Re: [ccp4bb] Poor correlation coefficient of model to cryo-EM map.

[Reza Khayat]

The clash score is also impressively high. Your map may have the incorrect 
hand. Have you tried flipping it?

Best wishes
Reza

From: CCP4 bulletin board  on behalf of Martyn Winn - 
STFC UKRI <7c0f4d7fc2b7-dmarc-requ...@jiscmail.ac.uk>
Sent: 10 January 2024 4:51 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [EXTERNAL] Re: [ccp4bb] Poor correlation coefficient of model to 
cryo-EM map.


You can also try the CCPEM list for more cryoEM-orientated advice 
https://www.jiscmail.ac.uk/CCPEM
 and look in CCP-EM for more fitting, refinement, validation tools.



Certainly, the map doesn’t look 3A, unless you have filtered it for these 
pictures. The CC for the middle and C-terminal domains is not just low, but 
essentially zero. And as Basil points out, a map-model FSC of 22.3A at 0.5. So 
I think you need to look again at the initial fitting.



HTH

Martyn





From: CCP4 bulletin board  On Behalf Of Basil Greber
Sent: 10 January 2024 08:12
To: ccp4bb 
Subject: Re: [ccp4bb] Poor correlation coefficient of model to cryo-EM map.



Are you confident that your 3 Å resolution is correct? The map in the picture 
you supplied looks more like 5 Å, and the model vs. map FSC at 0.5 is 
apparently 20 Å (?).



Basil


Gesendet mit der mobilen Mail App

Am 10.01.24 um 05:57 schrieb Ketul Saharan

Von: "Ketul Saharan" 
Datum: 10. Januar 2024
An: CCP4BB@JISCMAIL.AC.UK
Cc:
Betreff: [ccp4bb] Poor correlation coefficient of model to cryo-EM map.

Dear CCP4 community,

I am building a structure model from ~3.0 Å resolution cryo-EM map. The 
structure consists of seven chains, with each chain containing an N-, middle, 
and C-terminal domain. Although I attempted to directly fit the Alfa-fold 
model, it became evident that the protein exhibited some movement, leading to 
poor fitting of N-terminal. To improve the fitting, I segmented the alfa-fold 
model into two parts: i) the N-terminal and ii) the middle and C-terminal 
domain. These fragments were then fitted into the map. After a few rounds of 
refinement using coot and phenix, the model effectively fitted all seven chains.

The refinement resulted in a model to map correlation (CC mask) of over 60% for 
the N-terminus. However, even though the model appeared to fit well inside the 
map, particularly in the middle and C-terminus regions, the refining 
consistently resulted in a map to model correlation of 0%.

For your perusal, I have included the snapshot of the phenix refinement 
results, the correlation graph, and the fitted model within the map (displaying 
one chain out of seven).

I am not able to figure out why the correlation is so poor even after fine 
fitting of model to map.

Any support in resolving this issue would be much appreciated.



Thank,

Ketul Saharan


--

Ketul Saharan

Senior Research Fellow (Ph.D. Scholar)

Laboratory of Macromolecular Crystallography (Lab-8)

Institute of Life Sciences

Nalco Square, Chandrasekharpur

Bhubaneswar – 751023

Odisha State, INDIA



Phone: +91 8708290889

[https://ssl.gstatic.com/docs/doclist/images/icon_10_generic_list.png] 
124.png[Image
 removed by sender.]

[https://ssl.gstatic.com/docs/doclist/images/icon_10_generic_list.png] 
correlation.tif[Image
 removed by sender.]

[https://ssl.gstatic.com/docs/doclist/images/icon_10_generic_list.png] map to 
model.tif[Image
 removed by sender.]





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Re: [ccp4bb] Validation of structure prediction

[Reza Khayat]

Hi,


Thank you for the help. I've addressed some of the concerns raised here in 
another thread. "Validation" referred to checking geometric parameters; 
however, outstanding geometric parameters do not indicate a structure that is 
comparable to an experimentally determined structure. The structures were 
predicted with the Robetta server and all have, as expected, geometry better 
than most experimental structures.


Best wishes,
Reza


Reza Khayat, PhD
Associate Professor
City College of New York
Department of Chemistry and Biochemistry
New York, NY 10031

From: CCP4 bulletin board  on behalf of Krieger, James M 

Sent: Tuesday, December 21, 2021 7:14 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] Re: [ccp4bb] Validation of structure prediction

There are also dedicated homology modelling validation tools such as ANOLEA 
(ANOLEA (Atomic Non-Local Environment Assessment) 
(melolab.org)<https://urldefense.proofpoint.com/v2/url?u=http-3A__melolab.org_anolea_=DwMF-g=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0=wygUyYLV7I0tm87EBKcsmtMhtRx3xWnT36HOD04_BCo=Y379nBTcaulXSuY9Y7ZQJsQSkQtJGpI8jj-fuT7THVU=>).

Best wishes
James

From: CCP4 bulletin board  on behalf of Nicholas Clark 

Sent: Tuesday, December 21, 2021 11:57 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Validation of structure prediction

Reza,

Thus far, it seems we’ve all assumed this was an AlphaFold or RobettaFold 
model. If this is not indeed the case, it may be worthwhile to “validate” your 
mode by running your sequence through one of these two and using the validation 
from them.

The AlphaFold DB can be found here, with a number of predicted structures:

https://alphafold.ebi.ac.uk<https://urldefense.proofpoint.com/v2/url?u=https-3A__nam12.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Falphafold.ebi.ac.uk-252F-26data-3D04-257C01-257Ckriegerj-2540PITT.EDU-257Ca05aa26f114e4fb7e6c108d9c479140b-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C637756847599764985-257CUnknown-257CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0-253D-257C3000-26sdata-3D1szS78fj9859h8AR0Mt5YtgUXeW1JmsB2YU0j5Hhy5U-253D-26reserved-3D0=DwMF-g=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0=wygUyYLV7I0tm87EBKcsmtMhtRx3xWnT36HOD04_BCo=OM7PDWl19Mz3PGjngC_uwF797ZeTV5gVNCg5RXNrUV8=>

The AlphaFold colab can be found here, although the prediction is not as good 
as AlphaFold 2, as it does not use templates:

https://colab.research.google.com/github/deepmind/alphafold/blob/main/notebooks/AlphaFold.ipynb<https://urldefense.proofpoint.com/v2/url?u=https-3A__nam12.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fcolab.research.google.com-252Fgithub-252Fdeepmind-252Falphafold-252Fblob-252Fmain-252Fnotebooks-252FAlphaFold.ipynb-26data-3D04-257C01-257Ckriegerj-2540PITT.EDU-257Ca05aa26f114e4fb7e6c108d9c479140b-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C637756847599764985-257CUnknown-257CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0-253D-257C3000-26sdata-3Dd323My3Ix-252BQwDW3D7ZCe6RTlY7wvMqXl92ZGRiIiMSo-253D-26reserved-3D0=DwMF-g=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0=wygUyYLV7I0tm87EBKcsmtMhtRx3xWnT36HOD04_BCo=iJvvBod4oNFHS9YMaLMNdy0edv1eh47A2txWBV5IuoQ=>


RobettaFold can be found here:
https://robetta.bakerlab.org<https://urldefense.proofpoint.com/v2/url?u=https-3A__nam12.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Frobetta.bakerlab.org-252F-26data-3D04-257C01-257Ckriegerj-2540PITT.EDU-257Ca05aa26f114e4fb7e6c108d9c479140b-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C637756847599764985-257CUnknown-257CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0-253D-257C3000-26sdata-3DT3foQ-252FDRiOinSJjqeKwkPkw1UtSXc4PvyJRCTSwtUiI-253D-26reserved-3D0=DwMF-g=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0=wygUyYLV7I0tm87EBKcsmtMhtRx3xWnT36HOD04_BCo=8b0eA8ZDF8wlvyfoyNxzxxmGN0T58L9WsgHqEaxUoZY=>

Best,

Nick Clark

On Tue, Dec 21, 2021 at 6:20 AM Randy John Read 
mailto:rj...@cam.ac.uk>> wrote:
Just to add one point that I don’t think I’ve seen yet. If what the referee 
wants is a data-free assessment of the expected quality of the model, I think 
that the best assessment at the moment is the one done by AlphaFold2 (or indeed 
RoseTTAFold if you’re using one of their models). The machine-learning 
algorithm is pretty good at assessing how good of a job it has done, either 
overall (predicted TM score) or locally (predicted lDDT score for AlphaFold2 or 
predicted RMSD for RoseTTAFold). There are cases of false positives (poor 
models that think they’re good) and false negatives (good models that think 
they’re bad), but these are in the minority from what I’ve seen so

Re: [ccp4bb] [EXTERNAL] [ccp4bb] Fwd: [ccp4bb] Validation of structure prediction

[Reza Khayat]

​Dear all,


Thank you for the responses. My question regarding validation was not directed 
at weather the prediction was correct (i.e. sufficiently comparable to the 
experimentally determined structure). I realize this question in itself is not 
easily answered. I also agree with what everyone has written below. My question 
was regarding the geometry of the structure. I also understand that outstanding 
geometry does not indicate an accurately predicted structure -the helix example 
indicated by Tristian. Nonetheless, the server links sent earlier are extremely 
helpful.


Reza Khayat, PhD
Associate Professor
City College of New York
Department of Chemistry and Biochemistry
New York, NY 10031

From: CCP4 bulletin board  on behalf of F.Xavier 
Gomis-Rüth 
Sent: Tuesday, December 21, 2021 5:04 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] [ccp4bb] Fwd: [ccp4bb] Validation of structure prediction

Dear all,
this is by far not the general case in our hands. Depending on which AlphaFold 
protocol is used, the resulting models have locally disfavourable
geometries–including clashes–, impossible chain crossovers, etc. I would 
definitively recommend everybody to go through the model in detail and perform
a final geometry minimization with Coot and/or Phenix/Refmac. And in these 
cases, general geometry validation as provided by MolProbity
provides a final proof of the computational model.
Best,
Xavier


 Forwarded Message 
Subject:Re: [ccp4bb] Validation of structure prediction
Date:   Tue, 21 Dec 2021 09:43:37 +
From:   Vollmar, Melanie (DLSLtd,RAL,LSCI) 
<64fe7ccc6b4d-dmarc-requ...@jiscmail.ac.uk><mailto:64fe7ccc6b4d-dmarc-requ...@jiscmail.ac.uk>
Reply-To:   Vollmar, Melanie (DLSLtd,RAL,LSCI) 
<mailto:melanie.voll...@diamond.ac.uk>
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>


Tristan is spot on. All the predicted structures have near perfect geometry, so 
commonly used validation tools like MolProbity can no longer be applied.

What you need to consider is biological relevance of the predicted model. Does 
the model correctly reflect residue arrangement in the active site? Are domains 
in correct relative orientation to allow for interactions and movements, 
perhaps found by some other assay? Is there appropriate room to fit a 
ligand/cofactor? Are transmembrane helices, if there are any, correctly found?

You need to map the knowledge you have of your protein to the structure and see 
if the atom positions and what you know support each other.

