Re: [ccp4bb] MR for coiled coil structure

[Shengyang Jin]

Dear All,

Thank you very much for your valued suggestions. I am trying them out and will 
let you know how they work ASAP.
If successful I will write a summary and post to ccp4bb.


Best,
Shengyang



From: Conners, Becky [mailto:r.conn...@exeter.ac.uk]
Sent: 10 July 2019 16:29
To: Shengyang Jin 
Subject: Re: MR for coiled coil structure

Hi Shengyang,

It is from a few years ago now, and there may be better methods now, but you 
could have a look at:

EMBO J. 2008 Jun 
18;27(12):1779-89. doi: 10.1038/emboj.2008.101. Epub 2008 May 22.
The Moraxella adhesin UspA1 binds to its human CEACAM1 receptor by a deformable 
trimeric coiled-coil.
Conners 
R1,
 Hill 
DJ,
 Borodina 
E,
 Agnew 
C,
 Daniell 
SJ,
 Burton 
NM,
 Sessions 
RB,
 Clarke 
AR,
 Catto 
LE,
 Lammie 
D,
 Wess 
T,
 Brady 
RL,
 Virji 
M.

The structure was solved by a novel exploratory molecular replacement method 
using in excess of 20 different coiled-coil structures as search models in the 
program PHASER (McCoy et al., 2005). The coordinates of a single strand of the 
coiled coil of cortexellin (1D7M.pdb), which has no recognisable sequence 
identity to UspA1(527-665), was eventually successful despite being from a 
dimeric coiled coil. Solutions from PHASER had Z scores of 6 and 13 after 
rotation and translation respectively to find the first molecule in the 
asymmetric unit, and 5 and 9 for the second molecule. Initial phases were 
improved by mutation of the sequence to poly-Ala followed by "atoms update and 
refinement" mode of ARP/wARP (Perrakis et al., 2001). Symmetry operators were 
applied to the first monomer to create the two trimers. The structure was 
refined with iterative cycles of manual model-building using COOT (Emsley and 
Cowtan, 2004), restrained refinement with REFMAC5 (Murshudov et al., 1997) and 
density improvement with ARP/wARP as the correct sequence was gradually built 
into the improving electron density maps. Data collection and final refinement 
statistics are summarised in Table 1.
The structure was a trimeric coiled coil, but the model we were most successful 
with was actually from a dimeric coiled coil and had no sequence identity to 
our protein. The "atom update and refinement" option in Arp/wARP was excellent 
at improving our maps.

Good luck!

Becky


--

Dr Becky Conners

Postdoctoral Research Associate

University of Exeter



01392 727468

www.exeter.ac.uk

Office S03.07, Living Systems Institute, University of Exeter, Stocker Road, 
Exeter, EX4 4QD, UK


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From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Shengyang Jin 
mailto:jinshengy...@outlook.com>>
Sent: 10 July 2019 09:00
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] MR for coiled coil structure

Dear all,

We recently acquired a data set (2.0 A, P222) for a coiled coil protein 
(according to Itasser, QUARK, Robetta, and Phaser).
Matthews coefficient indicates 1 copy of protein per ASU. Sequence of the 
protein is quite novel with no apparent homolog in PDB.
We tried to to MR with various models (ab initio or homology based) but with 
little success.

We then tried to use AMPLE, but in ccp4 it always returned this error:
__main__.py: 

[ccp4bb] Two postdoctoral positions at the Walter and Eliza Hall Institute, Melbourne

[James Murphy]

Dear colleagues,
We are seeking two talented and enthusiastic postdocs to join teams within the 
Inflammation Division of the Walter and Eliza Hall Institute of Medical 
Research, Melbourne, Australia: one focused on structural biology+cell biology; 
and another postdoc to study protein-protein interactions.

1. A postdoc or senior postdoc to study the structural and cell biology of 
pseudokinases, with a focus on the necroptosis effector MLKL
https://www.wehi.edu.au/research-officer-senior-research-officer-postdoctoral-scientist
Enquiries to jam...@wehi.edu.au

2. A postdoc to study the interactions of signaling proteins using biophysics 
and proteomics.
https://www.wehi.edu.au/research-officer-postdoctoral-scientist
Enquiries to snichol...@wehi.edu.au

With thanks and best wishes
James

--
Assoc. Prof. James M. Murphy, PhD
Head, Inflammation Division
Walter and Eliza Hall Institute of Medical Research
1G Royal Parade
Parkville, VIC 3052, Australia

T: +61 3 93452407
F: +61 3 93470852

http://www.wehi.edu.au/people/james-murphy


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You must not disclose, forward, print or use it without the permission of the 
sender.

The Walter and Eliza Hall Institute acknowledges the Wurundjeri people of the 
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the continuing connection to country and community.
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Re: [ccp4bb] Fo-Fc density close to cysteine residue

[Prem Prakash]

Hi Lumbini,

There are couple of queries I have before commenting on this issue.

1) how many Cysteine residues are there in your protein

2) have you calculated their solvent accessible surface area ?

3) What is the final concentration of Sodium dithionite ?

Cysteine is highly prone to get thiol covalently modified if it is quite
accessible to the solvent in its free form. As you know, sodium dithionite
is having almost same chemical structure like BME. And you will find N
numbers of papers about how BME covalently modifies the accessible cysteine
in the protein. It seems to me that it modified by sodium dithionite by air
oxidation.

Please check the facts and try to fit with manually built derivative of the
sodium dithionite with this cysteine residue.

