Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] add ligand with AceDRG

[Diana Tomchick]

When you try to use LIG in Phenix it gives you an error message as it doesn’t 
know that LIG is obsoleted.

Diana

Sent from my iPhone

> On Apr 26, 2024, at 10:08 AM, Yong Wang 
> <3c4fc05cc53b-dmarc-requ...@jiscmail.ac.uk> wrote:
>
> EXTERNAL MAIL
>
> Dear all,
>
> Perhaps the issue is that the refinement program should be able to handle the 
> priority of ligand dictionary, i.e. your custom ligand ID and dictionary 
> overrides any existing in the default collection.  I have not had any trouble 
> with LIG or SX1, both existing in PDB ligands.  I also never had any trouble 
> depositing structures containing such ligand ID's, albeit a few years ago.  
> The capable staff at RCSB will always assign a new ligand ID based on its 
> structure, regardless of what ID you have in your pdb file.
>
> Best,
>
> Yong
>
> -Original Message-
> From: CCP4 bulletin board  On Behalf Of Gerard Bricogne
> Sent: Friday, April 26, 2024 11:47 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [EXTERNAL] Re: [ccp4bb] add ligand with AceDRG
>
> EXTERNAL EMAIL: Use caution before replying, clicking links, and opening 
> attachments.
>
>
>
>
> Dear Oliver and CCP4BB readers,
>
> This obsoleted LIG came from PDB entry 1JVP deposited in 2001 by Jean-Michel 
> Rondeau working at Novartis. He must simply have been the first person to 
> deposit a structure in which the ligand was called LIG ... . That molecule is 
> now called 89E, but as Phil showed, there are still remnants of its old 
> identity.
>
> Best wishes,
>
> Gerard.
>
> --
>> On Fri, Apr 26, 2024 at 04:31:13PM +0100, Oliver Smart wrote:
>> Hi Stefanie,
>>
>> If you want to try Grade2 (for a non-confidential ligand) then this is
>> easy to do using the Grade Web Server
>>
>> https://urldefense.com/v3/__https://grad/__;!!MznTZTSvDXGV0Co!CIY1cS7S0u6TVzIG6Dh20opNZM2FHbu_xJYD7NwCU3qrUh_75Gd5xd9DbpExqoRdxiNPtuJUy2ls-trztGTScQP12D1gski_Rlab9zxaesA9ZFs$
>> e.globalphasing.org%2F=05%7C02%7CWANG_YONG_Y%40LILLY.COM%7C00daf6
>> fed4844df87c9e08dc66082939%7C18a59a81eea84c30948ad8824cdc2580%7C0%7C0%
>> 7C638497432436392044%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQI
>> joiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C0%7C%7C%7C=mvPPcWbmv
>> N%2FCuXXB%2BacAmaHf%2BqZr0O9lCoFxc9p5plk%3D=0
>>
>> We have altered our default "3-letter code" (aka PDB chemical component ID) 
>> to "LIG".
>> The reserved PDB two letter codes 01 to 99 are also fine (as are INH and 
>> DRG).
>> We  understand from users that Grade2 restraint dictionaries work well
>> with REFMAC (as to AceDG) Checking my installation of CCP4 8.0 there are no 
>> distributed dictionaries for LIG, DRG or 01 to 99.
>>
>> There was a PDB component LIG that was obsoleted in 2021
>> https://urldefense.com/v3/__https://www/__;!!MznTZTSvDXGV0Co!CIY1cS7S0u6TVzIG6Dh20opNZM2FHbu_xJYD7NwCU3qrUh_75Gd5xd9DbpExqoRdxiNPtuJUy2ls-trztGTScQP12D1gski_Rlab9zxaEuNjNL0$
>>  .
>> ebi.ac.uk%2Fpdbe%2Fstatic%2Ffiles%2Fpdbechem_v2%2FLIG.cif=05%7C02
>> %7CWANG_YONG_Y%40LILLY.COM%7C00daf6fed4844df87c9e08dc66082939%7C18a59a
>> 81eea84c30948ad8824cdc2580%7C0%7C0%7C638497432436399432%7CUnknown%7CTW
>> FpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6
>> Mn0%3D%7C0%7C%7C%7C=mFCw%2BNkuhOvtx4ozk9IeaBryFaqU6dt0CLQcneSzpm
>> g%3D=0 And there was a restraint dictionary for this
>> distributed with CCP4 7.0.
>>
>> Perhaps you are picking up things from an old CCP4 installation
>>
>> Hope this helps,
>>
>> Oliver
>>
>>
>>
>>
 On 26 Apr 2024, at 11:21, FREITAG-POHL, STEFANIE 
  wrote:
>>>
>>> Dear all,
>>>
>>> thank you so much for all your suggestions.
>>>
>>> Unfortunately, nothing helped. Whatever 3-letter code I choose Refmac is 
>>> complaining that there is a clash with an already existing ligand (even 
>>> DRG, LIG, numbers) and AceDRG in CCP4i2 insists on 3 letters.
>>>
>>> I am not quite sure how to bypass this issue as I think Refmac is
>>> also insisting on 3-letters for ligands (???)
>>>
>>> I have not tried Grade yet.
>>>
>>> Any advise much appreciated.
>>>
>>> Best wishes,
>>> Stefanie
>>>
>>>
>>>
>>> Dr. Stefanie Freitag-Pohl (she/her)
>>> Durham University
>>> Department of Chemistry
>>> South Road, Durham
>>> DH1 3LE
>>> United Kingdom
>>> 0191 334 2596
>>> stefanie.freitag-p...@durham.ac.uk
>>> 
>>> From: CCP4 bulletin board >> > on behalf of FREITAG-POHL, STEFANIE
>>> >> >
>>> Sent: 25 April 2024 13:01
>>> To: CCP4BB@JISCMAIL.AC.UK 
>>> mailto:CCP4BB@JISCMAIL.AC.UK>>
>>> Subject: [ccp4bb] add ligand with AceDRG
>>>
>>> [EXTERNAL EMAIL]
>>> Hi all,
>>>
>>> I have trouble adding a ligand with AceDRG in CCP4i2 into my refinement:
>>>
>>> I put in a smilesstring and the ligand is written ok, but since I can only 
>>> chose already 'taken' 3-letter-codes the refinement always crashes as there 
>>> is a clash with existing library entries.
>>> Is there any way around this? How 

Re: [ccp4bb] add ligand with AceDRG

[Diana Tomchick]

Use the search tab at the RCSB web site, type in a 3 letter code, and if it has 
been used for a ligand then it will appear as a Chemical Component.

Thanks to Phil, I am on vacation in Mexico and cut and pasting URLs is a pain 
on an iPhone.

Diana


Sent from my iPhone

On Apr 26, 2024, at 9:07 AM, Deborah Harrus  wrote:



Hi Diana,

Could you please clarify where you are searching?

LIG is obsolete and definitely not in use.

Cheers,

Deborah

On 26/04/2024 15:40, Diana Tomchick wrote:
But I think that is a mistake, if you search for LIG in the PDB, it brings up a 
definite ligand that has that 3-letter code.

Diana

Sent from my iPhone

On Apr 26, 2024, at 8:04 AM, Deborah Harrus 
<mailto:dhar...@ebi.ac.uk> wrote:



Dear all,

Just to clarify, "LIG" is also a reserved code, so it's safe to use.

See 
https://www.wwpdb.org/news/news?year=2023#656f4404d78e004e766a96c6<https://urldefense.com/v3/__https://www.wwpdb.org/news/news?year=2023*656f4404d78e004e766a96c6__;Iw!!MznTZTSvDXGV0Co!AfUYvlrA8vP5MVBYtrpLM9ngnPKUQR90_5Jw6YGFmeUccyiJEyYjJIBepH4mugozZQSzLqpZiP0rVKHw73KmyZ_ryOpJ8Pw$>

Kind regards,

Deborah Harrus

PDBe

On 25/04/2024 16:04, Diana Tomchick wrote:
The PDB has reserved the following codes for unknown ligands:

DRG
INH
01 - 99

Using one of these should not cause you the described problems. I successfully 
used

99

just last week. If you try to use

999 or LIG

these will not work, there are ligands assigned to those codes.


Diana

Sent from my iPhone

On Apr 25, 2024, at 8:03 AM, Nicholas Clark 
<b2b1c7e93c2d-dmarc-requ...@jiscmail.ac.uk><mailto:b2b1c7e93c2d-dmarc-requ...@jiscmail.ac.uk>
 wrote:


EXTERNAL MAIL

Hi Stefanie,

Why can you only use "taken" three letter codes? In the "output monomer" box, 
you should be able to enter whatever you'd like for the "Three letter code for 
output monomer". In the attached image, this is shown as "DRG" but can be 
changed to any 3 letter code of your choice. Obviously, just make sure your 
existing selection does not exist in the PDB.

Best,

Nick Clark



On Thu, Apr 25, 2024 at 8:51 AM Maria Håkansson 
mailto:maria.hakans...@saromics.com>> wrote:
Hi Stefanie,
Can you manually edit the restraints file using TextEdit and find and replace 
and the pdb
file of course?
Other option is to use Grade or Grade2 and the smiles string if you have this 
software installed.
I often find this easier than ccp4i2.

Best regards and good luck!
Maria





On 25 Apr 2024, at 14:01, FREITAG-POHL, STEFANIE 
mailto:stefanie.freitag-p...@durham.ac.uk>> 
wrote:

Hi all,

I have trouble adding a ligand with AceDRG in CCP4i2 into my refinement:

I put in a smilesstring and the ligand is written ok, but since I can only 
chose already 'taken' 3-letter-codes the refinement always crashes as there is 
a clash with existing library entries.
Is there any way around this? How do I add a novel ligand?

Thanks so much for your help.

Best wishes,
Stefanie



Dr. Stefanie Freitag-Pohl (she/her)
Durham University
Department of Chemistry
South Road, Durham
DH1 3LE
United Kingdom
0191 334 2596
stefanie.freitag-p...@durham.ac.uk<mailto:stefanie.freitag-p...@durham.ac.uk>


To unsubscribe from the CCP4BB list, click the following link:
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Maria Håkansson, PhD,
Principal Scientist

SARomics Biostructures AB
Medicon Village
SE-223 81 Lund, Sweden

Mobile: +46 (0)76 8585706
Web: 
www.saromics.com<https://urldefense.com/v3/__http://www.saromics.com__;!!MznTZTSvDXGV0Co!CQY9Ex2w1tfRofs9ZzM1ost58GieUu-kuX4wZBJLmNpAxJ3y96S4g-3tG25Bp4dKhWslwfnWyCiVOtFpZij1ydt_5BqeNO54IDAL6k6Sf4INmI8$>








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Re: [ccp4bb] add ligand with AceDRG

[Diana Tomchick]

But I think that is a mistake, if you search for LIG in the PDB, it brings up a 
definite ligand that has that 3-letter code.

Diana

Sent from my iPhone

On Apr 26, 2024, at 8:04 AM, Deborah Harrus  wrote:



Dear all,

Just to clarify, "LIG" is also a reserved code, so it's safe to use.

See 
https://www.wwpdb.org/news/news?year=2023#656f4404d78e004e766a96c6<https://urldefense.com/v3/__https://www.wwpdb.org/news/news?year=2023*656f4404d78e004e766a96c6__;Iw!!MznTZTSvDXGV0Co!AfUYvlrA8vP5MVBYtrpLM9ngnPKUQR90_5Jw6YGFmeUccyiJEyYjJIBepH4mugozZQSzLqpZiP0rVKHw73KmyZ_ryOpJ8Pw$>

Kind regards,

Deborah Harrus

PDBe

On 25/04/2024 16:04, Diana Tomchick wrote:
The PDB has reserved the following codes for unknown ligands:

DRG
INH
01 - 99

Using one of these should not cause you the described problems. I successfully 
used

99

just last week. If you try to use

999 or LIG

these will not work, there are ligands assigned to those codes.


Diana

Sent from my iPhone

On Apr 25, 2024, at 8:03 AM, Nicholas Clark 
<b2b1c7e93c2d-dmarc-requ...@jiscmail.ac.uk><mailto:b2b1c7e93c2d-dmarc-requ...@jiscmail.ac.uk>
 wrote:


EXTERNAL MAIL

Hi Stefanie,

Why can you only use "taken" three letter codes? In the "output monomer" box, 
you should be able to enter whatever you'd like for the "Three letter code for 
output monomer". In the attached image, this is shown as "DRG" but can be 
changed to any 3 letter code of your choice. Obviously, just make sure your 
existing selection does not exist in the PDB.

Best,

Nick Clark



On Thu, Apr 25, 2024 at 8:51 AM Maria Håkansson 
mailto:maria.hakans...@saromics.com>> wrote:
Hi Stefanie,
Can you manually edit the restraints file using TextEdit and find and replace 
and the pdb
file of course?
Other option is to use Grade or Grade2 and the smiles string if you have this 
software installed.
I often find this easier than ccp4i2.

Best regards and good luck!
Maria





On 25 Apr 2024, at 14:01, FREITAG-POHL, STEFANIE 
mailto:stefanie.freitag-p...@durham.ac.uk>> 
wrote:

Hi all,

I have trouble adding a ligand with AceDRG in CCP4i2 into my refinement:

I put in a smilesstring and the ligand is written ok, but since I can only 
chose already 'taken' 3-letter-codes the refinement always crashes as there is 
a clash with existing library entries.
Is there any way around this? How do I add a novel ligand?

Thanks so much for your help.

Best wishes,
Stefanie



Dr. Stefanie Freitag-Pohl (she/her)
Durham University
Department of Chemistry
South Road, Durham
DH1 3LE
United Kingdom
0191 334 2596
stefanie.freitag-p...@durham.ac.uk<mailto:stefanie.freitag-p...@durham.ac.uk>


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Maria Håkansson, PhD,
Principal Scientist

SARomics Biostructures AB
Medicon Village
SE-223 81 Lund, Sweden

Mobile: +46 (0)76 8585706
Web: 
www.saromics.com<https://urldefense.com/v3/__http://www.saromics.com__;!!MznTZTSvDXGV0Co!CQY9Ex2w1tfRofs9ZzM1ost58GieUu-kuX4wZBJLmNpAxJ3y96S4g-3tG25Bp4dKhWslwfnWyCiVOtFpZij1ydt_5BqeNO54IDAL6k6Sf4INmI8$>








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--
Nicholas D. Clark (He/Him)
PhD Candidate
Malkowski Lab
University at Buffalo
Department of Structural Biology
Jacob's School of Medicine & Biomedical Sciences
955 Main Street, RM 5130
Buffalo, NY 14203



To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] add ligand with AceDRG

[Diana Tomchick]

The PDB has reserved the following codes for unknown ligands:

DRG
INH
01 - 99

Using one of these should not cause you the described problems. I successfully 
used

99

just last week. If you try to use

999 or LIG

these will not work, there are ligands assigned to those codes.


Diana

Sent from my iPhone

On Apr 25, 2024, at 8:03 AM, Nicholas Clark 
 wrote:


EXTERNAL MAIL

Hi Stefanie,

Why can you only use "taken" three letter codes? In the "output monomer" box, 
you should be able to enter whatever you'd like for the "Three letter code for 
output monomer". In the attached image, this is shown as "DRG" but can be 
changed to any 3 letter code of your choice. Obviously, just make sure your 
existing selection does not exist in the PDB.

Best,

Nick Clark



On Thu, Apr 25, 2024 at 8:51 AM Maria Håkansson 
mailto:maria.hakans...@saromics.com>> wrote:
Hi Stefanie,
Can you manually edit the restraints file using TextEdit and find and replace 
and the pdb
file of course?
Other option is to use Grade or Grade2 and the smiles string if you have this 
software installed.
I often find this easier than ccp4i2.

Best regards and good luck!
Maria





On 25 Apr 2024, at 14:01, FREITAG-POHL, STEFANIE 
mailto:stefanie.freitag-p...@durham.ac.uk>> 
wrote:

Hi all,

I have trouble adding a ligand with AceDRG in CCP4i2 into my refinement:

I put in a smilesstring and the ligand is written ok, but since I can only 
chose already 'taken' 3-letter-codes the refinement always crashes as there is 
a clash with existing library entries.
Is there any way around this? How do I add a novel ligand?

Thanks so much for your help.

Best wishes,
Stefanie



Dr. Stefanie Freitag-Pohl (she/her)
Durham University
Department of Chemistry
South Road, Durham
DH1 3LE
United Kingdom
0191 334 2596
stefanie.freitag-p...@durham.ac.uk


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

Maria Håkansson, PhD,
Principal Scientist

SARomics Biostructures AB
Medicon Village
SE-223 81 Lund, Sweden

Mobile: +46 (0)76 8585706
Web: 
www.saromics.com








To unsubscribe from the CCP4BB list, click the following link:
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--
Nicholas D. Clark (He/Him)
PhD Candidate
Malkowski Lab
University at Buffalo
Department of Structural Biology
Jacob's School of Medicine & Biomedical Sciences
955 Main Street, RM 5130
Buffalo, NY 14203



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Re: [ccp4bb] Mystery blob

[Diana Tomchick]

It’s the Easter Bunny!

But seriously, it looks like more than one compound, perhaps hydrogen bonded to 
each other.

Diana Tomchick

Sent from my iPhone

On Mar 7, 2024, at 7:32 AM, Eleanor Dodson 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:


EXTERNAL MAIL

Anyone ANY idea what this could be?? Beautiful density but...

I am told:
The possible/likely components of the protein solution/cryo/crystallisation 
buffer are:
NaCl, MES, a tiny bit of HEPES possibly, ligand ( GR8.027 = the pentasaccharide 
near catalytic residues Glu276 and Glu165), Et3N which is monomer TEA (a 
possible counterion for the ligand - see diagram below), glycerol (cryo)
There may also be carbonate ions present from the ligand.  Any thoughts 
appreciated!!



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Re: [ccp4bb] How to make GDP.BeF3 solution ?

[Diana Tomchick]

Be aware that beryllium is also quite toxic.

https://en.wikipedia.org/wiki/Acute_beryllium_poisoning

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
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diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

From: CCP4 bulletin board  on behalf of Dr. Kevin M Jude 

Sent: Friday, January 5, 2024 1:51 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] How to make GDP.BeF3 solution ?


EXTERNAL MAIL

Nb, dissolution of BeCl2 in water is quite exothermic and releases HCl vapor, 
you will want to prepare that stock in a fume hood.



Best wishes

Kevin



From: CCP4 bulletin board  on behalf of Matthew BOWLER 

Date: Thursday, January 4, 2024 at 2:19 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] How to make GDP.BeF3 solution ?

Dear Firdous,

beryllium fluoride is actually a ground state analogue of GTP as 
trifluoroberyllate is tetrahedral. To get a transition sate analogue you need 
either AlF4- or MgF3-. Preparation of these complexes is very easy. The great 
advantage of metal fluoride transition state analogue and ground state analogue 
complexes is the fact that all components are present in solution and readily 
self-assemble in the active site forming stable enzyme complexes that are 
relevant to the catalytic cycle. The inorganic metal fluoride salts (AlF3, and 
MgF2) are too insoluble to use; therefore, the fluoride anion and metal cation 
components must be added from separate stock solutions. Both ammonium fluoride 
and sodium fluoride are suitable as the source of fluoride and are readily 
soluble in water. Metal chlorides can be easily dissolved in water at high 
concentration (0.5 M) and the solutions conserved at -20°C. One of the critical 
aspects in preparing metal fluoride enzyme complexes is the pH of the resulting 
solution. In particular, solutions of AlCl3 and BeCl2 are highly acidic (pH 2) 
samples should be prepared in 100 mM unbuffered Tris base. The optimized 
sequence of component addition is to add fluoride to the prepared buffer first, 
then the metal chloride stock.

Hope this helps, Matt











On 02/01/2024 18:53, Firdous Tarique wrote:

Hi



I would appreciate it if someone could share with me a step by step protocol 
for making a stable GDP.BeF3 solution which is often used as a transition state 
analogue for structural studies of a protein complex ?



Or a vendor from where it can be purchased directly.



Regards



Firdous





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Project Leader MASSIF1@ESRF

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71 avenue des Martyrs

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France

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Re: [ccp4bb] the structures of Nucleic acid

[Diana Tomchick]

I have also found that there is significant anomalous signal from strontium 
near the Se K-edge, which is useful if one uses strontium chloride instead of 
potassium chloride (or in addition to KCl) during crystal growth.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)



On Sep 19, 2023, at 1:32 PM, Wagner, Armin (DLSLtd,RAL,LSCI) 
>
 wrote:


EXTERNAL MAIL

Dear Fu Xingke,

Indeed P-SAD is quite attractive but requires high resolution as the number of 
anomalous scatterers (1 P per nucleotide) is rather large, but the unit cells 
are typically quite small, resulting in a rather small number of anomalous 
differences per scatterer. Based on the very nice publication from Tom 
Terwilliger et al. ( 
https://doi.org/10.1107/S2059798315019269)
 we wrote a little web app to predict the success for S-SAD phasing at the 
long-wavelength beamline I23 at Diamond. 
https://www.diamond.ac.uk/Instruments/Mx/I23/resolution-requirement-phasing-app.html
While the predictions are pretty reliable for S-SAD, there is a (not heavily 
tested) option to also submit DNA or RNA sequences, which can give you a hint 
on what you are against, or what resolution you should aim for. Unfortunately, 
we have not had many successful examples to far, but basically all the P-SAD 
projects which were predicted not to work at the resolutions the crystals 
diffracted to didn’t solve, so we are very interested in projects which are 
predicted to work to fine tune this also for P-SAD as there will be continuous 
need for experimental phasing in particular for non-canonical RNA/DNA 
structures.

5-Br-U is a good alternative and works well, but we have also managed to solve 
RNA/DNA by K-SAD or Co-SAD 
(https://doi.org/10.1093/nar/gkaa439)
 and Ca could be attractive as a potential anomalous scatter as well.

Best regards,

Armin




From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Mark J. van Raaij 
mailto:mjvanra...@cnb.csic.es>>
Date: Tuesday, 19 September 2023 at 12:31
To: CCP4BB@JISCMAIL.AC.UK 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: Re: [ccp4bb] the structures of Nucleic acid
This just appeared and may be relevant:
https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkad726/7272628

Critical Reviews and Perspectives
When will RNA get its AlphaFold moment?
Mark van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)


On 18 Sep 2023, at 18:07, William G. Scott 
<2844d921eb97-dmarc-requ...@jiscmail.ac.uk>
 wrote:

The phosphorus absorption edge is about 5.8Å.

I've had much better luck with 5-Br-U for anomalous phasing.

Molecular replacement with sub-structural fragments can also work:



Yours sincerely,

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


On Sep 18, 2023, at 2:43 AM, Eleanor Dodson 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>
 wrote:

I am afraid most scientists will use the most straightforward technique!
If SAD is available the PHOSPHATE backbone of DNA will provide sufficient 
signal to allow SAD to work, and you get an unambiguous answer to 

Re: [ccp4bb] How to deal with tNCS?

[Diana Tomchick]

I would second what Eleanor has to say about the relationship of your C2221 
cell to the P212121 cell. When I have had cases where there is a peak in the 
self Patterson function that is as high as yours (99.471% is essentially 100%), 
it means that the peak at 1/2, 0, 0 in the self Patterson is equivalent in size 
to the origin peak of the self Patterson. This happened for me when I indexed 
the data with a unit cell that was twice the size of the correct cell. From 
what you present, it sounds as though you should try refining the model in the 
smaller, primitive cell.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)



On Jul 20, 2023, at 10:10 PM, Lande 
<8bc2565720d4-dmarc-requ...@jiscmail.ac.uk>
 wrote:


EXTERNAL MAIL


 Dear Eleanor and Jon,


Thank you for your replies. My good datasets are in P212121 space group and 
unit cell 56.67 60.53 67.51 90.00 90.00 90.00 ,2 copies in ASU. The tNCS one is 
in C2221 space group and unit cell 122.32 134.88  55.45  90.00  90.00  90.00. 
Matthews suggests 4 copies and I tried 4-6 copies to search in phaser (only 4 
copies in solutions).  For fractional coordinate, it is 1/2 and the Rfree/Rwork 
is 0.4877/0.4466. This is another topic I can hardly found in modern 
crystallography textbooks..



Here I also attached the self rotaion fuction. I guess there might be 8 peaks 
but honestly I have no idea how to read those graphs. There is another 
screenshot of "more tNCS" image.


Many thanks to your help.


regards,
Lande Fu







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Re: [ccp4bb] Easy/Silly question

[Diana Tomchick]

It's GLC

otherwise known as 4-ortho-alpha-D-Glucopyranosyl-D-glucose.

See PDB ID 1ANF

also

https://en.wikipedia.org/wiki/Maltose
[https://upload.wikimedia.org/wikipedia/commons/thumb/6/6a/Maltose2.svg/1200px-Maltose2.svg.png]
Maltose - Wikipedia
Maltose (/ ˈ m ɔː l t oʊ s / or / ˈ m ɔː l t oʊ z /), also known as maltobiose 
or malt sugar, is a disaccharide formed from two units of glucose joined with 
an α(1→4) bond.In the isomer isomaltose, the two glucose molecules are joined 
with an α(1→6) bond.Maltose is the two-unit member of the amylose homologous 
series, the key structural motif of starch.When alpha-amylase breaks ...
en.wikipedia.org
Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

From: CCP4 bulletin board  on behalf of Joel Tyndall 

Sent: Thursday, June 30, 2022 10:50 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Easy/Silly question


EXTERNAL MAIL

Hi folks,



I frustratingly cant find “maltose” as a ligand in the pdb or ccp4 database. 
Does any one have the code for the ligand? Surely its been used before.



Thanks in advance



J



Joel Tyndall | BSc(Hons) PhD

Associate Professor in Medicinal Chemistry
School of Pharmacy | He Rau Kawakawa
University of Otago | Te Whare Wānanga o Otāgo

PO Box 56 9054

Dunedin | Ōtepoti

New Zealand | Aotearoa

Ph: 64 3 479 7293
Skype: jtyndall

Website | pharmacy.otago.ac.nz







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Re: [ccp4bb] Has anyone successfully used RoseTTAFold or AF2 to guide crystallization?

[Diana Tomchick]

I would say that in a small survey (~5-6) of bacterial enzymes from our lab, 
AlphaFold2 predicted exactly the constructs that were discovered by limited 
proteolysis and/or sequence alignment for currently unpublished structures.

