php/s/J5cKnOpCC4vb1VC
>
> In the screenshot, the yellow color is the anomalous map and a barium
> ion is fitted into density near the positive green electron density.
>
> The oligonucleotide was purchased from IDT. The crystallization
> condition is 15% MPD, 120 mM BaCl2, and 30 mM NaCaC pH 6.4. I have tried
> modelling Ba2+ with coordinated waters, MPD, and sodium cacodylate into 
> the
> electron density, but none of those fit well.
>
> Any suggestions regarding the identity of this electron density is
> much appreciated. Thank you!
>
> Sincerely,
>
> Betty Chu
> Paukstelis Research Group
> Department of Chemistry and Biochemistry
> University of Maryland, College Park
>



 --

 ---
 Pradeep Pallan

>>>
>>>
>>
>
>

Re: [ccp4bb] Unknown positive electron density

[CRAIG A BINGMAN]

What is the data collection wavelength/energy? Would you expect significant 
anomalous diffraction from As at this wavelength?

On Aug 21, 2017, at 11:37 AM, Betty Chu 
> wrote:

Hi Shailesh,

When I modelled in the Barium ion with octahedrally coordinated waters and ran 
the refinement, the distances from the barium to some of the waters ended up 
being too close (<2.2 Angstroms). Also, the positive electron density is 
connected. If the density indicated barium with coordinated waters, would that 
mean there are multiple ones present in the positive density?

Here are more views of the connected positive density.

https://cbsostorage.chem.umd.edu/owncloud/index.php/s/j5CgZIJe6NKgUlZ

https://cbsostorage.chem.umd.edu/owncloud/index.php/s/YbiNFbT8AW1iHUQ

On Mon, Aug 21, 2017 at 12:00 PM, Shailesh Tripathi 
> wrote:
Looks like Ba2+. Since it exist with coordination number 6 or above check what 
geometry water is following there (trigonal bipiramidal or so on). Water might 
also be shared by symmetry related Ba cation.



Shailesh Kumar Tripathi,
Phone: 9686289668


On Mon, Aug 21, 2017 at 9:17 PM, Betty Chu 
> wrote:
Yes, I have. The cacodylate ion does not fit well into the density.

On Mon, Aug 21, 2017 at 11:44 AM, Pradeep Pallan 
> wrote:
Did you try modelling in a cacodylate ion (CH3)2AsO2-?

On Mon, Aug 21, 2017 at 10:19 AM, Betty Chu 
> wrote:
Dear ccp4bb,

I am refining a 1.40 Angstrom data set for a DNA oligonucleotide. While the 
model for the DNA fits very well into the density, there is a patch of positive 
electron density in the solvent space that we are having trouble with.

The screenshot can be viewed through this link:
https://cbsostorage.chem.umd.edu/owncloud/index.php/s/J5cKnOpCC4vb1VC

In the screenshot, the yellow color is the anomalous map and a barium ion is 
fitted into density near the positive green electron density.

The oligonucleotide was purchased from IDT. The crystallization condition is 
15% MPD, 120 mM BaCl2, and 30 mM NaCaC pH 6.4. I have tried modelling Ba2+ with 
coordinated waters, MPD, and sodium cacodylate into the electron density, but 
none of those fit well.

Any suggestions regarding the identity of this electron density is much 
appreciated. Thank you!

Sincerely,

Betty Chu
Paukstelis Research Group
Department of Chemistry and Biochemistry
University of Maryland, College Park



--

---
Pradeep Pallan

Re: [ccp4bb] Unknown positive electron density

[Betty Chu]

Hi Shailesh,

When I modelled in the Barium ion with octahedrally coordinated waters and
ran the refinement, the distances from the barium to some of the waters
ended up being too close (<2.2 Angstroms). Also, the positive electron
density is connected. If the density indicated barium with coordinated
waters, would that mean there are multiple ones present in the positive
density?