Good luck

P.P

On Tue, 9 Jul 2019, 04:32 Lumbini Yadav,  wrote:

> Dear all,
>
>
>
> We have found a huge Fo-Fc density close to cysteine residue (see attached
> image) in the structure with resolution of 1.2A. In the crystallization
> condition, we have PEG 3350, Potassium phosphate monobasic, glycerol and
> protein was in Tris and NaCl. Before freezing the crystals were soaked in
> mother liquor containing sodium dithionite.
>
>
>
> I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD,
> CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in
> all the screenings we do see some part of Fo-Fc density unaddressed at 3
> sigma.
>
>
>
> Does anyone have an idea about what this density could be? Covalent
> modification?
>
>
>
> Thanks.
>
>
>
> Kind regards,
>
> Lumbini
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Fo-Fc density close to cysteine residue

[Nukri Sanishvili]

Hi Jakob,
If there is radiation damage, for every "new" positive density there should
be a negative density in the original position. From the pictures we've
seen, it seems unlikely.
Best,
Nukri

On Wed, Jul 10, 2019 at 10:21 AM Keller, Jacob 
wrote:

> How about radiation-damaged/smashed Sulphur? You could test this by
> refining occupancy of the cys S.
>
>
>
> JPK
>
>
>
> +
>
> Jacob Pearson Keller
>
> Research Scientist / Looger Lab
>
> HHMI Janelia Research Campus
>
> 19700 Helix Dr, Ashburn, VA 20147
>
> Desk: (571)209-4000 x3159
>
> Cell: (301)592-7004
>
> +
>
>
>
> The content of this email is confidential and intended for the recipient
> specified in message only. It is strictly forbidden to share any part of
> this message with any third party, without a written consent of the sender.
> If you received this message by mistake, please reply to this message and
> follow with its deletion, so that we can ensure such a mistake does not
> occur in the future.
>
>
>
> *From:* CCP4 bulletin board  * On Behalf Of *Lumbini
> Yadav
> *Sent:* Wednesday, July 10, 2019 7:12 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] Fo-Fc density close to cysteine residue
>
>
>
> Thank you for the valuable input
>
>
>
> On Wed, Jul 10, 2019 at 4:25 PM Eleanor Dodson 
> wrote:
>
> Well - that more or less proves the residue is a CYS - there is a peak in
> the PHAN map right on the S.
>
> And that the extra density does not contain an anomalous scatterer so is
> probably not a metal or a sulphate or...
>
>
>
> But that still doesnt explain WHAT it is . Sorry not to be more help..
>
>
>
> Eleanor
>
>
>
> On Wed, 10 Jul 2019 at 11:32, Lumbini Yadav  wrote:
>
> No I am using ccp4i. I tried doing SAD refinement in refmac and the output
> image is attached below .
>
> I do not seen density near cysteine that was visible in Fo-Fc map
>
>
>
> On Wed, Jul 10, 2019 at 3:40 PM Eleanor Dodson 
> wrote:
>
> The key word for refmac is ANOM MAPONLY
>
> Are you using GUI2?
>
> Eleanor
>
>
>
> On Wed, 10 Jul 2019 at 09:32, Lumbini Yadav  wrote:
>
> I have soaked my crystals in  sodium dithionite a reducing agent. I have
> not done mass spec but sequence is confirmed
>
>
>
> On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel 
> wrote:
>
> Have you mass-speced the protein before crystallization to make sure it
> wasn’t derivatized during expression and/or purification, or compared the
> mass spec of the crystals verses purified protein? Any fancy reagents or
> other reductants used during purification?
>
> What about S-Acetyl-cysteine (3-letter code: SCY).
>
>
>
> Best,
>
>
>
> Dan
>
>
>
> Daniel A Bonsor PhD.
>
> Sundberg Lab
>
> Institute of Human Virology
>
> University of Maryland, Baltimore
>
> 725 W Lombard Street N370
>
> Baltimore
>
> Maryland
>
> MD 21201
>
> Tel: (410) 706-7457
>
>
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
> *Lumbini
> Yadav
> *Sent:* Tuesday, July 09, 2019 5:22 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] Fo-Fc density close to cysteine residue
>
>
>
> Dear all,
>
>
>
> We have found a huge Fo-Fc density close to cysteine residue (see attached
> image) in the structure with resolution of 1.2A. In the crystallization
> condition, we have PEG 3350, Potassium phosphate monobasic, glycerol and
> protein was in Tris and NaCl. Before freezing the crystals were soaked in
> mother liquor containing sodium dithionite.
>
>
>
> I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD,
> CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in
> all the screenings we do see some part of Fo-Fc density unaddressed at 3
> sigma.
>
>
>
> Does anyone have an idea about what this density could be? Covalent
> modification?
>
>
>
> Thanks.
>
>
>
>
>
> Kind regards,
>
> Lumbini
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> 
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> 
>
>
> --
>
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> 

Re: [ccp4bb] Fo-Fc density close to cysteine residue

[Keller, Jacob]

How about radiation-damaged/smashed Sulphur? You could test this by refining 
occupancy of the cys S.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

From: CCP4 bulletin board  On Behalf Of Lumbini Yadav
Sent: Wednesday, July 10, 2019 7:12 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Fo-Fc density close to cysteine residue

Thank you for the valuable input

On Wed, Jul 10, 2019 at 4:25 PM Eleanor Dodson 
mailto:eleanor.dod...@york.ac.uk>> wrote:
Well - that more or less proves the residue is a CYS - there is a peak in the 
PHAN map right on the S.
And that the extra density does not contain an anomalous scatterer so is 
probably not a metal or a sulphate or...

But that still doesnt explain WHAT it is . Sorry not to be more help..

Eleanor

On Wed, 10 Jul 2019 at 11:32, Lumbini Yadav 
mailto:lumbin...@gmail.com>> wrote:
No I am using ccp4i. I tried doing SAD refinement in refmac and the output 
image is attached below .
I do not seen density near cysteine that was visible in Fo-Fc map

On Wed, Jul 10, 2019 at 3:40 PM Eleanor Dodson 
mailto:eleanor.dod...@york.ac.uk>> wrote:
The key word for refmac is ANOM MAPONLY
Are you using GUI2?
Eleanor

On Wed, 10 Jul 2019 at 09:32, Lumbini Yadav 
mailto:lumbin...@gmail.com>> wrote:
I have soaked my crystals in  sodium dithionite a reducing agent. I have not 
done mass spec but sequence is confirmed

On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel 
mailto:dbon...@som.umaryland.edu>> wrote:
Have you mass-speced the protein before crystallization to make sure it wasn’t 
derivatized during expression and/or purification, or compared the mass spec of 
the crystals verses purified protein? Any fancy reagents or other reductants 
used during purification?
What about S-Acetyl-cysteine (3-letter code: SCY).

Best,

Dan

Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Lumbini Yadav
Sent: Tuesday, July 09, 2019 5:22 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Fo-Fc density close to cysteine residue

Dear all,

We have found a huge Fo-Fc density close to cysteine residue (see attached 
image) in the structure with resolution of 1.2A. In the crystallization 
condition, we have PEG 3350, Potassium phosphate monobasic, glycerol and 
protein was in Tris and NaCl. Before freezing the crystals were soaked in 
mother liquor containing sodium dithionite.