Cheers

M

From: CCP4 bulletin board <mailto:CCP4BB@JISCMAIL.AC.UK> 
on behalf of Tristan Croll <mailto:ti...@cam.ac.uk>
Sent: 21 December 2021 08:28
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 
<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] Validation of structure prediction

I agree with Dale. Tools like MolProbity are not the right approach to 
validating a structure prediction. To understand why, just consider that all 
you need to do to get a perfect MolProbity score is predict every structure as 
a single long alpha helix with ideal rotamers, with a kink at each proline.

To validate a predicted structure will require a completely different toolset - 
one that I’m not sure fully exists yet.

— Tristan

> On 20 Dec 2021, at 18:47, Dale Tronrud 
> <mailto:de...@daletronrud.com> wrote:
>
>   I don't see any reason to believe that software designed to validate 
> crystallographic or NMR models would have any utility validating AlphaFold 
> predicted models.  Doesn't the prediction software already ensure that all 
> the indicators used by Molprobity are obeyed?  I'm afraid that the tools to 
> validate any new technique must be designed specifically for that technique. 
> (And when they become available they will be useless for validating 
> crystallographic models!)
>
> Dale E. Tronrud
>
>> On 12/20/2021 10:28 AM, Nicholas Clark wrote:
>> The Molprobity server can be run online and only requires the coordinates in 
>> PDB format: 
>> http://molprobity.biochem.duke.edu/<https://urldefense.proofpoint.com/v2/url?u=http-3A__molprobity.biochem.duke.edu_=DwMDaQ=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0=Ore7pZl_g57-Bha3m6bv3ayt12QpXPz1lbBlJYIx0rY=ekPRcBIEvpMpSOCQ3Iwa3WeIz5hew6AEXPmRUbWF9eg=>
>>  
>> <http://molprobity.biochem.duke.edu/<https://urldefense.proofpoint.com/v2/url?u=http-3A__molprobity.biochem.duke.edu_=DwMDaQ=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0=Ore7pZl_g57-Bha3m6bv3ayt12QpXPz1lbBlJYIx0rY=ekPRcBIEvpMpSOCQ3Iwa3WeIz5hew6AEXPmRUbWF9eg=>>.
>> Best,
>> Nick Clark
>> On Mon, Dec 20, 2021 at 11:10 AM Reza Khayat 
>> mailto:rkha...@ccny.cuny.edu> 
>

[ccp4bb] Validation of structure prediction

[Reza Khayat]

?Hi,

Can anyone suggest how to validate a predicted structure? Something similar to 
wwPDB validation without the need for refinement statistics. I realize this is 
a strange question given that the geometry of the model is anticipated to be 
fine if the structure was predicted by a server that minimizes the geometry to 
improve its statistics. Nonetheless, the journal has asked me for such a 
report. Thanks.

Best wishes,

Reza


Reza Khayat, PhD
Associate Professor
City College of New York
Department of Chemistry and Biochemistry
New York, NY 10031



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Re: [ccp4bb] [EXTERNAL] [ccp4bb] Searching Sequence Motif

[Reza Khayat]

?Try using the psi-blast package and search against the PDB.


Reza Khayat, PhD
Associate Professor
City College of New York
Department of Chemistry and Biochemistry
New York, NY 10031

From: CCP4 bulletin board  on behalf of Cryo EM 

Sent: Sunday, September 5, 2021 11:23 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] [ccp4bb] Searching Sequence Motif

Hi all,

I want to search NCBI/PDB for all E.coli proteins with a specific sequence 
motif.
Is there any server/software in which I can search and display all the proteins 
in E.coli with this specific sequence motif?
Suggestions are highly appreciated.

Thanks!





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Re: [ccp4bb] poly-dN model

[Reza Khayat]

Thank you Robbie.

Reza Khayat, PhD
Associate Professor
City College of New York
Department of Chemistry and Biochemistry
New York, NY 10031


From: CCP4 bulletin board  on behalf of Robbie Joosten 

Sent: Saturday, March 13, 2021 9:09 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] Re: [ccp4bb] poly-dN model

Hi Reza,

The equivalent for UNK (not ALA!) is residue N for RNA and DN for DNA.

HTH,
Robbie

> -Original Message-
> From: CCP4 bulletin board  On Behalf Of Reza
> Khayat
> Sent: Saturday, March 13, 2021 14:53
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] poly-dN model
>
> Hi,
>
>
>
>
>
> For DNA/RNA models, is there an equivalent to a poly-Ala model? Something
> like a poly-dN perhaps? This is in case you don't know the nucleic acid
> sequence. Thanks.
>
>
>
>
>
> Best wishes,
> Reza
>
>
>
>
>
> Reza Khayat, PhD
> Associate Professor
> City College of New York
> Department of Chemistry and Biochemistry New York, NY 10031
>
> 
>
>
> To unsubscribe from the CCP4BB list, click the following link:
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[ccp4bb] poly-dN model

[Reza Khayat]

Hi,


For DNA/RNA models, is there an equivalent to a poly-Ala model? Something like 
a poly-dN perhaps? This is in case you don't know the nucleic acid sequence. 
Thanks.


Best wishes,
Reza


Reza Khayat, PhD
Associate Professor
City College of New York
Department of Chemistry and Biochemistry
New York, NY 10031



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Re: [ccp4bb] AlphaFold: more thinking and less pipetting (?)

[Reza Khayat]

?Does anyone know how AlphaFold performs on sequences with little conservation? 
Virus and phage proteins are like this. Their structures are homologous, but 
sequence identity can be less than 10%.


Reza


Reza Khayat, PhD
Associate Professor
City College of New York
Department of Chemistry and Biochemistry
New York, NY 10031

From: CCP4 bulletin board  on behalf of Anastassis 
Perrakis 
Sent: Thursday, December 3, 2020 5:31 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] Re: [ccp4bb] AlphaFold: more thinking and less pipetting (?)

AlphaFold - or similar ideas that will surface up sooner or later - will beyond 
doubt have major impact. The accuracy it demonstrated compared to others is 
excellent.

"Our" target (T1068) that was not solvable by MR with the homologous search 
structure or a homology model (it was phased with Archimboldo, rather easily), 
is easily solvable with the AlphaFold model as a search model. In PHASER I get 
Rotation Z-score 17.9, translation Z-score 26.0, using defaults.


imho what remains to be seen is:

a. how and when will a prediction server be available?
b. even if training needs computing that will surely unaccessible to most, will 
there be code that can be installed in a "reasonable" number of GPUs and how 
fast will it be?
c. how do model quality metrics (that do not compared with the known answer) 
correlate with the expected RMSD? AlphaFold, no matter how impressive, still 
gets things wrong.
c. will the AI efforts now gear to ligand (fragment?) prediction with similarly 
impressive performance?

Exciting times.

A.




On 3 Dec 2020, at 21:55, Jon Cooper 
<488a26d62010-dmarc-requ...@jiscmail.ac.uk<mailto:488a26d62010-dmarc-requ...@jiscmail.ac.uk>>
 wrote:

Hello. A quick look suggests that a lot of the test structures were solved by 
phaser or molrep, suggesting it is a very welcome improvement on homology 
modelling. It would be interesting to know how it performs with structures of 
new or uncertain fold, if there are any left these days. Without resorting to 
jokes about artificial intelligence, I couldn't make that out from the CASP14 
website or the many excellent articles that have appeared. Best wishes, Jon 
Cooper.


Sent from ProtonMail mobile



 Original Message 
On 3 Dec 2020, 11:17, Isabel Garcia-Saez < 
isabel.gar...@ibs.fr<mailto:isabel.gar...@ibs.fr>> wrote:

Dear all,

Just commenting that after the stunning performance of AlphaFold that uses AI 
from Google maybe some of us we could dedicate ourselves to the noble art of 
gardening, baking, doing Chinese Calligraphy, enjoying the clouds pass or 
everything together (just in case I have already prepared my subscription to 
Netflix).

https://www.nature.com/articles/d41586-020-03348-4<https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_articles_d41586-2D020-2D03348-2D4=DwMGaQ=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0=5lc5MUokcPdJZuiKvx3xkaHGMFTkSQHuwMu3HoQZUNA=FjJEUNt1oYyfCSZk105Z-QvYSPRKxaj1NGZOmqJsXKw=>

Well, I suppose that we still have the structures of complexes (at the moment). 
I am wondering how the labs will have access to this technology in the future 
(would it be for free coming from the company DeepMind - Google?). It seems 
that they have already published some code. Well, exciting times.

Cheers,

Isabel


Isabel Garcia-Saez PhD
Institut de Biologie Structurale
Viral Infection and Cancer Group (VIC)-Cell Division Team
71, Avenue des Martyrs
CS 10090
38044 Grenoble Cedex 9
France
Tel.: 00 33 (0) 457 42 86 15
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[ccp4bb] Windows binary for probe and reduce

[Reza Khayat]

?Hi,


Has anyone been able to compile a windows version (non cygwin) of probe and 
reduce? I'm asking for the windows version of coot.  Thanks.


Best wishes,
Reza


Reza Khayat, PhD
Associate Professor
City College of New York
Department of Chemistry and Biochemistry
New York, NY 10031



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Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] Modeling ATP/ADP

[Reza Khayat]

Tried the homologues thing. There are homologues and I've done the fitting, but 
this is what I consider to be subjective. I'm certain the referee will ask: 
Given the quality of density for the nucleotide, how certain are the authors 
that a different fit is not possible? Have other fit poses been considered?


Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry and Biochemistry
New York, NY 10031

From: CCP4 bulletin board  on behalf of Jon Cooper 
<488a26d62010-dmarc-requ...@jiscmail.ac.uk>
Sent: Thursday, July 23, 2020 1:07 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] Re: [ccp4bb] Modeling ATP/ADP

Hello, do you have any homologues in the PDB with ATP, etc, bound as a guide? 
Coot is pretty good at fitting known ligands, and unknown ones, too!


 Original Message 
On 23 Jul 2020, 17:53, Reza Khayat < rkha...@ccny.cuny.edu> wrote:


Hi,


Can folks suggest programs for objectively docking ATP/ADP molecules into 
density? Our density is not so good, probably because of occupancy, and we'd 
like a less subjecting approach for modeling. Thanks.


Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry and Biochemistry
New York, NY 10031



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[ccp4bb] Modeling ATP/ADP

[Reza Khayat]

Hi,


Can folks suggest programs for objectively docking ATP/ADP molecules into 
density? Our density is not so good, probably because of occupancy, and we'd 
like a less subjecting approach for modeling. Thanks.


Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry and Biochemistry
New York, NY 10031



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Re: [ccp4bb] visual mask editor?

[Reza Khayat]

?You can generate maps using UCSF Chimera then convert these to binary masks. 
This means you can drag PDB around to generate a mask where ever you like.


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry and Biochemistry
New York, NY 10031

From: CCP4 bulletin board  on behalf of Soisson, Stephen 
M <338a268c4763-dmarc-requ...@jiscmail.ac.uk>
Sent: Thursday, May 28, 2020 12:57 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] Re: [ccp4bb] visual mask editor?

You used to be able to do this in O (dating myself)

From: CCP4 bulletin board  On Behalf Of Bernhard Rupp
Sent: Thursday, May 28, 2020 12:39 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] visual mask editor?