Here are more views of the connected positive density.

https://cbsostorage.chem.umd.edu/owncloud/index.php/s/j5CgZIJe6NKgUlZ

https://cbsostorage.chem.umd.edu/owncloud/index.php/s/YbiNFbT8AW1iHUQ

On Mon, Aug 21, 2017 at 12:00 PM, Shailesh Tripathi 
wrote:

> Looks like Ba2+. Since it exist with coordination number 6 or above check
> what geometry water is following there (trigonal bipiramidal or so on).
> Water might also be shared by symmetry related Ba cation.
>
>
>
> Shailesh Kumar Tripathi,
> Phone: 9686289668
>
>
> On Mon, Aug 21, 2017 at 9:17 PM, Betty Chu  wrote:
>
>> Yes, I have. The cacodylate ion does not fit well into the density.
>>
>> On Mon, Aug 21, 2017 at 11:44 AM, Pradeep Pallan > > wrote:
>>
>>> Did you try modelling in a cacodylate ion (CH3)2AsO2-?
>>>
>>> On Mon, Aug 21, 2017 at 10:19 AM, Betty Chu  wrote:
>>>
 Dear ccp4bb,

 I am refining a 1.40 Angstrom data set for a DNA oligonucleotide. While
 the model for the DNA fits very well into the density, there is a patch of
 positive electron density in the solvent space that we are having trouble
 with.

 The screenshot can be viewed through this link:
 https://cbsostorage.chem.umd.edu/owncloud/index.php/s/J5cKnOpCC4vb1VC

 In the screenshot, the yellow color is the anomalous map and a barium
 ion is fitted into density near the positive green electron density.

 The oligonucleotide was purchased from IDT. The crystallization
 condition is 15% MPD, 120 mM BaCl2, and 30 mM NaCaC pH 6.4. I have tried
 modelling Ba2+ with coordinated waters, MPD, and sodium cacodylate into the
 electron density, but none of those fit well.

 Any suggestions regarding the identity of this electron density is much
 appreciated. Thank you!

 Sincerely,

 Betty Chu
 Paukstelis Research Group
 Department of Chemistry and Biochemistry
 University of Maryland, College Park

>>>
>>>
>>>
>>> --
>>>
>>> ---
>>> Pradeep Pallan
>>>
>>
>>
>

Re: [ccp4bb] Protein quantitaion based on extiction coefficient versus peptide bond

[Thomas Cleveland]

Hi,

If you have the correct (experimentally-determined) extinction
coefficients, the accuracy is usually very good. Often, however, people use
calculated extinction coefficients, which can be off (usually not by more
than 25%, so still OK for many purposes).

One of the best practical ways to experimentally determine accurate
extinction coefficients is the Edelhoch method. You can read about it here:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2143013/pdf/8563639.pdf

-Tom

---

Thomas Cleveland
NIST Center for Neutron Research
Gaithersburg, MD

On Sun, Aug 20, 2017 at 10:14 AM, anita patil <
153960d6533c-dmarc-requ...@jiscmail.ac.uk> wrote:

>
>
>
>
> Dear Members,
> How well is extinction coefficient based protein concentration measurement
> accuracy established. It seems that several monograph based protein HPLC
> assays use 214 for the same. How accurate are extinction coefficients for
> the same proteins. Is there any consensus if which one is more accurate for
> estimation. Some glycoprotein  monographs use extinction coefficients.
>
> Thanks ahead
>
> Anita Ramdas Patil, Ph.D.
> Group Leader, ADL
> Wockhardt Research Center,
> Aurangabad,
> INDIA Email: patilramdasan...@yahoo.com
> Mobile# 91+8980217260
>
>
>

Re: [ccp4bb] Unknown positive electron density

[Shailesh Tripathi]

Looks like Ba2+. Since it exist with coordination number 6 or above check
what geometry water is following there (trigonal bipiramidal or so on).
Water might also be shared by symmetry related Ba cation.