It’s a useful tool but I’ve also had two cases where it incorrectly predicted 
the sequences of helices in a helical bundle—frameshift errors in the sequence, 
which were confirmed by SeMet SAD data. Caveat emptor.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)




On Apr 4, 2022, at 2:06 PM, Scott Classen 
mailto:sclas...@lbl.gov>> wrote:


EXTERNAL MAIL

Hello CCP4,

Has anyone successfully used the available ML/AI protein folding tools to guide 
crystallization construct design? Maybe you had a protein or domain that was 
resistant to crystallization efforts and the folding algorithms  predicted some 
loops or termini that were disordered? Then you trimmed or modified them in 
some way to aid in crystallization? Or if you haven’t done this yourself, are 
you aware of anyone who has?

Thanks,
Scott


~~
Scott Classen, Ph.D.
ALS-ENABLE
TomAlberTron Beamline 8.3.1
SIBYLS Beamline 12.3.1
Advanced Light Source
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MS6R2100
Berkeley, CA 94720
mobile 510.206.4418
desk 510.495.2697
beamline 510.495.2134
~~




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Re: [ccp4bb] mysterious density

[Diana Tomchick]

Contour the 2Fo-Fc map at lower sigma than 1.3, and see if the resulting 
density looks like MES. As others have said, if there is no specific pocket or 
interactions for the non-sulfonic acid head group, then I would expect that 
density to be weaker or smeared out due to positional disorder.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)




On Mar 13, 2022, at 9:19 AM, Amir Khan 
mailto:k...@crystal.harvard.edu>> wrote:


EXTERNAL MAIL

Hi,

I wonder whether anyone can advise on the ‘tadpole’ like density. The head is 
currently a phosphate
and I’ve placed a water in tail, but it obviously looks connected… would 
appreciate any help!

Green density at 5 sigma (Fo-fc), while blue is 2fo-fc at 1.3 sigma.

Crystallization condition is unknown, though it came from sparse matrix 
commercial (either Wizard, Pact Prem, or JCSG+).

Thanks!

Amir



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Re: [ccp4bb] Looking or Coot 0.9 or newer for OSX

[Diana Tomchick]

The default version of Coot included in the latest CCP4 download (version 
7.1.016) is 0.9.6.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)




On Oct 6, 2021, at 5:51 PM, Scott Pegan 
mailto:scott.pe...@medsch.ucr.edu>> wrote:


EXTERNAL MAIL

Been looking for Coot 0.9 or better package install files for OSX, anyone know 
where to look? I seem to only find 0.8 or less through the web.
Scott

--
Scott Pegan
Professor
Division of Biomedical Sciences
School of Medicine
University of California Riverside
205 SOM Research Building
900 University Avenue
Riverside, CA  92521-0001
(951) 827 7907
sco...@medsch.ucr.edu




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Re: [ccp4bb] Fluka-branded PEG 3000

[Diana Tomchick]

If you even still have a few milliliters of the original PEG solution, you 
could simply use that for mixing with the protein in the drop and use a 
different PEG solution for the reservoir, thereby ensuring a mother liquor that 
could either provide (or remove) a missing impurity.

Diana

From: CCP4 bulletin board  on behalf of Patrick Loll 

Sent: Monday, March 22, 2021 1:39 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Fluka-branded PEG 3000


EXTERNAL MAIL

I don’t think we have any of that PEG lying around (sorry), but you might also 
consider that PEGs are notorious for impurities of various types; perhaps the 
Fluka reagent has less of an impurity that is particularly vexing for your 
specific protein.

Of course, I suppose it might have more of a particular impurity that’s 
stimulating crystallization….

Here’s an older reference that talks about ways to clean up PEGs:

doi: 10.1016/0003-2697(85)90544-5

I honestly don’t know if “modern” PEG reagents are less likely to contain 
impurities than the materials available at the time this paper was written (but 
the intrinsic nature of the molecule hasn’t changed, so maybe not?).

Good luck.

Pat Loll

Patrick J.  Loll, PhD (he,his)
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St.
Philadelphia, PA 19102-1192 USA

On 22 Mar 2021, at 1:00 PM, Correy, Galen 
mailto:galen.cor...@ucsf.edu>> wrote:

Dear all,

I'm wondering if anyone could spare 50-100 g of Fluka branded PEG 3000 (see 
attached image)?

We have some crystals that diffract better when grown using Fluka PEG compared 
to other brands. Unfortunately, we've exhausted our supply of the Fluka PEG, 
and it's no longer commercially available (the same product code is available 
from Sigma - we've tested it, along with several other brands - but they don't 
seem to have the same magic as the Fluka PEG).

We'll happily purchase a new bottle of Sigma-branded PEG 3000 to replace any 
sent our way.

Many thanks,
Galen


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Re: [ccp4bb] Student Question--Negative Difference Density in some Histidine side chains in Iron Coordination complex in 2XGF T4 Phage Model Structure

[Diana Tomchick]

Depending upon the amount of radiation exposure, the iron might be reduced (or 
more likely, in a mixed oxidation state) by the X-ray beam.


Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

From: CCP4 bulletin board  on behalf of Nukri Sanishvili 

Sent: Monday, February 22, 2021 12:33 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Student Question--Negative Difference Density in some 
Histidine side chains in Iron Coordination complex in 2XGF T4 Phage Model 
Structure


EXTERNAL MAIL

Sorry, in my previous email I was oblivious to the fact that there was Fe. Iron 
is almost certainly oxidized. So, if you started collecting data with reduced 
iron, but it got oxidized during data collection, you are probably using the 
parametrization for the reduced rather than oxidized one in the refinement. I 
wonder if it is so and if going to the reduced state would make a difference.
Best,
N.

On Mon, Feb 22, 2021 at 12:23 PM Pavel Afonine 
mailto:pafon...@gmail.com>> wrote:
Hi,

as others pointed out electron rich elements tend to amplify imperfections 
visible in Fo-Fc maps. Consider:
- refining occupancy of Fe, the site may be partially occupied;
- refining f' and f'' if data are anomalous;
- surrounding histidines may 'see' this Fe as a nonbonded interaction and thus 
repulsion terms may kick in and push Fe from its position. Try defining weak 
coordination bonds between Fe and respective nitrogens;
- refining anisotropic ADP of Fe only.

Good luck!
Pavel

On Mon, Feb 22, 2021 at 7:27 AM Patrick Needham 
mailto:needh...@miamioh.edu>> wrote:



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Re: [ccp4bb] Challenging Molecular Replacement

[Diana Tomchick]

There are a large number of PAK clashes, I would regard this "solution" with 
caution. Have you inspected the solution to see where these clashes are 
occurring? If they are from long flexible loops or the termini, perhaps if you 
would trim the model to remove those clashes and re-run the MR calculation, you 
might wind up with a better solution that will refine.

I also second the re-building option. Run density modification with the MR 
solution, then try both Phenix AutoBuild and also Buccaneer. For a 3.0 Å 
dataset, I would expect that Buccaneer would be more likely to work (and 
certainly give you an answer in less time). If Buccaneer can't give you a 
solution after using your "solution" for density modification and re-building, 
I would regard this as an unusable solution.

The R-factors you quote after refinement indicate that the solution is not 
correct, or at least not correct enough to converge to the true solution.

Since it is a multi-domain protein, perhaps try the MR search with individual 
domains.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

From: CCP4 bulletin board  on behalf of Sharan Karade 

Sent: Tuesday, February 16, 2021 12:38 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Challenging Molecular Replacement


EXTERNAL MAIL

Hi,

Looks like, you have the solution. Have you tried the Phenix AutoBuild module? 
sometimes it works well.

Best luck


On Tue, Feb 16, 2021 at 1:21 PM Muhammad Bashir Khan 
mailto:m...@ualberta.ca>> wrote:
Dear All;

I have data set of about 3.0A. It's a multidomain protein. I tried several 
options with MR but it's was not working. I trimmed one of the search models, 
it does not give any solution using CCP4 molrep.

I used Phenix phaser it gives a solution after several hours with the following 
output.

** SINGLE solution

** Solution written to PDB file:  MgtA_phaser.1.pdb
** Solution written to MTZ file:  MgtA_phaser.1.mtz
   Solution annotation (history):
   SOLU SET  RFZ=2.7 TFZ=6.3 PAK=6 LLG=62 TFZ==6.5 RFZ=2.2 TFZ=28.0 PAK=9 
LLG=1207 TFZ==42.0 LLG=1370 TFZ==41.8 PAK=9
LLG=1370 TFZ==41.8
   SOLU SPAC P 21 2 21
   SOLU 6DIM ENSE ense_1 EULER  269.0   80.4  177.3 FRAC  0.24 -0.22 -0.07 BFAC 
-1.51 #TFZ==6.5
   SOLU 6DIM ENSE ense_1 EULER   89.0   80.4  177.3 FRAC  0.26 -1.01 -0.07 BFAC 
 2.26 #TFZ==41.8
   SOLU ENSEMBLE ense_1 VRMS DELTA -0.7291 #RMSD  1.50 #VRMS  1.23

Running Phenix refine on the solution gives a breakup map and the R factors are 
not decreasing below 47.61 and 50.94.

Thank you for any suggestions in advance

Regards;

Bashir


--
--
Muhammad Bashir Khan, Ph.D.
Research Associate
Department of Biochemistry
Medical Science Bldg.
Lab 3-27
University of Alberta
Edmonton AB, T6G 2H7

Phone: 780-492-4577-
e-mail: m...@ualberta.ca



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Re: [ccp4bb] TNCS and oligomeric state

[Diana Tomchick]

One of the most lucid explanations of how translational NCS results in peaks in 
a self-Patterson map is provided in Phil Evans’ classic paper, “Rotations and 
rotation matrices”, which is an Open Access publication. In particular, check 
out Figure 4, which illustrates a non-crystallographic dyad axis parallel to a 
crystallographic screw dyad.

Acta Christ. (2001) D57, 1355-1359.
https://journals.iucr.org/d/issues/2001/10/00/ba5006/index.html

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)




On Feb 3, 2021, at 10:33 AM, Jon Cooper 
<488a26d62010-dmarc-requ...@jiscmail.ac.uk>
 wrote:


EXTERNAL MAIL

Hello, in the self-rotation function the peaks arising from the NCS rotation 
axes are good at hiding under the peaks for the crystallographic rotations, 
which is because they are actually translational NCS elements, I think. Also, 
sorry Eleanor, a double-check on wikipedia confirms the space group ambiguity 
is actually between I422 and your original choice I4(1)22. Did you do something 
special to get good anomalous to 2.7A e.g. wavelength, covalent modification, 
etc? Hope this helps. Cheers, Jon.C.


Sent from ProtonMail mobile



 Original Message 
On 3 Feb 2021, 13:59, Eleanor Dodson < 
176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>
 wrote:

I would look VERY carefully at your data processing. CCP4I2 report is a good 
place to start.
The self rotation with the ring around the edge is hard to reconcile with two 
molecules in the asymmetric unit.

Are the images clean or streaky? Do you have a photo of the crystal?
And by the way - you cannot decide on spacegroup I422 or I 42 2 2 till you 
solve the structure..
Eleanor

On Wed, 3 Feb 2021 at 12:12, Randy John Read 
mailto:rj...@cam.ac.uk>> wrote:
Dear John,

It’s hard to be absolutely certain from the reproduction, but it looks like you 
have equally high 2-fold axes all around the xy plane in the self-rotation 
function.  Do you have an explanation for that?

It would be helpful to know the heights of the Patterson peaks relative to the 
origin peak.  However, assuming that the ones you have listed are all 
relatively high, this can be interpreted in terms of a commensurate modulation 
or, in alternative terminology, a multiple tNCS vector.  All of the peaks are 
approximate multiples of the same translation vector, i.e. 1/2, 1/2, 1/6.

1/2, 1/2, 1/6
2/2, 2/2, 2/6, equivalent to 0, 0, 1/3
3/2, 3/2, 3/6, equivalent to 1/2, 1/2, 1/2

The next three in this series would be the same vectors in the opposite 
direction, i.e. related by the inversion operator in the Patterson.  If the 
Matthews coefficient suggests 2 copies, you could probably have 3 in the 
asymmetric unit related by multiples of the same vector, but not 6.

So in Phaser you would express this as
TNCS NMOL 3
TNCS TRA VECTOR 0.5 0.5 0.169

Best wishes,

Randy Read

> On 3 Feb 2021, at 11:34, leo john 
> mailto:ljohn16012...@gmail.com>> wrote:
>
> Sticking to the same doubt:
>
> Just to check the correct symmetry and space group, I have now processed my 
> data in P1 and I4 followed by running MOLREP for self-rotation function 
> (Figure attached).
>
> Can we conclude now on the correct space group and oligomeric state.
>
> Thank You all
> John
>
> On Wed, Feb 3, 2021 at 8:34 AM leo john 
> mailto:ljohn16012...@gmail.com>> wrote:
> Dear All:
>
> I have a peptide that is crystallized in the space group I4122 with cell 
> dimensions 40,40, 200 Ang.
> Mathews Coefficient suggests 2 molecules/ ASU.
> Since I do not have a starting model for MR, I went for experimental phasing. 
> However, unsuccessful so far.
>
> Details about my dataset:
>
> 1) It has been collected at 2 Ang and has an Anomalous signal till 2.7
> 2) It has got Translational NCS.
> 3) I checked the self-rotation function using MOLREP (figure attached), but 
> not able to make anything out of it. Please suggest.
> 4) I got the translational and rotational peaks from the log file of MOLREP
>
> Peak 1: trans.vector /ort/ :19.27019.27098.550
>trans.vector /frac/: 0.500 0.500 0.500
>Peak 2: trans.vector /ort/ :19.27019.27033.326
>trans.vector /frac/: 0.500 0.500 0.169
>Peak 3: trans.vector /ort/ :-0.000-0.00065.224
>trans.vector /frac/: 0.000 0.000 0.331
>
> Sol_Rf   1 0.000.000.000.000.000.000.4448E+06  
> 4.53
> Sol_Rf   289.99  112.50  180.000.00  180.00  -45.000.3561E+06  

Re: [ccp4bb] Tiny rocks on my CX100 shipping dewar

[Diana Tomchick]

Interesting observation. In our home X-ray lab, we constructed a liquid 
nitrogen dumping station from a large garbage can filled with sand to about 12 
inches. To keep the sand out of the dewars we topped the sand with a large wad 
of screen-door netting. All items can easily be purchased from a local home 
improvement store, and the screen-door netting is super cheap (plus it makes it 
easy to remove the stray bits of garbage that mistakenly find their way into 
the nitrogen dump/garbage can (you can put a sign that states, “not for 
garbage” on a garbage can, but you can’t get 100% compliance, LOL).

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)




On Dec 4, 2020, at 10:42 AM, Nukri Sanishvili 
mailto:sannu...@gmail.com>> wrote:


EXTERNAL MAIL

Hi John,
I think I know what might have happened:
Many of the MX beamlines at the APS use some sort of filler in the containers 
where the LN2 is dumped. If I remember correctly, one of the beamlines is using 
fine gravel for this purpose. Also, it is required that before shipping, the 
dewars are emptied - i.e. don't contain liquid. Now, imagine somebody dumping 
the liquid into the grave-filled container without removing the blue cap and 
without holding the dewar in the air - i.e. the top of the dewar with the cap 
on is slightly buried into the gravel. Upon straightening the dewar up, the 
blue cap would scoop up a little bit of the gravel. Distribution of the pebbles 
on your picture is also noteworthy. It suggests the side where the pebbles are 
was the side dipped into the gravel.
You might want to discuss this with your beamline host.
Best,
Nukri

On Fri, Dec 4, 2020 at 10:05 AM Tanner, John J. 
mailto:tanne...@missouri.edu>> wrote:
When we opened our CX100 shipping dewar returned from APS via FedEx this week, 
we observed what appears to be tiny rocks on the rim below the foam neck core:

https://www.dropbox.com/s/ky09a1vbm9t0mrl/CX100withrocks.png?dl=0

Has anyone seen this before? Is this perhaps the absorbent material from the 
inside of the dewar?

Thanks,

Jack

John J. Tanner
Professor of Biochemistry and Chemistry
Associate Chair of Biochemistry
Department of Biochemistry
University of Missouri
117 Schweitzer Hall
503 S College Avenue
Columbia, MO 65211
Phone: 573-884-1280
Email: tanne...@missouri.edu
https://cafnrfaculty.missouri.edu/tannerlab/
Lab: Schlundt Annex rooms 3,6,9, 203B, 203C
Office: Schlundt Annex 203A



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Re: [ccp4bb] Stabilizing Mitegen reusable bases/mounts

[Diana Tomchick]

We no longer use the reusable bases for the same reasons, but we do re-use our 
bases. If you use superglue to glue the pins into the bases, you can remove the 
pins from the bases by soaking them in acetone. This works about 75% of the 
time, especially if you are careful not to use too much superglue when you glue 
the pin into the base.

It goes without saying that the use of acetone will dissolve the loop, but then 
the main reason we remove the pin is due to imperfections in the loop, so no 
big deal.

Whenever we see the motions that you describe, we postulate that somehow the 
pins were damaged and they are loose in the base. Occasionally this motion is 
ascribed to ice between the base of the pin and the magnetic mount, but this 
happens very rarely in our experience.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

From: CCP4 bulletin board  on behalf of Tao-Hsin Chang 

Sent: Sunday, November 15, 2020 3:07 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Stabilizing Mitegen reusable bases/mounts


EXTERNAL MAIL

Hi Patrick,

I have met the same issue and that is very bad for any micro-focus beam and 
small crystals. I have talked with Mitegen and they are developing a new type 
of reusable base having an improvement for this issue. You may get some update 
from them. But, I do not use the reusable bases anymore.

Best wishes,
Tao-Hsin

On Nov 15, 2020, at 3:45 PM, Patrick Loll 
mailto:pjl...@gmail.com>> wrote:

Hi everyone,

I’ve become very fond of the Mitegen reusable bases for mounting crystals, 
since the reusable aspect savse me from having to discard the base every time I 
break a microloop. However, once the crystals arrive at the synchroteon, I 
observe motions of the loops (some gradual, some sporadic). The the amplitudes 
of these motions are becoming significant as I take data from smaller and 
smaller crystals. I don’t think I’m imagining this, since the good folks at 
NSLS-2/AMX have warned me about this very issue.

I’m writing to ask if anyone has any clever ideas about stabilizing these 
assemblies. Obviously, I can epoxy the pins in place, but then I’ll probably 
need to discard the entire assembly when I break a loop, and I’d prefer not to 
waste more money than necessary. I’ve considered putting a bead of wax at the 
point where the pin enters the base (although I haven’t yet checked to see if 
that will survive immersion in liquid nitrogen). Does anyone have any other 
(better) ideas?

Much obliged in advance,

Pat
__

Patrick J.  Loll, PhD
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St.
Philadelphia, PA 19102-1192 USA

(215) 762-7706
pj...@drexel.edu



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Re: [ccp4bb] AW: [ccp4bb] over-fitting? over-refinement?

[Diana Tomchick]

I too have seen some horrendous low-resolution models, with correspondingly bad 
validation statistics. A little time spent cleaning up the outliers (geometric 
and others) rarely results in large reductions in R(free) for these types of 
datasets & models, but ultimately we as a community need to emphasize that the 
R(free) is not the be all and end all as a quality metric.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

From: CCP4 bulletin board  on behalf of Tristan Croll 

Sent: Tuesday, October 20, 2020 7:11 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] AW: [ccp4bb] over-fitting? over-refinement?


EXTERNAL MAIL

I'd like to append a very important caveat to this discussion: most of the talk 
on Rfree as protection against overfitting is perfectly correct, if your 
dataset is high enough resolution. Remember that Rfree only provides protection 
against one form of overfitting: that is, fitting of atoms into random noise. 
What it doesn't protect well against is fitting the wrong atoms into real 
density. Remember, all your x-ray data ultimately says is "there are electrons 
here" - your R-factors don't care where those electrons come from, as long as 
they're present in about the right numbers (with some fudge-room for B-factors 
and occupancies). If you browse through the back catalogue of >3A models, 
you'll find some with horrendous geometry statistics but remarkably good 
R-factors (both work and free) - ultimately, I think, because the model atoms 
have been "overstuffed" into density that is real according to both the working 
and free data. In quite a few such cases I find that even after extensive 
reworking I'm unable to beat the original R-free, despite every other metric 
improving markedly.

Best regards,

Tristan

From: CCP4 bulletin board  on behalf of Barone, Matthias 

Sent: 20 October 2020 12:59
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] AW: [ccp4bb] over-fitting? over-refinement?


Eleanor rises a very important practical point here..."sidechains at the 
solvent interface have multiple conformations, and that as a result the water 
networks should also have partial occupancies". I was fighting with such a 
model for half a year and also tested XSHEL (there was a thread in here for 
that..). Coupling partial occupancies of sidchains with waters and other 
sidchains is a horrendously time-consuming task...and in the end, as Eleanor 
said, "correcting these details does not change the Rfactors at all". You just 
get fed up with that puzzle and stop right there.

best, matthias


Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Eleanor Dodson 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>
Sent: Tuesday, October 20, 2020 12:40:19 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] AW: [ccp4bb] over-fitting? over-refinement?

It is always hard to know when to stop tweaking a model.. We know from high 
resolution studies that many sidechains at the solvent interface have multiple 
conformations, and that as a result the water networks should also have partial 
occupancies. But usually correcting these details does not change the Rfactors 
at all - nor contribute much to the biological relevance of your structure!
So often the point to stop is when you get fed up, Phil Evans said years ago - 
I spend 95% of my time on 5% of the structure, most of which is unimportant..
In practice I let the difference maps decide when to stop - 10 Sigma peak - 
think why - lots of 5 Sigma positive and negative ones not so important
Eleanor

On Tue, 20 Oct 2020 at 11:27, Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>> wrote:

A practice that was very popular before the Rfree came around was to fit a 
water molecule in every noise peak. One would get spectacular low Rfactors this 
way, but I cannot imagine that anyone would believe that this would be fitting 
and not over-fitting.



Best,

Herman



Von: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
Im Auftrag von Sam Tang
Gesendet: Dienstag, 20. Oktober 2020 05:27
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] over-fitting? over-refinement?



Hi, the question may be a bit weird, but how do you define 'over-fitting' in 
the context of structure refinement? From users' perspective the practical 
aspect is to 'fit' the model into the density. So there comes this question 
from our juniors: fit is fit, how is a model over-fit?



BRS



Sam





To 

Re: [ccp4bb] dimeric tag to induce the homodimerization of protein

[Diana Tomchick]

Any dimeric tag should work if you add a long enough linker to satisfy your 
distance criterion.

GST, for example. Download the coordinates and get a rough idea how long the 
linker would have to be for your protein.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Sep 22, 2020, at 12:08 PM, Srivastava, Dhiraj 
mailto:dhiraj-srivast...@uiowa.edu>> wrote:


EXTERNAL MAIL

Hi
I want to make my protein dimeric to increase its affinity for its 
interaction partner which is a dimer. does anyone know a suitable tag/fusion 
protein which can be used as C terminal fusion for this purpose? I can not use 
any of the leucine zipper as I am looking for the distance between the c 
terminus to be around 30-40 A.


Thank you
Dhiraj


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Re: [ccp4bb] polarizer

[Diana Tomchick]

If only glass is placed between the polarizer and analyzer, the crystal will 
not show artificial colors (try it in a 9-well Pyrex depression plate). The 
artificial colors come from the diffraction of visible light from the plastic 
ware, which depending upon the type of plastic and the way the plate is 
manufactured, will have some preferred orientation of the polymer chains. 
Although it could have more to do with the method of manufacture of the plate.

I would love to hear a different explanation from someone that either sells or 
manufactures crystallization plastic ware.

Diana

**
Diana R. Tomchick
Department of Biophysics, Rm. ND10.214A
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Dallas, TX 75061 USA
214-645-6383 (office)

On Aug 16, 2020, at 12:19 PM, Nukri Sanishvili  wrote:


EXTERNAL MAIL

Hi All,

Adding some more details to what's been said already. Only because I've seen 
too many times the polarizers being used incorrectly.
First, you need two polarization filters which are typically called polarizer 
and analyzer. First one (the polarizer) lets through only the light waves of a 
certain polarization. Then one needs to rotate the other one (analyzer) until 
there is no more light getting through. At this point the analyzer blocks the 
light that was let through by the polarizer This is what Diane referred to as 
90 degrees. Please note that the polarizer-analyzer plates stay parallel to 
each other. After that, a crystal is placed between them and is rotated. Unless 
it is a crystal with cubic symmetry, at some angles it will light up in 
beautiful colors and at some angles it will not. This is because the crystal 
changes the polarization of the light passing through and "90 degree setup" of 
the polarizer/analyzer pair is no longer valid for newly polarized light.
Please note that using plastic plates in this context is not quite appropriate. 
The plastic polymer itself changes the polarization as well and therefore it 
breaks the main principle of this method. With plastic interference, it will be 
impossible to reach complete darkening of the field of view. I can almost hear 
a lot of people saying that they've used it with plastic plates without a 
problem. I believe it to be the case but it still doesn't make it right.
Best,
Nukri

On Sun, Aug 16, 2020 at 9:15 AM Matthias Zeug 
mailto:matthias.z...@gmx.de>> wrote:
Hi all,

The polarizer-microscope in our facility is not working properly, and I have to 
check my plates using a standard stereo-microscope. As a workaround, I thought 
about buying one at Amazon, placing it on top of the plates and rotating it to 
still test for birefringence.

The product is linked below. Does anyone have some experience with this kind of 
"homemade" system? And also (this might be a stupid question), does the product 
even work? As far as I know, the polarizers in the microscopes are linear 
polarizers, whereas the product linked below is a circular polarizer. I would 
also be happy for product recommendations (optimally available at the German 
Amazon).

Cheers

Matthias

https://www.amazon.de/dp/B00XNMXYBY/ref=cm_sw_r_cp_apa_i_5YsoFbFQXTBP9

___
Buchmann Institute of Molecular Life Sciences
Goethe University Frankfurt



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Re: [ccp4bb] polarizer

[Diana Tomchick]

It's my understanding that you have two polarizers on your 
polarizer-microscope--one in the base, and the one that attaches to the 
magnifying lens. When you rotate the one on the lens so that it is 90 degrees 
to the one in the base, no (or very little) light should pass through to your 
eyes, unless there is a crystal that plane polarizes the light at an angle that 
differs from the two on the microscope.