EXTERNAL EMAIL - Use caution with any links or file attachments.
Brief question: Does something like a visual density mask editor exist?
Thx, BR
--
Bernhard Rupp
http://www.hofkristallamt.org/<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.hofkristallamt.org_=DwMGaQ=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0=7QfAmFyI5MFldHxM12Yqegu7ZtXisfwGxAHNyqpr8KM=-4yBBEZaabWsrzmtBh3Qhr4C2Kn6qRri8CnSSCkrzuA=>
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+43 676 571 0536
--
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[ccp4bb] Arp/wARP

[Reza Khayat]

Hi,


I've installed the entire CCP4 7.1 package (ShelX and arpWarp); however, I 
can't find the arp/Warp binaries/scripts (e.g. auto_em.sh). Are these not 
included with the CCP4 package? If not, why not? Thanks.


Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry and Biochemistry
New York, NY 10031



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Re: [ccp4bb] Searching similar local structural conformations

[Reza Khayat]

?Hi Lei,


Rosetta uses something very similar to this for designing proteins that bind to 
ligands/proteins. Perhaps look there.


Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

From: CCP4 bulletin board  on behalf of Zheng, Lei 

Sent: Wednesday, January 8, 2020 10:57 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] [ccp4bb] Searching similar local structural conformations

Dear CCP4ers,

We identify a potential ion binding site formed by four residues in a 
structure. I want to search if any other structures have a similar local 
residual geometry. Is there any programs to perform such a searching? For 
example, to search in Protein Data Bank using coordinates of the binding site 
residues? I appreciate your suggestions.

Happy New Year!
Lei





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Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] ITC question -dimer vs monomer

[Reza Khayat]

?Isn't the entire idea of using ITC that you are measuring an equilibrium 
constant? Wouldn't this eliminate the nuances of what you're looking for (i.e. 
kinetics)? Perhaps you should use SPR to tease out this model?


Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

From: CCP4 bulletin board  on behalf of Bernhard Rupp 

Sent: Thursday, October 3, 2019 11:51 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] Re: [ccp4bb] ITC question -dimer vs monomer

I am not looking for anything yet - I wonder what - if any - the consequences 
of doing it one way or the other would be.
I am reasonably certain that any difference affects the analysis.

Thx, BR

From: CCP4 bulletin board  On Behalf Of Keller, Jacob
Sent: Thursday, October 3, 2019 17:41
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

I don't understand what you are trying to do-are you trying to show, by the 
difference in ITC response, that the predictions you made about the 
oligomerization are true?

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 Bernhard Rupp
Sent: Thursday, October 3, 2019 11:06 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] ITC question -dimer vs monomer

Hi Fellows,

please let me ask the respective experts an ITC question: I have 2 proteins, 
stable and dialyzed in identical buffer.
A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer 
will form.

Intuitively, it should make a difference whether I titrate the dimer with the 
monomer or vice versa.
In the first case, a momomer would initially meet a lot of free dimers, and I 
would expect that randomly, a AB2 complex
is more likely to form than a A2B2 (let's disregard any more complex 
colligative/cooperative effects).

If I drip the dimer into the monomer pool, it is quite likely that the B dimer 
meets 2 free As, and I get right away a higher population of A2B2s.

Maybe at dilutions of ITC and with sufficient equilibration that is not an 
issue at all (again, absent any cooperative effects that might alter the first 
Kd vs. the second, despite the sites on the dimer are at least initially 
equivalent).

Can someone guide me towards literature about this or perhaps share some 
first-hand experience?

Many thanks, BR

--
Bernhard Rupp
http://www.hofkristallamt.org/<https://urldefense.proofpoint.com/v2/url?u=https-3A__urldefense.com_v3_-5F-5Fhttp-3A_www.hofkristallamt.org_-5F-5F-3B-21oCotSwSxbw8-21SE9mT6grUy1tHFSKLCraXt4bhlDri03OEMEyqQUCLAVSLsg3vwn0GTQtxbStgtNvxBs-24=DwMFAg=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0=7ZuSeTWtLa-zqSUcsmMCVR2swEqxCFprx15uq21CKHk=Lxm2SJmbFVHs4eyFZrLgqJ3_PkrVzZDYhsWUJPWKpdE=>
b...@hofkristallamt.org<mailto:b...@hofkristallamt.org>
+1 925 209 7429
+43 676 571 0536
--
Many plausible ideas vanish
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--




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Re: [ccp4bb] Reg: water pentagon at dimer interface

[Reza Khayat]

?The interface doesn't appear to be extensive. What is the buried surface area? 
Is the molecule a dimer in solution, or is the dimerization caused by crystal 
packing?


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

From: CCP4 bulletin board  on behalf of Ronald E. 
Stenkamp 
Sent: Friday, September 27, 2019 11:22 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] Re: [ccp4bb] Reg: water pentagon at dimer interface

I don't know about the myth thing, but I remember Martha Teeter describing 
pentagons of waters in crambin.

Here's a reference:

Water Structure of a Hydrophobic Protein at Atomic Resolution: Pentagon Rings 
of WaterMolecules in Crystals of Crambin  M. M. Teeter  Proceedings of 
the National Academy of Sciences of the United States of America, 1984, 81(1), 
6014-6018.

Ron

From: CCP4 bulletin board  On Behalf Of Vijaykumar 
Pillalamarri
Sent: Friday, September 27, 2019 4:34 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Reg: water pentagon at dimer interface

Dear Community,

I solved the structure of a protein from vibrio. There are two molecules in the 
asymmetric unit of this protein. At the dimer interface, the C-termini of both 
the chains interact with each other with the help of five water molecules that 
form a pentagon. I have attached an image showing both the chains and stereo 
image of dimer interface in the inset. I was wondering if there is any 
significance to this or if there is any relevant literature that explains this 
behavior.

Thank you
Vijaykumar Pillalamarri
C/O: Dr. Anthony Addlagatta
Principal Scientist
CSIR-IICT, Tarnaka
Hyderabad, India-57
Mobile: +918886922975



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Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] challenges in structural biology

[Reza Khayat]

Biophysical techniques used to screen samples (e.g. SEC, SEC-MALS, DLS, SAXS, 
CD...) before freezing are not as promising as many hope for. There are lots of 
examples where samples behave beautiful by multiple biophysics methods and then 
crash and burn on a cryo-EM grid. Consequently, screening freezing conditions 
become the bottle neck of cryo-EM.

?
Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

From: CCP4 bulletin board  on behalf of Patrick Shaw 
Stewart 
Sent: Tuesday, July 23, 2019 1:35 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] Re: [ccp4bb] challenges in structural biology



On a completely different tack, isn't the most pressing requirement in current 
structural biology a really good method of characterizing macromolecular 
samples before they are put onto cryoEM grids - ie analysing and screening them 
in solution.

For one thing I'm told those huge microscopes are quite prone to breaking down, 
which makes the queues (lines) to get onto them even longer.

That method might be (micro-scale) DLS - or something completely different.

Thx, Patrick


On Mon, Jul 15, 2019 at 8:44 PM Holton, James M 
<270165b9f4cf-dmarc-requ...@jiscmail.ac.uk<mailto:270165b9f4cf-dmarc-requ...@jiscmail.ac.uk>>
 wrote:
Hello folks,

I have the distinct honor of chairing the next Gordon Research
Conference on Diffraction Methods in Structural Biology (July 26-31
2020).  This meeting will focus on the biggest challenges currently
faced by structural biologists, and I mean actual real-world
challenges.  As much as possible, these challenges will take the form of
friendly competitions with defined parameters, data, a scoring system,
and "winners", to be established along with other unpublished results
only at the meeting, as is tradition at GRCs.

But what are the principle challenges in biological structure
determination today?  I of course have my own ideas, but I feel like I'm
forgetting something.  Obvious choices are:
1) getting crystals to diffract better
2) building models into low-resolution maps (after failing at #1)
3) telling if a ligand is really there or not
4) the phase problem (dealing with weak signal, twinning and
pseudotranslation)
5) what does "resolution" really mean?
6) why are macromolecular R factors so much higher than small-molecule ones?
7) what is the best way to process serial crystallography data?
8) how should one deal with non-isomorphism in multi-crystal methods?
9) what is the "structure" of something that won't sit still?

What am I missing?  Is industry facing different problems than
academics?  Are there specific challenges facing electron-based
techniques?  If so, could the combined strength of all the world's
methods developers solve them?  I'm interested in hearing the voice of
this community.  On or off-list is fine.

-James Holton
MAD Scientist




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[ccp4bb] DNA or RNA

[Reza Khayat]

Hi,


Sorry for the non-crystallography question. We have a protein complex with 
nucleic acid and would like to know if the nucleic acid is DNA or RNA. Is there 
a sensitive method for doing this where we don't need buckets of the sample? 
Thanks.


Best wishes,
Reza

?
Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031



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[ccp4bb] Sequence variability mapped on a PDB

[Reza Khayat]

Hi,

Thanks. Several suggestions have been made:
1. UCSF Chimera can plot %ID and %Similarity
2. Consurf can plot conservation scores
3. AL2CO can also plot conservation scores

I have a protein with a fast molecular clock, so I need to think about this a 
bit more. 

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031


From: Clarke, Oliver 
Sent: Saturday, March 16, 2019 8:36 PM
To: Reza Khayat
Subject: [EXTERNAL] Re: CCP4BB Digest - 15 Mar 2019 to 16 Mar 2019 (#2019-81)

Hi Reza

You can do more or less this using the ConSurf server - or just load up your 
sequence alignment in Chimera, then you can assign %Id or similarity as a per 
residue attribute.

Cheers
Oli

> On Mar 16, 2019, at 8:03 PM, CCP4BB automatic digest system 
>  wrote:
>
> There is 1 message totaling 85 lines in this issue.
>
> Topics of the day:
>
>  1. Sequence variability mapped on a PDB
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
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>
> --
>
> Date:    Sat, 16 Mar 2019 23:42:18 +
> From:Reza Khayat 
> Subject: Sequence variability mapped on a PDB
>
> ?Hi,
>
>
> Can someone direct me to a program that can replace the B-factors of a PDB 
> with sequence variability calculated from a sequence alignment? I'm have 
> tried the PVS server, but my sequence alignment is too large for its liking. 
> Thanks.
>
>
> Best wishes,
> Reza
>
>
> Reza Khayat, PhD
> Assistant Professor
> City College of New York
> Department of Chemistry
> New York, NY 10031
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
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> End of CCP4BB Digest - 15 Mar 2019 to 16 Mar 2019 (#2019-81)
> 



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[ccp4bb] Off topic question

[Reza Khayat]

?Hi,


Happy new year to all!  A bit of an off topic question.  Does anyone know of a 
method/program to extract the most distinct "n" (n>2) sequences from a sequence 
alignment?  Thanks.


Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031



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Re: [ccp4bb] RNA pdb molecular weight

[Reza Khayat]

Thanks everyone.  I did two things:

1.   Extract the MW from the cif file as suggested by David (see below)

2.   I extracted the RNA sequence from the cif file and ran it through an 
oligo calculator with ssRNA as the option

Both give similar results.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
Department of Chemistry
City College of New York
85 Saint Nicholas Terrace, CDI 2.318
New York, NY 10031
http://www.khayatlab.org/
212-650-6070

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Eleanor 
Dodson
Sent: Friday, November 16, 2018 12:29 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] RNA pdb molecular weight

There is a CCP4 basic program rwcontents

Run it as
rwcontents xyzin rna.pdb
It lists no_of_C m no_of_O etc.. and sums their mass
If you have the H atoms in the pdb that will give you the total mass.