Shailesh Kumar Tripathi,
Phone: 9686289668


On Mon, Aug 21, 2017 at 9:17 PM, Betty Chu  wrote:

> Yes, I have. The cacodylate ion does not fit well into the density.
>
> On Mon, Aug 21, 2017 at 11:44 AM, Pradeep Pallan 
> wrote:
>
>> Did you try modelling in a cacodylate ion (CH3)2AsO2-?
>>
>> On Mon, Aug 21, 2017 at 10:19 AM, Betty Chu  wrote:
>>
>>> Dear ccp4bb,
>>>
>>> I am refining a 1.40 Angstrom data set for a DNA oligonucleotide. While
>>> the model for the DNA fits very well into the density, there is a patch of
>>> positive electron density in the solvent space that we are having trouble
>>> with.
>>>
>>> The screenshot can be viewed through this link:
>>> https://cbsostorage.chem.umd.edu/owncloud/index.php/s/J5cKnOpCC4vb1VC
>>>
>>> In the screenshot, the yellow color is the anomalous map and a barium
>>> ion is fitted into density near the positive green electron density.
>>>
>>> The oligonucleotide was purchased from IDT. The crystallization
>>> condition is 15% MPD, 120 mM BaCl2, and 30 mM NaCaC pH 6.4. I have tried
>>> modelling Ba2+ with coordinated waters, MPD, and sodium cacodylate into the
>>> electron density, but none of those fit well.
>>>
>>> Any suggestions regarding the identity of this electron density is much
>>> appreciated. Thank you!
>>>
>>> Sincerely,
>>>
>>> Betty Chu
>>> Paukstelis Research Group
>>> Department of Chemistry and Biochemistry
>>> University of Maryland, College Park
>>>
>>
>>
>>
>> --
>>
>> ---
>> Pradeep Pallan
>>
>
>

Re: [ccp4bb] Unknown positive electron density

[Betty Chu]

Yes, I have. The cacodylate ion does not fit well into the density.

On Mon, Aug 21, 2017 at 11:44 AM, Pradeep Pallan 
wrote:

> Did you try modelling in a cacodylate ion (CH3)2AsO2-?
>
> On Mon, Aug 21, 2017 at 10:19 AM, Betty Chu  wrote:
>
>> Dear ccp4bb,
>>
>> I am refining a 1.40 Angstrom data set for a DNA oligonucleotide. While
>> the model for the DNA fits very well into the density, there is a patch of
>> positive electron density in the solvent space that we are having trouble
>> with.
>>
>> The screenshot can be viewed through this link:
>> https://cbsostorage.chem.umd.edu/owncloud/index.php/s/J5cKnOpCC4vb1VC
>>
>> In the screenshot, the yellow color is the anomalous map and a barium ion
>> is fitted into density near the positive green electron density.
>>
>> The oligonucleotide was purchased from IDT. The crystallization condition
>> is 15% MPD, 120 mM BaCl2, and 30 mM NaCaC pH 6.4. I have tried modelling
>> Ba2+ with coordinated waters, MPD, and sodium cacodylate into the electron
>> density, but none of those fit well.
>>
>> Any suggestions regarding the identity of this electron density is much
>> appreciated. Thank you!
>>
>> Sincerely,
>>
>> Betty Chu
>> Paukstelis Research Group
>> Department of Chemistry and Biochemistry
>> University of Maryland, College Park
>>
>
>
>
> --
>
> ---
> Pradeep Pallan
>

Re: [ccp4bb] Unknown positive electron density

[Pradeep Pallan]

Did you try modelling in a cacodylate ion (CH3)2AsO2-?

On Mon, Aug 21, 2017 at 10:19 AM, Betty Chu  wrote:

> Dear ccp4bb,
>
> I am refining a 1.40 Angstrom data set for a DNA oligonucleotide. While
> the model for the DNA fits very well into the density, there is a patch of
> positive electron density in the solvent space that we are having trouble
> with.
>
> The screenshot can be viewed through this link:
> https://cbsostorage.chem.umd.edu/owncloud/index.php/s/J5cKnOpCC4vb1VC
>
> In the screenshot, the yellow color is the anomalous map and a barium ion
> is fitted into density near the positive green electron density.
>
> The oligonucleotide was purchased from IDT. The crystallization condition
> is 15% MPD, 120 mM BaCl2, and 30 mM NaCaC pH 6.4. I have tried modelling
> Ba2+ with coordinated waters, MPD, and sodium cacodylate into the electron
> density, but none of those fit well.
>
> Any suggestions regarding the identity of this electron density is much
> appreciated. Thank you!
>
> Sincerely,
>
> Betty Chu
> Paukstelis Research Group
> Department of Chemistry and Biochemistry
> University of Maryland, College Park
>



-- 

---
Pradeep Pallan

[ccp4bb] Unknown positive electron density

[Betty Chu]

Dear ccp4bb,

I am refining a 1.40 Angstrom data set for a DNA oligonucleotide. While the
model for the DNA fits very well into the density, there is a patch of
positive electron density in the solvent space that we are having trouble
with.