What you have is just one polarizing lens. Not sure how that would work, even 
if it is a circular polarizer.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

From: CCP4 bulletin board  on behalf of Matthias Zeug 

Sent: Sunday, August 16, 2020 9:05 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] polarizer


EXTERNAL MAIL

Hi all,



The polarizer-microscope in our facility is not working properly, and I have to 
check my plates using a standard stereo-microscope. As a workaround, I thought 
about buying one at Amazon, placing it on top of the plates and rotating it to 
still test for birefringence.



The product is linked below. Does anyone have some experience with this kind of 
"homemade" system? And also (this might be a stupid question), does the product 
even work? As far as I know, the polarizers in the microscopes are linear 
polarizers, whereas the product linked below is a circular polarizer. I would 
also be happy for product recommendations (optimally available at the German 
Amazon).



Cheers



Matthias



https://www.amazon.de/dp/B00XNMXYBY/ref=cm_sw_r_cp_apa_i_5YsoFbFQXTBP9



___

Buchmann Institute of Molecular Life Sciences

Goethe University Frankfurt



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Re: [ccp4bb] number of frames to get a full dataset?

[Diana Tomchick]

There are lots of places where you could find this information (many textbooks, 
articles, etc.) but one that I use for classes is quite good due to ease of 
understanding. It’s part of the Proceedings of the CCP4 Study Weekend on Data 
Collection and Processing. There are other quite excellent articles in that 
issue, and all are Open Access.

Dauter, Z. (1999) “Data-collection strategies” Acta Cryst. D55, 1703-1717.

https://journals.iucr.org/d/issues/1999/10/00/ba0020/index.html

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Jun 22, 2020, at 5:03 PM, Murpholino Peligro 
mailto:murpholi...@gmail.com>> wrote:


EXTERNAL MAIL

Hi.
Quick question...
I have seen *somewhere* that to get a 'full dataset we need to collect n 
frames':
at least 180 frames if symmetry is X
at least 90 frames if symmetry is Y
at least 45 frames if symmetry is Z
Can somebody point where is *somewhere*?

...also...
what other factors can change n... besides symmetry and radiation damage?

Thanks



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Re: [ccp4bb] Any advice on specs for a new Mac Laptop to run CCP4 and other Xtal Software?

[Diana Tomchick]

Make sure the 13” display is large enough to display all the GUIs for program 
suites that you are interested in.

For example, in the past, the 13” Mac laptops couldn’t display the HKL2000 GUI. 
Requirements are found here:

https://hkl-xray.com/hardware-operating-systems

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Jun 8, 2020, at 10:56 AM, Scott Pegan 
mailto:scott.d.pe...@gmail.com>> wrote:


EXTERNAL MAIL

Finally looking to replace my 2014 Mac Pro lap (duo core; 16 gb RAM. I would 
welcome any input or being put in the direction of some related to the 
following:

13" versus 16"

I know the 16" has a better and standalone GPU.  Is there anyone using a 13" 
that finds that it works well? If so, what were the specs

Memory for the 16"

Torn between 16 gb and 32 gb RAM for the next computer.  Apple asks a premium 
for the 32 gb, but some say that now with the SSD's it's not as big of a deal. 
Any thoughts?

GPU

For the 15", apple has two 4 gb and an 8 gb. Does anyone have any experience on 
these for running CCP4 and other Xtal software on these?

Thanks for your help in advance, looking to get something that is a Mac, robust 
to last a while spec wise.  However, not looking to buy something that is the 
equivalent of a small car in price and overkill.

Scott






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Re: [ccp4bb] disinfecting keyboards

[Diana Tomchick]

?100% ethanol or isopropanol work really well on the microscopes, I soak a 
Kimwipe and then clean the eyepieces and the knobs for changing magnification 
and focus, as well as the door handles, bench tops, etc.


Diana


**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

From: CCP4 bulletin board  on behalf of Diana Tomchick 

Sent: Wednesday, April 29, 2020 2:00 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] disinfecting keyboards

EXTERNAL MAIL

?You could try doing what my technician does with her keyboard; she wraps it in 
a clear, thin food wrap that can be taped to the back of the keyboard. This is 
usually done to keep food and other things (liquids) from damaging the 
keyboard, but you could simply replace the wrap every time someone else uses it.


Personally I like using a Kimwipe soaked with 100% isopropanol, I've never yet 
encountered a keyboard that suffered from having the writing removed with that 
or 100% ethanol. Both work and as long as they are 100% (no water), the 
keyboard and mouse have no issues.


Diana


**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

From: CCP4 bulletin board  on behalf of Tim Gruene 

Sent: Wednesday, April 29, 2020 1:53 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] disinfecting keyboards

Dear all,

can you make suggestions for how to disinfect computer keyboards, and
instrument panels?

Our facility is going to reboot next week, with shifts so that people
don't meet. The main interface will be the computer keyboards, as well
as the door of our X-ray diffractometer and the mounting of the
crystals.

The keyboard labels may not like alcohols (and the efficiency of
injecting disinfecting through the USB cable is also under discussion,
so I heard).

One way would be to use individual keyboards, and wearing gloves for
replugging, and to use gloves for mounting crystals.

But maybe there are other ways that won't require gloves?

Best regards,
Tim

--
--
Tim Gruene
Head of the Centre for X-ray Structure Analysis
Faculty of Chemistry
University of Vienna

Phone: +43-1-4277-70202

GPG Key ID = A46BEE1A



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Re: [ccp4bb] disinfecting keyboards

[Diana Tomchick]

?You could try doing what my technician does with her keyboard; she wraps it in 
a clear, thin food wrap that can be taped to the back of the keyboard. This is 
usually done to keep food and other things (liquids) from damaging the 
keyboard, but you could simply replace the wrap every time someone else uses it.


Personally I like using a Kimwipe soaked with 100% isopropanol, I've never yet 
encountered a keyboard that suffered from having the writing removed with that 
or 100% ethanol. Both work and as long as they are 100% (no water), the 
keyboard and mouse have no issues.


Diana


**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

From: CCP4 bulletin board  on behalf of Tim Gruene 

Sent: Wednesday, April 29, 2020 1:53 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] disinfecting keyboards

Dear all,

can you make suggestions for how to disinfect computer keyboards, and
instrument panels?

Our facility is going to reboot next week, with shifts so that people
don't meet. The main interface will be the computer keyboards, as well
as the door of our X-ray diffractometer and the mounting of the
crystals.

The keyboard labels may not like alcohols (and the efficiency of
injecting disinfecting through the USB cable is also under discussion,
so I heard).

One way would be to use individual keyboards, and wearing gloves for
replugging, and to use gloves for mounting crystals.

But maybe there are other ways that won't require gloves?

Best regards,
Tim

--
--
Tim Gruene
Head of the Centre for X-ray Structure Analysis
Faculty of Chemistry
University of Vienna

Phone: +43-1-4277-70202

GPG Key ID = A46BEE1A



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The future of medicine, today.




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Re: [ccp4bb] Raw diffraction images for SARS-CoV-2 related structures

[Diana Tomchick]

​Frankly this is a great idea for datasets on any projects. I've been so busy 
the last 3-4 years that I have been unable to do it for my projects, but now I 
should be able to get it done. Not to mention trying to solve a couple of those 
pesky structures that have proven tough nuts to crack.


Diana


**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

From: CCP4 bulletin board  on behalf of Eleanor Dodson 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>
Sent: Wednesday, March 18, 2020 5:33 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Raw diffraction images for SARS-CoV-2 related structures

EXTERNAL MAIL

What a great idea? Have you approached the depositors? Eleanor

On Wed, 18 Mar 2020 at 22:31, Gerard Bricogne 
mailto:g...@globalphasing.com>> wrote:
Dear colleagues,

Perusal and some initial (re-)refinement of the various SARS-CoV-2 protease
structures in the PDB seems to indicate that that there might be potential
to improve these if refinements could be repeated after some reprocessing
and further analysis of the raw diffraction images, rather than against the
deposited merged data. This statement should in no way be construed as a
criticism of the remarkable achievements of the research groups concerned,
who have been operating under tremendous time pressure, but as an exciting
opportunity to push methods to their limits on a uniquely significant class
of structures.

Another consideration is that the various logistical problems created by
COVID-19 may soon make it increasingly difficult to collect new diffraction
data on potential drug targets relevant to the fight against SARS-CoV-2,
underlining the importance of ensuring that the best results be obtained
from every dataset actually collected, and that the most useful conclusions
be drawn from the analysis of those datasets towards improving the quality
of subsequent data collections.

On this basis we would like to propose that special efforts be made to grant
public access to the raw image data associated with any SARS-CoV-2 related
structure that is deposited into the PDB. This can be done by (1) archiving
these raw image data using resources such as 
data.sbgrid.org, zenodo.org,
proteindiffraction.org or any other cloud-based 
data-sharing service, and
(2) communicating the corresponding DOIs to the wwPDB centres. This idea
could be extended to datasets that investigators would like to offer to
interested methods developers or expert users at the pre-deposition stage.

Experts making use of those raw data would be encouraged to document, in as
much detail as possible, how particular programs or workflows could be used
on those structures/datasets to obtain the best results. This would be a
kind of "virtual workshop", a particularly valuable collective activity at
the present time when several in-person workshops (e.g. RapiData) have been
cancelled and many meetings are in limbo for several months.

The latter activity would benefit from having a centralised facility set up
for the experts to post their results and annotations: we could create such
a facility, but other, larger groups might want to consider doing so.


With best wishes,

Clemens & Gerard.



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Re: [ccp4bb] Hydrogens in PDB File

[Diana Tomchick]

My structure contains riding hydrogens because (surprise!) the protein contains 
hydrogens, no matter the resolution of the data. Hydrogens should contribute to 
the calculated structure factors, because they do contribute to the observed 
structure factors. If I delete the hydrogens from the coordinates and re-refine 
the model (even keeping the B-factors constant), the geometry will be worse.

When using Refmac, you can opt to explicitly write out the riding hydrogen 
atoms to the final mmCIF file, why don’t you try that and see if it works for 
you?

From the Phenix documentation web site 
(https://www.phenix-online.org/documentation/reference/refine_gui.html)

Optimization methods and other 
options<https://www.phenix-online.org/documentation/reference/refine_gui.html#id9>

Several other options are available for the types of optimization used; these 
typically apply globally, except as noted.

  *   Automatically add hydrogens to model runs phenix.ready_set to add 
hydrogen atoms (or deuteriums, if performing neutron crystallography) where 
appropriate. This usually only affects the R-factors at high resolution, but 
can be very helpful for improving geometry at any resolution. We recommend 
using explicit hydrogens on protein, nucleic acid, and ligand molecules 
throughout refinement. (We do not reocmmend adding hydrogens to waters unless 
you have exceptionally high resolution.) Hydrogen atoms will still be defined 
using the "riding" model unless otherwise requested, so they do not add 
parameters during refinement. (Note that this option can be left on if you 
already have hydrogen atoms in place and are refining as "riding"; if you are 
refining against neutron data and/or allowing hydrogen atoms to refine 
individually, you should uncheck the box, as it will otherwise replace the 
existing atoms.)

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu<mailto:diana.tomch...@utsouthwestern.edu>
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Feb 28, 2020, at 2:23 PM, Alexander Aleshin 
mailto:aales...@sbpdiscovery.org>> wrote:


EXTERNAL MAIL

Diana,
Your structure 6PWD contains riding hydrogens even though the resolution is  
only 2.5A. So, the lack of differences between Depositor and DCC values does 
not contradict my point. Now, try to delete the hydrogens and recalculate 
R/Rfree without re-refining B-factors. You’ll see a noticeable difference. It 
is because hydrogens contribute to calculated structural factors.

Alex

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Diana Tomchick 
mailto:diana.tomch...@utsouthwestern.edu>>
Reply-To: Diana Tomchick 
mailto:diana.tomch...@utsouthwestern.edu>>
Date: Friday, February 28, 2020 at 11:52 AM
To: "CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>" 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: Re: [ccp4bb] Hydrogens in PDB File

[EXTERNAL EMAIL]
Here is just one of many examples that illustrates what I was trying to say 
about deposition of the final mmCIF file from the final output of Phenix 
refinement. This is from the freely downloadable PDB validation report for PDB 
ID 6PWD. Search the PDB on my last name and download several of the validation 
reports, and if you find one that diverges a lot between the Depositor and DCC 
values, let me know. They should be so close to the same as to be meaningless, 
at least for depositions within the last 3-5 years (older ones may indeed 
diverge a lot, but we all can’t be held responsible for the outputs of 
now-obsolete software).

Diana




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Re: [ccp4bb] Hydrogens in PDB File

[Diana Tomchick]

If you deposit an mmCIF file that contains both the observed and calculated 
structure factors from your final round of refinement, then the PDB 
auto-validation reports the same (or so close to the same as to be negligible) 
R factors.

Phenix outputs all of this automatically for you if you click the correct radio 
button in the GUI.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Feb 28, 2020, at 12:51 PM, Ethan A Merritt 
mailto:merr...@uw.edu>> wrote:

EXTERNAL MAIL

On Thursday, 27 February 2020 16:34:50 PST Alexander Aleshin wrote:
Ethan wrote:
   - If you are not making claims about hydrogens but just want to
describe what you did during refinement, I'd go with taking them out

I've noticed that REFMAC and Phenix use riding hydrogens to calculate the
refinement statistics, and their exclusion affects R/Rfree. As a result, it
is not clear what values should be reported.

In my opinion, riding
hydrogens play same role as the TLS parameters, which we keep in a pdb
submission. So, I am not convinced their omission is a good idea.
I think PDB curators should provide a guidance  how to deal with issues like
a resolution of anisotropic or incomplete data sets, riding hydrogens,
sequence numbering etc.

Alex:

You are right that the PDB auto-validation step of recalculating R factors
from the deposited model and observed F's is far from perfect.
I have not looked at the DCC source code, but my impression from the
R factors it spits back at me during deposition:
- it ignores TLS records
- it ignores the header record specifying choice of solvent model
- it does use scattering factors f' and f" from the mmcif coordinate file
- I have no idea what it does with twinning descriptions

As a result there is often a noticeable discrepancy between the R-factors
from "Depositor" and "DCC" in the validation reports.

Regards,
Alex




On 2/27/20, 4:05 PM, "CCP4 bulletin board on behalf of Ethan A Merritt"
mailto:CCP4BB@JISCMAIL.AC.UK> on behalf of 
merr...@uw.edu> wrote:

   [EXTERNAL EMAIL]

   On Thursday, 27 February 2020 15:35:05 PST Whitley, Matthew J wrote:

Hello all,



I am nearly finished refining the structures of two mutant proteins
from
crystals that diffracted to very high resolution, 1 Å and 1.2 Å,
respectively.  Refinement was conducted in the presence of explicit
hydrogens on the models.  I am preparing to deposit these models into
the
PDB but am unsure about whether to retain or remove the hydrogens for
deposition.  On one hand, these hydrogens were explicitly used during
refinement, so that makes me want to keep them, but on the other hand,
they
were added at theoretical positions by MolProbity’s reduce tool
for refinement and were not positioned on the basis of experimentally
observed electron density, so that makes me want to delete them from
the
experimental model.  Which is the preferred option for this
situation?


   The order of operations you describe is unclear.

   If you explicitly refined hydrogens then their final positions are
indeed
based on experimentally determined data.
   The fact that you initially placed them into ideal geometry is not
really
any different from the non-H atoms of individual protein residues
in your model, whose original positions were also based on known
stereochemistry.
   On the other hand, if you mean that the hydrogens you used for
refinement
were deleted and replaced during validation by Molprobity
(which I think it may do by default) that's not good.  You should rather
keep the hydrogen positions from refinement, not the ones from Molprobity.

   Assuming (since this is ccp4bb) you refined with refmac...
   - If you are at the level of investigating hydrogen positions, you may
want
to consider taking the refinement into shelxl.
   - If you are not making claims about hydrogens but just want to
describe
what you did during refinement, I'd go with taking them out and
settling for the standard record in the resulting PDB file:
 REMARK   3  HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
   which looks like this in the corresponding mmcif file:
 _refine.details   'Hydrogens have been added in their riding
positions'

   Ethan




Thanks,
Matthew



---
Matthew J. Whitley, Ph.D.
Research Instructor
Department of Pharmacology & Chemical Biology
University of Pittsburgh School of Medicine




##
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Re: [ccp4bb] What resolution - X-ray diffraction round this time

[Diana Tomchick]

But that wouldn’t incorporate other useful information, such as percentage 
completeness in the outer shell and whether inclusion of the reflections in the 
outer shell for refinement actually effects the resulting structural model and 
map to a significant degree.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Feb 28, 2020, at 10:03 AM, Phoebe A. Rice 
mailto:pr...@uchicago.edu>> wrote:

EXTERNAL MAIL

Can we get some momentum for the "standard table 1" including TWO numbers - 
outer limit used in refinement, and nominal resolution based on some standard 
such as I/sigI =2 (or 3, or whatever the community can agree on)?  That would 
hopefully cut down on all the reviewer complaints of overstated resolution.

~~~
Phoebe A. Rice
Dept. of Biochem & Mol. Biol. and
 Committee on Microbiology
https://voices.uchicago.edu/phoebericelab/


On 2/28/20, 6:56 AM, "CCP4 bulletin board on behalf of Malý Martin" 
mailto:CCP4BB@JISCMAIL.AC.UK> on behalf of 
martin.m...@ibt.cas.cz> wrote:

   Dear colleagues,

   I agree with all the previous responses, it is a pity to throw away
   useful high-resolution data. The problem of high-resolution cutoff
   estimation is also nicely summarized in another paper by Andrew Karplus
   and Kay Diederichs "Assessing and maximizing data quality in
   macromolecular crystallography"
   https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684713/ . It is suggested
   using CC1/2 for the selection of the cutoff for data processing (not
   I/sigI or R_whatever). Later on, the decision should be validated
   performing the paired refinement protocol.

   Good luck with the argumentation.
   Martin


   On 2/28/20 11:08 AM, LMB wrote:
Ask the referee - (apart from the other suggestions here)

‘How would removing data Improve my model?”

Sent from my iPad

On 28 Feb 2020, at 08:22, dusan turk 
mailto:dusan.t...@ijs.si>> wrote:

Hi,

Browsing through the recent discussion on EM data resolution cutoff
it occurred to me that the X-ray diffraction community isn’t that
unanimous either.

My stand:

When the default resolution cutoff provided with the data processing
software in electron density map calculation and refinement delivers
quality maps noisier than expected and/or too high R-factors I start
adjusting the resolution cutoff by lowering the resolution and trying
alternative space group. Hence, I allow the data processing programs
to suggest where to draw the line (be it CC1/2, I/sigI, R merge, R
sym, R p.i.m. and R r.i.m, …) , unless there are problems.

Doing so, I came into a dispute with a referee who shaped his request:

"It is well accepted that the criteria for resolution cutoff should
consider both I/SigI and Rmerge for the outer most shell. For data
sets collected at synchrotron sources, the criteria of I/SigI > 5 and
Rmerge <50% can be taken as a good practical reference.”

So where do we stand? Which are the most objective criteria for
resolution cutoff to be used in diffraction data processing? Which
number of shells to use when calculating the statistics? Do we have a
consensus?

best wishes,

dusan turk



Dr. Dusan Turk, Prof.
Head of Structural Biology Group http://stef.ijs.si/
Head of Centre for Protein and Structure Production
Centre of excellence for Integrated Approaches in Chemistry and
Biology of Proteins, Scientific Director
http://www.cipkebip.org/
e-mail: dusan.t...@ijs.si
phone: +386 1 477 3857 Dept. of Biochem.& Mol.& Struct. Biology
fax: +386 1 477 3984 Jozef Stefan Institute
Jamova 39, 1 000 Ljubljana,Slovenia
Skype: dusan.turk (voice over internet: www.skype.com



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   -

   Upozornění: Není-li v této zprávě výslovně uvedeno jinak, má tato e-mailová 
zpráva nebo její přílohy pouze informativní charakter. Tato zpráva ani její 
přílohy v žádném ohledu Biotechnologický ústav AV ČR, v. v. i. k ničemu 
nezavazují. Text této zprávy nebo jejích příloh není návrhem na uzavření 
smlouvy, ani přijetím případného návrhu na uzavření smlouvy, ani jiným právním 
jednáním směřujícím k uzavření jakékoliv smlouvy a nezakládá předsmluvní 
odpovědnost Biotechnologického ústavu AV ČR, v. v. i.

   Disclaimer: If not expressly stated otherwise, this e-mail message 
(including any attached files) is intended purely for informational purposes 
and 

Re: [ccp4bb] powdery residue on pucks?

[Diana Tomchick]

I believe my technician decided that it was due to the breakdown of the 
adsorbent foam in our oldish shipping dewar, as it stopped happening once we 
replaced that particular dewar.

But I would be open to other explanations.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Feb 21, 2020, at 6:33 PM, Emilia C. Arturo (Emily) 
mailto:ecgart...@gmail.com>> wrote:


EXTERNAL MAIL

Hi All,

We have been noticing lately that our crystal pucks return from different 
beamlines coated with the same powdery material. For the record, we typically 
undo our pucks, clean the assemblies and dry out the pucks within a day of 
receiving the shipping dewar, but notice the same thing regardless of how long 
between receiving the dewar and disassembling the pucks. Have any of you 
experienced this sort of thing, or have suggestions of what might be the cause 
and/or the powdery material?

Regards,
Emily.

--
"Study as if you were going to live forever; live as if you were going to die 
tomorrow." - Maria Mitchell

"I'm not afraid of storms for I'm learning to sail my ship."  - Louisa May 
Alcott




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Re: [ccp4bb] Macromolecular Crystallography workshop in South America 2020

[Diana Tomchick]

Then ask more “junior women.” This isn’t rocket science, after alll.

Diana

**
Diana R. Tomchick
Department of Biophysics, Rm. ND10.214A
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Dallas, TX 75061 USA
214-645-6383 (office)

On Feb 5, 2020, at 12:09 PM, Goldman, Adrian  wrote:


EXTERNAL MAIL

Phoebe and all,

What I heard recently (I have no idea whether it applies in this particular 
case…) is that organisers of conferences/meetings often have considerable 
difficulty getting women speakers in the first place - apparently 85% of the 
XYs asked say “yes” and only 50% (less?) of the XXs. Presumably not to GRCs, or 
to keynote a major international symposium - but ? Precisely for this kind of 
event. I was told this is because the senior women get asked more often, as 
there are (qv) fewer of them, and so there is meeting-attendance-burnout.

Adrian

On 5 Feb 2020, at 18:00, Phoebe A. Rice 
mailto:pr...@uchicago.edu>> wrote:

While there is some truth to that argument, the problem is that it is harder to 
achieve an international reputation in the first place while being routinely 
overlooked.

~~~
Phoebe A. Rice
Dept. of Biochem & Mol. Biol. and
  Committee on Microbiology
https://voices.uchicago.edu/phoebericelab/


From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Andrew Leslie 
mailto:and...@mrc-lmb.cam.ac.uk>>
Reply-To: Andrew Leslie 
mailto:and...@mrc-lmb.cam.ac.uk>>
Date: Wednesday, February 5, 2020 at 11:56 AM
To: "CCP4BB@JISCMAIL.AC.UK" 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: Re: [ccp4bb] Macromolecular Crystallography workshop in South America 
2020

Dear All,

  In fairness to the organisers, I would like to point out that 
there is nothing that is “lazy” about organising these workshops. It involves a 
considerable effort both in arranging the course, the venue and especially in 
attracting funds to support the workshop (it is important to note that CCP4 
does not supply all the funds). In addition, it is unfair to single out this 
particular workshop for criticism, as I believe it has long been the case that 
these workshops have not had a good gender balance in terms of the tutors. It 
is also important to realise that the gender imbalance does NOT extend to 
choice of the students, where as far as I am aware the gender balance is always 
very good.

One difficulty the organisers face is that funding will typically depend on 
having tutors with an international reputation in the areas in which they are 
teaching, ideally having been involved in developing the software that is being 
used. Unfortunately, this inevitably leads to gender bias.

While I would agree that this is an issue that is worthy of being raised, and I 
feel sure that this point will be taken on board by future organisers, it is 
also important to realise the practical difficulties that organisers face and 
the considerable effort that is involved in running these workshops.

Regards,

Andrew Leslie


On 5 Feb 2020, at 00:30, Alejandro Buschiazzo 
mailto:ale...@pasteur.edu.uy>> wrote:

Dear colleagues,

We are pleased to announce the 8th South American Macromolecular 
Crystallography School:

Macromolecular Crystallography School 2020
"Structural Biology to enhance high impact research in health and disease”

To be held at the Institut Pasteur de Montevideo (Uruguay) - September 9-19, 
2020
http://pasteur.uy/novedades/mx2020/
The application deadline is July 9, 2020. For further inquiries : 
mx2...@pasteur.edu.uy


Main Topics:

•   data processing;

•   phasing and structure determination;

•   model refinement and validation;

•   introduction to crystallography + cryo-electron microscopy integration

Confirmed speakers and tutors (so far... a few more will join the crew):

Alejandro Buschiazzo (Institut Pasteur de Montevideo, Uruguay)
Paul Emsley (Laboratory of Molecular Biology MRC, Cambridge, UK)
Rafael Junqueira Borges (Instituto de Biociências UNESP, Botucatu, Brazil)
Ronan Keegan (STFC Rutherford Appleton Lab - CCP4, Didcot, UK)
Eugene Krissinel (STFC Rutherford Appleton Lab - CCP4, Didcot, UK)
Joāo Muniz (Instituto de Fisica de São Carlos, Brazil)
Garib Murshudov (Laboratory of Molecular Biology MRC, Cambridge, UK)
Colin Palmer (STFC Rutherford Appleton Lab - CCP-EM, Didcot, UK)
James Parkhurst (Diamond Light Source, Didcot, UK)
Randy Read (University of Cambridge, UK)
Kyle Stevenson (STFC Rutherford Appleton Lab - CCP4, Didcot, UK)
Clemens Vonrhein (Global Phasing Ltd, Cambridge, UK)

Please find the application form and further contact information at 
http://pasteur.uy/novedades/mx2020/
(this www site will be updated regularly, so stay tuned!)