I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD, 
CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in all the 
screenings we do see some part of Fo-Fc density unaddressed at 3 sigma.

Does anyone have an idea about what this density could be? Covalent 
modification?

Thanks.


Kind regards,
Lumbini



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Re: [ccp4bb] Fo-Fc density close to cysteine residue

[Sergei Strelkov]

Dear Lumbini,


Certainly not a proof but your density looks like (di)methyl-arsinoyl-cysteine 
(PDB residue type CAF). This modification can happen when the protein has been 
exposed to cacodylate buffer. You can find such residues (and maps) in the PDB 
entries 3LPT or 5MDI.


Best wishes,

Sergei


Prof. Sergei V. Strelkov Laboratory for Biocrystallography Department of 
Pharmaceutical Sciences, KU Leuven O, Campus Gasthuisberg, Herestraat 49 bus 
822, 3000 Leuven, Belgium Phone: +32 16 33 08 45, mobile: +32 486 29 41 32 Lab 
pages: 
http://pharm.kuleuven.be/Biocrystallography


From: CCP4 bulletin board  on behalf of Lumbini Yadav 

Sent: Wednesday, July 10, 2019 13:12
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Fo-Fc density close to cysteine residue

Thank you for the valuable input

On Wed, Jul 10, 2019 at 4:25 PM Eleanor Dodson 
mailto:eleanor.dod...@york.ac.uk>> wrote:
Well - that more or less proves the residue is a CYS - there is a peak in the 
PHAN map right on the S.
And that the extra density does not contain an anomalous scatterer so is 
probably not a metal or a sulphate or...

But that still doesnt explain WHAT it is . Sorry not to be more help..

Eleanor

On Wed, 10 Jul 2019 at 11:32, Lumbini Yadav 
mailto:lumbin...@gmail.com>> wrote:
No I am using ccp4i. I tried doing SAD refinement in refmac and the output 
image is attached below .
I do not seen density near cysteine that was visible in Fo-Fc map

On Wed, Jul 10, 2019 at 3:40 PM Eleanor Dodson 
mailto:eleanor.dod...@york.ac.uk>> wrote:
The key word for refmac is ANOM MAPONLY
Are you using GUI2?
Eleanor

On Wed, 10 Jul 2019 at 09:32, Lumbini Yadav 
mailto:lumbin...@gmail.com>> wrote:
I have soaked my crystals in  sodium dithionite a reducing agent. I have not 
done mass spec but sequence is confirmed

On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel 
mailto:dbon...@som.umaryland.edu>> wrote:
Have you mass-speced the protein before crystallization to make sure it wasn't 
derivatized during expression and/or purification, or compared the mass spec of 
the crystals verses purified protein? Any fancy reagents or other reductants 
used during purification?
What about S-Acetyl-cysteine (3-letter code: SCY).

Best,

Dan

Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Lumbini Yadav
Sent: Tuesday, July 09, 2019 5:22 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Fo-Fc density close to cysteine residue

Dear all,

We have found a huge Fo-Fc density close to cysteine residue (see attached 
image) in the structure with resolution of 1.2A. In the crystallization 
condition, we have PEG 3350, Potassium phosphate monobasic, glycerol and 
protein was in Tris and NaCl. Before freezing the crystals were soaked in 
mother liquor containing sodium dithionite.

I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD, 
CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in all the 
screenings we do see some part of Fo-Fc density unaddressed at 3 sigma.

Does anyone have an idea about what this density could be? Covalent 
modification?

Thanks.



Kind regards,
Lumbini



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Re: [ccp4bb] Error in data reduction!

[Andrew Leslie]

I think Helen’s answer is absolutely right. I asked Phil Evans about this when 
I saw him yesterday, and he said that error usually arises when it is not 
possible to write the xml file, either because a disk is full, or because you 
do not have permission to write to that area.

Andrew

> On 3 Jul 2019, at 15:09, Mike Xishan  wrote:
> 
> Dear all,
> 
> Sorry for asking for a very naive question that might be asked before. 
> 
> After integration, I am trying to do "data reduction" through Aimless, but 
> task fails with an error message as
> 
>  #CCP4I TERMINATION STATUS 0 "FILEIO: cannot open file 
> /Processing1_8_pointless.xml
> 
> I really appreciate for your opinion and help to fix this problem.
> 
> Thanks, 
> 
> Mike
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
> 



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Re: [ccp4bb] Fo-Fc density close to cysteine residue

[Lumbini Yadav]

Thank you for the valuable input

On Wed, Jul 10, 2019 at 4:25 PM Eleanor Dodson 
wrote:

> Well - that more or less proves the residue is a CYS - there is a peak in
> the PHAN map right on the S.
> And that the extra density does not contain an anomalous scatterer so is
> probably not a metal or a sulphate or...
>
> But that still doesnt explain WHAT it is . Sorry not to be more help..
>
> Eleanor
>
> On Wed, 10 Jul 2019 at 11:32, Lumbini Yadav  wrote:
>
>> No I am using ccp4i. I tried doing SAD refinement in refmac and the
>> output image is attached below .
>> I do not seen density near cysteine that was visible in Fo-Fc map
>>
>> On Wed, Jul 10, 2019 at 3:40 PM Eleanor Dodson 
>> wrote:
>>
>>> The key word for refmac is ANOM MAPONLY
>>> Are you using GUI2?
>>> Eleanor
>>>
>>> On Wed, 10 Jul 2019 at 09:32, Lumbini Yadav  wrote:
>>>
 I have soaked my crystals in  sodium dithionite a reducing agent. I
 have not done mass spec but sequence is confirmed

 On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel <
 dbon...@som.umaryland.edu> wrote:

> Have you mass-speced the protein before crystallization to make sure
> it wasn’t derivatized during expression and/or purification, or compared
> the mass spec of the crystals verses purified protein? Any fancy reagents
> or other reductants used during purification?
>
> What about S-Acetyl-cysteine (3-letter code: SCY).
>
>
>
> Best,
>
>
>
> Dan
>
>
>
> Daniel A Bonsor PhD.
>
> Sundberg Lab
>
> Institute of Human Virology
>
> University of Maryland, Baltimore
>
> 725 W Lombard Street N370
>
> Baltimore
>
> Maryland
>
> MD 21201
>
> Tel: (410) 706-7457
>
>
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf
> Of *Lumbini Yadav
> *Sent:* Tuesday, July 09, 2019 5:22 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] Fo-Fc density close to cysteine residue
>
>
>
> Dear all,
>
>
>
> We have found a huge Fo-Fc density close to cysteine residue (see
> attached image) in the structure with resolution of 1.2A. In the
> crystallization condition, we have PEG 3350, Potassium phosphate
> monobasic, glycerol and protein was in Tris and NaCl. Before freezing
> the crystals were soaked in mother liquor containing sodium dithionite.
>
>
>
> I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC,
>  CSD, CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide.
> But in all the screenings we do see some part of Fo-Fc density
> unaddressed at 3 sigma.
>
>
>
> Does anyone have an idea about what this density could be? Covalent
> modification?
>
>
>
> Thanks.
>
>
>
>
>
> Kind regards,
>
> Lumbini
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>

 --

 To unsubscribe from the CCP4BB list, click the following link:
 https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

>>>



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Re: [ccp4bb] Fo-Fc density close to cysteine residue

[Eleanor Dodson]

Well - that more or less proves the residue is a CYS - there is a peak in
the PHAN map right on the S.
And that the extra density does not contain an anomalous scatterer so is
probably not a metal or a sulphate or...

But that still doesnt explain WHAT it is . Sorry not to be more help..

Eleanor

On Wed, 10 Jul 2019 at 11:32, Lumbini Yadav  wrote:

> No I am using ccp4i. I tried doing SAD refinement in refmac and the output
> image is attached below .
> I do not seen density near cysteine that was visible in Fo-Fc map
>
> On Wed, Jul 10, 2019 at 3:40 PM Eleanor Dodson 
> wrote:
>
>> The key word for refmac is ANOM MAPONLY
>> Are you using GUI2?
>> Eleanor
>>
>> On Wed, 10 Jul 2019 at 09:32, Lumbini Yadav  wrote:
>>
>>> I have soaked my crystals in  sodium dithionite a reducing agent. I have
>>> not done mass spec but sequence is confirmed
>>>
>>> On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel 
>>> wrote:
>>>
 Have you mass-speced the protein before crystallization to make sure it
 wasn’t derivatized during expression and/or purification, or compared the
 mass spec of the crystals verses purified protein? Any fancy reagents or
 other reductants used during purification?

 What about S-Acetyl-cysteine (3-letter code: SCY).



 Best,



 Dan



 Daniel A Bonsor PhD.

 Sundberg Lab

 Institute of Human Virology

 University of Maryland, Baltimore

 725 W Lombard Street N370

 Baltimore

 Maryland

 MD 21201

 Tel: (410) 706-7457





 *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf
 Of *Lumbini Yadav
 *Sent:* Tuesday, July 09, 2019 5:22 AM
 *To:* CCP4BB@JISCMAIL.AC.UK
 *Subject:* [ccp4bb] Fo-Fc density close to cysteine residue



 Dear all,



 We have found a huge Fo-Fc density close to cysteine residue (see
 attached image) in the structure with resolution of 1.2A. In the
 crystallization condition, we have PEG 3350, Potassium phosphate
 monobasic, glycerol and protein was in Tris and NaCl. Before freezing
 the crystals were soaked in mother liquor containing sodium dithionite.



 I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC,
  CSD, CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide.
 But in all the screenings we do see some part of Fo-Fc density
 unaddressed at 3 sigma.



 Does anyone have an idea about what this density could be? Covalent
 modification?



 Thanks.





 Kind regards,

 Lumbini


 --

 To unsubscribe from the CCP4BB list, click the following link:
 https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

>>>
>>> --
>>>
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>>
>>



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Re: [ccp4bb] Fo-Fc density close to cysteine residue

[Lumbini Yadav]

No I am using ccp4i. I tried doing SAD refinement in refmac and the output
image is attached below .
I do not seen density near cysteine that was visible in Fo-Fc map

On Wed, Jul 10, 2019 at 3:40 PM Eleanor Dodson 
wrote:

> The key word for refmac is ANOM MAPONLY
> Are you using GUI2?
> Eleanor
>
> On Wed, 10 Jul 2019 at 09:32, Lumbini Yadav  wrote:
>
>> I have soaked my crystals in  sodium dithionite a reducing agent. I have
>> not done mass spec but sequence is confirmed
>>
>> On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel 
>> wrote:
>>
>>> Have you mass-speced the protein before crystallization to make sure it
>>> wasn’t derivatized during expression and/or purification, or compared the
>>> mass spec of the crystals verses purified protein? Any fancy reagents or
>>> other reductants used during purification?
>>>
>>> What about S-Acetyl-cysteine (3-letter code: SCY).
>>>
>>>
>>>
>>> Best,
>>>
>>>
>>>
>>> Dan
>>>
>>>
>>>
>>> Daniel A Bonsor PhD.
>>>
>>> Sundberg Lab
>>>
>>> Institute of Human Virology
>>>
>>> University of Maryland, Baltimore
>>>
>>> 725 W Lombard Street N370
>>>
>>> Baltimore
>>>
>>> Maryland
>>>
>>> MD 21201
>>>
>>> Tel: (410) 706-7457
>>>
>>>
>>>
>>>
>>>
>>> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf
>>> Of *Lumbini Yadav
>>> *Sent:* Tuesday, July 09, 2019 5:22 AM
>>> *To:* CCP4BB@JISCMAIL.AC.UK
>>> *Subject:* [ccp4bb] Fo-Fc density close to cysteine residue
>>>
>>>
>>>
>>> Dear all,
>>>
>>>
>>>
>>> We have found a huge Fo-Fc density close to cysteine residue (see
>>> attached image) in the structure with resolution of 1.2A. In the
>>> crystallization condition, we have PEG 3350, Potassium phosphate
>>> monobasic, glycerol and protein was in Tris and NaCl. Before freezing
>>> the crystals were soaked in mother liquor containing sodium dithionite.
>>>
>>>
>>>
>>> I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD,
>>> CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in
>>> all the screenings we do see some part of Fo-Fc density unaddressed at
>>> 3 sigma.
>>>
>>>
>>>
>>> Does anyone have an idea about what this density could be? Covalent
>>> modification?
>>>
>>>
>>>
>>> Thanks.
>>>
>>>
>>>
>>>
>>>
>>> Kind regards,
>>>
>>> Lumbini
>>>
>>>
>>> --
>>>
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>>
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>