The screenshot can be viewed through this link:
https://cbsostorage.chem.umd.edu/owncloud/index.php/s/J5cKnOpCC4vb1VC

In the screenshot, the yellow color is the anomalous map and a barium ion
is fitted into density near the positive green electron density.

The oligonucleotide was purchased from IDT. The crystallization condition
is 15% MPD, 120 mM BaCl2, and 30 mM NaCaC pH 6.4. I have tried modelling
Ba2+ with coordinated waters, MPD, and sodium cacodylate into the electron
density, but none of those fit well.

Any suggestions regarding the identity of this electron density is much
appreciated. Thank you!

Sincerely,

Betty Chu
Paukstelis Research Group
Department of Chemistry and Biochemistry
University of Maryland, College Park

Re: [ccp4bb] BP3 error

[Wei Ding]

Thank you for your quick reply.
Crank2 seems to work very well when the job running.
Thanks again.



--

Wei Ding
P.O.Box 603
The Institute of Physics,Chinese Academy of Sciences
Beijing,China
100190
Tel: +86-10-82649083
E-mail: ding...@iphy.ac.cn

At 2017-08-21 15:02:53, "Navraj Pannu"  wrote:

Dear Wei,


Thanks for reporting the error.  Although it does not answer your question, I 
would strongly recommend to use crank2 over crank: it provides much better 
results.  In fact, Pavol Skubak and I will probably shortly decide to only 
distribute crank2.


I send this email with ccp4bb (cc'ed) just to let the community know to run 
crank2 instead of crank, but I'll still look into your problem and provide a 
fix.


Best wishes, Navraj


On Mon, Aug 21, 2017 at 5:54 AM, Wei Ding  wrote:

Dear all,

Running BP3 for SAD data, but fail, the error message shown as follow:
Dose anyone know how to fix it?



crank->Error:crank::crank binary and crank XML version do not match





***
* Information from CCP4Interface script
***
The program run with command: 
/home/dingwei/software/IPCAS_CENTOS6.5/Cryst_software/ccp4-7.0/bin/tclsh 
/home/dingwei/software/IPCAS_CENTOS6.5/Cryst_software/ccp4-7.0/bin/crank 
/sugon1/dingwei/ccp4tmp/adic_1_1_com.tmp 
has failed with error message
crank::crank binary and crank XML version do not match
while executing
"error $message"
(procedure "crank_error" line 8)
invoked from within
"crank_error "crank::crank binary and crank XML version do not match""
invoked from within
"if { [info exists XMLParse([join "crank parameters version" __])] } {
   if { $version != $XMLParse([join "crank parameters version" __]) } {
  cr..."
(file 
"/home/dingwei/software/IPCAS_CENTOS6.5/Cryst_software/ccp4-7.0/bin/crank" line 
71)
***




#CCP4I TERMINATION STATUS 0 "crank::crank binary and crank XML version do not 
match while executing "error $message" (procedure "crank_error" line 8) 
invoked from within "crank_error "crank::crank binary and crank XML version 
do not match"" invoked from within "if { [info exists XMLParse([join "crank 
parameters version" __])] } {if { $version != $XMLParse([join "crank 
parameters version" __]) } {   cr..." (file 
"/home/dingwei/software/IPCAS_CENTOS6.5/Cryst_software/ccp4-7.0/bin/crank" line 
71)"
#CCP4I TERMINATION TIME 21 Aug 2017  09:26:09
#CCP4I TERMINATION OUTPUT_FILES   /sugon1/dingwei/ccp4/1_crank
#CCP4I MESSAGE Task failed










--

Wei Ding
P.O.Box 603
The Institute of Physics,Chinese Academy of Sciences
Beijing,China
100190
Tel: +86-10-82649083
E-mail: ding...@iphy.ac.cn




 

Re: [ccp4bb] BP3 error

[Navraj Pannu]

Dear Wei,

Thanks for reporting the error.  Although it does not answer your question,
I would strongly recommend to use crank2 over crank: it provides much
better results.  In fact, Pavol Skubak and I will probably shortly decide
to only distribute crank2.