This Workshop is supported by the Collaborative Computational Project Nº4 
(CCP4, UK) & Science and Technology Facilities Council (UK); the Centro de 
Biologia Estructural del 

Re: [ccp4bb] Mixed oligomeric states in crystallo

[Diana Tomchick]

Here’s two examples of heterooligomers that crystallized in a lattice with an 
extra monomer of one of the proteins. In both cases this was an unexpected 
result, but easily explained due to the low micro molar affinities for the 
complex.

4PKY
2CJS

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Jan 31, 2020, at 9:23 AM, Kluenemann, Thomas 
mailto:thomas.kluenem...@helmholtz-hzi.de>> 
wrote:


EXTERNAL MAIL

Dear all,

We recently solved a the structure of a small c-type cytochrome. We observed, 
that of the eleven chains in the asymmetric unit ten form 3D domain swapped 
dimers by exchanging an α-helix. The eleventh  chain is present as a monomer. 
Based on the anomalous iron signal and the chain tracing we are sure that no 
chain was missed.
I tried to find other examples in the PDB, were one crystal is made of 
different homo- or heterooligomers.  I only found proteins with partial 
occupied peptide binding sites, which is not what I am looking for. Does anyone 
know of a case were the presence of different homo- or heterooligomers is 
required to form the crystal?

Best regards,
Thomas Klünemann





Helmholtz-Zentrum für Infektionsforschung GmbH | Inhoffenstraße 7 | 38124 
Braunschweig | www.helmholtz-hzi.de

Vorsitzende des Aufsichtsrates: Frau MinDir'in Prof. Dr. Veronika von Messling
Stellvertreter: MinDirig Rüdiger Eichel, Niedersächsisches Ministerium für 
Wissenschaft und Kultur
Geschäftsführung: Prof. Dr. Dirk Heinz; Silke Tannapfel
Gesellschaft mit beschränkter Haftung (GmbH)
Sitz der Gesellschaft: Braunschweig
Handelsregister: Amtsgericht Braunschweig, HRB 477



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Re: [ccp4bb] A crystallisation screen pH query.

[Diana Tomchick]

Although it sounds as though this condition was not pH adjusted by the company, 
here are a couple of suggestions that are applicable to ANY commercial screen 
condition.


1) Try looking at the company's web site for details on how the solution was 
made, or failing that, try calling the company where you bought the screen and 
ask (you don't specify the company in your question).

2) Take a small drop of the screen solution from the reservoir well and apply 
it to pH paper, it'll give you an approximate idea.


Don't forget that the protein and the contents of the protein buffer solution 
will also affect the pH in the mother liquor.


Diana


**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

From: CCP4 bulletin board  on behalf of Jon Cooper 
<0c2488af9525-dmarc-requ...@jiscmail.ac.uk>
Sent: Saturday, October 5, 2019 4:09 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] A crystallisation screen pH query.

WARNING: This email was sent from an external source. Please be cautious of 
links or attachments, and validate the sender's email address before replying.

Does anyone know the pH of JCSG+ condition A3 which is stated as 0.2 M ammonium 
citrate dibasic, 20% (v/v) PEG3350. I can't really measure it myself, so any 
help much appreciated!



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Re: [ccp4bb] Intergrown crystals and excess of nucleation

[Diana Tomchick]

In addition to all the excellent suggestions to perform seeding, you could also 
try sitting drop vapor diffusion, and also different temperatures. Sometimes 
moving from 20 to 4 degrees does the trick.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Sep 9, 2019, at 10:22 AM, Nikolas 
mailto:nikolas.ca...@gmail.com>> wrote:

Dear crystalgrowers,

I am currently working with a protein that appeared to be friendly but turned 
out it was not the case.
I found myself to face -in the scale up- the opposite of the usual problem of 
nucleation (I really love how this topic finds new ways to make fun of me). In 
24-well plates, hanging-drop, for the same condition but in different drops I 
found few big but intergrown crystals and/or a full with microcrystals. 
Sometimes also in the same well, when having more drops.
I already decreased the concentration to less than 4mg/mL, made small 
adjustments in the optimizations - both with apo and ligand samples, used Al's 
oil.

I have read about the "containerless crystallization" but since I cannot obtain 
the sample myself I would like to know if there are any experiences and/or if 
there are suggestions for solving this problem.

Many thanks!

Best regards,
Nikolas



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Re: [ccp4bb] tNCS incompatible with cell dimensions

[Diana Tomchick]

Your native Patterson indicates pseudo C-centering. Are you sure you don’t have 
space group C222(1)?

If your space group is correct, it’s still pseudo C-centered. You should see 
that in the intensity-weighted reciprocal lattice.

You could try re-indexing on just the most intense spots to give you a data set 
indexed in a C-centered lattice. Use that data to solve via MR, then convert to 
the data indexed in the actual space group.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On May 31, 2019, at 3:09 PM, Kevin Jude 
mailto:kj...@stanford.edu>> wrote:

Hello community, I wonder if I could solicit advice about a problematic 
dataset. I plan to solve the structure by molecular replacement and expect that 
the protein is relatively compact, ie not elongated. SAXS data supports this 
expectation.

The crystals diffract to 2.6 Å resolution and appear to be in P 21 21 2 with a 
= 49, b = 67, c = 94, which should fit <=2 molecules in the ASU with 40% 
solvent. The native Patterson shows a large peak (12 sigma) suggesting a tNCS 
vector of {0.5, 0.5, 0}.

If you're sharper than me, you may have already spotted the problem - c is the 
long axis of the unit cell, but tNCS constrains the proteins to a plane 
parallel to the a,b plane. Indeed, molecular replacement attempts using Phaser 
will not give a solution in any orthorhombic space group unless I turn off 
packing, and then I get large overlaps in the a,b plane and huge gaps along c.

Since I believe that my model is good (or at least the correct shape, based on 
SAXS), I wonder if I'm misinterpreting my crystallographic data. Any insights 
into how to approach this problem would be much appreciated.

--
Kevin Jude, PhD
Structural Biology Research Specialist, Garcia Lab
Howard Hughes Medical Institute
Stanford University School of Medicine
Beckman B177, 279 Campus Drive, Stanford CA 94305
Phone: (650) 723-6431




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Re: [ccp4bb] how many crystallographers are there?

[Diana Tomchick]

Try asking the IUCr for information on crystallographers.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On May 29, 2019, at 1:54 PM, Scott Horowitz 
mailto:horow...@umich.edu>> wrote:

Hi all, I was recently asked how many biological crystallographers plus cryo-EM 
users there are in the world (in relation to how many people could therefore be 
theoretically impacted by Foldit electron density tools, for the purposes of 
grant funding). I'm a bit at a loss as to even what order of magnitude to 
provide. Any thoughts about how to estimate a number?

Thanks,
Scott


Scott Horowitz, Ph.D.
Assistant Professor
Department of Chemistry & Biochemistry
Knoebel Institute for Healthy Aging
University of Denver



ECS Building
2155 E. Wesley Ave
Denver, CO 80208
Phone: 303-871-4326
Fax: 303-871-7915
Email: scott.horow...@du.edu
Office: Room 561   Lab: Room 505




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Re: [ccp4bb] pointless warning - space group ambiguity

[Diana Tomchick]

You didn’t tell us what method and computer programs you are using for phasing.

If you are using Molecular Replacement and not getting a solution, it could be 
that your model isn’t close enough to the actual solution.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Apr 19, 2019, at 1:11 PM, Almudena Ponce Salvatierra 
mailto:maps.fa...@gmail.com>> wrote:


Dear all,

I'm processing my data with XDS and with DIALS and at first sight it would seem 
reasonable to assume that the Laue group is

either orthorrombic or monoclinic. Autoprocessing brings me always to P212121, 
but I have tried also to solve the
structure in space groups 1, 3, 4, 16, 17 and 18, without success.

Pointless gives the following warning:

WARNING:  The data were integrated on a primitive lattice, but show some 
apparent features of a centered

  lattice of type I, Mean(E^2) for lattice absences = 1.15

 However, it might be pseudo-centered, due to translational 
non-crystallographic symmetry

 Mn(I/sigI) for potentially absent reflections in the first  7 resolution bins 
= 4.76 is significantly above 0.0

Rough estimated probability of true centering = 0.714

NB: I lattice was chosen for further analysis rather than lattice type I



Best Solution:space group P 21 21 21



   Reindex operator:   [h,k,l]

   Laue group probability: 0.762

   Systematic absence probability: 0.682

   Total probability:  0.520

   Space group confidence: 0.478

   Laue group confidence   0.702


However XDS doesn't give me option to choose any other lattices if not those in 
which I have already failed to

solve the structure, those marked with starts that are consistent with the 
observed locations of the diffraction spots.


So, I don't know what to do, I'm guessing it could be that it is C222, C2221 or 
C2 as well... but when I try space groups 5, 20 or 21 XDS
gets stalled and DIALS can not proceed with the step of refinement.


I'll be very thankful for any suggestions on how to continue on this venture...


Have a nice weekend and a happy Easter!

Best,


Almudena



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Re: [ccp4bb] High Rfree in last Shell

[Diana Tomchick]

Ah, even though it seems a long time ago, approximately one out of every two 
referee comments that I receive complain about our modern methods for 
processing, scaling and reporting our data collection/processing/refinement 
statistics. I’m afraid it’s an ongoing effort at education.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Apr 16, 2019, at 3:09 PM, Frank von Delft 
mailto:frank.vonde...@sgc.ox.ac.uk>> wrote:

Jan, tell your reviewer to join us all in the 21st century.

Diederichs and Karplus, Science, about 2 decades ago.  (Technically, 
2012,
 but it really is a long long time ago now.)


On 16/04/2019 18:06, Tim Gruene wrote:

Dear Jan,

You statistics look quite solid.

R-factors are not good criteria to judge the resolution cut-off. The weighting
schemes in refinement programs have much improved since the late 1990s. A good
starting point to learn more is
Rupp's "Against Method: Table 1 -Cui Bono?", https://doi.org/10.1016/j.str.
2018.04.013

Best regards,
Tim

On Tuesday, April 16, 2019 6:57:06 PM CEST Jan van Agthoven wrote:


Hi everyone,
I’m trying to publish two structures at 3.1Å resolution with the following
refinement statistics:

Resolution range (Å)   49.2-3.1
49.3-3.1 Rfactor (%)
24.0 (32.4)  23.4 (32.0) Rfree (%)
26.6 (29.2)
26.3 (31.6)

Data collection
Completeness  100 (100)
  100 (100)

Redundancy6.9 (7.0)
6.2 (6.3)

Molecules in asymmetric unit  1
1

Average I/σ 14.1 (1.7)
 15.3 (2.0)

Rmerge (%)  14.9 (100)
12.7 (100)

Rmeas (%)16.2 (100)
 13.9 (100)

Rsym (%)   6.2 (68.6)
5.5 (57.1) Wilson B-factor
   65.662.7

I’ve been told that the Rfree factor in the last shell are too high. Does
anyone know how I can improve these Rfree factors other then cutting the
resolution, which already is rather low?


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Re: [ccp4bb] High Rfree in last Shell

[Diana Tomchick]

Sorry, I meant to write

Why are the values for R(free) in parentheses LOWER than R(factor) in 
parentheses?

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu<mailto:diana.tomch...@utsouthwestern.edu>
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Apr 16, 2019, at 1:21 PM, Diana Tomchick 
mailto:diana.tomch...@utsouthwestern.edu>> 
wrote:

Why are the the values for R(free) in parentheses higher than R(factor) in 
parentheses? I would not expect that to happen, if the same resolution limits 
for the last shell are used for both values. And I echo the previous 
commentator, please provide the resolution limits for the values in parentheses.

What exactly is R(factor) in this case? The R(work), or the [R(work) + 
R(free)]? Please define.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu<mailto:diana.tomch...@utsouthwestern.edu>
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Apr 16, 2019, at 11:57 AM, Jan van Agthoven 
mailto:janc...@gmail.com>> wrote:

Hi everyone,
I’m trying to publish two structures at 3.1Å resolution with the following 
refinement statistics:

Resolution range (Å)   49.2-3.1 
 49.3-3.1
Rfactor (%)24.0 (32.4)  
23.4 (32.0)
Rfree (%)  26.6 (29.2)  
26.3 (31.6)

Data collection
Completeness  100 (100) 
   100 (100)
Redundancy6.9 (7.0) 
 6.2 (6.3)
Molecules in asymmetric unit  1 
 1
Average I/σ 14.1 (1.7)  
  15.3 (2.0)
Rmerge (%)  14.9 (100)  
 12.7 (100)
Rmeas (%)16.2 (100) 
  13.9 (100)
Rsym (%)   6.2 (68.6)   
 5.5 (57.1)
Wilson B-factor 65.6
62.7
I’ve been told that the Rfree factor in the last shell are too high. Does 
anyone know how I can improve these Rfree factors other then cutting the 
resolution, which already is rather low?



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Re: [ccp4bb] High Rfree in last Shell

[Diana Tomchick]

Why are the the values for R(free) in parentheses higher than R(factor) in 
parentheses? I would not expect that to happen, if the same resolution limits 
for the last shell are used for both values. And I echo the previous 
commentator, please provide the resolution limits for the values in parentheses.

What exactly is R(factor) in this case? The R(work), or the [R(work) + 
R(free)]? Please define.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Apr 16, 2019, at 11:57 AM, Jan van Agthoven 
mailto:janc...@gmail.com>> wrote:

Hi everyone,
I’m trying to publish two structures at 3.1Å resolution with the following 
refinement statistics:

Resolution range (Å)   49.2-3.1 
 49.3-3.1
Rfactor (%)24.0 (32.4)  
23.4 (32.0)
Rfree (%)  26.6 (29.2)  
26.3 (31.6)

Data collection
Completeness  100 (100) 
   100 (100)
Redundancy6.9 (7.0) 
 6.2 (6.3)
Molecules in asymmetric unit  1 
 1
Average I/σ 14.1 (1.7)  
  15.3 (2.0)
Rmerge (%)  14.9 (100)  
 12.7 (100)
Rmeas (%)16.2 (100) 
  13.9 (100)
Rsym (%)   6.2 (68.6)   
 5.5 (57.1)
Wilson B-factor 65.6
62.7
I’ve been told that the Rfree factor in the last shell are too high. Does 
anyone know how I can improve these Rfree factors other then cutting the 
resolution, which already is rather low?



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Re: [ccp4bb] beryllium chloride

[Diana Tomchick]

No, that should read

[cid:2D6B8E73-AD29-4B8C-972A-06201D871588]

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Apr 1, 2019, at 5:54 PM, Keller, Jacob 
mailto:kell...@janelia.hhmi.org>> wrote:

Is that 4+ an April fools’ joke? Pretty crazy if not…can’t think of another ion 
with such a charge, well except things like DNA and proteins, but not single 
atoms.

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
+

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From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Aaron Finke
Sent: Monday, April 1, 2019 6:45 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] beryllium chloride

American Elements sells BeCl2 but you’d have to check with them on what scale 
they sell it at. They tend to do custom manufacturing.

https://www.americanelements.com/beryllium-chloride-7787-47-5

BeCl2 dissociates in aqueous solution to form Be(H2O)4+ 2Cl-.

Aaron
--
Aaron Finke
Staff Scientist, MacCHESS
Cornell University
e-mail: af...@cornell.edu

On Apr 1, 2019, at 17:07, Alexandra Deaconescu 
mailto:alexandra_deacone...@brown.edu>> wrote:
Hello,

Is anyone aware of a company that sells Beryllium chloride in the US? Sigma 
does not carry it any longer, and a quick Google search failed to reveal 
alternatives.

Thank you very much,

Alexandra


--
Alexandra Deaconescu, B.E., Ph.D.
Assistant Professor
Brown University

Office: (401) 863-3215
Wet Lab: (401) 863-6729
Computational Lab: (401) 863-7031

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Re: [ccp4bb] acceptable difference between Average B-factor and Wilson B

[Diana Tomchick]

>   I do not believe comparing the average B to the Wilson B has any
> utility at all.
>
> Dale Tronrud

There are not many times that reading a CCP4bb posting makes me laugh out loud, 
but this one made my day. Thanks!

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)




UT Southwestern


Medical Center



The future of medicine, today.



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Re: [ccp4bb] Mandatory mmCIF format for crystallographic depositions to the PDB

[Diana Tomchick]

The PDB extract tool can be used to create the proper format mmCIF file from 
coordinates uploaded in PDB format.

https://pdb-extract.wwpdb.org

https://pdb-extract.wwpdb.org/tutorial.html

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Feb 20, 2019, at 1:09 PM, Tim Grüne 
mailto:tim.gru...@psi.ch>> wrote:

Dear John, dear CCP4,
Is there a converter from CIF to mmCIF?
Some structures are refined with Shelxl, which can output CIF.
A converter would be very useful, even if they make only a small fraction of 
the PDB.
Best regards,
Tim


On February 20, 2019 9:06:22 AM GMT+01:00, John Berrisford 
mailto:j...@ebi.ac.uk>> wrote:

From July 1st 2019 onward, mmCIF format files will become mandatory for 
crystallographic depositions to the Protein Data Bank. PDB format files will no 
longer be accepted for deposition of structures solved by these techniques.

PDBx/mmCIF will be the only format accepted for deposition of PDB structures 
resulting from macromolecular crystallography (MX), including X-ray, neutron, 
fiber, and electron diffraction methods via OneDep starting July 1st 2019. The 
deposition of PDBx/mmCIF format files will improve the efficiency of the 
deposition process and enhance validation through capture of the more extensive 
experimental metadata supported by PDBx/mmCIF, compared to the legacy PDB 
format.

For information about these changes, please visit the wwPDB news 
page. If 
you have any queries or comments regarding these changes, please contact the 
wwPDB consortium via 
deposit-h...@mail.wwpdb.org.

The wwPDB consortium



--
John Berrisford
PDBe
European Bioinformatics Institute (EMBL-EBI)
European Molecular Biology Laboratory
Wellcome Trust Genome Campus
Hinxton
Cambridge CB10 1SD UK
Tel: +44 1223 492529

http://www.pdbe.org
http://www.facebook.com/proteindatabank
http://twitter.com/PDBeurope




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--
Sent from my Android device with K-9 Mail. Please excuse my brevity.


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Re: [ccp4bb] [EXTERNAL] [ccp4bb] refmac same residue different names

[Diana Tomchick]

The scenario I presented earlier works like a charm in refinement with the 
Phenix program suite. For example, I have used it for both mixed populations of 
ligands (e.g., AAMP and BADP) as well as phosphorylation (ASER and BSEP).

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Feb 6, 2019, at 11:19 AM, Edwin Pozharski 
mailto:pozharsk...@gmail.com>> wrote:

Herman,

thanks - however, it seems like I have poorly worded my question.  I do know 
how to generate alternate conformers, what the PDB ATOM record format is etc.  
The point was that when I have alternate conformers that carry the same residue 
ID but different residue types, Refmac exits with the error.  The question was 
whether there is a "native" solution to this that does not include some pdb 
file acrobatics (i.e. separating the alternative type into a separate residue 
and enforcing connectivity using elaborate LINK records).   Based on what I see 
so far, there likely isn't any such native option.  Whether these situations 
are common enough to warrant (possibly elaborate) software changes is a 
separate question.

Cheers,

Ed.

---
I don't know why the sacrifice didn't work. The science seemed so solid.
Julien XIII, Lord of the Lemurs

On Wed, Feb 6, 2019 at 2:36 AM 
mailto:herman.schreu...@sanofi.com>> wrote:
Dear Edwin,

I do not know whether your question has been answered already, but the answer 
is simple: you have to define alternative conformations. Easiest is to generate 
them in coot with the “add alternate conformation” option in the right panel. 
You may have to delete the original unlabeled alternative conformation first 
though.
Alternatively, if you want to keep the original coordinates, or if the 
alternative residue is different: say a Leu versus a Phe you can open the pdb 
file with an editor and generate the alternative conformation yourself:
One of the residues gets an “A” in front of the residue name, e.g. ALEU, and 
the alternative residue a “B”, say BLEU. You also have to reset the occupancies 
to 0.5 for both conformations (or different fractions which add up to one).

Good luck!
Herman

Von: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
Edwin Pozharski
Gesendet: Montag, 4. Februar 2019 22:35
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] refmac same residue different names

Belated happy 2019, everyone.

For whatever obscure reason, I need to refine a model that has two different 
residue types as alternate conformers with the same residue ID.  Presented with 
a pdb file that has such feature, Refmac fails saying this

 ERROR: in chain A residue: 443
different residues have the same number

There is an error in the input coordinate file
At least one the chains has 2 residues with the same number
Check above to see error
===> Error: Problem with coordinate file

There are several ways of getting around this I can think of.  Perhaps 
duplicate chain with strict NCS for all but the residue in question could work. 
 Perhaps adding this residue as two separate chains and then adding enough LINK 
records to keep things in place could.  Either solution here is inelegant and 
requires reformating pdb file back to sanity prior to deposition.

Is there some way to allow different geometries for alternate conformers that 
is native to Refmac?

Cheers,

Ed.

PS.  I know that phenix.refine takes the mixed name pdb file straight up.  I 
still want to be able to refine such structure with refmac (and buster, 
actually, but that's a question I already asked in the appropriate forum.


Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
-- / Lao Tse /



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Re: [ccp4bb] AW: [EXTERNAL] [ccp4bb] refmac same residue different names

[Diana Tomchick]

If you have the odd case where one residue (of the same number in the 
polypeptide chain) is a Leu and the alternative residue is a Phe, then it would 
be ALEU and BPHE, both residues would have the same residue number, and reset 
the occupancies to fractions that sum to 1.0.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Feb 6, 2019, at 1:36 AM, 
herman.schreu...@sanofi.com wrote:

Dear Edwin,

I do not know whether your question has been answered already, but the answer 
is simple: you have to define alternative conformations. Easiest is to generate 
them in coot with the “add alternate conformation” option in the right panel. 
You may have to delete the original unlabeled alternative conformation first 
though.
Alternatively, if you want to keep the original coordinates, or if the 
alternative residue is different: say a Leu versus a Phe you can open the pdb 
file with an editor and generate the alternative conformation yourself:
One of the residues gets an “A” in front of the residue name, e.g. ALEU, and 
the alternative residue a “B”, say BLEU. You also have to reset the occupancies 
to 0.5 for both conformations (or different fractions which add up to one).

Good luck!
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Edwin 
Pozharski
Gesendet: Montag, 4. Februar 2019 22:35
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] refmac same residue different names

Belated happy 2019, everyone.

For whatever obscure reason, I need to refine a model that has two different 
residue types as alternate conformers with the same residue ID.  Presented with 
a pdb file that has such feature, Refmac fails saying this

 ERROR: in chain A residue: 443
different residues have the same number

There is an error in the input coordinate file
At least one the chains has 2 residues with the same number
Check above to see error
===> Error: Problem with coordinate file

There are several ways of getting around this I can think of.  Perhaps 
duplicate chain with strict NCS for all but the residue in question could work. 
 Perhaps adding this residue as two separate chains and then adding enough LINK 
records to keep things in place could.  Either solution here is inelegant and 
requires reformating pdb file back to sanity prior to deposition.

Is there some way to allow different geometries for alternate conformers that 
is native to Refmac?

Cheers,

Ed.

PS.  I know that phenix.refine takes the mixed name pdb file straight up.  I 
still want to be able to refine such structure with refmac (and buster, 
actually, but that's a question I already asked in the appropriate forum.


Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
-- / Lao Tse /



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Re: [ccp4bb] visualising anis B factors

[Diana Tomchick]

ORTEP = Oak Ridge Thermal Ellipsoid Program

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu<mailto:diana.tomch...@utsouthwestern.edu>
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Jan 23, 2019, at 3:50 PM, Keller, Jacob 
mailto:kell...@janelia.hhmi.org>> wrote:

What’s the story with the funny name—does it have to do with its being petro 
backwards?

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 mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Mooers, Blaine H.M. (HSC)
Sent: Wednesday, January 23, 2019 4:17 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] visualising anis B factors

Dear Eleanor,

Ethan Merritt's rastep program is a sort of color version of ortep.
It gives beautiful figures.

http://skuld.bmsc.washington.edu/raster3d/html/rastep.html

Best regards,

Blaine

Blaine Mooers, Ph.D.
Associate Professor
Department of Biochemistry and Molecular Biology
College of Medicine
University of Oklahoma Health Sciences Center
S.L. Young Biomedical Research Center (BRC) Rm. 466
975 NE 10th Street, BRC 466
Oklahoma City, OK 73104-5419


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] 
on behalf of Diana Tomchick 
[diana.tomch...@utsouthwestern.edu<mailto:diana.tomch...@utsouthwestern.edu>]
Sent: Wednesday, January 23, 2019 3:01 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [EXTERNAL] Re: [ccp4bb] visualising anis B factors
Hi Bob,

You might recognize this manual.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu<mailto:diana.tomch...@utsouthwestern.edu>
(214) 645-6383 (phone)
(214) 645-6353 (fax)



On Jan 23, 2019, at 2:07 PM, Sweet, Robert 
<27e0eb9d20ec-dmarc-requ...@jiscmail.ac.uk<mailto:27e0eb9d20ec-dmarc-requ...@jiscmail.ac.uk>>
 wrote:

Please remember Carrol Johnson and colleagues. In 1965 from Oak Ridge National 
Lab they released a FORTRAN program, ORTEP, that would write instructions for a 
CalComp plotter, not only to make a molecular drawing showing thermal 
ellipsoids, but also to create them IN STEREO! This program revolutionized 
crystallographic publishing, not so much for the ellipsoids, but for the easy 
visualization of ones molecules in three dimensions. It seems so easy now.