If not you will have to estimate the no of H yourself..
Eleanor




On Fri, 16 Nov 2018 at 16:46, David Armstrong 
mailto:dav...@ebi.ac.uk>> wrote:

Dear Reza,



The calculated molecular weight for each molecule in a PDB entry is given in 
the archive PDBx/mmCIF file. The weight in Daltons is given in the 
_entity.formula_weight category. Please see 
http://www.ebi.ac.uk/pdbe/entry-files/4tna.cif<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.ebi.ac.uk_pdbe_entry-2Dfiles_4tna.cif=DwMFaQ=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0=5itxEGXDI6EW8_C_MVWsmgoRHhNnrOxgjPKbNUqqCzQ=nYiEO2JLKYNAkliZUFwcaU3u07gcmV5ow99_BJl3KhE=>
 for an example file.



Kind Regards,

David

On 16/11/18 16:19, Reza Khayat wrote:
Hi,

I’m not an RNA person. Can anyone suggest a method to calculate the mass of a 
RNA PDB?  I’d like the protons to also be considered in the calculation. Thanks.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
Department of Chemistry
City College of New York
85 Saint Nicholas Terrace, CDI 2.318
New York, NY 10031
http://www.khayatlab.org/<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.khayatlab.org_=DwMFaQ=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0=5itxEGXDI6EW8_C_MVWsmgoRHhNnrOxgjPKbNUqqCzQ=wFjZkIMrlyTPrQL2Ru2KdEVYyDSeelB34KTeiS9qvz0=>
212-650-6070




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--

David Armstrong

Outreach and Training Coordinator

PDBe

European Bioinformatics Institute (EMBL-EBI)

European Molecular Biology Laboratory

Wellcome Trust Genome Campus

Hinxton

Cambridge CB10 1SD UK

Tel: +44 1223 492544



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[ccp4bb] RNA pdb molecular weight

[Reza Khayat]

Hi,

I'm not an RNA person. Can anyone suggest a method to calculate the mass of a 
RNA PDB?  I'd like the protons to also be considered in the calculation. Thanks.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
Department of Chemistry
City College of New York
85 Saint Nicholas Terrace, CDI 2.318
New York, NY 10031
http://www.khayatlab.org/
212-650-6070




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[ccp4bb] Practices for publishing a crystal structure

[Reza Khayat]

Hi,

?

I apologize for not doing the legwork for answering this question. Can an 
article be identified describing the acceptable statistics for publishing a 
crystal structure?  This includes processing the reflections from a crystal and 
refining a structure to the reflection. Thanks.


Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

[ccp4bb] Structure of dextran sulfate

[Reza Khayat]

Hi,

Can anyone point me to the correct structure of dextran sulfate -if there is 
one. Some reviews draw it as an unbranched glucose polysaccharide with sulfates 
on C2, C3, and C4. Others draw it as having sulfates on C2 and C4. Yet a third 
group draws it as branched polysaccharide.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
Department of Chemistry
City College of New York
85 Saint Nicholas Terrace, CDI 2.318
New York, NY 10031
http://www.khayatlab.org/
212-650-6070

[ccp4bb] EM voxel size correction

[Reza Khayat]

?Hi,


Has anyone established a robust method to calibrate the pixel/voxel size of a 
cryo-EM image reconstructions using a known PDB? Thanks.


Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

Re: [ccp4bb] Electrostatic Potential: Poisson-Boltzmann

[Reza Khayat]

I think this is an excellent opportunity to combine MD calculations with your 
structures to see what role(s) this flexible region may play in your structure. 

Reza

Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Dale Tronrud 
[de...@daletronrud.com]
Sent: Saturday, December 2, 2017 1:16 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Electrostatic Potential: Poisson-Boltzmann

   I don't know anything about the practicalities of PDB2PQR but it
would seem to me that you have to calculate a potential for the molecule
with each conformation.  Then you would say "This is the potential with
the A altloc and THIS is the potential with B".  There will be no
individual molecule with the average potential so the average has no
chemical meaning.

   Of course life gets even harder when you have multiple side chains
with multiple conformations.  The combinatorials grow quickly.  If you
don't believe that the conformations are all tied to each other you have
to say that the ensemble of conformations leads to an ensemble of
potentials.  Somehow your chemistry has to work in the presence all this
variability and THAT is the interesting question you have to answer.

   I don't know what an ensemble of potentials looks like so one would
have to calculate some and see what their properties are.  I'm not aware
that anyone has done this, but my literature search has been very limited.

Dale Tronrud

On 12/2/2017 5:51 AM, Sam Tang wrote:
> To add to the discussion, could I raise a relevant question about
> generating ESP (Apologies to Jiri if this distracts too much from your
> initial thread).
>
> In our structure in hand, the density for two conformations of the side
> chain are clearly seen and they could be modeled. This brings a bit of
> problem because the positive charge becomes more prominent with two
> conformations there than with one. So what do we usually do when
> generating ESP for such structures with alternate conformations? Do we
> remove one before the calculation?
>
> PS - I use online PDB2PQR server to do my calculation with PARSE field.
> I did notice from some old archived discussion on the Web that it
> ignores one conformation by default. But this seemingly is not the case
> in newer versions?
>
> Regards
>
> Sam
>
> School of Life Sciences, CUHK
>
> On 2 December 2017 at 02:59, Robbie Joosten <robbie_joos...@hotmail.com
> <mailto:robbie_joos...@hotmail.com>> wrote:
>
> If you cannot trust the surface of your protein, perhaps you should
> not look at the the potential on the surface. Instead you can look
> at the field around your protein. This is less precise, but also
> less sensitive to local errors. If you want to know how your peptide
> finds your protein, this is actually more informative anyway.
>
> There must be several programs that do this. I have done this for
> MHC in the past with YASARA. It really explained nicely how he
> peptide moved in.
>
>
>
> Cheers,
>
> Robbie
>
>
>
> Sent from my Windows 10 phone
>
>
>
> 
> *From:* CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK
> <mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Dale Tronrud
> <de...@daletronrud.com <mailto:de...@daletronrud.com>>
> *Sent:* Friday, December 1, 2017 7:29:01 PM
> *To:* CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
> *Subject:* Re: [ccp4bb] Electrostatic Potential: Poisson-Boltzmann
>
>These are not easy questions to answer.  Certainly atoms,
> particularly ones that are charged, even with fractional charges, have a
> strong effect on the ESP.  If you delete them because you don't know
> exactly where they are you will get a different answer than if you put
> them in in some reasonable but unsupported location (as you have found).
>  This result indicates that the peptide does affect the ESP
> significantly and you have to consider it.
>
>You could build lots of models with the peptide in different
> conformations and average all the maps.  This misses the point.  You
> have uncertainty in your model which means that you have uncertainty in
> your electrostatic potential.  Any particular ESP that you calculate and
> draw conclusions from will have a large uncertainty and you must
> consider that uncertainty when deciding between your potential
> conclusions.  (I'm not sure if the pun is intended or not!)
>
>I suppose you could believe that each possible conformation exists to
&

[ccp4bb] RMSD calculation for large assemblies

[Reza Khayat]

Hi,


I'm analyzing the RMSD between 60 subunits of a virus. Can someone identify a 
program that can generate a spread for the RMSD between equivalent C-alpha 
atoms? For example, the C-alpha atom for amino acid 39 may have RMSD values 
from 0.1 to 1.5. Coot does a nice job of automatically detecting and 
calculating RMSD, but I'd like to have the spread for each atom in the final 
graph that Coot generates. Thank you.


Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

[ccp4bb] Biotin binding protein

[Reza Khayat]

Hi,

Sorry for the non crystallography question. Is anyone aware of monomeric 
proteins (<20kDa) that can bind to biotin? We need this for a couple of 
different projects where biotin covalently modifies a ligand of interest. We'd 
like to complex the biotin to a protein Thanks.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

[ccp4bb] Unbranched polysaccharide chain

[Reza Khayat]

Hi,

Is there a server/program that can generate a chemical picture/PDB from the 
sequence of a polysaccharide and the type of connections? I'm not so interested 
in the minimized structure at this point. Thanks.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
Department of Chemistry
City College of New York
85 Saint Nicholas Terrace, CDI 2.318
New York, NY 10031
http://www.khayatlab.org/
212-650-6070

[ccp4bb] Coot no GUI

[Reza Khayat]

Hi,

Is it possible to run Coot without the use of GUI?  We have some Coot scripts 
for a pipeline and we'd like to do without the GUI. Thanks.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
Department of Chemistry
City College of New York
85 Saint Nicholas Terrace, CDI 2.318
New York, NY 10031
http://www.khayatlab.org/
212-650-6070

Re: [ccp4bb] Weak density of RNA in a complex structure

[Reza Khayat]

Dear Chen,

Is this an icosahedral virus crystal structure, or a highly symmetric structure 
where the RNA may have different binding modes to each subunit and thus 
averaged out in the crystal?

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Nicolas FOOS 
[nicolas.f...@esrf.fr]
Sent: Wednesday, June 21, 2017 4:45 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Weak density of RNA in a complex structure

Dear Chen,

I will answer with some question :

- How is the refinement going ? Is the RNA properly taking in count ? Which 
soft do you use ?

- How do you solve the structure ? MR ? If it's MR did you use a model which 
contain both protein and RNA ? Did you try to solve with the protein only ? 
Maybe you are "forging" RNA.

- Depending of your data (wavelenght and redundancy) you can try to calculate 
an anomalous difference map to see the phosphorus of the RNA to be more 
confident.

- Last point, it could be the occupancy connected with the stability of the 
complex, maybe sometimes RNA is here sometimes not. I see that one time with 
two complex in the same ASU, one has one missing partner.

For my side, I really like to refine my DNA-protein complex with Buster from 
global Phasing. It gives you a very nice and informative density map. If you 
have this possibility let's try.

Hope to help.

Nicolas

Nicolas Foos
PhD
Structural Biology Group
European Synchrotron Radiation Facility (E.S.R.F)
71, avenue des Martyrs
CS 40220
38043 GRENOBLE Cedex 9
+33 (0)6 76 88 14 87
+33 (0)4 76 88 45 19


On 21/06/2017 09:34, Chen WeiFei wrote:

Dear All,


We have get a complex crystal and the resolution can be refined to nearly 1.84 
Å. But the electron density of the RNA is very weak.

In some datasets we can't find any density of the RNA and in other datasets we 
can see more or less some RNA density.

For now we can build 6-7 nucleotides but we can't distinguish the rigth 
sequence.

If anyone has the same problem and how to solve this problem.

Best Regards,


Dr Wei-Fei Chen

College of Life Sciences,

Northwest A University

[ccp4bb] Rhodamine in FPLC

[Reza Khayat]

Hi,

Sorry for the non crystallography question. We're using Rhodamine for labeling 
some material that we need to run through our FPLC. It seems that the Rhodamine 
is getting stuck to the PEEK tubing. Anyone has experience with successfully 
removing the Rhodamine from the tubing? Thanks.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

Re: [ccp4bb] Structure comparison

[Reza Khayat]

Hi,

My initial e-mail may have been a bit vague so I'll try to be more specific. 
Superposing the structures and comparing them against one another, while 
appropriate, is a subjective way to do the analysis as I would have to 
subjectively define a threshold that would indicate a difference between the 
structures. My threshold may be grossly different than someone else's 
threshold. I am interested in an objective criterion. One where strong emphasis 
has been put on error analysis and error modeling in terms of both the refined 
structure and the underlying data. I realize that defining such criterion is by 
no means trivial. Thanks again for the help.