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Re: [ccp4bb] Fo-Fc density close to cysteine residue

[Eleanor Dodson]

The key word for refmac is ANOM MAPONLY
Are you using GUI2?
Eleanor

On Wed, 10 Jul 2019 at 09:32, Lumbini Yadav  wrote:

> I have soaked my crystals in  sodium dithionite a reducing agent. I have
> not done mass spec but sequence is confirmed
>
> On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel 
> wrote:
>
>> Have you mass-speced the protein before crystallization to make sure it
>> wasn’t derivatized during expression and/or purification, or compared the
>> mass spec of the crystals verses purified protein? Any fancy reagents or
>> other reductants used during purification?
>>
>> What about S-Acetyl-cysteine (3-letter code: SCY).
>>
>>
>>
>> Best,
>>
>>
>>
>> Dan
>>
>>
>>
>> Daniel A Bonsor PhD.
>>
>> Sundberg Lab
>>
>> Institute of Human Virology
>>
>> University of Maryland, Baltimore
>>
>> 725 W Lombard Street N370
>>
>> Baltimore
>>
>> Maryland
>>
>> MD 21201
>>
>> Tel: (410) 706-7457
>>
>>
>>
>>
>>
>> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of
>> *Lumbini Yadav
>> *Sent:* Tuesday, July 09, 2019 5:22 AM
>> *To:* CCP4BB@JISCMAIL.AC.UK
>> *Subject:* [ccp4bb] Fo-Fc density close to cysteine residue
>>
>>
>>
>> Dear all,
>>
>>
>>
>> We have found a huge Fo-Fc density close to cysteine residue (see
>> attached image) in the structure with resolution of 1.2A. In the
>> crystallization condition, we have PEG 3350, Potassium phosphate
>> monobasic, glycerol and protein was in Tris and NaCl. Before freezing
>> the crystals were soaked in mother liquor containing sodium dithionite.
>>
>>
>>
>> I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD,
>> CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in
>> all the screenings we do see some part of Fo-Fc density unaddressed at 3
>> sigma.
>>
>>
>>
>> Does anyone have an idea about what this density could be? Covalent
>> modification?
>>
>>
>>
>> Thanks.
>>
>>
>>
>>
>>
>> Kind regards,
>>
>> Lumbini
>>
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Error in Aimless task!

[Helen Ginn]

Hello Mike,

This is just a guess. From the error message it seems that it’s trying to write 
to the root directory of your filesystem.

I am assuming you are using the old ccp4i interface. It might be because the 
ccp4i project directory you’re running it under has been set to / or is empty. 
If so, best set it to a user directory you can write to.

Best wishes
Helen Ginn

> Date:Mon, 8 Jul 2019 12:21:39 +0530
> From:Mike Xishan 
> Subject: Error in Aimless task!
> 
> Hello everyone,
> 
> Data reduction with Aimless from (CCP4i) fails with error message as below
> FILEIO: cannot open file /Processing1_8_pointless.xml
> 
> How can this error be fixed?  Comment please...
> 
> Thanks
> 
> Mike
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1



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[ccp4bb] PostDoc Position at the Wohl Institute of King’s College London

[doriano lamba]

Dear Members of the CCP4bb,

On behalf of Prof. Annalisa Pastore, I am forwarding the following 
Post-Doctoral Job Advertisement at the Wohl Institute of King’s College 
London.


Please direct any enquire to:

annalisa.past...@crick.ac.uk

Kindest regards
Doriano Lamba


DA: "Annalisa Pastore" 
A: "Doriano Lamba" 
INVIATO: Mercoledì, 10 luglio 2019 7:38:12
OGGETTO: con preghiera di diffusione

We are looking for a young and dynamic person post-PhD willing to carry
out an interesting and challenging project of Structural Biology at the
Wohl Institute of King’s College London. The Wohl is an institute
opened in 2015 and devoted to the study of Neuroscience from a
multidisciplinary point of view. Our group works within the tradition of
Structural Biology established at King’s College London which sees
among its alumni Rosalind Franklin, Maurice Wilkins, Alex Stokes,
Herbert Wilson and John Randall. We aim to solve the structure of
ARPP21, a protein involved in neuronal development and, when mutated, in
amyotrophic latheral schlerosis, and determine its properties in
aggregation. ARPP21 is a mixed folded protein which contains potentially
structured domains and intrinsically unfolded regions. The successful
candidate will have access to all the superb facilities of the Wohl,
Randal and Crick Institutes. These include several NMR spectrometers
ranging from 600 to 950 MHz, advanced optical spectroscopies,
crystallization robots and many other tools in support of structural and
integrated biology.

Enquiries to annalisa.past...@crick.ac.uk

 The Francis Crick Institute Limited is a registered charity in England
and Wales no. 1140062 and a company registered in England and Wales no.
06885462, with its registered office at 1 Midland Road London NW1 1AT

--
Dr. Doriano Lamba
Senior Scientist

Istituto di Cristallografia - C.N.R.
Struttura Secondaria di Trieste
Area Science Park - Basovizza
Edificio Q1 - Stanza 106
Strada Statale 14 - Km. 163.5
I-34149 Trieste - Italy

Phone Office: ++39+040-375-7527
Phone Lab: ++39+040-375-7521/7536/7537
Phone XRD-1 Beamline: ++39+040-375-8355/8349
Skypename: doriano.lamba
http://www.ic.cnr.it/ic4/staff/doriano-lamba/
E-mail: doriano.la...@ic.cnr.it or/and doriano.la...@elettra.eu

Home:
Via dei Giustinelli 6
I-34124 Trieste - Italy
Phone: ++39+040-2038589

___
WoS ResearchID: http://www.researcherid.com/rid/L-9808-2018
ORCID ID: http://orcid.org/-0001-6859-7868
Google Scholar: 
http://scholar.google.it/citations?hl=en=YzLT5gUJ

Research Gate: https://www.researchgate.net/profile/Doriano_Lamba2
Scopus ID: https://www.scopus.com/authid/detail.url?authorId=7003374970



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[ccp4bb] Research Fellow Position in Singapore

[hans song]

We are seeking an enthusiastic and self-motivated postdoctoral research
fellow to study proteins involved in maintaining genome stability and the
Hippo signaling pathway. We take a combinatorial approach of molecular
biology, biochemical and biophysical methods, and structural biology
(crystallography and cryo-EM). ­Significant aspects of our projects have
translational potential. Candidates with an interest in the development of
small molecules/peptides for cancer therapeutics are also welcome.