Bob


  Robert M. Sweet   E-Dress:  
sw...@bnl.gov<mailto:sw...@bnl.gov>
  Deputy Director, LSBR: The Life Science and
   Biomedical Technology Research Center at NSLS-II
  Photon Sciences and Biology Dept
  Brookhaven Nat'l Lab.
  Upton, NY  11973 U.S.A.
  Phones:631 344 3401  (Office)
 631 338 7302  (Mobile)



From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Eleanor Dodson 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk<mailto:176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>>
Sent: Wednesday, January 23, 2019 1:46:29 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] visualising anis B factors

Thank you! I still love the BB
Eleanor

On Wed, 23 Jan 2019 at 16:24, Christian Roth 
mailto:christianroth...@gmail.com>> wrote:
In ccp4mg it is the representation tab --> Thermal ellipsoids



On Wed, Jan 23, 2019 at 4:51 PM Oliviero Carugo 
mailto:oliviero.car...@univie.ac.at>> wrote:
You might be interested in Chimera too (--> Tools --> Structure Analysis
--> Thermal ellipsoids)




On 23.01.2019 16:17, Eleanor Dodson wrote:
> Is there any easy way to do this?
>
> Coot? ccp4mg?
>
> Eleanor Dodson
>
> -
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1<https://urldefense.p

Re: [ccp4bb] Long term storage for raw images/ crystallographic data sets

[Diana Tomchick]

I hope you are compressing your images, typically that makes them 1/4 the 
original size.

SBGrid and Wladek Minor also have image archival services. As I am replying 
from my cell phone while on vacation, the links to those services are not handy 
to me. But they have been mentioned many times on this bulletin board, and 
should be easily findable by an Internet search.

Diana

**
Diana R. Tomchick
Department of Biophysics, Rm. ND10.214A
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Dallas, TX 75061 USA
214-645-6383 (office)

On Nov 29, 2018, at 3:54 PM, Lieberman, Raquel L 
mailto:raquel.lieber...@chemistry.gatech.edu>>
 wrote:

Dear All,

How do your labs handle long-term raw data backups? My lab is maxing out our 
6TB RAID backup (with two off-site mirrors) so I am investigating our next long 
term solution. The vast majority of the data sets are published structures 
(i.e. processed data deposited in PDB) or redundant/unusable so immediate 
access is not anticipated, but the size of data sets is increasing quickly with 
time, so I am looking for a scalable-yet-affordable solution.

Would be grateful for input into various options, e.g. bigger HD/RAIDs, cloud 
backup, tape, anything else.

I will compile.

Thank you,

Raquel
--
Raquel L. Lieberman, Ph.D.
Professor
School of Chemistry and Biochemistry
Georgia Institute of Technology





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Re: [ccp4bb] suggestions for cryoprotectant

[Diana Tomchick]

Condition #3 isn’t far from the saturated conditions for lithium sulfate. 
Perhaps slightly increasing the lithium sulfate concentration will work, or 
using a related lithium salt.

See

Cryosalts: suppression of ice formation in macromolecular crystallography
Acta Cryst D
Volume 56| Part 
8| August 
2000| Pages 996-1001
doi:10.1107/S090744497587

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Oct 19, 2018, at 4:57 PM, Firdous Tarique 
mailto:kahkashantari...@gmail.com>> wrote:

Dear members

I have got beautiful crystal hits in SaltRx screens which are not diffracting 
to a good resoultion. All of them are salt based condition and I am not able to 
formulate a good cryoprotectant for these crystals. I also think that in my 
case the poor resolution is due to a poor cryoprotectant selection.

The conditions are as follows:

1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0
2>0.5M KCN 100mM Tris pH8.5
3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0
4>4M Sodium Nitrate 100mM Tris pH8.5
5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6

There are few more conditions but so far not able to see good diffraction with 
using lower peg and glycerol based cryoprotectants.

Can anybody suggest me good cryos conditions for salt based crystallization 
conditions or anything good for SaltRx crystallization hits.

Thanks

Firdous



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Re: [ccp4bb] Literature on crystallization screen ingredients not showing up in crystal structure

[Diana Tomchick]

Not being in a model does not show that it's really not there.

This is especially true when considering crystals that diffract to rather low 
resolution (~2.8 - 3.5+ Å).

You shouldn’t expect in that case to uniquely identify very many buffer 
components.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Sep 11, 2018, at 7:14 AM, Robbie Joosten 
mailto:robbie_joos...@hotmail.com>> wrote:

Hi Tobias,

I don't know of any specific claims made about this but you'd better check the 
data yourself if you find an example. A nice paper by Bill Hunter and his team 
(http://scripts.iucr.org/cgi-bin/paper?S1744309111005835) showed a case where 
Co2+ was an essential ion for crystallisation , but it was overlooked by the 
original depositors of the model. Not being in a model does not show that it's 
really not there.

Cheers,
Robbie

-Original Message-
From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Tobias
Beck
Sent: Tuesday, September 11, 2018 13:42
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Literature on crystallization screen ingredients not
showing up in crystal structure

Thanks for the replies I have received so far (on and off list).

I would like to emphasize an important aspect: What about crystallization
trials where the ingredients do not show up, but cannot be omitted for the
crystallization, e.g. buffers to control pH, as mentioned in one response - I
am also thinking of specific ions, that are required, but are disordered - any
general reference for this?


Can't you just point out that crystallization is a purification method
Yes, that would be one way to put it.

Thanks, Tobias.



On Tue, Sep 11, 2018 at 12:38 PM Patrick Shaw Stewart
mailto:patr...@douglas.co.uk> 
 > wrote:



Can't you just point out that crystallization is a purification method?
E.g. look at the number of proteins in the Sigma catalogue that have been
purified by crystallization.  Clearly crystallization often excludes other 
solutes
in the mix.

Best wishes, Patrick


On Tue, 11 Sep 2018 at 08:41, Tobias Beck 
mailto:tobiasb...@gmail.com>
 > wrote:


Dear all,

I am looking for some general references regarding the fact
that for a crystallization condition not all ingredients of the crystallization
cocktail will show up in the crystal structure. Ions could be disordered,
buffer components located in the solvent regions, etc.


I think this is rather common knowledge, but maybe there is,
especially regarding protein crystallization, a more general reference (apart
from text books on solubility) for this and I am just not using the right search
terms...


Thank you!

Best, Tobias.

--

___

Dr. Tobias Beck
- independent group leader -
RWTH Aachen University
Institute of Inorganic Chemistry
Landoltweg 1, office: 304N
52056 Aachen, Germany
phone:  +49-241-80-90057
fax:   +49-241-80-99003
web:  http://www.ac.rwth-aachen.de/extern/beck/
___






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 Douglas
Instruments Ltd.
 Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
 Directors: Peter Baldock, Patrick Shaw Stewart

 http://www.douglas.co.uk
 Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034
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--

___

Dr. Tobias Beck
- independent group leader -
RWTH Aachen University
Institute of Inorganic Chemistry
Landoltweg 1, office: 304N
52056 Aachen, Germany
phone:  +49-241-80-90057
fax:   +49-241-80-99003
web:  http://www.ac.rwth-aachen.de/extern/beck/
___






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Re: [ccp4bb] crystals that dont diffract :( :(

[Diana Tomchick]

May I add the comment that despite the fact that it would be great if all of 
the samples that I worked with would be stable at room temperature for 3 weeks, 
if I had applied that as a criterion for crystallization screening and 
subsequent structure determination, then my publication record would be about 
1/5 the current length. Most of the really interesting and high-impact 
structures that I’ve worked on over the last 18 years were not that stable, and 
several were ones that were unstable enough that I couldn’t allow the 
crystallization setups to sit for longer than one week without seeing 
degradation of the diffraction pattern.

When you notice this phenomenon, it focuses the mind to search for the most 
optimum conditions for rapid, quality crystal growth. It also relieves one of 
having to wait weeks to months for the best diffracting crystals. You take what 
you can get, and if 3.5 Å is the best you’ll see but it answers your biological 
questions, you’ll be happy with what you have.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Aug 26, 2018, at 1:39 PM, James Holton 
mailto:jmhol...@slac.stanford.edu>> wrote:


An excellent review on improving diffraction that has not been mentioned yet 
is: https://doi.org/10.1107/S0907444905032130

As for how often it happens?  At my beamline we do see this fairly 
infrequently, but often enough that it no longer surprises me. I suppose that 
brings some comfort to the user, but not a lot.

Something important to do in these cases is take an exposure with 180 deg of 
rotation or more on a single image.  This is because you might have a salt 
crystal and happen to have an orientation where no hkls are on the Ewald 
sphere.  Doing the wide sweep will make sure any salt reflections are observed. 
 If you see a beautiful, symmetric pattern of very bright spots, but none 
anywhere near the beamstop, then you've got salt.  Mind you some salts have 
unit cells as long as 10 A or more, but again the wide sweep gives you an upper 
limit on the unit cell size and therefore the unit cell volume and molecular 
weight.  Disappointing to be sure, but better to know that try to optimize it.  
So, in a way, doing a 180-deg shot and still seeing no spots at all is a good 
sign.  Means you have a protein crystal.

Of course, it is possible your 0,0,1 reflections are there and you just can't 
seem them because of the beamstop.  But making the beamstop smaller is probably 
not going to make you any happier.

As for how a protein crystal can not diffract at all?  Yes, it is difficult to 
imagine how a crystal lattice can even exist if the "atomic displacements" are 
so large as to extinguish even the lowest-order reflections.  Those 
"displacements" need to be at least as big as a unit cell or larger to do that. 
 However, for visible light (5000 A wavelength) the unit cell of a protein 
crystal is not very big at all, and movements of a unit cell or more are still 
not enough to perturb an optical photon enough to make the crystal start to 
turn brown from all the little micro-cracks.  So, it is possible to have no 
order in the x-ray range and optical clarity in the visible.  That said, it is 
still hard to imagine how a crystal could _grow_ this way.  It is probably 
something that happens after the lattice forms.  And that is good news.

How does it get that way?  Yes, sitting on the bench for 3 weeks might be why 
your protein is denaturing in the lattice.  Remember, there is nothing magical 
about a crystal lattice.  Yes, they tend to hold things in place, but if your 
protein falls apart after sitting in a tube on the bench after 3 weeks then it 
is not surprising that it might do the same inside a crystal.  One of my user 
labs actually does this as a pre-crystallization assay: leave it out on the 
bench for a few weeks, and then run a gel filtration column again and see if 
its still good.  If not, then you need to work on stability before setting up 
trays.

So, the short answer is that optimization is key.  It is very rare in 
crystallography to get away with not having to do any optimization at all, and 
in those situations you should really worry because your competitor is also 
having an easy time.  It is when things don't go well that being a 
critically-thinking scientist is an advantage.  And that, I hope, is also good 
news.

Good luck!

-James Holton
MAD Scientist


On 8/14/2018 2:58 AM, Careina Edgooms wrote:
I got the most beautiful crystals I have ever seen and they don't diffract at 
all. Not poor diffraction, NO diffraction. Anyone know why this could be and 
how I can go about fixing it? I had three beautiful crystals and not one 
diffracted. I 

Re: [ccp4bb] Structure solution - hexapeptide

[Diana Tomchick]

I’ll second the use of ACORN. I was able to solve an RNA duplex structure with 
1.4 Å data using the program, and it only took about 20 minutes of CPU time on 
an old (~5 years) Mac Pro.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Aug 2, 2018, at 9:00 AM, Mark J. van Raaij 
mailto:mjvanra...@cnb.csic.es>> wrote:


When I was at Santiago de Composrela Univ., we have had success with the CCP4 
program ACORN even at around 1.2 Å resolution. Also peptides, no heavy atoms.
Didn't do this myself though I have to admit, but could put you in contact with 
the people who did.

Mark J van Raaij
CNB-CSIC
wwwuser.csic.es/~mjvanraaij


Mark J van Raaij
CNB-CSIC
wwwuser.csic.es/~mjvanraaij

 Original message 
From: Kristof Van Hecke 
mailto:kristofrg.vanhe...@gmail.com>>
Date: 02/08/2018 14:53 (GMT+01:00)
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Structure solution - hexapeptide

Dear all,

I’m trying to solve a structure of a (modified) hexapeptide:
- inhouse (very decent) data up to 0.8 Angstrom
- average redundancy = 10
- according to the Matthews coefficient of 1.88 with 34.77 %solvent, there 
should be 3 Nmol/asym
- ‘large’ unit cell of about a=54, b=54, c=12
- SG = P3(1)12 or P3(2)12

As there’s (presumably) only C, H, N and O in the structure, I’m not able to 
solve this via Direct Methods, Charge Flipping etc,.
Trying MR (with Phaser) doesn’t give any results either, as there’s hardly any 
homologous models


Has anyone encountered a similar problem please, and could provide any possible 
solutions?
(building in heavy atoms isn’t my first option at the moment,. )


Thank you very much

Regards

Kristof


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Re: [ccp4bb] teaching question: graphics for chromebook?

[Diana Tomchick]

If the chromebook has a USB port, and if the laptop can be booted using an 
external drive, you could boot the laptop with a Linux operating system and the 
educational version of PyMOL installed on a USB stick.

Or you could just pay the $269/year for the Laboratory & Classroom 
subscription, which would take you less time and hassle.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On May 23, 2018, at 3:01 PM, Phoebe A. Rice 
> wrote:

One for fellow educators:
For years, I’ve given students pymol-heavy homework assignments.  Today I found 
out that at least one has a chromebook and can’t install Pymol.
I gather that these chrome thingies are becoming more and more popular with 
students, so I’m wondering if someone out there already come up with a good 
solution – either an easy way to install pymol, or an alternative 
undergraduate-user-friendly, free macromolecular graphic program that will work 
for chromebook users.
  Thanks,
  Phoebe

~~~
Phoebe A. Rice
Dept. of Biochem & Mol. Biol. and
  Committee on Microbiology
https://voices.uchicago.edu/phoebericelab/




UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] Difficult experimental phasing

[Diana Tomchick]

Estimating isomorphism by eyeballing the difference in unit cell lengths may 
mask significant non-isomorphism that is present. Iodine is a large atom and it 
is notoriously difficult to get an iodine derivative that is isomorphous to a 
native protein.

Depending upon the chemistry of your mother liquor, it is very possible that 
you no longer have a significant amount of I3C around, so not seeing a triangle 
of iodines does not mean that your data is worthless.

Instead, I would try MR-SAD again with Phaser, and try on individual and merged 
datasets. If you are merging datasets from multiple crystals, this might be 
problematic if the level of heavy atom substitution is different between 
crystals. You can also try as input the heavy atom solution from HySS or SHELX. 
Try to limit the resolution of your heavy atom search to the resolution of 
measurability for the anomalous signal.

Iodine soaks, especially quick soaks, might not be the best HA derivative in 
your particular case. Try tantalum bromide, that cluster is less likely to 
degrade and will give a stronger signal at lower resolution.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Apr 30, 2018, at 11:28 AM, Abris Bendes  wrote:

Dear all,

I’m trying to solve the structure of a putative homodimer, but despite having 
collected good quality data, it has proven to be rather difficult.

I have collected native and derivative datasets (2.2-4Å, C2 SG, 
165/35/115Å//104° unit cell) with acceptable merging statistics. Xtriage, 
Aimless does not report any apparent twinning, tNCS or significant anisotropy. 
Based on Matthews coefficient, two monomers should be present in the ASU. 
Self-rotation function doesn’t give a clear idea on NCS, I see a few weak peaks 
at Chi=180°. Crystal was checked against contaminant with SDS-PAGE, MS and 
ContaMiner.

MR approaches so far have failed due to low sequence homology (~20%). I have 
tried MoRDa, MrBUMP, Balbes or ensembles of different models as homodimer, 
monomer or divided into smaller domains. I can place the dimer or a monomer 
with high TFZ score (6-30), but with smaller domains MR usually thrashes. The 
solution always has a rather high R factor (over 0.5), unclear electron density 
and does not improve on subsequent intensive rebuilding. Trials in P1 (or in 
other possible spacegroups) have failed too.

I have recently collected multiple good datasets from a single I3C soaked 
crystal with measurability extending to 4-7Å on individual datasets or 3-4Å on 
merged multi-dataset data. No apparent radiation damage or other pathologies 
present. Moderate isomorphism with native data (<0.4% unit cell difference).

Any attempt to solve the substructure or get interpretable maps has so far 
failed even with exhaustive SHELX, CRANK2, HySS, Autosol, Phaser or 
AutoRickshaw runs. MR-SAD or SIRAS approaches have so far failed too. Despite 
promising HySS solutions (CC>0.35) I can´t see the expected triangle 
arrangement of the I atoms, can’t discern the hands and it always result in 
uninterpretable low FOM maps.

I have a few weaker datasets (not as isomorphous though) with different HA 
soaks (signal up to 7Å), so I could try MIR or multi-crystal averaging, though 
I have no experience with these approaches.

I can provide more details if needed and I would appreciate any kind of help 
with my problem.

Thank you very much.

Best regards,
Abris




UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] Rfree going up after refinement

[Diana Tomchick]

?Hi,


I have seen this type of behavior happen often, albeit in phasing runs with 
Buccanneer and/or Phenix. Check the model statistics immediately after phasing, 
and compare them to the model statistics after refinement in Refmac. When I 
have observed this phenomenon, it's likely due to the fact that the model 
statistics after phasing are not great--poor Ramachandran and bond and angle 
statistics, and places with poor fit to the electron density. After a round of 
positional refinement, it's not uncommon to see the model geometry improve and 
then the R-free go up.


It doesn't have to mean that your phasing model is incorrect, it just means you 
need to inspect your electron density. Maybe you just need to fix the obvious 
problems with side chains, incorrect portions of the model in loops with weak 
density, etc., to improve the model and lower the Rfree.


Diana


**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

From: CCP4 bulletin board  on behalf of Eleanor Dodson 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>
Sent: Saturday, April 21, 2018 1:36 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Rfree going up after refinement

I really need more information to comment properly.
However just check that you are using the same reflections for both Rfree 
calculations.

An Rfree of 26% seems low to me from a Se met phased structure at 2.1A - there 
are usually small corrections to be made, What is the Rwork?

Maybe attach the REFMAC log file?
Eleanor

On 21 April 2018 at 03:24, ??? 
<21620150150...@stu.xmu.edu.cn> wrote:

Dear all

I have got my se-phase structure with ccp4-CRANK2-SAD, and its Rfree is 0.26 
when CRANK2 output, but the Rfree is higher to 0.36 when I refine it with 
refmac or phenix-refinement. does it mean I got a wrong phase, or I didn't got 
the right parameters when refinement.BTW, the resolution is 2.1A.THANKS A LOT

Best Regards

shijun




UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] According correct space group assignment...

[Diana Tomchick]

Several years ago I solved a small RNA structure by direct methods using the 
CCP4 program ACORN, and built the model using Nautilus. This was with data to 
only 1.25 Å resolution, collected at the Se edge, so there was no significant 
anomalous signal.

I think that this worked like a charm because the crystal probably diffracted 
to around 1 Å resolution, but due to beam line hardware issues, I decided it 
wasn’t worth it to try to collect the higher resolution data.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Apr 20, 2018, at 2:35 PM, Randy Read  wrote:

I think that starting with a direct methods program like shelxt would be fun.  
If that doesn’t work, it could be interesting to try to solve it by molecular 
replacement with fragments varying from a tetraplex, a base pair or even just 
single bases.  (Assuming that Phoebe’s concern about twinning does not turn out 
to be correct…)

Best wishes,

Randy Read

-
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical ResearchTel: +44 1223 336500
Wellcome Trust/MRC Building Fax: +44 1223 336827
Hills RoadE-mail: 
rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.   
www-structmed.cimr.cam.ac.uk

> On 20 Apr 2018, at 20:09, Tim Gruene  wrote:
>
> Dear Rafal,
>
> shelxt does not require the space group, it only needs the Laue group. If it
> finds a decent solution, it'll find also the space group for you.
>
> Best,
> Tim
>
> On Friday, April 20, 2018 3:30:36 PM CEST Rafal Dolot wrote:
>> Dear CCP4BB,
>>
>> I've recently collected data for 11mer build of DNA (9xG, 2xT). XDS, and
>> DIALS gave me similar solution - SG: I2(1)2(1)2(1) or I222, with cell
>> dimension 20.65, 22.96, 43.37, 90, 90, 90, what is too small for this
>> size of the molecule. 11mer is rich in G, so we expect the G-tetraplex
>> formation. Data were collected to almost 1 A, so it should be enough for
>> trials with direct methods/ab initio solution. What I should do first to
>> find correct SG and/or cell parameters?
>>
>> Best regards,
>>
>> Rafal
>
> --
> --
> Paul Scherrer Institut
> Tim Gruene
> - persoenlich -
> OSUA/204
> CH-5232 Villigen PSI
> phone: +41 (0)56 310 5297
>
> GPG Key ID = A46BEE1A




UT Southwestern


Medical Center



The future of medicine, today.

[ccp4bb] Research Specialist/Cryo-Electron Microscopist, UT Southwestern Medical Center

[Diana Tomchick]

Research Specialist/Cryo-Electron Microscopist
A Cryo-Electron Microscopist position is available in the Structural Biology 
Laboratory at
UT Southwestern Medical Center in Dallas, Texas. The person selected for this 
position will apply single particle cryo-electron microscopy to the study of 
protein structures.

Primary Responsibilities:
·  Work with other team members on sample grid preparation and grid 
screening
·  Ensure high quality data acquisition on Talos Arctica and Titan Krios
·  Provide guidance in the use of current image processing programs and in 
structure determination and validation
·  Assist with final manuscript preparation

Required Education and Qualifications:
·  Ph.D. degree in biochemistry, biology, chemistry, physics or a related 
field
·  Expertise in the use of current cryo-electron microscopy methods
·  Good technical, communication, and teaching skills

Other Useful Skills:
·  Facility with related biophysical techniques such as cryo-EM tomography, 
X-ray crystallography, NMR, small-angle X-ray scattering, isothermal titration 
calorimetry, analytical ultracentrifugation and protein expression and 
purification is highly desirable.

UT Southwestern has recently established a new state-of-the-art cryo-electron 
microscopy facility housing a 200kV Talos Arctica for cryo-EM single particle 
screening and a 300kV Titan Krios for high-resolution structure determination. 
A sample grid lab is fully equipped with two Vitrobot robots, a Safematic HV 
Compact Coating Unit and assorted instruments for sample preparation. We are 
seeking a motivated individual to join a team of scientists in the Structural 
Biology Laboratory at UT Southwestern and participate in the research projects 
that utilize single particle cryo-electron microscopy technology for protein 
structure determination.

About the UT Southwestern Structural Biology Laboratory
The Structural Biology Laboratory (Diana Tomchick, Director) is a platform that 
allows researchers with different expertise to come together in a collaborative 
setting and tackle biological problems in a comprehensive way. Our publications 
are representative of the success of this strategy: 
http://www3.utsouthwestern.edu/sbl/Publications.html
This position will be filled at the Research Specialist level with a salary 
commensurate with qualifications and experience.   The Research Specialist will 
be employed by the Howard Hughes Medical Institute (HHMI), which collaborates 
with UT Southwestern in conducting advanced biomedical research.
To Apply: please submit an application and provide a curriculum vitae, a brief 
statement of research and technical experience, and a list of three references 
at www.hhmi.org/Careers.  HHMI is an Equal Opportunity Employer.  Women, 
minorities, veterans and individuals with disabilities are encouraged to apply.

For further inquiries, please contact:

Diana Tomchick, PhD
Director, Structural Biology Laboratory
Department of Biophysics, Rm. ND10.214A
UT Southwestern Medical Center
5323 Harry Hines Blvd
Dallas TX 75390-8816
Tel. +1 214 645 6383
Email: 
diana.tomch...@utsouthwestern.edu<mailto:diana.tomch...@utsouthwestern.edu>

Youxing Jiang, PhD
Professor of Physiology and Howard Hughes Medical Institute Investigator
Department of Physiology, Rm. ND13.300A
UT Southwestern Medical Center
5323 Harry Hines Blvd
Dallas TX 75390-
Tel. +1 214 645 6027
Email: youxing.ji...@utsouthwestern.edu
​
**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)




UT Southwestern


Medical Center



The future of medicine, today.

[ccp4bb] Research Specialist/Cryo-Electron Microscopist, UT Southwestern Medical Center

[Diana Tomchick]

Research Specialist/Cryo-Electron Microscopist

A Cryo-Electron Microscopist position is available in the Structural Biology 
Laboratory at

UT Southwestern Medical Center in Dallas, Texas. The person selected for this 
position will apply single particle cryo-electron microscopy to the study of 
protein structures.



Primary Responsibilities:

·  Work with other team members on sample grid preparation and grid 
screening

·  Ensure high quality data acquisition on Talos Arctica and Titan Krios

·  Provide guidance in the use of current image processing programs and in 
structure determination and validation

·  Assist with final manuscript preparation

Required Education and Qualifications:

·  Ph.D. degree in biochemistry, biology, chemistry, physics or a related 
field

·  Expertise in the use of current cryo-electron microscopy methods

·  Good technical, communication, and teaching skills

Other Useful Skills:

·  Facility with related biophysical techniques such as cryo-EM tomography, 
X-ray crystallography, NMR, small-angle X-ray scattering, isothermal titration 
calorimetry, analytical ultracentrifugation and protein expression and 
purification is highly desirable.

UT Southwestern has recently established a new state-of-the-art cryo-electron 
microscopy facility housing a 200kV Talos Arctica for cryo-EM single particle 
screening and a 300kV Titan Krios for high-resolution structure determination. 
A sample grid lab is fully equipped with two Vitrobot robots, a Safematic HV 
Compact Coating Unit and assorted instruments for sample preparation. We are 
seeking a motivated individual to join a team of scientists in the Structural 
Biology Laboratory at UT Southwestern and participate in the research projects 
that utilize single particle cryo-electron microscopy technology for protein 
structure determination.

About the UT Southwestern Structural Biology Laboratory

The Structural Biology Laboratory (Diana Tomchick, Director) is a platform that 
allows researchers with different expertise to come together in a collaborative 
setting and tackle biological problems in a comprehensive way. Our publications 
are representative of the success of this strategy: 
http://www3.utsouthwestern.edu/sbl/Publications.html

This position will be filled at the Research Specialist level with a salary 
commensurate with qualifications and experience.   The Research Specialist will 
be employed by the Howard Hughes Medical Institute (HHMI), which collaborates 
with UT Southwestern in conducting advanced biomedical research.

To Apply: please submit an application and provide a curriculum vitae, a brief 
statement of research and technical experience, and a list of three references 
at www.hhmi.org/Careers<http://www.hhmi.org/Careers>.  HHMI is an Equal 
Opportunity Employer.  Women, minorities, veterans and individuals with 
disabilities are encouraged to apply.

For further inquiries, please contact:

Diana Tomchick, PhD

Director, Structural Biology Laboratory

Department of Biophysics, Rm. ND10.214A

UT Southwestern Medical Center

5323 Harry Hines Blvd

Dallas TX 75390-8816

Tel. +1 214 645 6383

Email: 
diana.tomch...@utsouthwestern.edu<mailto:diana.tomch...@utsouthwestern.edu>



Youxing Jiang, PhD

Professor of Physiology and Howard Hughes Medical Institute Investigator

Department of Physiology, Rm. ND13.300A

UT Southwestern Medical Center

5323 Harry Hines Blvd

Dallas TX 75390-

Tel. +1 214 645 6027

Email: youxing.ji...@utsouthwestern.edu<mailto:youxing.ji...@utsouthwestern.edu>

?

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)



UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] A question related to Fe-S proteins

[Diana Tomchick]

Is it possible that you have a case of domain swapping that causes the trimeric 
assembly?

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Mar 19, 2018, at 3:07 PM, PULSARSTRIAN 
> wrote:

Dear all,
Sorry for the slightly off-topic question.
   I am working on a non-native, de novo [4Fe-4S] protein, 
designed as a four-helix bundle. The in vitro reconstituted protein assembles 
with [4Fe-4S] (confirmed by EPR) and exists in monomer-dimer configuration 
(confirmed by SEC). These results have been already published.
   Recently we could get the [4Fe-4S] assembly directly from the E. 
coli (in vivo assembly). Everything is as expected (compared to reconstituted 
protein), except the oligomerization state. The protein assembles as trimer, in 
contrast to monomer-dimer configuration of the reconstituted protein. The 
trimeric nature of the in vivo assembled protein has been confirmed by SEC, 
SEC-SLS and SAXS.

 So, my question is, has anyone encountered such situation, where 
the As-purified Fe-S protein having a completely different oligomeric state 
compared to the in vitro reconstitution protein?

Looking forward to hearing for some examples and/or references.

Regards,
Bhanu




UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] protein quasicrystals?

[Diana Tomchick]

Some of the high intensity, high resolution spots are possibly from ice.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Feb 13, 2018, at 3:09 PM, Keller, Jacob 
> wrote:

I would say definitely not a quasicrystal, just multiple crystals, but the 
spots look like salt spots to me, being very sharp and intense. The puzzling 
thing is that the low-resolution zone is so populated, which would argue a bit 
against salt.

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: yu@sanofi.com [mailto:yu@sanofi.com]
Sent: Tuesday, February 13, 2018 4:03 PM
To: Keller, Jacob >; 
CCP4BB@JISCMAIL.AC.UK
Subject: RE: [ccp4bb] protein quasicrystals?

As asked by a few people, here are the images of crystals and diffraction.

Thanks,
Yu

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, 
Jacob
Sent: Tuesday, February 13, 2018 4:00 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] Re: [ccp4bb] protein quasicrystals?

I also would love to see an image….

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 [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of James 
Phillips
Sent: Tuesday, February 13, 2018 3:03 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] protein quasicrystals?

There are programs which are good at indexing patterns from multiply twinned 
crystals. Bruker AXS has one, to my knowledge. There may be other sources. I 
suggest you try that first before you invoke a quasicrystal explanation.




James Phillips

On Tue, Feb 13, 2018 at 12:07 PM, Takanori Nakane 
> wrote:
Hi,

"dials.reciprocal_space_viewer" is very useful to identify multiple lattices.
For quasicrystal and modulated crystals, "dials.rs_mapper" is also very
useful.

Best regards,

Takanori Nakane

> Have you tried microseeding of these sphere crystals? It may help to get
> better crystals.
>
>
> Burak
>
> 
> From: CCP4 bulletin board 
> > on behalf of Yu Qiu
> >
> Sent: 13 February 2018 15:09:43
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] protein quasicrystals?
>
>
> Hi,
>
>
>
> I have been trying to crystallize a protein complex and keep getting
> sphere shape crystals. The diffraction is around 3 angstrom, but looks
> like multiple lattices. I am wondering if it could be a quasi crystal? Is
> there anyone has such experience?
>
>
>
> Thanks,
>
> Yu
>




UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] "Atomic resolution"

[Diana Tomchick]

In the “good old days” we used to refer to “atomic resolution” as higher than 
1.0 Å (or more specifically, at least 0.8 Å resolution), e.g. the resolution 
obtained by most small molecule structures, where you get at least 10 times the 
number of observables per refined parameter, and thus could do full matrix 
least squares refinement of your model (including anisotropic thermal parameter 
refinement on all non-hydrogen atoms).

But then, I guess that just shows my age,

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Jan 11, 2018, at 1:59 PM, Thomas Edwards 
> wrote:

Dear Jacob,

Ah... this old chestnut!

Current EM people say that they are at atomic resolution because they are 
building atomic models (naive??).

I have been criticised in the past for using the term with say 2.2A diffraction 
data. By co-authors and reviewers alike. When I was young and naive.

My (current) definition would be yours - visible with data.
I think 1.5A is about right for X-ray. Maybe higher res?

I’m sure there are lots of rigorous ways to think. I probably haven’t taken 
that route. However, I think it is a semantic problem that might benefit from 
some disambiguation rather than rigour.

It depends why you are asking the question...

Sorry..!

Ed is: Out and about...
Sent from iPhone6sPlus.

On 11 Jan 2018, at 19:31, Keller, Jacob 
> wrote:

Dear Crystallographers,

Has there been a consensus as to what is meant by “atomic resolution?” Seems 
like the term is taken by various practitioners to mean different things.

A related question: at what resolution are atoms “visible” using only the data? 
I have an empirical feeling that this would be around 1.5 Ang Bragg spacings, 
but on the other hand, one can contour up most maps and see individual atom 
peaks. I would be interested to hear a more rigorous way to think about this.

All the best,

Jacob Keller

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

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.




UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] Na-Binding Protein?

[Diana Tomchick]

The human branched-chain alpha-ketoacid dehydrogenase requires a K+ ion to 
stabilize the binding of the cofactor thiamine diphosphate and to achieve 
maximum catalytic efficiency. See

https://www.ncbi.nlm.nih.gov/pubmed/10745006

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Jan 9, 2018, at 8:42 AM, Keller, Jacob 
> wrote:

Dear Crystallographers,

Is anyone aware of a soluble protein which changes large-scale conformation +/- 
Na+, and is specific for Na+ per se, or at least ignores K+ and Ca++? E.g., Rb+ 
or Li+ might be okay. Structural info would be a plus, but not a sine qua non.

Similarly, what about with K+ or Cl- specificities, but oblivious to similar 
common ions?

All the best,

Jacob Keller

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

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.




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Medical Center



The future of medicine, today.

Re: [ccp4bb] Overlapping ligand electron density

[Diana Tomchick]

Matt,

Modeling two molecules that occupy overlapping binding sites in a structure 
simply involves designating them as alternate conformers, with the same chain 
and residue number, and an occupancy that sums to 1.0. For example, if you have 
an AMP and an ADP that occupy the same binding site, you would define them as

AAMP B 501
BADP B 501

and initially set the occupancies for the atoms in each conformer to the ratio 
(50:50, 30:70, etc.) that you observe in the density.

Refinement in this manner is straightforward in PHENIX.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Dec 13, 2017, at 1:11 PM, Matthew Bratkowski 
> wrote:

Hello all,

I am working on a ligand binds near the active site of the protein, such that 
part of the ligand would clash with part of the natural substrate.  I recently 
co-crystallized the enzyme with both molecules and solved the crystal structure 
to high resolution (around 1.4 angstrom).  Surprisingly, the structure appears 
to contain both molecules.  A few atoms from both molecules are located only 
~1.4 A apart and are clashing (although not overlapping).  The electron density 
between them looks connected, but based on the two groups that are clashing (a 
methyl group and a carbonyl oxygen), I do not think that a covalent adduct 
occurs.  I had a few questions.

1) My guess is that the crystal is "sampling" two different conformational 
states and that both are visible due to the high diffraction resolution.  The 
substrate contains a ring that shows a characteristic "hole" in the electron 
density and binds in the exact substrate binding site, suggesting that it is 
not a different molecule (no molecules with ring structures were included in 
the sample, crystallization buffer, or cry-protectant).  One of the two 
proteins in the ASU contains electron density for whole substrate, while the 
other site has only density around the ring.  However, a sizable amount of red 
FoFc density is present around the substrate, suggesting that it is only 
partially occupied.

Does this explanation seem plausible?

2) How would I go about modeling these two molecules in the structure?  Should 
I include both molecules (in their entirety) in the structure?  I suspect that 
neither the ligand nor substrate are completely occupied, so should I modify 
the occupancies to reflect this?

Thanks,
Matt




UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] new ContaMiner features

[Diana Tomchick]

It is also possible that it co-purifies via a nickel IMAC column purification 
step. The affinity of many E. coli proteins for such columns is well known.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Dec 1, 2017, at 6:31 PM, Ivan Shabalin  
wrote:

Dear All,

To add my voice to those who wrote crystallization artifacts happen - we just 
witnessed one today. A postdoc from another lab tried crystallizing a protein 
for months. Today, during data collection from his poorly diffracting crystals, 
one of our guys (Dr. Porebski) scaled the data and checked if there is a 
structure with the same Sp Gr and unit cell parameters in the PDB. And there 
was one - 4ZNZ (Escherichia coli carbonic anhydrase (YadF) in complex with Zn - 
an artifact of purification), deposited by our group two years ago. Quick 
molecular replacement showed that it is indeed the protein we had in the beam 
today.

The protein looked good on the SDS gel. The band for YadF was definitely less 
than 1% of the protein sample (less than one percent!!! if the gel would not be 
overloaded, nobody would see the band), but it still crystallized. We speculate 
that YadF from Escherichia coli was co-purified with the target protein due to 
relatively strong protein-protein interactions despite multiple purification 
steps.

We did not use ContaMiner, though. Instead, we just used the search of the unit 
cell parameters in the PDB, which is much faster (but works only if the 
structure of this particular artifact in the same SpGr is in PDB! otherwise, 
one should use ContaMiner or similar service). This feature is implemented in 
HKL3000, but it can also be done quickly on the PDB webpage. The procedure, as 
well as cases with other crystallization artifacts, is described in:

Protein purification and crystallization artifacts: The tale usually not told. 
(2016) Protein Sci. 25: 720-733

Today's example clearly shows that depositing these artifacts can greatly help 
others (it took just a few minutes to realize we had an artifact). Therefore, I 
would like to encourage everyone to deposit their artifacts to the PDB, 
especially if these artifacts were crystallized with unit cell parameters not 
reported previously. An increase in the size of the library of crystallization 
artifact structures deposited to the PDB can make troubleshooting of new 
artifacts much easier, and save much effort for those who are new (or not very 
new!) to such cases.


With best regards,
Ivan Shabalin, Ph.D.
Research Scientist,
Department of Molecular Physiology and Biological Physics,
University of Virginia,
1340 Jefferson Park Avenue, Pinn Hall,Room 4223,
Charlottesville, VA 22908

On 11/23/2017 02:35 PM, r...@mrc-lmb.cam.ac.uk wrote:
> Dear Stefan,
> Just a couple of thoughts:
> - first of all I think that Gerard is absolutely right, it would have been
> nice to raise such issues first with the developers. In my experience,
> Staraniso does a fantastic job if used correctly.
> - but if you're OK with public trials, may I ask: why on Earth would anybody
> need ContaMiner? Are you trying to offer some sort of computational cure for
> sloppy biochemistry? There is zero point in crystallizing crap samples, sorry
> to say this. In my 17 or so years in Strubi I've never heard of anybody
> crystallizing a "contaminant", being it a purification tag or whatever.
> I suppose this might have happened to somebody you know, hence the motivation
> to spend time on the bizarre ContaMiner. Which is a pity, a silly outcome
> would only teach people to do their job (or train their robots) properly.
> Best wishes,
> Radu






UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] high TFZ score but 50% of Rfree

[Diana Tomchick]

Do the following.

1) Perform density modification (NCS averaging and solvent flattening)
2) Run the new phases through a model building program (Buccaneer works for all 
resolutions, Arp/wArp works well for better than ~2.7 Å or better resolution)

If this doesn’t give you a better quality model (meaning one that has 
identifiable secondary structure and an R-free below about 45%), then you need 
to go back to square one and examine your assumptions regarding data quality, 
Laue symmetry and space group, MR solution, etc.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Nov 17, 2017, at 2:23 PM, Yue Li 
<17ef008f608c-dmarc-requ...@jiscmail.ac.uk>
 wrote:

Dear all,

I have several datasets (one best resolution reaching 2.2A), giving C2 space 
group, two molecules in an asymmetry unit (65.1% of solvent content).  When 
running MR using a template (<20% sequence identify to the target molecule), I 
got a solution with high TFZ (23.7) and LLG (842). However, the Rfree sticks to 
50% in the structural refinement using phenix. There is no complain in Xtriage 
- no twin, no translational NCS.  I think that the structure solution looks 
reasonable, which can explain the three disulfide bond formations through the 
sequence threading.  I tried to search three molecules in the asymmetry unit, 
but the final solution gives me two molecules.

Do you have any suggestion for this high Rfree problem?

Thank you very much for your help.

All the very best,

Simon




UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] High R/Rfree after MR

[Diana Tomchick]

I sent this on Friday to Gianluca but forgot to also send it to the bulletin 
board, so I’m posting it now, as it relates to Eleanor’s response.

__

It would also be worthwhile to consider the possibility of out-of-register 
errors in the sequence assignment for your model. I have seen cases at a 
similar resolution limit and anisotropic that had high free-R values due to 
this problem. A sequence assignment error in 10-15% of the the total scattering 
mass can be the difference between free R-values of 38% versus 32%, for 
example. With low resolution models, one frequently has one or two (or more) of 
what I like to refer to as “disembodied helixes”—I.e., helixes that have both 
N- and C- termini with insufficient electron density to connect them to the 
rest of the protein. These are great candidates for out-of-register errors.

I find Buccaneer to be a great program for re-building an initial MR model and 
correcting such errors. It won’t fix everything, especially if the local 
electron density is poor, but it does a surprisingly good job with maps in the 
2.8-3.6 Angstrom range. And if you’re reasonably sure of the sequence 
assignment in the majority of the structure solution, you can fix the portion 
that you think is correct and ask it to re-build the questionable part.

It’s my go-to method for these type of problematic structures,

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Oct 14, 2017, at 9:10 AM, Eleanor Dodson 
> wrote:

I would be worried by a structure with these R values, whether or not there is 
anisotropy to consider.

Several times when this has happened to me the spacegroup turns out to be wrong.

You do not give any details of the solution but especially if there is some 
non-crystallographic translation you can assign the space group wrongly - get a 
MR solution which fits some of the amplitudes perfectly ( eg h k 2l, and the 
rest approximately so the maps look reasonable.

Have you run MR in all possible spacegroup for your point group?

Eleanor

On 13 October 2017 at 22:08, Gerard Bricogne 
> wrote:
Dear Randy,

On Fri, Oct 13, 2017 at 04:11:44PM +0100, Randy Read wrote:
> Just to add to this point.  The MR algorithms in Phaser are now able
> to make better use of intensity data, which is particularly
> important when you have any very weak data.  Having weak data can’t
> be avoided when you have serious anisotropy (or tNCS or a
> combination of the two).  Unfortunately, if you use amplitudes that
> have been through the French & Wilson (truncate) algorithm, the real
> variation in intensity is partially masked because the posterior
> amplitude values are computed on the prior assumption that all the
> reflections in a resolution shell have the same underlying intensity
> distribution.

 Your "unfortunately" calsue may be the case with the UCLA server,
but your statement is not true about what the STARANISO server (or
STARANISO as invoked within autoPROC) does: as I already indicated in
a reply to you a few months ago in this BB, the version of TRUNCATE in