Best wishes,
Reza 

Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031


From: Reza Khayat
Sent: Sunday, April 9, 2017 6:07 PM
To: CCP4 bulletin board
Subject: Structure comparison

Hi,

I have refined several structures of a protein from different space groups and 
would like to compare them to one another. Is there a program/software suite 
that would provide an objective comparison of the structures and identify 
regions where the structures are sufficiently different from one another to 
warrant a closer look? I think the most important aspect of the analysis would 
be defining a threshold (possibly based on resolution and structure statistics) 
that would identify sufficient difference between structures. Thanks.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

[ccp4bb] Structure comparison

[Reza Khayat]

Hi,

I have refined several structures of a protein from different space groups and 
would like to compare them to one another. Is there a program/software suite 
that would provide an objective comparison of the structures and identify 
regions where the structures are sufficiently different from one another to 
warrant a closer look? I think the most important aspect of the analysis would 
be defining a threshold (possibly based on resolution and structure statistics) 
that would identify sufficient difference between structures. Thanks.

Best wishes,
Reza 

Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

[ccp4bb] Off topic instrumentation

[Reza Khayat]

Hi,

Sorry for another non crystallography question. Can someone suggest a fraction 
collector to collect fractions from a CsCl/glycerol/sucrose/... gradient from 
an ultracentrifugation run? Thanks.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

Re: [ccp4bb] Completely Off-Topic

[Reza Khayat]

I don't think this is taught in Biochem101. You didn't miss it. The cytoplasm 
is quite viscous, like jello. 



Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Tim Gruene 
[tim.gru...@psi.ch]
Sent: Thursday, January 12, 2017 3:55 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Completely Off-Topic

Dear JPK,

I was not aware of the absolute numbers, but maybe they are little suprising:
when your tinned food contains 'yeast extract' it is equivalent to monosodium
glutamate, which is commonly used as flavour enhancing agent.

I am not a chemist to worry about it, but yeast seems to have a fullfilling
life with it.

Best,
Tim

On Thursday, January 12, 2017 12:45:03 AM CET Keller, Jacob wrote:
> Dear Crystallographers,
>
> Was anyone else aware that in E coli the intracellular glutamate
> concentration is ~100 mM? Also other cell types (yeast, mammalian) are 10s
> mM. Anything to say about this? I learned of this just recently, and have
> been amazed about it for more than a week. Did I miss this in Biochem 101?
> Does it matter?
>
> JPK
>
> ***
> Jacob Pearson Keller, PhD
> Research Scientist
> HHMI Janelia Research Campus / Looger lab
> Phone: (571)209-4000 x3159
> Email: kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>
> ***

--
--
Paul Scherrer Institut
Tim Gruene
- persoenlich -
OFLC/102
CH-5232 Villigen PSI
phone: +41 (0)56 310 5297

GPG Key ID = A46BEE1A

[ccp4bb] Off-topic question about SEC

[Reza Khayat]

Hi,


Sorry for the off-topic question. Can a protein in lower [NaC] run faster on a 
SEC than at higher [NaCl] (i.e. elute at an earlier volume)? The protein elutes 
well within the resolution limits of the SEC with a symmetric gaussian A280 
profile. I know that at lower [NaCl] the protein can elute later because it may 
interact with the matrix.  Thanks.


Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

[ccp4bb] Detecting chlorides in x-tals

[Reza Khayat]

Hi,

With the hopes of not reigniting the heated "Chloride or water" CCP4 discussion 
in Jan. 2015, what experimental method is suggested for identifying chlorides 
in a x-tal?

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

Re: [ccp4bb] visible side-chains but breaks in main-chain

[Reza Khayat]

I would have to agree with Tim. I'm a little surprised that your R/Rf is 27/32. 
Are the images representative of the entire model/map fits, or a small section 
of it?

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
Department of Chemistry
City College of New York
85 Saint Nicholas Terrace, CDI 2.318
New York, NY 10031
http://www.khayatlab.org/
212-650-6070

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tim Gruene
Sent: Thursday, December 01, 2016 9:19 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] visible side-chains but breaks in main-chain

Dear V,

your map still looks quite poor and disconnected. Maybe you could improve your 
overall model before you worry about such detailed questions like main chain 
vs. side chain density.

If your resolution is not too poor, you could try rebuilding your model with 
shelxe, or buccanner, or phenix, or ...

Regards,
Tim

On Thursday, December 01, 2016 02:35:21 PM Veronica Fiorentino wrote:
> Dear bb-ers,
> Is it uncommon to see side-chains for PHE/ARG visible in density but 
> the main-chain density breaking (say within a beta-strand)? I have 
> attached 1.FEM (map) 2&3. Experimental phase map. If I refine the 
> model as Poly-ala, the side-chains appear green in difference map. I 
> therefore assume that my sequence assignments are correct(?). Is this 
> symptomatic of some big mistake?
> 
> R/Rfree are around 27/32 % respectively. The phase problem was solved 
> by SeMet phasing.
> 
> Many thanks
> V[image: Inline images 1]
> 
> [image: Inline images 2]
> 
> [image: Inline images 3]
--
--
Paul Scherrer Institut
Dr. Tim Gruene
- persoenlich -
Principal Investigator
Biology and Chemistry
OFLC/102
CH-5232 Villigen PSI

Phone: +41 (0)56 310 5297

GPG Key ID = A46BEE1A

Re: [ccp4bb] FW: [ccp4bb] Fwd: [ccp4bb] just out of totally idle curiosity ...

[Reza Khayat]

I Agree with Edward. No discussion of politics, religion, or sex at work.


Best wishes,

Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Gloria Borgstahl 
<gborgst...@gmail.com>
Sent: Wednesday, November 9, 2016 1:24 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] FW: [ccp4bb] Fwd: [ccp4bb] just out of totally idle 
curiosity ...

Eddie Snell for President

On Wed, Nov 9, 2016 at 11:02 AM, Edward Snell 
<esn...@hwi.buffalo.edu<mailto:esn...@hwi.buffalo.edu>> wrote:
As a Brexit and Trumpet affected person having a foot in both countries ,this 
topic is too far off the normal discussion on CCP4 and probably better taken up 
privately.  CCP4 is not a political discussion site. With CCP4 the signal is 
unusually high and the noise low when compared to any discussion board. I for 
one would like to keep it there. Political views aside, we're all trying to 
achieve the same scientific goals. Let's remember that and keep that the focus.

Edward Snell Ph.D.
President and CEO Hauptman-Woodward Medical Research Institute
Assistant Prof. Department of Structural Biology, University at Buffalo
700 Ellicott Street, Buffalo, NY 14203-1102
Phone: (716) 898 8631<tel:%28716%29%20898%208631> Fax: (716) 898 
8660<tel:%28716%29%20898%208660>
Skype:  eddie.snell Email: 
esn...@hwi.buffalo.edu<mailto:esn...@hwi.buffalo.edu>
[cid:image003.png@01D23A81.3A9485D0]
Heisenberg was probably here!

Re: [ccp4bb] Superpose program in CCP4

[Reza Khayat]

I've done this sort of analysis in the past and it can be very useful depending 
on your story is. I'm not sure if any software offers this. My solution was to 
write a script to do the comparison after the superposition was done. 
Unfortunately I can't find the script.


Best wishes,
Reza


Reza Khayat, PhD

Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Edward Berry 
<ber...@upstate.edu>
Sent: Sunday, October 30, 2016 9:13 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Superpose program in CCP4

WenHe,
I'm not sure if you want to superimpose a number of structures (as rmsd would 
imply) or just compare two structures.
If you want a complete list of the distances between corresponding atoms in two 
pdb files of identical sequence,
you can use the fortran program http://www.cytbc1.net/berry/for/pdbdist2b.for
(linux executable http://www.cytbc1.net/berry/for/pdbdist2b ).
You need to superpose the structures (if necessary) with something else, and 
save the two files.
Grep out C-alphas to two new files if you only care about them.

The program asks you for the names of the two files and the residue number at 
which
to start (this latter is to synch the files in case they have different start 
residues. If
sequences are identical, just give the first residue number)
It also asks for last residue to compare in first file - just give a large 
number to do all.
Then it asks for a threshold - only distances larger will be printed. Put -1 to 
print all.

You can put all parameters on the command line if you run it with the shell 
script pdbd2b:
echo 'Find distances greater than threshold between corresponding atoms in 2 
PDB files'
echo 'Usage: pdbd2b file1 file2 startres# [thresh]'
pdbdist2b <>> WENHE ZHONG <wenhezhong.xmu@gmail.com> 10/29/16 11:49 AM >>>
Dear all,

I always use the SUPERPOSE tool in CCP4 to superpose molecules. This time I 
want to use the RMSD values of superposed C-alpha atoms to plot a RMSD graph 
(instead of using the graph automatically made by the program). However, there 
are many atoms missing in the RMSD list.

In the settings I chose "Superpose specific atoms/residues", checked "Output 
all distances to a file", fit "C-alpha atoms". The superposed structures have 
exactly the same sequence.

My question is: is there any way to get the completed list of RMSD value for 
each C-alpha atom? Or is there any other program for this purpose?

Thank you!

Kind regards,
Wenhe

[ccp4bb] ssDNA

[Reza Khayat]

Hi,

Sorry for the non-crystallography question, does anyone know how to produce 
milligram quantities of single stranded DNA? Thanks.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
Department of Chemistry
City College of New York
85 Saint Nicholas Terrace, CDI 12318
New York, NY 10031
http://www.khayatlab.org/
212-650-6070

Re: [ccp4bb] How to fit BioSAXS shape to the Structure

[Reza Khayat]

That's interesting. Enantiomer differences can be detected at worse than 
20Angstrom resolution in EM reconstructions. What do you think is the reason 
for this? 


Reza Khayat, PhD
Assistant Professor
Department of Chemistry
City College of New York
85 Saint Nicholas Terrace, CDI 12318
New York, NY 10031
http://www.khayatlab.org/
212-650-6070

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Zbyszek 
Otwinowski
Sent: Friday, June 26, 2015 11:43 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] How to fit BioSAXS shape to the Structure

At low resolution, without interpretable anomalous signal, neither SAXS nor 
molecular replacement with SAXS model, can distinguish correct from inverted 
solution. So inverted model will fit crystal data equally well.

Only phase extension to much higher resolution (e.g. 5A) can help.




 Yes, SAXS has an enantiomer problem - mirror image DAMMIN/F 
 reconstructions will give the same fit to the raw scattering data, 
 whereas your protein structure will only fit one hand.

 SUPCOMB can certainly deal with this problem, as detailed in 
 http://www.embl-hamburg.de/biosaxs/manuals/supcomb.html




 [image: David Briggs on about.me]

 David Briggs
 about.me/david_briggs
   http://about.me/david_briggs

 On 26 June 2015 at 12:04, Reza Khayat rkha...@ccny.cuny.edu wrote:

  Hi,

  Follow up question on SAXS. Does SAXS have an enantiomer problem 
 like electron microscopy? In other words, does the calculated model 
 possess the correct handedness or can both handedness of a model fit 
 the scattering profile just as well?