Singapore is an excellent environment to do research with many research
groups close by in Biopolis [https://www.a-star.edu.sg/imcb]. A*STAR offers
highly competitive salaries with benefits including housing allowance and
1-3 months performance bonus.



Requirements:

·   PhD in biochemistry, biophysics, molecular and cell biology or  a
related field

·  Solid practical experience in Cryo-EM and/or X-ray crystallography

·  Practical skills in molecular biology, protein expression (preferably insect
and/or mammalian cells) and purification, and various biochemical techniques

·   Good publication record

·   Competent in scientific writing



Interested applicants should send a CV, publication list, a brief
description of research interests and experience, and three confidential
letters of reference to: Prof. Haiwei Song (Email: hai...@imcb.a-star.edu.sg
),
Institute of Molecular and Cell Biology, A*STAR, Singapore.



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Re: [ccp4bb] MR for coiled coil structure

[Claudia Millán Nebot]

Dear Mark and Shengyang,

indeed, as Mark pointed out, we have a special mode in ARCIMBOLDO_LITE
devised to tackle coiled-coil structures. Trough the ccp4i interface this
will be available as an option you can tick when running an ARCIMBOLDO_LITE
job. A tutorial is available on our website (
http://chango.ibmb.csic.es/tutorial_coiled) and you can also contact us
directly if you have any questions regarding its use.

As for Mark's comment on beta structures, they are always more challenging
for a number of reasons, but we have been successful in solving some of
them using our libraries of general folds in ARCIMBOLDO_BORGES. There is
also an example on our website (http://chango.ibmb.csic.es/tutorial) but if
you have any particular question regarding parameterization or you need to
generate a library of a fold that it is still not available trough CCP4 you
can contact us and well be happy to help.

Best wishes,

Claudia




Claudia Millán (cmn...@ibmb.csic.es)

Crystallographic Methods Group

http://chango.ibmb.csic.es

Institut de Biologia Molecular de Barcelona (IBMB-CSIC)

Barcelona, Spain

LinkedIn: es.linkedin.com/in/claudiamillan/


ResearchGate:
https://www.researchgate.net/profile/Claudia_Millan?ev=hdr_xprf

Twitter: https://twitter.com/cheshireminima




El mié., 10 jul. 2019 a las 10:01, Mark J van Raaij ()
escribió:

> This sounds like a case for the Arcimboldo program developed by the Uson
> group (http://chango.ibmb.csic.es/), of which three versions are in CCP4
> (Borges, Lite and Shredder).
> This will use short secondary structure elements in a mixed MR/ab initio
> approach.
> We almost never get it to work with our well-diffracting beta-structured
> proteins :-(, but for alpha-helical proteins it apparently has a really
> good success rate.
>
> a recent paper on use of Arcimboldo for coiled coils is here:
> https://journals.iucr.org/d/issues/2018/03/00/cb5097/
>
> Mark J van Raaij
> Dpto de Estructura de Macromoleculas
> Centro Nacional de Biotecnologia - CSIC
> calle Darwin 3
> E-28049 Madrid, Spain
> tel. (+34) 91 585 4616
>
>
> On 10 Jul 2019, at 10:00, Shengyang Jin  wrote:
>
> Dear all,
>
> We recently acquired a data set (2.0 A, P222) for a coiled coil protein
> (according to Itasser, QUARK, Robetta, and Phaser).
> Matthews coefficient indicates 1 copy of protein per ASU. Sequence of the
> protein is quite novel with no apparent homolog in PDB.
> We tried to to MR with various models (ab initio or homology based) but
> with little success.
>
> We then tried to use AMPLE, but in ccp4 it always returned this error:
> __main__.py: error: unrecognized arguments: -use_arpwarp True
> (if we untick arpwarp and choose buccaneer instead, it returns -use_arpwarp
> False)
>
> Could anyone help?
>
> Thank you very much.
>
>
> Shengyang Jin
> Nanyang Technological University
> Singapore
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> <1.jpg>
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] MR for coiled coil structure

[Mark J van Raaij]

This sounds like a case for the Arcimboldo program developed by the Uson group 
(http://chango.ibmb.csic.es/ ), of which three 
versions are in CCP4 (Borges, Lite and Shredder).
This will use short secondary structure elements in a mixed MR/ab initio 
approach.
We almost never get it to work with our well-diffracting beta-structured 
proteins :-(, but for alpha-helical proteins it apparently has a really good 
success rate.

a recent paper on use of Arcimboldo for coiled coils is here:
https://journals.iucr.org/d/issues/2018/03/00/cb5097/ 


Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616


> On 10 Jul 2019, at 10:00, Shengyang Jin  wrote:
> 
> Dear all,
> 
> We recently acquired a data set (2.0 A, P222) for a coiled coil protein 
> (according to Itasser, QUARK, Robetta, and Phaser). 
> Matthews coefficient indicates 1 copy of protein per ASU. Sequence of the 
> protein is quite novel with no apparent homolog in PDB.   
> We tried to to MR with various models (ab initio or homology based) but with 
> little success. 
> 
> We then tried to use AMPLE, but in ccp4 it always returned this error:  
> __main__.py: error: unrecognized arguments: -use_arpwarp True
> (if we untick arpwarp and choose buccaneer instead, it returns -use_arpwarp 
> False)
> 
> Could anyone help?
> 
> Thank you very much.
> 
> 
> Shengyang Jin
> Nanyang Technological University
> Singapore
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
> <1.jpg>




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[ccp4bb] Problems in installing XDSapp in macOS Moiave 10.14.5

[Valentina Furlanetto]

Dear all,

I am using OS Moiave 10.14.15, and after installing XDS I would need also
XDSAPP. Did anyone successfully installed XDSAPP on Moiave?
Following the installation instructions, I faced some problems with
linkapps and with the installation of pyqwt.
Do you have any suggestions on how to fix the problem?