I send this email with ccp4bb (cc'ed) just to let the community know to run
crank2 instead of crank, but I'll still look into your problem and provide
a fix.

Best wishes, Navraj

On Mon, Aug 21, 2017 at 5:54 AM, Wei Ding  wrote:

> Dear all,
> Running BP3 for SAD data, but fail, the error message shown as follow:
> Dose anyone know how to fix it?
>
> crank->Error:crank::crank binary and crank XML version do not match
>
>
> 
>
>
> ***
>
> * Information from CCP4Interface script
>
> ***
>
> The program run with command: 
> /home/dingwei/software/IPCAS_CENTOS6.5/Cryst_software/ccp4-7.0/bin/tclsh 
> /home/dingwei/software/IPCAS_CENTOS6.5/Cryst_software/ccp4-7.0/bin/crank 
> /sugon1/dingwei/ccp4tmp/adic_1_1_com.tmp
>
> has failed with error message
>
> crank::crank binary and crank XML version do not match
>
> while executing
>
> "error $message"
>
> (procedure "crank_error" line 8)
>
> invoked from within
>
> "crank_error "crank::crank binary and crank XML version do not match""
>
> invoked from within
>
> "if { [info exists XMLParse([join "crank parameters version" __])] } {
>
>if { $version != $XMLParse([join "crank parameters version" __]) } {
>
>   cr..."
>
> (file 
> "/home/dingwei/software/IPCAS_CENTOS6.5/Cryst_software/ccp4-7.0/bin/crank" 
> line 71)
>
> ***
>
>
>
> #CCP4I TERMINATION STATUS 0 "crank::crank binary and crank XML version do not 
> match while executing "error $message" (procedure "crank_error" line 
> 8) invoked from within "crank_error "crank::crank binary and crank XML 
> version do not match"" invoked from within "if { [info exists 
> XMLParse([join "crank parameters version" __])] } {if { $version != 
> $XMLParse([join "crank parameters version" __]) } {   cr..." (file 
> "/home/dingwei/software/IPCAS_CENTOS6.5/Cryst_software/ccp4-7.0/bin/crank" 
> line 71)"
>
> #CCP4I TERMINATION TIME 21 Aug 2017  09:26:09
>
> #CCP4I TERMINATION OUTPUT_FILES   /sugon1/dingwei/ccp4/1_crank
>
> #CCP4I MESSAGE Task failed
>
>
>
>
>
>
>
>
> --
> Wei Ding
> P.O.Box 603
> The Institute of Physics,Chinese Academy of Sciences
> Beijing,China
> 100190
> Tel: +86-10-82649083 <+86%2010%208264%209083>
> E-mail: ding...@iphy.ac.cn 
>
>
>
>

Re: [ccp4bb] Co-purified ligand present in protein crystal

[Tomas Malinauskas]

Dear Wenhe,
we had a similar case and extraction using CHCl3 plus CH3OH (2:1
ratio, v/v) (65 °C, 30 min) before mass spectrometry worked very well:
https://www.ncbi.nlm.nih.gov/pubmed/21743455
Hope that helps,
Tomas