STARANISO applies the French & Wilson procedure with a prior Wilson
probability whose expectation value for the intensity is modulated by
the anisotropy of the dataset. This is clearly explained on the server
- see

   http://staraniso.globalphasing.org/staraniso_about.html#step16


 With best wishes,

  Gerard.

--
> The UCLA server actually uses Phaser under the hood — what they add is to 
> turn the anisotropic B-values into suggested resolution limits in the 
> different directions.  However, I don’t think they allow you yet to submit 
> intensities, which would be better.
>
> Best wishes,
>
> Randy Read
>
> -
> Randy J. Read
> Department of Haematology, University of Cambridge
> Cambridge Institute for Medical ResearchTel: +44 1223 
> 336500
> Wellcome Trust/MRC Building Fax: +44 1223 
> 336827
> Hills RoadE-mail: 
> rj...@cam.ac.uk
> Cambridge CB2 0XY, U.K.   
> www-structmed.cimr.cam.ac.uk
>
> > On 13 Oct 2017, at 10:29, vincent Chaptal 
> > > wrote:
> >
> > Dear Gia and Paul,
> >
> > about anisotropy, one point to keep in mind is that it is not necessarily 
> > linked to the difference in resolution limits.
> > In fact I am at the moment working 

Re: [ccp4bb] Fix sidechain outliers

[Diana Tomchick]

Fitmunk works pretty well in the initial stages of model fitting. You will 
still need to manually check the results of any automated model fitting program 
to see if it makes sense; your brain is still your best tool, in the final 
analysis.

Diana

> On Sep 4, 2017, at 1:29 PM, Dominika Borek  wrote:
>
> You may try Fitmunk for side chains:
>
> http://kniahini.med.virginia.edu/fitmunk/server/
>
> D.
>
>> On 2017-09-04 09:36 AM, rohit kumar wrote:
>> Dear All,
>> Could anyone help me, how I can fix my sidechain outliers or
>> clashscore in coot or in CCP4 tools.
>> Or by using any online severs.
>> Its urgent
>> Thank You in advance
>> --
>> WITH REGARDS
>> Dr. Rohit Kumar Singh
>> School of Life Sciences,
>> Jawaharlal Nehru University
>> New Delhi -110067
>
> --
> Dominika Borek, Ph.D. *** UT Southwestern Medical Center
> 5323 Harry Hines Blvd. *** Dallas, TX 75390-8816
> 214-645-9577 (phone) *** 214-645-6353 (fax)



UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] pseudo-centering or twinning problem in P3

[Diana Tomchick]

Maybe, but it may also indicate that the problem isn’t twinning, but simply 
pseudo symmetry.

Zanuda is one way to double-check, and it takes approximately a day to 
re-process the data, run the MR and run Zanuda.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Aug 8, 2017, at 1:27 PM, Eleanor Dodson 
> wrote:

That seems hard work to me! Data quality suffers when you discard multiplicity..
 Eleanor

On 8 August 2017 at 17:15, Sudipta Bhattacharyya 
>
 wrote:
Hi Giorgio,

I also suspect the presence of twining and as Eleanor has suggested the L test 
is the best test to detect that (if also t-NCS is not present). You can 
reprocess your data in P1, do MR in P1 and then feed your P1 model and mtz file 
to zanuda to see what SG it suggests. That trick once worked for me.

Cheers,
Sudipta.


Sudipta Bhattacharyya,
Postdoctoral fellow
Department of Molecular Biosciences
University of Texas at Austin
Texas, USA.


On Tue, Aug 8, 2017 at 8:27 AM, Eleanor Dodson 
> wrote:
First of all - check whether your crystal data shows twinning - thee is an 
L-test plot which usually gives a clear indication and if you are using GUI2 
the report suggests if this is so.


If you have twinning the most likely SG is P31 (or P32 ) - you cant tell the 
difference till the structure is solved.

Check the merging stats in that point group -
Then try MR searches in the two spacegroups.

The C2 and I2 SGs are probably sub-group possibilities if you reindex - there 
are common cell dimensions -pointless output tells you how to do that - but in 
general one always chooses the highest symmetry that gives reasonable merging 
statistics and is sensible.

Eleanor



On 8 August 2017 at 14:15, Giorgio Giardina 
> wrote:
Hello everybody,

I think I have a pseudo-centering problem.

I have a 1.6 ang. dataset of a mutant  protein that is an homodimer in solution.
Data processing gives the following SG:

Space group: P 31 2 1
Average unit cell:  111.36  111.36   28.56   90.00   90.00  120.00
Average mosaicity:   0.24
Rmerge Overall 0.07

However with this SG the unit Cell is too small and monomer doesn't fit the in 
the AU.

I reprocessed the data in other possible SG (including P6) and finally I got 2 
equivalent SG's in which I can get a correct Molecular Replacement solution:

Space group: I 1 2 1
Average unit cell:   57.07  111.11  192.90   90.00   90.07   90.00

and

Space group: C 1 2 1
Average unit cell:  201.10  111.11   57.07   90.00  106.42   90.00

Both with Average mosaicity:   0.38 and Rmerge Overall 0.06,
but the corresponding MR solutions do not refine, with R-factor stuck to 45-47%.

From what I understand, in the I121 SG I have the NC two-fold axis of the dimer 
at 1/2a and this originates the pseudo centering and the small P3 Cell

Largest Patterson peak with length larger than 15 Angstrom:
 Frac. coord.  :0.5000.0000.000
 Distance to origin:   28.566
 Height relative to origin :   53.830 %

I'm really not so good with symmetry, so I'll be grateful for any 
suggestion/help/solution from you out there.
Many thanks,
Giorgio







UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] refmac output

[Diana Tomchick]

Yes, I agree! This (“Please look at my structure, and here are my files from 
the last cycle of refinement") happens to me almost every week. :)

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Aug 1, 2017, at 10:48 AM, James Holton  wrote:

As someone who uses those "superfluous" columns all the time, I would like to 
chime in in favor of keeping the default output columns of refmac.  If only I 
had a nickle for every time someone asked me to "look at" a structure and only 
gave me the output files of refinement.  Kind of ties your hands.

I have always been a fan of erring on the side of providing information in 
output files.  How hard is it to delete something? How hard is it to get it 
back after you deleted it?

My two cents,

-James Holton
MAD Scientist

On 7/31/2017 8:57 AM, Edwin Pozharski wrote:
> I know space is cheap these days, but is there a reason for Refmac to 
> generate all those extra columns in the output mtz file?  Refmac (as well as 
> phenix.refine and buster-tnt) output mtz file is almost always used for only 
> one purpose - look at the map in coot.  You only need 4 columns for that, not 
> 14.  Other columns are useful for testing, but why not make them optional?
>
> This would certainly be a low priority - one can easily delete extra columns 
> using, say, sftools.
>
> Cheers,
>
> Ed.
>
> ---
> Hurry up, before we all come to our senses!
> Julien, King of Lemurs
>




UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] Buccaneer places residues in different asymmetric units

[Diana Tomchick]

Or use Coot to generate the symmetry mates, then write out the symmetry-related 
coordinates.

Many ways to achieve the same outcome,

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Jul 24, 2017, at 3:56 AM, Lingxiao Zeng 
> wrote:

Dear All,

I tried to use buccaneer to build a model. The starting model is a partial 
model, after model building the Rwork and Rfree are reasonable but buccaneer 
places residues in different asymmetric units and the model looks really weird.

Is there any way to build the model into the same ASU or put different parts 
together after model building? Thanks!



Best,

Alice

--
Lingxiao Zeng
PhD candidate
School of Biomedical Sciences
The University of Hong Kong





UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] Fine Phi Slicing

[Diana Tomchick]

And the instrument makers (e.g., Rigaku and Bruker) currently sell multi-axis 
goniostats for in-house data collection--and not just for small-molecule 
purposes.

Diana

P.S. Ron, we still call 'em goniometer heads and goniostats at UT Southwestern.

> On Jul 14, 2017, at 1:00 PM, "stenk...@u.washington.edu" 
>  wrote:
>
> I'm not understanding much of this discussion, but I've noticed the use of 
> words reminiscent of old issues concerning omega-two theta scans on 
> four-circle goniostats (see Stout and Jensen, pages 168-173, second edition).
>
> p.s.  In my upbringing, crystals were placed on goniometer heads so they 
> could be placed on goniometers or goniostats.
>
>> On Fri, 14 Jul 2017, Harry Powell wrote:
>>
>> hi folks
>> Just my two ha'porth - the small molecule crystallographers have been doing 
>> multi-orientation data collections
>> since they moved from point detectors to area detectors in the early 1990's, 
>> for the very reasons that Gerard
>> gives (their cusps are huge compared to ours...). Since they were perfectly 
>> used to using multi-axis goniostats*,
>> this wasn't a big psychological jump for them.
>> * I prefer calling the thing that the crystals sit on a "goniostat" because 
>> a "goniometer" is correctly something
>> for measuring angles (however, a "positioning goniometer" appears to be a 
>> specialised kind of goniostat);
>> wikipedia tells me that crystallographers seem to be the only group of 
>> people who confuse the two (but I didn't
>> read the article very carefully so IMWBW).
>> On 14 Jul 2017, at 15:15, Gerard Bricogne wrote:
>>  Dear Leo,
>>  What seems to have happened is that an existing thread where fine
>>  phi (actually: omega!) slicing was discussed, among many other things,
>>  digressed into a discussion of data collection protocols using more
>>  than one instrumental setting (either using a 2-theta motion of the
>>  detector, or a chi reorientation of the crystal). Briefly, my two
>>  cents on that topic: a 2-theta movement may help use different pixels
>>  on the detector, and could be valuable in filling the wide horizontal
>>  gaps on a Pilatus or Eiger, but it will leave the cusp in the same
>>  place and therefore will not fill it. Reorienting the crystal, on the
>>  other hand, can help cure all the known ills of single-sweep datasets
>>  (gaps and cusp in particular).
>>  On the matter of multi-orientation data collection, the idea and
>>  the practice go back (at least, in my memory) to Alan Wonacott, the
>>  co-creator of the Arndt-Wonacott rotation camera in the early 1970's.
>>  It was all done with gonio arcs. As each crystal had to be aligned
>>  manually in order to continue data collection where the previous one
>>  had left off, these arcs were in constant use, and there was always an
>>  extra cusp-filling collection at the end. Nowadays data collection has
>>  speeded up so much, and has become so dominated by automation, that
>>  multi-axis goniometry has been sidestepped because using it properly
>>  would have had to involve non-automated steps that are difficult to
>>  standardise (a notable exception being the protocol with 8 different
>>  values of Chi, using the PRIGo goniometer on the PX-III beamline at
>>  the SLS, that has been "instrumental" in enabling large structures to
>>  be experimentally phased by native SAD at 6keV).
>>  It is great to see that there are many developments underway in
>>  both hardware and software, leading gradually towards a reinstatement
>>  of multi-orientation data collection as an off-the-shelf option for
>>  those who are prepared to spend a bit more time to reliably get much
>>  better data. The Proxima-1 beamline scientists at SOLEIL have always
>>  been among the believers that the time would come when these efforts
>>  would bear fruit, and what my group has been able to do in this area
>>  owes a great deal to them.
>>  With best wishes,
>>   Gerard.
>>  --
>>  On Fri, Jul 14, 2017 at 01:18:35PM +, CHAVAS Leonard wrote:
>>Reading back my email, when I mentioned 'just introduced', it is 
>> not giving justice to
>>the reality and those who came up with the concept. I should have 
>> mentioned 'just
>>reminded us', as the concept has been introduced quite a long 
>> time ago and few tens of
>>communications. It is therefore a reminder that when coming to 
>> the will to collect good,
>>clean and complete data, things aren't as simple as they would 
>> seem. Automation at our
>>favourite beamlines do help by providing much more time thinking 
>> properly of the
>>necessary strategies when coming to these difficult crystals so 
>> important to our hearts.
>>Sorry again for the confusion. No 

Re: [ccp4bb] Granada Crystallization Boxes?

[Diana Tomchick]

?If it weren't for the fact that trying to 3-D print translucent plastic is 
still problematic, you would think that this would be a perfect application of 
that technology.


Diana


**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

From: CCP4 bulletin board  on behalf of Gloria Borgstahl 

Sent: Thursday, July 13, 2017 7:56 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Granada Crystallization Boxes?

We probably would be interested in used ones.

On Thu, Jul 13, 2017 at 4:18 AM, Patrick Shaw Stewart 
> wrote:

Gloria, would you be interested in used ones?  I don't actually have any - we 
threw them out a few months ago, I've just checked - but someone might have 
some.

Best wishes, Patrick


On 11 July 2017 at 19:04, Gloria Borgstahl 
> wrote:
I have recently found out that these are no longer being manufactured or sold 
commercially.  But, as fortune has it, we have just been funded to fly some 
large quartz capillaries crystallization experimente up to the International 
Space Station for neutron crystallography.  Our experimental design is to fly 
the experiments in the Granada Crystallation Boxes!  NASA has already approved 
the 3x10x0.5 cm plastic Granada boxes for flights.   Does anyone have any in 
their lab supplies that they do not plan to use?  We would be willing to buy 
them from you!  Thanks, Gloria



--
 patr...@douglas.co.ukDouglas Instruments Ltd.
 Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
 Directors: Peter Baldock, Patrick Shaw Stewart

 http://www.douglas.co.uk
 Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034
 Regd. England 2177994, VAT Reg. GB 480 7371 36




UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] Granada Crystallization Boxes?

[Diana Tomchick]

Sorry, that’s Triana Science & Technology (darn automatic spell-check!).

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu<mailto:diana.tomch...@utsouthwestern.edu>
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Jul 11, 2017, at 1:39 PM, Diana Tomchick 
<diana.tomch...@utsouthwestern.edu<mailto:diana.tomch...@utsouthwestern.edu>> 
wrote:

You can continue to buy new ones from Tirana Science & Technology, which is a 
Spanish company.

http://www.trianatech.com/index.php?option=com_content=article=65=88=en

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu<mailto:diana.tomch...@utsouthwestern.edu>
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Jul 11, 2017, at 1:04 PM, Gloria Borgstahl 
<gborgst...@gmail.com<mailto:gborgst...@gmail.com>> wrote:

I have recently found out that these are no longer being manufactured or sold 
commercially.  But, as fortune has it, we have just been funded to fly some 
large quartz capillaries crystallization experimente up to the International 
Space Station for neutron crystallography.  Our experimental design is to fly 
the experiments in the Granada Crystallation Boxes!  NASA has already approved 
the 3x10x0.5 cm plastic Granada boxes for flights.   Does anyone have any in 
their lab supplies that they do not plan to use?  We would be willing to buy 
them from you!  Thanks, Gloria




UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] Granada Crystallization Boxes?

[Diana Tomchick]

You can continue to buy new ones from Tirana Science & Technology, which is a 
Spanish company.

http://www.trianatech.com/index.php?option=com_content=article=65=88=en

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Jul 11, 2017, at 1:04 PM, Gloria Borgstahl 
> wrote:

I have recently found out that these are no longer being manufactured or sold 
commercially.  But, as fortune has it, we have just been funded to fly some 
large quartz capillaries crystallization experimente up to the International 
Space Station for neutron crystallography.  Our experimental design is to fly 
the experiments in the Granada Crystallation Boxes!  NASA has already approved 
the 3x10x0.5 cm plastic Granada boxes for flights.   Does anyone have any in 
their lab supplies that they do not plan to use?  We would be willing to buy 
them from you!  Thanks, Gloria




UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] Protein or DNA crystals

[Diana Tomchick]

It’s been my experience that protein crystals with that high a percentage of 
tryptophan residues (>2%) should give a very clearly positive result from a UV 
microscope such as a Jansi UVEX.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Jun 19, 2017, at 9:20 AM, Joseph Ho  wrote:

Dear all:

I would like to seek your opinion on our crystal hits. We are working
on protein/dsDNA complex. By changing different protein and DNA
(14-22bp) constructs, we recently got some hits from commercial
screens using sitting drop vapor diffusion (very small xtals). The
precipitant is PEG and the picture of crystals are attached. In this
particular condition, it is 30%PEG3350, sodium succinate pH5.5 and
100mM NaCl. The crystal seems floating and sit in the bottom. We do
some test shot from other conditions and it is not salt crystals. The
crystals can suck in izit dye.  I do some google and it seems izit dye
also turns dsDNA crystal into blue. We also do UV/Vis microscope but
no Trp fluorescence (6 Trp in 256 aa). It may due to low Trp.

This is our first time to work on protein/DNA complex crystals and we
are not certain if this is just DNA or protein/DNA crystals. Can you
provide your comments on our hits?

Thank you for your help

Joseph





UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] preparing DL-Malic acid stock solution

[Diana Tomchick]

As the pKa1 of malic acid is 3.4, it may be that the partially neutralized 
malic acid salt is less soluble than the acid or the fully neutralized salt.

The pKa2 of malic acid is 5.1.

Contact the people at Hampton Research

https://www.hamptonresearch.com/contact_us.aspx

and ask them. They sell it as a 3.0 M solution, pH neutralized to 7.0.

It is possible that you need to fully neutralize it before it turns clear, or 
that you may also need to gently heat it.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Jun 8, 2017, at 9:54 AM, Sebastiano Pasqualato 
> wrote:


Dear all,
we’ve recently having trouble preparing a 3 M stock solution of DL-Malic Acid, 
pH 7 (which we had, so it’s doable!).
When we reach pH 3 - 4 the solution turns milky white and does not goes back to 
a clear solution even when the pH is raised.
Does anybody have any advice on how to get a clear solution? Has anyone gone 
through the same?
Thanks in advance,
ciao,
Sebastiano


--
Sebastiano Pasqualato, PhD
Crystallography Unit
Department of Experimental Oncology
European Institute of Oncology
IFOM-IEO Campus
via Adamello, 16
20139 - Milano
Italy

tel +39 02 9437 5167
fax +39 02 9437 5990
web http://is.gd/IEOXtalUnit





UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] [phenixbb] comfortable OS X level

[Diana Tomchick]

Hahahaha, that was a typo, I meant to write

10.12.5

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Jun 7, 2017, at 11:25 AM, Scott Classen <sclas...@lbl.gov> wrote:

10.2 ?
That OS is 15 years old.

> On Jun 7, 2017, at 8:27 AM, Diana Tomchick 
> <diana.tomch...@utsouthwestern.edu> wrote:
> 
> I’ve not had any problems; the current version is 10.2.5, so it’s pretty 
> stable now.
> 
> Diana
> 
> **
> Diana R. Tomchick
> Professor
> Departments of Biophysics and Biochemistry
> University of Texas Southwestern Medical Center
> 5323 Harry Hines Blvd.
> Rm. ND10.214A
> Dallas, TX 75390-8816
> diana.tomch...@utsouthwestern.edu
> (214) 645-6383 (phone)
> (214) 645-6353 (fax)
> 
> On Jun 7, 2017, at 10:14 AM, Patrick Loll <pat.l...@drexel.edu> wrote:
> 
> I’m still running Yosemite on my Macs, both because I’m change-averse and 
> because folks reported problems with some crystallographic software upon 
> upgrading the OS.
> 
> These reports have now faded into the haze of the past, and so I ask, have 
> the issues been resolved? Is it safe to move to Sierra?
> 
> Thanks as always,
> 
> Pat
> 
> ---
> Patrick J. Loll, Ph. D.
> Professor of Biochemistry & Molecular Biology
> Drexel University College of Medicine
> Room 10-102 New College Building
> 245 N. 15th St., Mailstop 497
> Philadelphia, PA  19102-1192  USA
> 
> (215) 762-7706
> pat.l...@drexelmed.edu
> 
> 
> ___
> phenixbb mailing list
> pheni...@phenix-online.org
> http://phenix-online.org/mailman/listinfo/phenixbb
> Unsubscribe: phenixbb-le...@phenix-online.org
> 
> 
> 
> 
> UT Southwestern
> 
> 
> Medical Center
> 
> 
> 
> The future of medicine, today.
> 

Re: [ccp4bb] [phenixbb] comfortable OS X level

[Diana Tomchick]

I’ve not had any problems; the current version is 10.2.5, so it’s pretty stable 
now.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Jun 7, 2017, at 10:14 AM, Patrick Loll  wrote:

I’m still running Yosemite on my Macs, both because I’m change-averse and 
because folks reported problems with some crystallographic software upon 
upgrading the OS.

These reports have now faded into the haze of the past, and so I ask, have the 
issues been resolved? Is it safe to move to Sierra?

Thanks as always,

Pat

---
Patrick J. Loll, Ph. D.
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu


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[ccp4bb] Fwd: biotech opportunity

[Diana Tomchick]

From: Steven McKnight 
>
Subject: favor
Date: June 2, 2017 at 3:21:39 PM CDT
To: Michael Rosen 
>
Cc: Joseph Ready 
>

Mike – could you share this e-mail message with late-stage graduate students 
and postdoctoral fellows working in your department.  I’d also appreciate it if 
you would share this with labs outside of your department that do X-ray 
crystallography.  Much thanks – Steve.

Dear trainees in the fields of structural biology and enzymology:

Together with Joe Ready, Andrew Pieper, Marc Freeman I have co-founded a 
biotechnology company dedicated to the discovery and development of 
neuroprotective chemicals.  The company has been funded by The Column Group, a 
top-flight venture capital firm out of San Francisco led by Dave Goeddel.  The 
company is named Neuro-protective Therapeutics and will be located in the San 
Francisco Bay Area.

We are keen to hire one or more young scientists skilled in the fields of 
enzymology, protein chemistry and X-ray crystallography.  Were we to find the 
proper candidate here at UT Southwestern Medical Center, the person could work 
closely with Drs. Ready and McKnight over the next 3-6 months before moving to 
California when the company laboratories are ready for occupancy.

Interested candidates should send an e-mail directly to me (Steve McKnight) and 
Joe Ready anytime during the month of June.  All we need is a brief, one page 
description of your research experience, a curriculum vitae, and the contact 
information of at least two faculty-level scientists familiar with your work.  
Drs. Ready and I will respond quickly to any and all inquiries – thanks for 
your consideration of this opportunity – Steve McKnight.





UT Southwestern


Medical Center



The future of medicine, today.

[ccp4bb] Research Track Assistant Professor Positions

[Diana Tomchick]

Assistant Professor, Research Track

Department of Biophysics





The Structural Biology Laboratory at UT Southwestern Medical Center currently 
has two openings at the non-tenure track Research Assistant Professor level for 
highly-motivated individuals who enjoy working in a collaborative, 
multi-disciplinary setting.



·  Position #1: Cryo-electron microscopist with extensive experience in 
high resolution single-particle reconstruction, including sample grid 
preparation, data collection, the use of modern software for particle picking 
and classification, map generation, model building and structure validation. 
Experience in cryo-EM tomography is a plus.

·  Position #2: X-ray crystallographer with extensive experience in modern 
methods of crystallization, data collection using home and synchrotron sources, 
structure solution via various experimental techniques, refinement and 
structure validation.



For both positions, a Ph.D. in biochemistry, chemistry, physics or a related 
field is required. Facility with related techniques for each position such as 
cryo-electron microscopy, X-ray crystallography, NMR, small-angle X-ray 
scattering, isothermal titration calorimetry, analytical ultracentrifugation 
and protein expression and purification is highly desirable.



Job requirements include a significant amount of both formal and informal 
teaching, and teaching experience is highly desirable. Salary is competitive 
and commensurate with experience and qualifications.



About the UT Southwestern Structural Biology Laboratory

The Structural Biology Laboratory (Diana Tomchick, Director) is a platform that 
allows researchers with different expertise to come together in a collaborative 
setting and tackle biological problems in a comprehensive way. Our publications 
are representative of the success of this strategy: 
http://www3.utsouthwestern.edu/sbl/Publications.html



State-of-the-art equipment is currently installed, operational and shared 
between the members in the Structural Biology community at UT Southwestern. 
Cryo-EM equipment consists of one 300 kV Titan Krios microscope, one 200 kV 
Talos Arctica microscope, and a cryo-Scios DualBeam for cryo-FIB milling. In 
addition, the Structural Biology Laboratory has a dedicated lab for cryo-EM 
grid preparation, with an FEI Vitrobot and assorted instruments for sample 
prep. Computational facilities include a campus high-performance computing 
cluster. Crystallographic equipment includes an Art Robbins Gryphon 
crystallization robot and a Scorpion liquid handling robot, a Jansi UVEX UV 
microscope, a Rigaku FRE superbright rotating anode X-ray generator with an 
ACTOR sample mounting robot, and 30 days of guaranteed synchrotron beam time at 
Sector 19 of the Advanced Photon Source.



These facilities are supplemented by a variety of biophysical instruments 
supporting the study of macromolecules using NMR, CD, dynamic light scattering, 
analytical ultracentrifugation, stopped-flow kinetics, isothermal titration 
calorimetry, microscale thermophoresis and mass spectrometry.



Applicants should submit a curriculum vita and three letters of reference by 
July 10, 2017 to Diana Tomchick, Chair, Structural Biology Laboratory Search 
Committee, U.T. Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 
75390-8816.  UT Southwestern is an Affirmative Action/Equal Opportunity 
Employer.  Women, minorities, veterans and individuals with disabilities are 
encouraged to apply.



For further inquiries, please contact:

Diana Tomchick

Department of Biophysics, Rm. ND10.214A

UT Southwestern Medical Center

5323 Harry Hines Blvd

Dallas TX 75390-8816

Tel. +1 214 645 6383

Email: 
diana.tomch...@utsouthwestern.edu<mailto:diana.tomch...@utsouthwestern.edu>



?

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)



UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] Control the crystallization process in the presence of small volatile organic molecules

[Diana Tomchick]

 Another thing to try when cryoprotecting any crystals grown at room temp using 
a volatile precipitant is to first transfer the crystal tray to 4 degrees, then 
quickly add a cold aliquot of a viscous cryoprotectant to the drop. Evaporation 
is considerably slower at 4 degrees, and I prefer to use glycerol as 
cryoprotectant because it significantly slows down the random walk of the 
crystals through the drop. It makes it so much easier to fish crystals out of 
the drop if they're not moving at lightning speed...

Place the tray at 4 degrees about 20-30 minutes prior to opening the drop. Any 
longer incubation at lower temp can result in extra nucleation or crystal 
dissolution.

BTW, good luck trying to use a really volatile organic as a cryoprotectant. 
Every few years I try it and the crystals just get blown out of the cryoloop by 
the nitrogen gas stream.

Diana

On Jan 24, 2014, at 5:31 PM, Mark van der Woerd 
mjvdwo...@netscape.netmailto:mjvdwo...@netscape.net wrote:

Chen,

Dioxane is not easy to work with, exactly for the reasons you describe.

There is one thing you did not mention, which I know to be an additional issue: 
the quality of the dioxane. I do not know if you need good quality (whatever 
that is) but it is a fact that crystallization works with dioxane from some 
manufacturer/lots and not with others. I have never figured out why this is so.

For a paper on kinetics and reservoir volume discussion, I would read the work 
by Forsythe et al 
(http://journals.iucr.org/d/issues/2002/10/01/ic0013/vidsup.html, 
doi:10.1107/S0907444902014208)http://dx.doi.org/10.1107/S0907444902014208
Basically you will find that this paper says that well volume does not matter 
much. Content of your drop matters a lot. In your case, having a very volatile 
component, equilibration would be really fast.

One way to get away from all that is to do batch experiments, where there is no 
active transport from a drop into a well. But your question remains very good: 
you need an oil (?) that will not permit water or a volatile organic 
substance to escape. I am not sure what would do the trick.

Dioxane is a decent cryo-protectant by itself. I would think about adding 
gooey things like low molecular weight PEG to try to get the drops better 
behaved. Evaporation of the dioxane will still be an issue, but with a higher 
viscosity you may not have to chase your crystals through the drop.

Hope this helps a little.

Mark



-Original Message-
From: Chen Zhao c.z...@yale.edumailto:c.z...@yale.edu
To: CCP4BB CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Sent: Wed, Jan 22, 2014 11:00 pm
Subject: [ccp4bb] Control the crystallization process in the presence of small 
volatile organic molecules

Dear all,

I am now optimizing a hit which contains about 30% 1,4-dioxane using 
hanging-drop vapor diffusion at 25 degree. I am having a hard time to reproduce 
the results: most of the times the drops are either dry in one day or full of 
precipitate, and only occasionally could I get small crystals. Is there a way 
to control the vapor diffusion process, like using oil to seal the reservoir? 
(I know paraffin is permeable to dioxane) Also if someone could refer me to 
studies on the effects of reservoir volume and surface area to the 
crystallization kinetics, that would be very helpful.

I am also seeking for recommendations for freezing crystals in this condition. 
What kind of cryoprotectant has a higher chance? Another problem is that when I 
tried to freeze the crystals, the drop dries super rapidly, and the crystals 
will dissolve if I add reservoir buffer. But I would assume good cryoprotectant 
could do the job. On the other hand, this points back to my previous question 
on dioxane-impermeable oil. If this magic oil exists, I could use it to seal 
the drop when freezing.

Thank you for help!

Sincerely,
Chen



UT Southwestern Medical Center
The future of medicine, today.

[ccp4bb] Postdoctoral position available at University of Texas Southwestern Medical Center

[Diana Tomchick]

This message is posted for Eric Olson, Ph.D. (Chair of Molecular Biology), who 
is looking for a postdoctoral fellow to join his lab to work on a novel 
membrane protein (first described in Nature, in press). See project description 
below and if interested please contact him at:

Email: eric.ol...@utsouthwestern.edumailto:eric.ol...@utsouthwestern.edu

Eric N. Olson, Ph.D.
Chair and Professor
Department of Molecular Biology
UT Southwestern Medical Center at Dallas
5323 Harry Hines Blvd.
Dallas, TX 75390-9148
214-648-1187 phone
214-648-1196 fax
http://www4.utsouthwestern.edu/olsonlab/index.html

Project Description:
We have discovered a novel muscle-specific membrane protein that is necessary 
and sufficient to induce the fusion of muscle cells to form multinucleated 
muscle fibers.  The process of myoblast fusion has been the focus of intense 
interest for decades but, until now, no muscle-specific fusigen has been 
discovered.  This fusigenic protein is highly hydrophobic and localized to the 
plasma membrane.  One of our major goals is to understand at the structural 
level how such a protein can promote the merger of membranes between cells.  We 
hope to recruit a structural biologist to determine the crystal structure of 
this fusigenic protein and then to introduce mutations to perturb its structure 
to further understand the mechanistic basis of its actions.  This project has 
the potential to yield important new insights into general properties of 
membrane fusion.  This project will offer a unique opportunity for a young 
scientist to establish a reputation in an important area of cell biology and to 
independently extend this work in the future.

Facilities Description:
State-of-the-art equipment is shared between the members in the Structural 
Biology community at UT Southwestern and consists of one Rigaku FR-E 
SuperBright high brilliancy X-ray generator equipped with one set of 