First install homebrew:
   ruby -e "$(curl -fsSL
https://raw.githubusercontent.com/Homebrew/install/master/install)"
   brew doctor
The command to install homebrew works for the bash shell. If you use
another shell, switch temporarily to bash before installing brew, by
entering bash in your terminal.
Then the rest of the packages can be easily installed (this requires the
previous installation of xcode):
   brew install qt
   brew linkapps
   brew install sip
   export PYTHONPATH=/usr/local/lib/python2.7/site-packages:$PYTHONPATH
(this might have to be adapted to your python version and path)
   brew install pyqt
   brew install pyqwt
*In case pyqwt can not be found, run*
*   brew tap homebrew/boneyard*
and try again
   brew install pyqwt
Then procede with
   brew linkapps (just in case)
Copy the line
   export PYTHONPATH=/usr/local/lib/python2.7/site-packages:$PYTHONPATH
into your ~/.bashrc or ~/.bash_profile file (for the bash shell) for future
uses of XDSAPP.

the highlighted lines are given as
Error: No available formula with the name "..."

For pyqwt I tried typing
brew tap homebrew/boneyard
I registered to github, since they asked me username and password, but I
got this:
remote: Repository not found.
fatal: repository 'https://github.com/Homebrew/homebrew-boneyard/' not found
Error:Failure while executing; `git clone
https://github.com/Homebrew/homebrew-boneyard
/usr/local/Homebrew/Library/Taps/homebrew/homebrew-boneyard --depth=1`
exited with 128.

Thank you for your help!

Best regards,

Valentina Furlanetto
PhD student
KTH School of Engineering Sciences in Chemistry, Biotechnology and Health
Department of Industrial Biotechnology
Visiting address: AlbaNova, Roslagstullsbacken 21, SE-106 91 Stockholm,
Sweden
Delivery address: AlbaNova, Roslagsvägen 30B, SE-114 19 Stockholm, Sweden



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Re: [ccp4bb] MR for coiled coil structure

[Daniel Rigden]

Dear Shengyang


We'll be happy to help you sort this out. I suggest we discuss it 
between the two of us and then post a summary to the bulletin board.



Could you please tell me, privately not to the board, which gui you are 
using, which version of CCP4 and which options you are trying to select?



Best wishes


Dan


On 10/07/2019 09:00, Shengyang Jin wrote:

Dear all,

We recently acquired a data set (2.0 A, P222) for a coiled coil 
protein (according to Itasser, QUARK, Robetta, and Phaser).
Matthews coefficient indicates 1 copy of protein per ASU. Sequence of 
the protein is quite novel with no apparent homolog in PDB.
We tried to to MR with various models (ab initio or homology based) 
but with little success.


We then tried to use AMPLE, but in ccp4 it always returned this error:
__main__.py: error: unrecognized arguments: -use_arpwarp True
(if we untick arpwarp and choose buccaneer instead, it returns 
-use_arpwarp False)


Could anyone help?

Thank you very much.


Shengyang Jin
Nanyang Technological University
Singapore



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--
Prof Daniel Rigden
Institute of Integrative Biology
Room 101, Biosciences Building
University of Liverpool
Crown St., Liverpool, L69 7ZB

(+44) 151 795 4467
pcwww.liverpool.ac.uk/~drigden/




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Re: [ccp4bb] Fo-Fc density close to cysteine residue

[Lumbini Yadav]

I have soaked my crystals in  sodium dithionite a reducing agent. I have
not done mass spec but sequence is confirmed

On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel 
wrote:

> Have you mass-speced the protein before crystallization to make sure it
> wasn’t derivatized during expression and/or purification, or compared the
> mass spec of the crystals verses purified protein? Any fancy reagents or
> other reductants used during purification?
>
> What about S-Acetyl-cysteine (3-letter code: SCY).
>
>
>
> Best,
>
>
>
> Dan
>
>
>
> Daniel A Bonsor PhD.
>
> Sundberg Lab
>
> Institute of Human Virology
>
> University of Maryland, Baltimore
>
> 725 W Lombard Street N370
>
> Baltimore
>
> Maryland
>
> MD 21201
>
> Tel: (410) 706-7457
>
>
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
> *Lumbini
> Yadav
> *Sent:* Tuesday, July 09, 2019 5:22 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] Fo-Fc density close to cysteine residue
>
>
>
> Dear all,
>
>
>
> We have found a huge Fo-Fc density close to cysteine residue (see attached
> image) in the structure with resolution of 1.2A. In the crystallization
> condition, we have PEG 3350, Potassium phosphate monobasic, glycerol and
> protein was in Tris and NaCl. Before freezing the crystals were soaked in
> mother liquor containing sodium dithionite.
>
>
>
> I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD,
> CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in
> all the screenings we do see some part of Fo-Fc density unaddressed at 3
> sigma.
>
>
>
> Does anyone have an idea about what this density could be? Covalent
> modification?
>
>
>
> Thanks.
>
>
>
>
>
> Kind regards,
>
> Lumbini
>
>
> --
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[ccp4bb] MR for coiled coil structure

[Shengyang Jin]

Dear all,

We recently acquired a data set (2.0 A, P222) for a coiled coil protein 
(according to Itasser, QUARK, Robetta, and Phaser).
Matthews coefficient indicates 1 copy of protein per ASU. Sequence of the 
protein is quite novel with no apparent homolog in PDB.
We tried to to MR with various models (ab initio or homology based) but with 
little success.

We then tried to use AMPLE, but in ccp4 it always returned this error:
__main__.py: error: unrecognized arguments: -use_arpwarp True
(if we untick arpwarp and choose buccaneer instead, it returns -use_arpwarp 
False)

Could anyone help?

Thank you very much.


Shengyang Jin
Nanyang Technological University
Singapore



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Re: [ccp4bb] Fo-Fc density close to cysteine residue

[Eugene Osipov]

Lumbini,
2.5A is close to the coordination bond length of sulfur-metal. Did you use
IMAC during purification?