On Mon, Aug 21, 2017 at 4:41 AM, WENHE ZHONG
 wrote:
> Dear CCP4BB members,
>
> We would like to identify a ligand that is present in crystal structure 
> (according to strong positive densities at active site) but absent in 
> crystallization condition. We already have some candidates in mind based on 
> our knowledges on this protein but we need to validate further. The general 
> method we are using now is to use methanal to precipitate protein and extract 
> ligand from the precipitated protein. Then we analyse the methanol extraction 
> sample on LC-MS. One problem of this method is that the methanol extraction 
> will not be 100% efficient which means there is only a small portion of 
> bound-ligand can be extracted from the protein— particularly if the ligand 
> binds very tightly to the protein. So I would like to know whether anyone has 
> experience to efficiently extract tighly-bound ligands from protein for 
> downstream analysis. One method is to digest protein with protease such as 
> trypsin. Or use urea to denature the protein. However, these methods require 
> relatively long processing time which is not optimal when the ligand that we 
> want to analyse is unstable (degrade overtime). Anyone has more suggestions?
>
> Thank you!
>
> Kind regards,
> Wenhe

Re: [ccp4bb] Co-purified ligand present in protein crystal

[Klaus Fütterer]

Wenhe

Potentially, mass spectrometry in combination with electron capture 
dissociation and/or collision-induced dissociation might be helpful. See doi: 
10.1007/s13361-013-0662-5.

Best wishes, 

Klaus


===
 
Dr. Klaus Fütterer
Deputy Head of School
Undergraduate Admissions
Room 717, Biosciences Tower

School of Biosciences P: +44-(0)-121-414 5895
University of Birmingham  E: k.futte...@bham.ac.uk 
Edgbaston  T: @KFbrumbio 
Birmingham, B15 2TT, UK   W: http://tinyurl.com/futterer-lab
===





> On 21 Aug 2017, at 04:41,  
>  wrote:
> 
> Dear CCP4BB members,
> 
> We would like to identify a ligand that is present in crystal structure 
> (according to strong positive densities at active site) but absent in 
> crystallization condition. We already have some candidates in mind based on 
> our knowledges on this protein but we need to validate further. The general 
> method we are using now is to use methanal to precipitate protein and extract 
> ligand from the precipitated protein. Then we analyse the methanol extraction 
> sample on LC-MS. One problem of this method is that the methanol extraction 
> will not be 100% efficient which means there is only a small portion of 
> bound-ligand can be extracted from the protein— particularly if the ligand 
> binds very tightly to the protein. So I would like to know whether anyone has 
> experience to efficiently extract tighly-bound ligands from protein for 
> downstream analysis. One method is to digest protein with protease such as 
> trypsin. Or use urea to denature the protein. However, these methods require 
> relatively long processing time which is not optimal when the ligand that we 
> want to analyse is unstable (degrade overtime). Anyone has more suggestions?
> 
> Thank you!
> 
> Kind regards,
> Wenhe

Re: [ccp4bb] Co-purified ligand present in protein crystal

[Ruud Hovius]

Dear Wenhe,

Are there other ligands known for this protein ? Could these be 
chemically modified and still interact with your protein ?
Then you could use ligand affinity chromatography to purify your protein 
eluting with the same or other ligands.


For me this the only way to obtain sepiapterin with defined co-factor 
(cybacron blue column) and/or ligand (sulfapyridine column) occupations.


Alternatively soaking to exchange ligands in the crystal ?

Best of luck,

Ruud

On 21/8/17 05:41, WENHE ZHONG wrote:

Dear CCP4BB members,

We would like to identify a ligand that is present in crystal structure 
(according to strong positive densities at active site) but absent in 
crystallization condition. We already have some candidates in mind based on our 
knowledges on this protein but we need to validate further. The general method 
we are using now is to use methanal to precipitate protein and extract ligand 
from the precipitated protein. Then we analyse the methanol extraction sample 
on LC-MS. One problem of this method is that the methanol extraction will not 
be 100% efficient which means there is only a small portion of bound-ligand can 
be extracted from the protein— particularly if the ligand binds very tightly to 
the protein. So I would like to know whether anyone has experience to 
efficiently extract tighly-bound ligands from protein for downstream analysis. 
One method is to digest protein with protease such as trypsin. Or use urea to 
denature the protein. However, these methods require relatively long processing 
time which is not optimal when the ligand that we want to analyse is unstable 
(degrade overtime). Anyone has more suggestions?

Thank you!

Kind regards,
Wenhe


--

Ruud Hovius
EPFL SB ISIC LIP
BCH 4209
CH-1015 Lausanne
+41-21-693-9442
http://lip.epfl.ch