  Best wishes,
 Reza

Reza Khayat, PhD
 Assistant Professor
 City College of New York
 85 St. Nicholas Terrace CDI 12308
 New York, NY 10031
 (212) 650-6070
  www.khayatlab.org

  On Jun 26, 2015, at 6:50 AM, David Briggs drdavidcbri...@gmail.com
 wrote:

  SASTBX has an online tool for achieving this:
 http://sastbx.als.lbl.gov/cgi-bin/superpose.html



 [image: David Briggs on about.me]

 David Briggs
  about.me/david_briggs
   http://about.me/david_briggs

 On 26 June 2015 at 11:39, Ashok Nayak ashokgocrac...@gmail.com wrote:

Dear Weifei,

  It can also be done manually in Pymol by changing the mouse mode 
 from
 3
 button viewing to 3 button editing and later moving the envelope 
 onto the X-ray structure or vice-versa, however the best fit can be 
 achieved in SUPCOMB.

  regards
  Ashok Nayak
  CSIR-CDRI, Lucknow
  India







Zbyszek Otwinowski
UT Southwestern Medical Center at Dallas
5323 Harry Hines Blvd.
Dallas, TX 75390-8816
Tel. 214-645-6385
Fax. 214-645-6353

Re: [ccp4bb] How to fit BioSAXS shape to the Structure

[Reza Khayat]

Hi,

Follow up question on SAXS. Does SAXS have an enantiomer problem like electron 
microscopy? In other words, does the calculated model possess the correct 
handedness or can both handedness of a model fit the scattering profile just as 
well?

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
City College of New York
85 St. Nicholas Terrace CDI 12308
New York, NY 10031
(212) 650-6070
www.khayatlab.orghttp://www.khayatlab.org

On Jun 26, 2015, at 6:50 AM, David Briggs 
drdavidcbri...@gmail.commailto:drdavidcbri...@gmail.com wrote:

SASTBX has an online tool for achieving this: 
http://sastbx.als.lbl.gov/cgi-bin/superpose.html


http://about.me/david_briggs

[David Briggs on about.me]

David Briggs
about.me/david_briggs



On 26 June 2015 at 11:39, Ashok Nayak 
ashokgocrac...@gmail.commailto:ashokgocrac...@gmail.com wrote:
Dear Weifei,

It can also be done manually in Pymol by changing the mouse mode from 3 button 
viewing to 3 button editing and later moving the envelope onto the X-ray 
structure or vice-versa, however the best fit can be achieved in SUPCOMB.

regards
Ashok Nayak
CSIR-CDRI, Lucknow
India

Re: [ccp4bb] Protein precipitation

[Reza Khayat]

Hi,

Try using 100mM Ammonium Citrate pH 8.5 as the buffer for your lysis -add salts 
as well. The Citrate will inhibit metalloproteases and is Ni-NTA friendly. It 
solved our proteolysis problems and may help with yours.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
City College of New York
85 St. Nicholas Dr. CDI 12308
New York, NY 10031
(212) 650-6070
www.khayatlab.orghttp://www.khayatlab.org

On May 19, 2015, at 2:05 AM, Manjula Ramu 
manjula@gmail.commailto:manjula@gmail.com wrote:

Thanks all for the suggestions.
@ Pius,
I used bacterial expression system.Yes I centrifuged precipitated protein 
sample and found more amount in soluble form itself, however within a day 
protein gets degraded. If I have to further purify the protein I have to 
concentrate the IMAC elutes and concentrate using filters. In this step protein 
degradation was more and filter used to block and could not complete the process
.
Do you use batch method of elution or gradient???
Also do you add PMSF in the elution buffer???

Yes Nikhil I did batch elution and included PMSF in all the buffers. and I 
tried using 500mM NaCl too but no change was observed.


Thanks and Regards,
Manjula R
Research Scholar
Department of Biophysics
National Institute of Mental Health and Neurosciences
Bengaluru-29, Karnataka
INDIA
E-mail: manjula@gmail.commailto:manjula@gmail.com
Mobile no:+91-9538553356
http://www.nimhans.kar.nic.in/
https://www.nimhans.kar.nic.in/

On Tue, May 19, 2015 at 11:14 AM, PULSARSTRIAN 
bhanu.hydpri...@gmail.commailto:bhanu.hydpri...@gmail.com wrote:
Try increasing NaCl to 500 mM and glycerol to 10%..And try reduce bME to 1mM 
for IMAC using Ni-NTA. If your protein requires any additional co-factors (like 
Mg, Ca or Zn) add them as chloride salts upto 1 to 5 mM.

On Tue, May 19, 2015 at 10:19 AM, Manjula Ramu 
manjula@gmail.commailto:manjula@gmail.com wrote:
Hi all,

I am purifying a basic protein with pI 7.8. I used 50mM tris 8.8, 300 mm NaCl, 
5% glycerol,  0.1mM PMSFand 5mM beta ME  in the buffer and performed affinity 
purification. Eluted with 200mM imidazole.  While elution I could see slight 
turbid in eluted protein (I get pure protein, single band on SDS-PAGE). 
However, when I centrifuged I couldn't see any pellet. After some time(a day) 
protein got degraded. In other method I tried cation exchange purification of 
lysate with MES buffer pH 6.5. Here also I saw slight precipitation and later 
degradation.

After this again I tried with MES buffer pH 5.5 and PIPES buffer pH 6.0, here 
also same problem I faced.

Please suggest me a method where I can get stable protein.

Thanks and Regards,
Manjula R
Research Scholar
Department of Biophysics
National Institute of Mental Health and Neurosciences
Bengaluru-29, Karnataka
INDIA
E-mail: manjula@gmail.commailto:manjula@gmail.com
Mobile no:+91-9538553356
http://www.nimhans.kar.nic.in/
https://www.nimhans.kar.nic.in/



--
B4U

Re: [ccp4bb] Cleaved peptide density!

[Reza Khayat]

Hi Dipkar,

My understanding is that the majority of a protease's activity comes from the 
oxyanion hole activating the scissile bond. Mutating the nucleophile reduces 
activity, sometimes appreciably, but does not kill the enzyme. Were your 
SDS-PAGE experiments on the same time scale and buffer conditions as your 
crystallography experiments? Perhaps something in the crystallization buffer 
increases the activity of the enzyme, or all/some of the peptide is digested in 
the time it takes to attain crystals -or only crystals of the digested peptide 
complex can be attained.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
Department of Chemistry
City College of New York
New York, NY 10031
http://www.khayatlab.org/
212-650-6070

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Radisky, 
Evette S., Ph.D.
Sent: Wednesday, April 22, 2015 3:40 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Cleaved peptide density!

We had a similar situation with a catalytically dead serine protease.  
Initially I was excited to think we might be seeing residual catalytic activity 
of the mutant enzyme on a highly specific substrate; however, the activity 
turned out to result from contamination with a very small amount of wt enzyme, 
likely as a result of using the same affinity column to purify both.  When we 
incubated the enzyme preparation with PMSF (an inhibitor that covalently 
modifies the catalytic serine and would not have affected the mutant), we 
eliminated the residual activity of the enzyme preparation.  However, in our 
case we never were able to get crystals with the intact substrate, which 
apparently was not compatible with our crystal form.

Is there a covalent inhibitor of your cysteine protease that you could use to 
pre-treat your enzyme, to see if this eliminates the activity?  If so this 
might help distinguish between residual activity of the mutant vs. 
contamination with wt enzyme.

Good luck!
Evette

Evette S. Radisky, Ph.D.
Associate Professor and Senior Associate Consultant
Department of Cancer Biology
Mayo Clinic Cancer Center
Griffin Cancer Research Building, Rm 310
4500 San Pablo Road
Jacksonville, FL 32224
(904) 953-6372
http://www.mayo.edu/research/labs/proteases-cancer/

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dipankar 
Manna
Sent: Monday, April 20, 2015 2:42 PM
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Cleaved peptide density!

Dear Crystallographers,

I am working with a cysteine protease. I co-crystallized the protease with some 
small chemically synthesised peptides of 7 amino acid residues. I mutated the 
active Cysteine residue with Alanine to avoid the peptide cleavage so that I 
can get the whole peptide bound with my protein. But interesting I got the 
density for a cleaved peptide with 4 amino acids instead of the whole peptide. 
The resolution is 1.4 A, I can see the clear cleavage and the cleavage occurred 
exactly at the same peptide bond where it should. But I do not know how!

Now my question is why I am getting the cleaved peptide as I already mutated 
the active residue Cysteine with Alanine (this mutant did no show any activity 
when I checked with SDS-PAGE).

If anybody has the same kind of experience please advice me.

Thanks in advance.

Best,

Dipankar

--
Dipankar Manna
Research Scholar
Department of Chemistry
University of Oslo
Oslo, Norway

Re: [ccp4bb] on NCS restraint

[Reza Khayat]

Hi,

I have to agree with Steven. I was too haste in my reply to the initial e-mail. 
As Steven put it, it is best to run multiple experiments at the same time and 
identify which produces the best result.

On a follow up to Victor Lamin's reply, have the results been published?

Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
Department of Chemistry
City College of New York
New York, NY 10031
http://www.khayatlab.org/
212-650-6070

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Victor 
Lamzin
Sent: Tuesday, April 21, 2015 9:07 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] on NCS restraint


Hi all,

We have carried out a large-scale test of the use of Refmac's NCS-restraints 
during model building with ARP/wARP. We have found advantageous to have such 
restraints turned on with data resolution extending to as high as 1.5 A.

Victor



On 21/04/2015 14:46, Sheriff, Steven wrote:

All:



I strongly disagree with Reza's suggestion that one should abandon NCS at 
better than 2.5 Å resolution (or even Herman's suggestion at better than 2.0 Å 
resolution). Either of these may be true in any particular case, BUT one should 
do the experiment - Run parallel refinements from the same starting model with 
and without NCS restraints and compare R-free, the gap between R-free and 
R-work, and particular places where one knows or suspects that the local 
geometry is different, before deciding to abandon NCS restraints. This was 
certainly true in the bad old days when loose (as opposed to strict) NCS 
restraints were used and bound each chain more-or-less to a single chain's 
geometry, even though one was refining all extant chains. Using so-called 
loose NCS restraints, I once had a loop pulled out of electron density during 
refinement and the tip moved ~6 Å!



However, for the last 5 years or so, BUSTER, which I use, and REFMAC (and 
presumably PHENIX) have used LSSR (local secondary structure restraints) where 
the maximum pull to uniformity tops out at a certain value. I have not 
rigorously followed my own advice above to run parallel refinements, but I have 
yet to find a case where LSSR-type NCS restraints have hurt the refinement 
down to at least ~1.5 Å resolution.



To give credit where it is due, Oliver Smart, who implemented LSSR in BUSTER, 
attributed the concept to George Sheldrick.



Steven



--

Date:Mon, 20 Apr 2015 10:38:27 +

From:Reza Khayat rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edu

Subject: Re: [ccp4bb] on NCS restraint



Hi,



The purpose of NCS is to reduce the degrees of freedom in order to avoid over 
refinement -not only to expedite refinement. Strict or restrained NCS should be 
applied at lower resolutions (2.7Å) or data completeness. Forgo NCS If you 
have a complete and better than 2.5Å dataset. Also, you can define the regions 
where NCS is applied and thus avoid loops/regions where the NCS is violated.