ср, 10 июл. 2019 г. в 08:44, Eleanor Dodson <
176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>:

> I am serious about checking the anomalous map!!
> (trivial from REFMAC - key word anom map on)
> Then do a peak search and just check first that the rogue residue IS  CYS
> first - you should see the S as a peak..)
>
> Eleanor
>
>
> On Wed, 10 Jul 2019 at 07:38, Lumbini Yadav  wrote:
>
>> Thanks for the reply.
>> The distance between sulphur and centre of   Fo-Fc map is around 2.5A.
>> The density does not appear to be of hydrogen (attached image) anisotropy
>> refinement also does not help.
>>
>> On Tue, Jul 9, 2019 at 5:13 PM Anthony Addlagatta 
>> wrote:
>>
>>> Lumbini,
>>>
>>> It would useful to know the distance between the sulfur and center of
>>> the Fo-Fc map that you have shown. Since you have very high resolution
>>> data, the extra density could be for a hydrogen atom in CSO-H (sufeneic
>>> acid) or anisotropy of the oxygen atom.
>>>
>>> Anthony
>>> --
>>> *From: *176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk
>>> *To: *CCP4BB@JISCMAIL.AC.UK
>>> *Sent: *Tuesday, July 9, 2019 4:48:32 PM
>>> *Subject: *Re: [ccp4bb] Fo-Fc density close to cysteine residue
>>>
>>> Any anomalous diffraction?
>>>
>>> On Tue, 9 Jul 2019 at 10:32, Lumbini Yadav  wrote:
>>>
 Dear all,



 We have found a huge Fo-Fc density close to cysteine residue (see
 attached image) in the structure with resolution of 1.2A. In the
 crystallization condition, we have PEG 3350, Potassium phosphate
 monobasic, glycerol and protein was in Tris and NaCl. Before freezing
 the crystals were soaked in mother liquor containing sodium dithionite.



 I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC,
  CSD, CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide.
 But in all the screenings we do see some part of Fo-Fc density
 unaddressed at 3 sigma.



 Does anyone have an idea about what this density could be? Covalent
 modification?



 Thanks.



 Kind regards,

 Lumbini

 --

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>>>
>>> --
>>>
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>>>
>>>
>>>
>>>
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>>
>> --
>>
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-- 
Evgenii Osipov
Laboratory for Biocrystallography,
Department of Pharmaceutical Sciences,
KU Leuven O



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Re: [ccp4bb] Fo-Fc density close to cysteine residue

[Eleanor Dodson]

I am serious about checking the anomalous map!!
(trivial from REFMAC - key word anom map on)
Then do a peak search and just check first that the rogue residue IS  CYS
first - you should see the S as a peak..)

Eleanor


On Wed, 10 Jul 2019 at 07:38, Lumbini Yadav  wrote:

> Thanks for the reply.
> The distance between sulphur and centre of   Fo-Fc map is around 2.5A. The
> density does not appear to be of hydrogen (attached image) anisotropy
> refinement also does not help.
>
> On Tue, Jul 9, 2019 at 5:13 PM Anthony Addlagatta 
> wrote:
>
>> Lumbini,
>>
>> It would useful to know the distance between the sulfur and center of the
>> Fo-Fc map that you have shown. Since you have very high resolution data,
>> the extra density could be for a hydrogen atom in CSO-H (sufeneic acid) or
>> anisotropy of the oxygen atom.
>>
>> Anthony
>> --
>> *From: *176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk
>> *To: *CCP4BB@JISCMAIL.AC.UK
>> *Sent: *Tuesday, July 9, 2019 4:48:32 PM
>> *Subject: *Re: [ccp4bb] Fo-Fc density close to cysteine residue
>>
>> Any anomalous diffraction?
>>
>> On Tue, 9 Jul 2019 at 10:32, Lumbini Yadav  wrote:
>>
>>> Dear all,
>>>
>>>
>>>
>>> We have found a huge Fo-Fc density close to cysteine residue (see
>>> attached image) in the structure with resolution of 1.2A. In the
>>> crystallization condition, we have PEG 3350, Potassium phosphate
>>> monobasic, glycerol and protein was in Tris and NaCl. Before freezing
>>> the crystals were soaked in mother liquor containing sodium dithionite.
>>>
>>>
>>>
>>> I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD,
>>> CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in
>>> all the screenings we do see some part of Fo-Fc density unaddressed at
>>> 3 sigma.
>>>
>>>
>>>
>>> Does anyone have an idea about what this density could be? Covalent
>>> modification?
>>>
>>>
>>>
>>> Thanks.
>>>
>>>
>>>
>>> Kind regards,
>>>
>>> Lumbini
>>>
>>> --
>>>
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>>
>>
>> --
>>
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>>
>>
>>
>>
>> --
>>
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>
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Re: [ccp4bb] Fo-Fc density close to cysteine residue

[Lumbini Yadav]

Thanks for the reply.
The distance between sulphur and centre of   Fo-Fc map is around 2.5A. The
density does not appear to be of hydrogen (attached image) anisotropy
refinement also does not help.

On Tue, Jul 9, 2019 at 5:13 PM Anthony Addlagatta 
wrote:

> Lumbini,
>
> It would useful to know the distance between the sulfur and center of the
> Fo-Fc map that you have shown. Since you have very high resolution data,
> the extra density could be for a hydrogen atom in CSO-H (sufeneic acid) or
> anisotropy of the oxygen atom.
>
> Anthony
> --
> *From: *176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk
> *To: *CCP4BB@JISCMAIL.AC.UK
> *Sent: *Tuesday, July 9, 2019 4:48:32 PM
> *Subject: *Re: [ccp4bb] Fo-Fc density close to cysteine residue
>
> Any anomalous diffraction?
>
> On Tue, 9 Jul 2019 at 10:32, Lumbini Yadav  wrote:
>
>> Dear all,
>>
>>
>>
>> We have found a huge Fo-Fc density close to cysteine residue (see
>> attached image) in the structure with resolution of 1.2A. In the
>> crystallization condition, we have PEG 3350, Potassium phosphate
>> monobasic, glycerol and protein was in Tris and NaCl. Before freezing
>> the crystals were soaked in mother liquor containing sodium dithionite.
>>
>>
>>
>> I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD,
>> CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in
>> all the screenings we do see some part of Fo-Fc density unaddressed at 3
>> sigma.
>>
>>
>>
>> Does anyone have an idea about what this density could be? Covalent
>> modification?
>>
>>
>>
>> Thanks.
>>
>>
>>
>> Kind regards,
>>
>> Lumbini
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>
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>
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>
>
>
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