high-resolution and one set of high-intensity X-ray optics, two imaging-plate 
X-ray detectors (one R-Axis IV++ and one R-Axis IV), two X-Stream crystal 
cooling devices, and one automated sample-mounting device (ACTOR) for the 
unattended screening of crystals; a Phoenix crystallization robot, plus two 
Desktop Minstrel imaging systems and a Gallery-160 Plate Hotel; 30 days per 
year of beamtime at beamlines 19ID at the Advanced Photon Source (APS), 
Argonne, IL; several NMR spectrometers (one 800 MHz, three 600 MHz, two 500 
MHz), cryo-probes and robotic sample changer.  These facilities are 
supplemented by a variety of biophysical instruments supporting the study of 
macromolecules using CD, dynamic light scattering, analytical 
ultracentrifugation, stopped-flow kinetics, isothermal titration calorimetry, 
microscale thermophoresis and mass spectrometry. Several investigators in the 
UT Southwestern Structural Biology community are expert membrane protein 
crystallographers, and have access to specific equipment and expertise related 
to construct optimization, crystallization and structure solution of membrane 
proteins.

Diana

* * * * * * * * * * * * * * * * * * * * * * * * * * * *
Diana R. Tomchick
Professor
University of Texas Southwestern Medical Center
Department of Biophysics
5323 Harry Hines Blvd.
Rm. ND10.136EB (until the end of July)
Dallas, TX 75390-8816, U.S.A.
Email: 
diana.tomch...@utsouthwestern.edumailto:diana.tomch...@utsouthwestern.edu
214-645-6383 (phone)
214-645-6353 (fax)









UT Southwestern Medical Center
The future of medicine, today.

Re: [ccp4bb] Check PDB file for missing atoms

[Diana Tomchick]

The RCSB PDB Validation server will also report this info for you.

Diana

**
Diana R. Tomchick
Associate Professor
Department of Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214B
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Tiago Barros 
[ti...@me.com]
Sent: Tuesday, August 23, 2011 2:19 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Check PDB file for missing atoms

Dear all,

Does anyone know a program that will check a PDB file for missing atoms and 
output a list of the corresponding residues?

Many thanks in advance,

Tiago

***

Tiago Barros, PhD
Kuriyan lab - Molecular and Cellular Biology
University of California, Berkeley
527 Stanley Hall, QB3
Berkeley, CA 94720-3220
USA

tel:  + 1 510 643 0164
fax:  + 1 510 643 2352

***




UT Southwestern Medical Center
The future of medicine, today.

Re: [ccp4bb] Another paper structure retracted

[Diana Tomchick]

A quick glance at the header of the PDB file shows that there is one glaring 
discrepancy between it and the table in the paper that hasn't been mentioned 
yet in this forum. The data completeness (for data collection) reported in the 
paper is 95.7%, but in the header of the PDB file (actually, in both the 2QNS 
and the 3KJ5 depositions) the data completeness (for data collection) is 
reported as only 59.4%. The PDB header also contains an inconsistency, with the 
data completeness (for refinement) reported as 95.7%. Since the numbers of 
reflections reported for refinement versus data collection in the PDB header 
differ by less than 1%, it appears that there's been a bit of magical thinking 
that took place somewhere along the process from data processing to final model 
refinement. Small wonder that the refined geometry is so poor. Perhaps if these 
scientists had actually collected a complete dataset, we would not be having 
this conversation.

Diana

P.S. I have, on occasion, provided the coordinates and a map file to reviewers 
when they requested it. The last time it was requested was many years ago; I 
decided it was safer and easier if I provided as much information as possible 
in the manuscript (including better quality electron density figures than 
appear in this paper) to allow the reader to determine whether the work is 
valid or not.

* * * * * * * * * * * * * * * * * * * * * * * * * * * *
Diana R. Tomchick
Associate Professor
University of Texas Southwestern Medical Center
Department of Biochemistry
5323 Harry Hines Blvd.
Rm. ND10.214B
Dallas, TX 75390-8816, U.S.A.
Email: diana.tomch...@utsouthwestern.edu
214-645-6383 (phone)
214-645-6353 (fax)



On Aug 10, 2011, at 5:45 PM, Dale Tronrud wrote:

   I've made a quick look at the model and the paper - and it doesn't
 need more than a quick look.  The description of the model in
 the paper sounds great.  The problems in the model are clear.  My
 favorite is the quote Trp-477 of PTH1R makes several van der Waals
 contacts with Trp-339 and Lys-337 of G-beta-1   They are contacts
 all right.  The distances between the 477:CH2 and 337:CE is 2.75 A
 and between 477:NE1 and 339:CH2 is 2.26 A.  There are many more.

   In general the geometry of this entire model is terrible.  In
 Table 1 the bond length rmsd is listed at 1.64 A and the bond angles
 are 0.0078 deg!  Perhaps one is to presume the numbers should be
 swapped.  In any case, the values I calculate for the model are
 0.160 A and 4.46 deg!  Absolutely dreadful.  The PDB header lists
 the (swapped) values from the paper and then reports hundreds of
 outliers.

   The tools proposed by the Validation Task Force should cause a
 model like this to pop out clearly.  Even the old tools show this
 model is quite unreliable.  We just have to use them.

 Dale Tronrud

 On 08/10/11 14:35, Jacob Keller wrote:
 On the surface it doesn't seem as bad as others, i.e., it does not
 seem to be a real fake--perhaps just a strong form of wishful thinking
 and creative density interpretation. I wonder what would be a good
 metric in which to establish a cutoff for present/not present in
 density. CC, maybe?

 Jacob

 On Wed, Aug 10, 2011 at 4:01 PM, David Schuller dj...@cornell.edu wrote:
 Time to fuel up the gossip engines for the approaching weekend:


 http://www.sciencedirect.com/science/article/pii/S096921260800186X

 RETRACTED: Structure of the Parathyroid Hormone Receptor C Terminus Bound to
 the G-Protein Dimer Gβ1γ2
 Structure, Volume 16, Issue 7, 9 July 2008, Pages 1086-1094
 Structure 2QNS withdrawn.

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








UT Southwestern Medical Center
The future of medicine, today.

Re: [ccp4bb] Structures determined: breakdown of methods

[Diana Tomchick]

On Jan 17, 2011, at 11:46 AM, James Holton wrote:


 I am willing to bet that the earliest no method entries (particularly the 
 ones that lack a REMark 200 record) were probably MIR, since that was the 
 obvious method to solve a structure for some time.  Modern NULL entries 
 seem to be mostly what I call molecular replacement, which includes just 
 refining from a native, etc.  not necessarily running an MR search program.

I was taught to refer to this method of solution as isomorphous replacement, 
as long as the space group and/or unit cell had not changed so much that rigid 
body refinement could not lower the free R-factor enough for conventional 
refinement to proceed.

Diana

* * * * * * * * * * * * * * * * * * * * * * * * * * * *
Diana R. Tomchick
Associate Professor
University of Texas Southwestern Medical Center
Department of Biochemistry
5323 Harry Hines Blvd.
Rm. ND10.214B
Dallas, TX 75390-8816, U.S.A.
Email: diana.tomch...@utsouthwestern.edu
214-645-6383 (phone)
214-645-6353 (fax)



UT Southwestern Medical Center
The future of medicine, today.

Re: [ccp4bb] microseeding SeMet drops with native xtal seeds

[Diana Tomchick]

See the following publication for an interesting method to resolve  
pseudo-merohedral twinning in SeMet crystals:


Cansizoglu, A. E. and Chook, Y. M. (2007) Conformational heterogeneity  
of Karyopherinβ2 is segmental. Structure, 15(11):1431-1441.


In an effort to obtain single selenomethionine Kapβ2 crystals,  
mixtures of selenomethionine and native proteins were crystallized. A  
1:1 molar mixture of the two proteins gave single crystals...


Diana

On Sep 1, 2010, at 10:37 PM, amit sharma wrote:


Dear All,

My SeMet protein crystals have a needle-like morphology, worse than  
that of the native xtals. Also, although the cell dimensions of both  
forms is very much similar, the SeMet xtals are twinned(as indicated  
by Ctruncate plots). I was wondering if such cases were commonly  
seen, also is it alright to microseed my SeMet protein drops with  
seeds from the native xtal. I would be grateful if someone could  
shed light on this.


thanks in advance,
--
Amit Sharma,
Postdoctoral Fellow,
Department of Biophysics,
Johns Hopkins University,
Baltimore,
MD21218


* * * * * * * * * * * * * * * * * * * * * * * * * * * *
Diana R. Tomchick
Associate Professor
University of Texas Southwestern Medical Center
Department of Biochemistry
5323 Harry Hines Blvd.
Rm. ND10.214B   
Dallas, TX 75390-8816, U.S.A.   
Email: diana.tomch...@utsouthwestern.edu
214-645-6383 (phone)
214-645-6353 (fax)

[ccp4bb] Faculty position in Structural Biology at UT Southwestern Medical Center

[Diana Tomchick]

The Structural Biology Laboratory at UT Southwestern Medical Center  
currently has an opening at the Research Assistant Professor level for  
a highly motivated individual who enjoys working in a collaborative,  
multi-disciplinary setting. A Ph.D. in biochemistry, chemistry,  
physics or a related field is required. Experience in structural  
biology, particularly X-ray crystallography and protein expression/ 
purification is also required. Facility with related techniques such  
as NMR or cryo-EM spectroscopy, small-angle X-ray scattering,  
isothermal titration calorimetry or analytical ultracentrifugation is  
highly desirable.


Demonstrable experience in all areas of X-ray crystallography,  
including crystal growth and sample preparation, data collection on  
home and synchrotron sources, structure determination and refinement  
is required, and familiarity with the latest software is necessary.  
Job requirements include a significant amount of both formal and  
informal teaching, and travel to a synchrotron source with students  
may be required several times per year. Salary is competitive and  
commensurate with experience and qualifications.


About the Structural Biology Laboratory
The Structural Biology Laboratory (Diana Tomchick, Director) is a  
platform that allows researchers with different expertise to come  
together in a collaborative setting and tackle biological problems in  
a comprehensive way. We use a multitude of experimental approaches,  
mostly X-ray crystallography, but also NMR spectroscopy, complemented  
by a plethora of biophysical and molecular-biological techniques.


State-of-the-art equipment is shared between the members in the  
Structural Biology community at UT Southwestern and consists of one  
Rigaku FR-E SuperBright high brilliancy X-ray generator equipped with  
one set of high-resolution and one set of high-intensity X-ray optics,  
two imaging-plate X-ray detectors (one R-Axis IV++ and one R-Axis IV),  
two X-Stream crystal cooling devices, and one automated sample- 
mounting device (ACTOR) for the unattended screening of crystals; a  
Fluidigm microfluidics crystallization robot and imaging system; a  
Phoenix crystallization robot, plus two Desktop Minstrel imaging  
systems and a Gallery-160 Plate Hotel; 30 days per year of beamtime at  
beamlines 19ID and 19BM at the Advanced Photon Source (APS), Argonne,  
IL; several NMR spectrometers (one 800 MHz, three 600 MHz, two 500  
MHz), cryo-probes and robotic sample changer.  These facilities are  
supplemented by a variety of biophysical instruments supporting the  
study of macromolecules using CD, dynamic light scattering, analytical  
ultracentrifugation, stopped-flow kinetics, isothermal titration  
calorimetry and mass spectrometry.


The medical center has three degree-granting institutions: UT  
Southwestern Medical School, UT Southwestern Graduate School of  
Biomedical Sciences and UT Southwestern School of Health Professions.  
Nearly 4,200 students are trained each year in graduate and allied  
health programs, as residents and post doctoral fellows. Funding from  
the NIH, foundations, individuals and corporations provide more than  
$360 million annually for over 3,500 research projects.
Our faculty is comprised of many distinguished members, including:  
four Nobel laureates, three of whom are active faculty members; 18  
members of the National Academy of Sciences; and 18 members of the  
Institute of Medicine.


Interested applicants should submit a curriculum vita and three  
letters of reference by September 30, 2010 to Diana Tomchick, Chair,  
Biophysics Search Committee, UT Southwestern Medical Center.


recruit...@utsouthwestern.edu
UT Southwestern is an Equal Opportunity/Affirmative Action Employer.  
Women and minority candidates are encouraged to apply.



For further inquiries, please contact:
Diana Tomchick

Department of Biochemistry, Rm. ND10.214B

UT Southwestern Medical Center

5323 Harry Hines Blvd

Dallas TX 75390-8816

Tel. +1 214 645 6383

Email: diana.tomch...@utsouthwestern.edu

http://www8.utsouthwestern.edu/utsw/cda/dept16358/files/16776.html

http://www3.utsouthwestern.edu/sbl/


Some recent publications:


Scheuermann T.H. et al. (2009). Artificial ligand binding within the  
HIF2a PAS-B domain of the HIF2 transcription factor. PNAS, 106:450-455.


Davis L. et al. (2009). Localization and structure of the ankyrin- 
binding site on b2-spectrin. J. Biol. Chem., 284:6982-6987.


Yu, B. et al. (2010). Structural and energetic mechanisms of  
cooperative autoinhibition and activation of Vav1. Cell, 140:246-256.


Shin O-H. et al. (2010). Munc13 C2B domain is an activity-dependent  
Ca2+ regulator of synaptic exocytosis. Nat Struct Mol Biol 17:280-288.


Tian W. et al. (2010). Structural and functional analysis of the YAP- 
binding domain of human TEAD2. PNAS, 107:7293-7298.


Luong P. et al. (2010). Kinetic and structural insights into the  
mechanism of AMPylation

[ccp4bb] Postdoctoral Research Position available at UT Southwestern Medical Center

[Diana Tomchick]

This is posted as a favor for a collaborator, please do not respond to  
me but directly to Neal Alto.


---

A postdoctoral research position is available in the laboratory of Dr.  
Neal M. Alto, in the department of Microbiology, to study the  
molecular mechanisms of bacterial type III effectors and specifically,  
their ability to hijack human signal transduction cascades regulated  
by Ras-family GTPases and actin cytoskeletal dynamics. The postdoc  
will be responsible to build upon current biochemical and protein  
interaction data to express, purify, crystallize, and perform  
structural analyses involving bacterial type III effectors in complex  
with human substrates.  These studies will aim to complement ongoing  
research using techniques in cell biology and microbial  
pathogenesis.   Additional project information can be found in [Alto  
NM et al., Cell 2006] and [Alto NM et al., Journal of Cell Biology  
2007].


The University of Texas Southwestern (UTSW) Medical Center is a world- 
renowned research institute that is built upon foundations of  
Biochemistry, Structural Biology, and Biomedical Research.  Structural  
studies will be performed in collaboration with the UTSW Structural  
Biology Laboratory (SBL), a state of the art facility equipped with  
modern X-ray crystallography equipment including a high-brilliance  
Rigaku FR-E X-ray generator with an ACTOR crystal-mounting robot, an  
integrated Phoenix crystallization robot and imaging system, and a  
Fluidigm microfluidics crystallization robot and imaging system. The  
Structural Biology Group at UTSW also has guaranteed access to 30 days  
a year of beamtime at the Structural Biology center (SBC) at the  
Advanced Photo Source (APS).


The minimal requirement is a Doctoral degree in protein chemistry or  
molecular biology as well as prior experience in techniques of  
molecular cloning, recombinant protein expression, and X-ray  
crystallography.  The successful applicant must be self-motivated,  
enthusiastic and work well in a collaborative environment.   
Competitive salary and fringe benefits will be provided based on UTSW  
pay scale.


Interested individuals should submit a CV, a summary of research  
achievements, and names of three references to:


Dr. Neal Alto (neal.a...@utsouthwestern.edu)



* * * * * * * * * * * * * * * * * * * * * * * * * * * *
Diana R. Tomchick
Associate Professor
University of Texas Southwestern Medical Center
Department of Biochemistry
5323 Harry Hines Blvd.
Rm. ND10.214B   
Dallas, TX 75390-8816, U.S.A.   
Email: diana.tomch...@utsouthwestern.edu
214-645-6383 (phone)
214-645-6353 (fax)

Re: [ccp4bb] protein crystallography universe

[Diana Tomchick]

Does this mean that you are now King of the protein crystallography  
universe?


I am confident that you will be a kind and benevolent ruler,

Diana

On Oct 14, 2008, at 2:47 PM, Paul Swepston wrote:


Folks:

While attending the ICSG meeting in Oxford last month it struck me  
that there might be a need for a general protein crystallography  
portal containing information about software, websites, jobs,  
journals, organizations, education, etc.  While I realize that many  
people have created great lists of related websites for protein  
crystallography, I decided to create a new one and maintain it going  
forward.  It can be found at www.pxuniverse.com.  The inspiration  
for the name came from Ian Wilson's discussions about the structural  
biology universe and the impetus to create it came from Ian Wilson's  
discussion of  XtalPred, Derewenda's talk on SERp, and Sussman's  
great presentation about Proteopedia; it is difficult to keep up  
with all the innovations and pxuniverse is an attempt to give easier  
access to all the available tools.


If you have any links that you would like to see added please send  
them directly to me.  If you have any suggestions about the  
organization, I will consider all suggestions.


Thanks - Paul Swepston


* * * * * * * * * * * * * * * * * * * * * * * * * * * *
Diana R. Tomchick
Associate Professor
University of Texas Southwestern Medical Center
Department of Biochemistry
5323 Harry Hines Blvd.
Rm. ND10.214B   
Dallas, TX 75390-8816, U.S.A.   
Email: [EMAIL PROTECTED]
214-645-6383 (phone)
214-645-6353 (fax)

Re: [ccp4bb] Quick-soak

[Diana Tomchick]

Or you could try crystallizing the protein in the presence of KI or  
NaI, and collect some in-house SAD data. You could also try to boost  
the concentration and number of ordered halide sites by quick soaking  
the crystals with a higher concentration of the iodide salt.


In my limited experience with iodide soaks, the crystals often crack  
with high concentrations (close to 1.0 M) of the iodide salt, but  
lower concentrations (close to 0.1-0.2 M) of the salt don't result in  
a high enough anomalous signal to phase the structure. I've never  
tried it with such a small protein, so perhaps you will be successful.


Diana

On Sep 25, 2008, at 6:03 AM, amit sharma wrote:



Dear CCP4bbers,
I have a protein molecule(~9.0 kDa) that crystallized in the  
presence of 0.15M KBr and HEPES pH 7.0. Since there is no  
homologuous structure present, I intend to perform heavy metal  
derivatization. I read some literature which suggested that I could  
carry out quick soak with 0.5M Sodium iodide. I was wondering if I  
could soak my crystals with a similar concentration of NaBr and  
collect some low resolution data at the in-house source? In addition  
should I try to also introduce other heavy metals? it might be worth  
mentioning that my protein carries no methionine/cys residues.
I have no prior experience in doing this. So, it would  be of great  
help if I could be directed towards literature/protocols pertaining  
to this. Any advice/suggestions would be greatly appreciated.


Thanks in advance
--
Amit Sharma


* * * * * * * * * * * * * * * * * * * * * * * * * * * *
Diana R. Tomchick
Associate Professor
University of Texas Southwestern Medical Center
Department of Biochemistry
5323 Harry Hines Blvd.
Rm. ND10.214B   
Dallas, TX 75390-8816, U.S.A.   
Email: [EMAIL PROTECTED]
214-645-6383 (phone)
214-645-6353 (fax)

Re: [ccp4bb] off-topic: Enzymology textbook recommendations?

[Diana Tomchick]

I have a copy of that textbook, and I like it. I had the privilege to  
sit in on Perry Frey's lectures at the University of Wisconsin, and  
the text is as clear and informative as his lectures. Plus it's easier  
to read than my old lecture notes (and more up-to-date).


Diana

On Sep 11, 2008, at 4:55 PM, Donnie Berkholz wrote:


Our resident enzymologist (who just retired) recently picked up a copy
of a current book that claims to update Jencks and Walsh, since they
were both published before 1980. It's by Frey  Hegeman:
http://www.oup.com/us/catalog/he/subject/Chemistry/Biochemistry/ProteinsandEnzymes/?view=usaci=9780195122589

If you try it out, I'd love to hear how it goes.

--
Thanks,
Donnie

Donnie Berkholz
Developer, Gentoo Linux
Blog: http://dberkholz.wordpress.com


* * * * * * * * * * * * * * * * * * * * * * * * * * * *
Diana R. Tomchick
Associate Professor
University of Texas Southwestern Medical Center
Department of Biochemistry
5323 Harry Hines Blvd.
Rm. ND10.214B   
Dallas, TX 75390-8816, U.S.A.   
Email: [EMAIL PROTECTED]
214-645-6383 (phone)
214-645-6353 (fax)

Re: [ccp4bb] Na Acetate Buffer volatility

[Diana Tomchick]

One important consideration with acetate buffer is volatility. All the  
care in the world to painstakingly reproduce the pH of the buffer will  
not help you if store your acetate buffer for long periods of time in  
a container that doesn't minimize evaporation (so all of you out there  
trying to reproduce an acetate buffered condition from a commercial  
crystallization screen that was stored in plastic 15-ml tubes, beware!).


Diana

On Jul 23, 2008, at 10:26 AM, Nadir T. Mrabet wrote:

Michael's comments are correct and follow indeed appropriate  
chemistry theory.
In practice however, as said earlier, it is less likely to read a  
correct pH value with a pH-meter (unless extensive care and  
adjustments are taken beforehand) than to predict amounts of acid  
and base to use reliably based on the HH equation.


I take this opportunity to correct my mistyping in my previous mail:  
In the second case, the HH is written 4.5 = 4.76 + log([NaOH])/(25  
- [NaOH])
should be rewritten into In the second case, the HH is written 4.5  
= 4.76 + log([NaOH]/(25 - [NaOH])).


Greetings,

Nadir

--

Pr. Nadir T. Mrabet
  Cellular  Molecular Biochemistry
  INSERM U-724
  Nancy University, School of Medicine
  9, Avenue de la Foret de Haye, BP 184
  54505 Vandoeuvre-les-Nancy Cedex
  France
  Phone: +33 (0)3.83.68.32.73
  Fax:   +33 (0)3.83.68.32.79
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R.M. Garavito wrote:


Buffer making is very much an empirical process, but there is a  
comment that needs to be made about the use of the H-H equation and  
pKa values. I have to teach our department's biochemistry  
laboratory, and I sadly would have to take off points from all the  
discussions as the H-H equation and pKa won't give the right answer  
as pKa is for an ideal (i.e., infinitely dilute) solution. A 25 mM  
Na acetate solution is not dilute. You need to use the pKa' which  
sadly changes as the concentration of the buffer increases or  
decreases: while the pKa of acetic acid is 4.76 (@25˚C), at 100mM,  
the pKa' is 4.60. The National Bureau of Standards (now NIST) has a  
detailed list of standard buffer recipes and pKa' values for most  
of the common buffers (e.g., see Bates, J. Natl. Bur. Stand. 66A,  
179, 1962).


That said, Nadir's method is a fine way to make a buffer of a known  
pH (using a well calibrated pH meter) at a known temperature, and  
it will allow you to make a buffer with the same pH value almost  
every time (depending on how your room temperature changes  
throughout the year). Being able to consistently and reliably  
repeat the buffer formulation is the most important point.


Cheers,

Michael

//

/R. Michael Garavito, Ph.D./

/Professor of Biochemistry  Molecular Biology/

/513 Biochemistry Bldg. /

/Michigan State University /

/East Lansing, MI 48824-1319/

/Office:// //(517) 355-9724 Lab: (517) 353-9125/

/FAX: (517) 353-9334 Email: [EMAIL PROTECTED]  
mailto:[EMAIL PROTECTED]/


//



On Jul 22, 2008, at 11:20 AM, Nadir T. Mrabet wrote:

I bet it is more difficult to adjust a pH-meter than to use the  
Henderson-Hasselbalch equation
and still get the expected pH with a pretty good accuracy  
especially if your work near the pKa.


There are actually two ways to prepare this 25 mM buffer, pH 4.5.

The pKa of acetate is 4.76 at 25 °C (with dpKa/° C = +0.0002, so  
don't worry too much about this).
Reference is Buffers for pH and Metal Ion Control, Perrin   
Dempsey, Chapman  Hall, NY, ISBN 0 412 21890 9.


High-grade glacial acetic acid (99-100%) is 18 N.
Make a stock solution of 250 mM (eg 3.472 mL for 1.0 L final).  
Keep is a dark, tightly closed bottle.


Make a stock solution of 250 mM sodium acetate (if you use FW, not  
MW, to calculate mass to use, then no worry about anhydrous or not  
since water is also taken into account if present)


or

make a stock solution of 5N NaOH. Keep is a dark, tightly closed  
bottle.


Use then the Henderson-Hasselbalch equation (HH), pH = pKa + log  
([A-]/[AH]).


In the first case, you write it : 4.5 = 4.76 + log ([sodium  
acetate]/[acetic acid])

Second equation is [sodium acetate] + [acetic acid] = 25 mM
which gives [sodium acetate] = 8.886 mM and [acetic acid] = 16.134  
mM.
For 1.0 L buffer, mix adequate volumes of stock solutions of  
sodium acetate and acetic acid and complete with water (add acid  
after un first fill with water to ~ 800 mL).


In the second case, the HH is written 4.5 = 4.76 + log([NaOH])/(25  
- [NaOH]),
which gives [NaOH] = 8.886 mM (same result as above for sodium  
acetate which was then the base).


The added advantage of using HH and stock solutions is that even  
if your pH is not exactly 4.5, say 4.55, if you make a new buffer  
the next day or even the next month,
your buffer will have the same pH value. I don't expect you can  
ever achieve such a repeatability using a pH-meter.


HTH,

Re: [ccp4bb] is it Ok to freeze

[Diana Tomchick]

Every small molecule dataset I collected as a graduate student in  
chemistry back in the mid to late 1980's was at 100K. I never had to  
worry about crystal slippage during collection, organic solvent  
evaporation, air oxidation of the sample (organometallic metal  
clusters) or secondary radiation damage.


When I switched to protein crystallography, I was absolutely amazed  
when told that you can not cool a protein crystal below 4 degrees C  
for data collection.


How times have changed,

Diana

On Jun 19, 2008, at 9:03 AM, Ian Tickle wrote:

I would go along with Harry  friends, I used crystal cooling when I  
was

at Aafje Vos' Struktuurchemie lab in Groningen in 1972, when the
technique had already been in routine use there for at least 10 years,
in order to study compounds that are liquid at ambient temp (of course
it was custom-built kit using a collection of liq N2 Dewar vessel 
tubes, nothing as fancy as a Cryostream!).  The Groningen group really
pioneered the use of low temp for small molecule structures and I  
don't
recall increased mosaicity ever being an issue.  Occasionally you  
would

get a compound with a phase transition on the way down and the crystal
would literally explode in a puff of powder before your eyes!  The
motive for using low temp was of course to reduce the thermal motion  
and

libration effects, and thus greatly improve the accuracy of the
molecular geometry, and low temp is pretty well essential if you're  
into

valence density deformation maps, again in order the minimise the
contribution from thermal motion.

-- Ian


-Original Message-
From: [EMAIL PROTECTED]
[mailto:[EMAIL PROTECTED] On Behalf Of harry powell
Sent: 19 June 2008 14:05
To: Remy Loris
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] is it Ok to freeze

Hi

Without wishing to start an argument, I've been checking with
some of my colleagues who are chemical crystallographers -
the reply I get is that, for routine structural analysis,
pretty well all datasets are collected at 100K unless the
crystals fall apart at low T, or if the cryostream is broken.

I should point out that the first production Cryostream that
I came across (serial number 2, which I think may have been
the first one sold!) was in the Cambridge Department of
Chemistry in about 1985. They didn't become common until the
mid-1990's in PX labs, when they were already
well-established as a bit of pretty well essential kit for
small molecule work.

So although what Remy says is true, the practice is to
cryocool most of the time.


On 19 Jun 2008, at 12:08, Remy Loris wrote:


Typically crystals of small organic compounds do not
require freezing as there are no solvent channels. They do in
general not suffer from radiation damage at room temperature
the way protein crystals do. Occasionally they are mounted in
a capillary instead of simply glueing them to a goniometer if
they are air sensitive. In principle freezing should not
damage the crystals, but one still may have to be carefull if
the crystals are large. I think you risk increasing
mosiacity, and any manipulation that is not needed will on
average only reduce the quality of the specimen rather than improve  
it


Remy Loris
Vrije Univesiteit Brussel

Jayashankar wrote:

Dear Scientists and Friends,
I am not sure, whether  organic crystals  need
to be in cryo stream necessarily during data  collection from
an  in house
xray machine .
How most of the organic crystals have been
solved mostly?
--
S.Jayashankar
(A bit confused new generation researcher).
Research Student
Institute for Biophysical Chemistry
Hannover Medical School
Germany


Harry
--
Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC
Centre, Hills Road, Cambridge, CB2 2QH








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Re: [ccp4bb] font size CCP4i

[Diana Tomchick]

I don't know, I kind of liked the larger font size. I thought it was  
an improvement.


Maybe that's because I'm getting older and need new glasses...

Diana


On May 23, 2008, at 7:47 AM, Kristof Van Hecke wrote:


Dear all,

I recently installed the CCP4 Program Suite 6.0.2., together with  
the CCP4Interface 1.4.4.2 under an Intel Mac Book Pro (running  
Leopard 10.5.2).


All works fine, except for a minor issue:
The font size in the GUI seems quit 'big' on both sides of the 'List  
of jobs', e.g. on the 'Data Reduction', 'Experimental  
Phasing',...buttons.

The 'List of jobs' itself shows a 'normal' font size.

This is probably due to some minor settings, which are not properly  
adjusted.

But somehow I don't find where to adjust these...??


When installing under my (old) PPC running Tiger, this problem does  
not occur.



Does anyone knows how to solve this please..?


Many thanks

Regards

Kristof



--
Kristof Van Hecke, PhD
Biomoleculaire Architectuur
Celestijnenlaan 200 F
B-3001 Heverlee (Leuven)
Tel: +32(0)16327477
--





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* * * * * * * * * * * * * * * * * * * * * * * * * * * *
Diana R. Tomchick
Associate Professor
University of Texas Southwestern Medical Center
Department of Biochemistry
5323 Harry Hines Blvd.
Rm. ND10.214B   
Dallas, TX 75390-8816, U.S.A.   
Email: [EMAIL PROTECTED]
214-645-6383 (phone)
214-645-6353 (fax)

[ccp4bb] Question about Phoenix crystallization robot

[Diana Tomchick]

BACKGROUND:  Recently we acquired an Art Robbins Phoenix  
crystallization robot. This instrument is in a shared environment,  
accessible to labs with projects that range from small, well-behaved  
soluble cytosolic proteins to large complexes and integral membrane  
proteins. Many of our users obtain only small quantities (a few  
hundred microliters) of purified proteins, and they are always  
looking for ways to maximize the number of crystallization screens  
they can set up with their samples.


QUESTION:  Several users have recently asked if they could use a  
protocol that allows them to aspirate enough protein into the  
Nanoneedle (the needle used for dispensing protein) to set up 3-5 or  
more different 96-condition screens. They feel this would minimize  
any sample waste and maximize their time. Our concern is that this  
might result in clogging of the Nanoneedle due to evaporation and  
subsequent precipitation of their protein, as the amount of time  
required for such a protocol would be greater than 10 minutes. Our  
local Art Robbins representative has agreed with us that this is not  
a recommended protocol. We are in a bit of a dilemma as we do not  
have enough experience with this robot to definitively say that the  
users should not follow this kind of protocol, but perhaps there is a  
better way to achieve their desired goal.


Does anyone out there have any practical suggestions and experience  
that could help us accommodate such user requests?


Thank you in advance, I'll post a summary of responses,

Diana

* * * * * * * * * * * * * * * * * * * * * * * * * * * *
Diana R. Tomchick
Associate Professor
University of Texas Southwestern Medical Center
Department of Biochemistry
5323 Harry Hines Blvd.
Rm. ND10.214B   
Dallas, TX 75390-8816, U.S.A.   
Email: [EMAIL PROTECTED]
214-645-6383 (phone)
214-645-6353 (fax)