Best wishes,

Reza



Reza Khayat, PhD

Assistant Professor

City College of New York

160 Convent Ave, MR-1135

New York, NY 10031

(212) 650-6070

rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edu%3cmailto:rkha...@ccny.cuny.edu



--

On Apr 20, 2015, at 4:01 AM, 
herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.com%3cmailto:herman.schreu...@sanofi.com
 wrote:



Dear Smith,



There used to be something called strict NCS which meant that instead of many 
identical subunits, only one average subunit was refined, which would speed 
up the refinement significantly, at the expense of requiring that all subunits 
are exactly identical.



I do not think that this option is used anymore and most refinement programs 
would require NCS related subunits to be similar, but not identical to each 
other. As Robbie Joosten pointed at, this can help a lot, especially when you 
do not have high resolution data. So for data with better than 2.0 Å 
resolution, including NCS restraints would probably not make a big difference, 
but otherwise I would switch them on.



Best,

Herman



--

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Smith Liu

Gesendet: Freitag, 17. April 2015 06:02

An: 
CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK%3cmailto:CCP4BB@JISCMAIL.AC.UK

Betreff: Re: [ccp4bb] on NCS restraint



Dear Jurgen,



My understanding is that NCS restraint can significantly enhance the speed of 
calculation, but considering the subunits even with the eactly same sequence 
may not be identical, to have NCS restraint may be not necessary or may be not 
good for the refinement, am I right?



Smith



At 2015-04-17 09:09:05, Jurgen Bosch 
jbos...@jhu.edumailto:jbos...@jhu.edumailto:jbos...@jhu.edu%3cmailto:jbos...@jhu.edu
 wrote:



yes.

Have two sets of NCS operators one that describe the four subunits and one 
describing

Re: [ccp4bb] [ccp4bb] on NCS restraint

[Reza Khayat]

Hi,

The purpose of NCS is to reduce the degrees of freedom in order to avoid over 
refinement -not only to expedite refinement. Strict or restrained NCS should be 
applied at lower resolutions (2.7Å) or data completeness. Forgo NCS If you 
have a complete and better than 2.5Å dataset. Also, you can define the regions 
where NCS is applied and thus avoid loops/regions where the NCS is violated.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
City College of New York
160 Convent Ave, MR-1135
New York, NY 10031
(212) 650-6070
rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edu


On Apr 20, 2015, at 4:01 AM, 
herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.com wrote:

Dear Smith,

There used to be something called “strict NCS“ which meant that instead of many 
identical subunits, only one “average” subunit was refined, which would speed 
up the refinement significantly, at the expense of requiring that all subunits 
are exactly identical.

I do not think that this option is used anymore and most refinement programs 
would require NCS related subunits to be similar, but not identical to each 
other. As Robbie Joosten pointed at, this can help a lot, especially when you 
do not have high resolution data. So for data with better than 2.0 Å 
resolution, including NCS restraints would probably not make a big difference, 
but otherwise I would switch them on.

Best,
Herman



Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Smith Liu
Gesendet: Freitag, 17. April 2015 06:02
An: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] on NCS restraint

Dear Jurgen,

My understanding is that NCS restraint can significantly enhance the speed of 
calculation, but considering the subunits even with the eactly same sequence 
may not be identical, to have NCS restraint may be not necessary or may be not 
good for the refinement, am I right?

Smith




At 2015-04-17 09:09:05, Jurgen Bosch 
jbos...@jhu.edumailto:jbos...@jhu.edu wrote:

yes.
Have two sets of NCS operators one that describe the four subunits and one 
describing the two subunits. If during the refinement of your structure you 
should find out that the subunits are not identical to each other you can relax 
the NCS weights.

Jürgen
..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry  Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742tel:%2B1-410-614-4742
Lab:  +1-410-614-4894tel:%2B1-410-614-4894
Fax:  +1-410-955-2926tel:%2B1-410-955-2926
http://lupo.jhsph.eduhttp://lupo.jhsph.edu/

On Apr 16, 2015, at 9:02 PM, Smith Lee 
0459ef8548d5-dmarc-requ...@jiscmail.ac.ukmailto:0459ef8548d5-dmarc-requ...@jiscmail.ac.uk
 wrote:


Dear All,

If a protein contains 6 subunits, 4 subunits from the same sequence (subunit A, 
B, C, D all from the same sequence), each of the 2 other subunits from 2 
diffrent sequences (subunit E from the second sequence, subunit F from the 
third sequence), in this situation should I use NCS restraint or not?

If my protein contains 2 subunits, both of the 2 subunits composed of the 
eaxctly same sequence, however supposing the 2 subunits have a little diffrent 
conformation, in this situation should we use NCS retraint or not?

Smith

Re: [ccp4bb] Protein expression

[Reza Khayat]

Hi,

I’ve received a number of concurring suggestions. Some have requested more 
detail about the experiments. Here are the details.

1. Cells: BL21(DE3)
2. Plasmid: pET28a (T7 promoter)
3. Media: TB
4. Protein: cytoplasmic
5. Expression: Grow at 37degC until OD600=0.6, cool to 20degC, induce with 1mM 
IPTG, grow 16hrs at 20degC, centrifuge.
6. Lysis method: Sonication via micro-tip/macro-tip. Small culture produces 
soluble protein where as large culture produces insoluble protein. We first 
thought it may be due to poor lysis, thus equivalent amount of cells from 3 and 
500ml cultures were lysed with the micro-tip. Same results were observed.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
City College of New York
160 Convent Ave, MR-1135
New York, NY 10031
(212) 650-6070
rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edu


On Mar 19, 2015, at 11:15 PM, John Fisher 
johncfishe...@gmail.commailto:johncfishe...@gmail.com wrote:

Hi Reza.
Clearly nobody needs to know anything about what protein you are specifically 
working on; that being said, in order to avoid a potentially endless email 
string of expert advices, please include everything detail-wise regarding your 
expression system, culture conditions, induction, and lysis method for BOTH the 
3 ml and 500 ml expression trials. You will get, I imagine, amazing advices 
likely specific enough to solve the problem without your having to chase your 
tail.
Best,
John Fisher

John C. Fisher, M.D./PhD

On Thu, Mar 19, 2015 at 2:33 PM, Reza Khayat 
rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edu wrote:
Hi,

We can express quite a bit of soluble protein when growing 3ml cultures. 
However, the protein becomes insoluble (inclusion bodies) when we scale up to 
500ml cultures. Has anyone experienced such a problem, and found a solution to 
it? Thanks.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
Department of Chemistry
City College of New York
New York, NY 10031
http://www.khayatlab.org/
212-650-6070tel:212-650-6070

Re: [ccp4bb] Protein expression

[Reza Khayat]

Hi,

Cultures are being properly cooled prior to induction (water bath supplemented 
with ice).

Reza

Reza Khayat, PhD
Assistant Professor
Department of Chemistry
City College of New York
New York, NY 10031
http://www.khayatlab.org/
212-650-6070

From: lieh low [mailto:liehy...@gmail.com]
Sent: Friday, March 20, 2015 8:06 AM
To: Reza Khayat
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Protein expression

Reza,
someone might have mentioned this, it takes longer time to cool down larger 
culture, we turn down the temp of the shaker when OD is about 0.4. For some 
protocol, we even use ice to cool the flask down before induction. You might 
also want to consider a lower induction temp, like 16degC. Maybe your protein 
is just temp sensitive.

ray

On Fri, Mar 20, 2015 at 6:56 AM, Reza Khayat 
rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edu wrote:
Hi,

I’ve received a number of concurring suggestions. Some have requested more 
detail about the experiments. Here are the details.

1. Cells: BL21(DE3)
2. Plasmid: pET28a (T7 promoter)
3. Media: TB
4. Protein: cytoplasmic
5. Expression: Grow at 37degC until OD600=0.6, cool to 20degC, induce with 1mM 
IPTG, grow 16hrs at 20degC, centrifuge.
6. Lysis method: Sonication via micro-tip/macro-tip. Small culture produces 
soluble protein where as large culture produces insoluble protein. We first 
thought it may be due to poor lysis, thus equivalent amount of cells from 3 and 
500ml cultures were lysed with the micro-tip. Same results were observed.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
City College of New York
160 Convent Ave, MR-1135
New York, NY 10031
(212) 650-6070tel:%28212%29%20650-6070
rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edu

On Mar 19, 2015, at 11:15 PM, John Fisher 
johncfishe...@gmail.commailto:johncfishe...@gmail.com wrote:

Hi Reza.
Clearly nobody needs to know anything about what protein you are specifically 
working on; that being said, in order to avoid a potentially endless email 
string of expert advices, please include everything detail-wise regarding your 
expression system, culture conditions, induction, and lysis method for BOTH the 
3 ml and 500 ml expression trials. You will get, I imagine, amazing advices 
likely specific enough to solve the problem without your having to chase your 
tail.
Best,
John Fisher

John C. Fisher, M.D./PhD

On Thu, Mar 19, 2015 at 2:33 PM, Reza Khayat 
rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edu wrote:
Hi,

We can express quite a bit of soluble protein when growing 3ml cultures. 
However, the protein becomes insoluble (inclusion bodies) when we scale up to 
500ml cultures. Has anyone experienced such a problem, and found a solution to 
it? Thanks.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
Department of Chemistry
City College of New York
New York, NY 10031
http://www.khayatlab.org/
212-650-6070tel:212-650-6070

Re: [ccp4bb] non-specific disulfide bonds in crystal structures

[Reza Khayat]

Hi Todd,

A concern of mine is that you may be looking at an artifact 
of the domain. Whatever assays you decide to do, make sure 
that they are also performed for the full sized protein.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070
www.khayatlab.org


 Original message 
Date: Sat, 20 Dec 2014 10:44:25 -0800
From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on 
behalf of Christine Gee chr...@gmail.com)
Subject: Re: [ccp4bb] non-specific disulfide bonds in 
crystal structures  
To: CCP4BB@JISCMAIL.AC.UK

   Hi Todd,
   I used to work on PNMT which also is supposedly
   monomeric and formed a disulfide between monomers in
   the crystals.
See 
http://www.sciencedirect.com/science/article/pii/S157096390
5000968?via=ihub
   We showed that it was irrelevant to activity.
   Cheers
   Christine
   Sent from my iPad
   On 20 Dec 2014, at 9:52 am, Todd Jason Green
   tgr...@uab.edu wrote:

 Hello All-

 I have recently determined a domain structure of a
 larger protein. The structure shows a clear
 disulfide bond between two monomers in the
 asymmetric unit. I'm trying to figure out if this
 is an artifact of the crystal packing or has
 biological relevance. The protein has been
 reported to function as a monomer. If I look at
 the pool of protein on a SDS-PAGE gel under
 non-reducing conditions, I see that a smaller
 percentage (~15-20%) of the protein runs as a
 dimer. In the structure, the association has
 2-fold symmetry with about 29% of the monomeric
 surface area buried between the dimer. Can anyone
 point me in the direction of a paper describing a
 non-specific disulfide in a crystal, or perhaps a
 criteria for assessing specificity? I will do some
 functional studies, but I'm looking for some info
 on a lazy saturday.

 Thanks in advance. Best-
 Todd

[ccp4bb] cryoprotectant

[Reza Khayat]

Hi,

Has anyone used citrate as the sole cryoprotectant? If so, 
what concentration was needed?

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070
www.khayatlab.org

Re: [ccp4bb] large empty spaces in solved crystal structure

[Reza Khayat]

Hi,

I'm inclined to say that you may have indexed your data 
with the wrong space group -your description violates the 
definition of a crystal. What happens if you index in P1 
and resolve the x-tal using the structure from your current 
phases as an MR search model? This is assuming that you 
have reasonable phases to build a reasonable model. 
 
Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070
www.khayatlab.org


 Original message 
Date: Thu, 27 Nov 2014 13:33:26 -0500
From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on 
behalf of kuan hu hukuan0570...@gmail.com)
Subject: [ccp4bb] large empty spaces in solved crystal 
structure  
To: CCP4BB@JISCMAIL.AC.UK

   Dear all,

    I ran into a problem when I was solving a
   structure using SAD. After determining phase and
   building an initial model using AutoSol, I suddenly
   found that every two well ordered protein molecule
   layers were separated by a large empty space that
   extended across the entire crystal. The void space
   is so large that another two layers of molecules
   could be fit in. In that case, the unit cell was
   extremely extended across the empty space and the
   solvent content was very high. How does a crystal
   pack like that? or did I do something wrong during
   phasing to make such kind of artifact?

   Best,

   David

[ccp4bb] Pipettes

[Reza Khayat]

Hi,

This is way off topic but relevant. Does anyone routinely 
use the Rainin Classic Pipet series? If so, what are your 
thoughts and how often do they need to be calibrated?

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070
www.khayatlab.org

[ccp4bb] PDB Domain parser

[Reza Khayat]

Hi,

Can people direct me to programs capable of detecting non-
contiguous protein domains from a PDB file? I realize that 
some structures may be problematic.
 
Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070
www.khayatlab.org

[ccp4bb] Seeking postdoctoral candidate

[Reza Khayat]

Hi,

The research group of Dr. Reza Khayat at the City College of New York 
(www.khayatlab.org) is seeking candidate postdoctoral fellows to study host 
pathogen interaction using a combination of cryo-electron microscopy (cryo-
EM), X-ray crystallography, biophysics and biochemistry. The research group is 
particularly interested in elucidating the structural mechanism by which 
enveloped and nonenveloped viruses and bacteriophages interact with their 
hosts to: acquire cellular entry, hijack cellular machinery, disassemble, 
replicate, 
assemble and egress. The group is also interested in determining the 
mechanism by which host antibodies and synthetic DNA aptamers neutralize the 
viruses.

The group has dedicated access to a JEOL 2100 and Zeiss EM902 for grid 
preparation and screening, and to the resources at the New York Structural 
Biology Center (NYSBC) that include facilities for cryo-EM grid and sample 
preparation, JEOL 3200FSC, Tecnai F20, JEOL 2100F, JEOL 1230 and a FEI Helios 
650. The group also has dedicated access to state-of-art instrumentation to be 
housed at the Advanced Science and Research Center (ASRC). Both NYSBC and 
ASRC are located in adjacent buildings and within five minute walking distance 
from the laboratory (GPS: 40.815927, -73.951201).

The group has open access to multiple Gryphon LCP robots for conducting 
crystallization experiments at multiple temperatures, Alchemist DT for screen 
optimization, and Minstrel HT for crystal imaging and data storage. The group 
shares the following instrumentation: microplate readers, ITC, DLS, CD, and 
bioreactors.

Candidates must have a Ph.D., be highly motivated, exhibit independence, and 
have experience with protein purification. Salary will be competitive for the 
NYC 
area and be commensurate with experience and prior success. Please send a 
cover letter, CV, and the names with contact information for three references 
to 
rkha...@ccny.cuny.edu.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070
www.khayatlab.org

Re: [ccp4bb] Removing PEG3350

[Reza Khayat]

Hi,

I managed to significantly reduce the viscosity of the PEG solution via buffer 
exchange using a 100kDa MWCO ultrafiltration device. The following papers have 
fantastic tables of solutes with their hydrodynamic radii. Definitely worth a 
read, followed by printing and posting of the tables on walls next to the FPLC 
:)

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3055910/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1304934/


Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070
www.khayatlab.org


 Original message 
Date: Wed, 20 Aug 2014 18:57:07 +
From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Alexander 
Aleshin aales...@sanfordburnham.org)
Subject: Re: [ccp4bb] Removing PEG3350  
To: CCP4BB@JISCMAIL.AC.UK

 I meant application of GF as an ion exchange
 column.

   Oh, my goodness! Ion exchange is something else!
   It should read buffer-exchange = desalting column.
   On Aug 20, 2014, at 11:48 AM, Alexander Aleshin
   wrote:

 Dear Remie,
 I meant application of GF as an ion exchange
 column. You can use special ion exchange columns,
 but our lab often uses preparative GF columns for
 this task.  We just load the column, keeping
 sample volume   the void volume. Thus, we do not
  concentrate a protein before an ion exchange,
 only after it. But that is inevitable. When I am
 afraid to loose a protein during its
 concentrating, I concentrate shoulders of the
 eluted peak first, then add a central part.
 My point was that it might be okay to exchange
 buffers by concentrating a protein, but other
 molecules like Peg3K would not penetrate the
 membrane as well as water or salts do, as a result
 their reduction in concentration will be
 unreliable. Like, you do a 10 fold
 concentrating/delusion of a solution, but the
 final concentration of PEG3K will drop only by 3
 fold...
 Alex
 On Aug 19, 2014, at 9:42 AM, Remie wrote:

   Hi Alex,
   I disagree with you even though GF is always the
   last step in my purifications.
   Because it involves concentration before and
   after the GF so during the concentration you can
   already be doing the buffer exchange.
   You use GF when you want to purify other protein
   impurities if they are different sizes. Of
   course it has other uses too. But not quite
   practical for just changing buffer also
   considering the amount of protein you could be
   loosing along the process. If one is careful,
   centripreps are best for concentrating and
   changing the buffer. I tell you this from
   experience with large hard to express proteins.
   Best of luck,
   Remie
   On Aug 19, 2014, at 10:45 AM, Alexander Aleshin
   aales...@sanfordburnham.org wrote:

 Remie,
 Actually, concentrating of a protein solution
 is not the best approach to removing low MW
 impurities, gel filtration chromatography is
  more reliable and ... faster.
 Regards,
 Alex
 On Aug 19, 2014, at 7:03 AM, Remie Fawaz-Touma
 wrote:

   Hi Reza, I had to do this before.
   This protocol works for any PEG and any
   chemical to be removed from a solution:
   buffer exchange into the new buffer you want
   your protein to be in. There are ways to do
   that by 15 mL Amicon concentrators from
   millipore for large volumes, or if your
   protein is already concentrated, there are
   some small 0.5 mL concentrators from
   millipore as well.
   The key is to keep your spinning at low
   speeds (concentrators manuals will tell you)
   so you don’t precipitate or loose
   your protein. Check your protein
   concentration every 2 hours just to make
   sure you are not loosing it on concentrator
   surfaces and so on.
   Good Luck,
   Remie
   On Aug 19, 2014, at 9:55 AM, Reza Khayat
   rkha...@ccny.cuny.edu wrote:

 Hi,

 Does anyone have a protocol for getting
 rid of PEG3350 from a protein sample?

 Best wishes,
 Reza

 Reza Khayat, PhD
 Assistant Professor
 The City College of New York
 Department of Chemistry, MR-1135
 160 Convent Avenue
 New York, NY  10031
 Tel. (212) 650-6070
 www.khayatlab.org

[ccp4bb] Removing PEG3350

[Reza Khayat]

Hi,

Does anyone have a protocol for getting rid of PEG3350 from a protein sample? 

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070
www.khayatlab.org

Re: [ccp4bb] Enigmatic electron density attached to Cys residue

[Reza Khayat]

Hi,

I guessed glycerol by looking at the figs and not reading your text. Having 
read your text afterwards, you do have glycerol in the solution. My guess is 
glycerol.
 
Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070
www.khayatlab.org


 Original message 
Date: Tue, 19 Aug 2014 16:39:36 +0200
From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Mark J van 
Raaij mjvanra...@cnb.csic.es)
Subject: Re: [ccp4bb] Enigmatic electron density attached to Cys residue  
To: CCP4BB@JISCMAIL.AC.UK

What odds to you give us?
...I bet on disordered DTT covalently bound to the protein.

Mark J van Raaij
Lab 20B
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/~mjvanraaij







On 19 Aug 2014, at 16:12, Bernhard Loll wrote:

 Dear all,
  
 We are currently working on a small GTPase. The structure has been solved to 
 1.4 A with two molecules in the ASU. In the difference electron density we 
 can clearly see difference density (in one monomer) attached to a Cys 
 residue.
  
 The protein has been expressed in E. coli. For crystallization experiments 
 the GTPase was incubated with GMPPNP, that had been dissolved in 20 mM HEPES 
 pH 7.5 and 50 mM NaCl. Prior to crystallization
 the protein was stored in a buffer composed of 20 mM HEPES pH 7.5, 200 mM 
 NaCl, 5 mM, Mg acetate, 2 mM DTT and 2% (v/v) glycerol.
  
 The protein crystallized under the following conditions:
 28% (v/v) PEG 200, 5% (w/v) PEG 3000 and 100 mM MES buffer at pH 6.0
  
 The anomalous signal is too weak to judge the positions of the sulphur 
 atoms. We have performed MS analysis on the protein before crystallization 
 and on dissolved protein crystals. MS revealed a mass difference of about 
 135 Da, indicating that some chemistry must have went on in the 
 crystallization drop.
  
 The extra electron density has a planar shape and is quite symmetric. We 
 have placed some dummy water molecules in the density. Distances are given 
 in A in the PNG file.
  
 Attached files
  
 coot1.png: 2FoFc electron density map (blue) @ sigma=1 and FoFc electron 
 density map (blue) @ sigma=+3 after phenix.refine
 coot2.png: same electron densities as in coot1.png, with dummy atoms placed
 coot3.png: same electron densities as in coot1.png, side view
  
 Thanks for your time and efforts.
  
 Cheers,
  
 Bernhard
 -- 
 Dr. Bernhard Loll
 Freie Universitaet Berlin
 Fachbereich Biologie, Chemie, Pharmazie
 Institut fuer Chemie und Biochemie
 AG Strukturbiochemie
 Takustr. 6
 D-14195 Berlin
 Germany
 
 Phone: +49 (0) 30 838-57348
 Fax:   +49 (0) 30 838-457348
 Email: 
 l...@chemie.fu-berlin.de
 
 Homepage: 
 http://www.bcp.fu-berlin.de/chemie/bc/ag/agwahl/
 coot1.pngcoot2.pngcoot3.png

Re: [ccp4bb] dynapro DLS cuvettes

[Reza Khayat]

Hi,

A crazy solution may be to make a cuvette with a 3D printer. I'm not sure if 
the available resins are transparent to DLS. 

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070
www.khayatlab.org


 Original message 
Date: Thu, 14 Aug 2014 15:13:48 -0700
From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Daniel 
Anderson d...@mbi.ucla.edu)
Subject: Re: [ccp4bb] dynapro DLS cuvettes  
To: CCP4BB@JISCMAIL.AC.UK

Hello, Gloria and everybody,

I'm typing most of this reply from memory.

When I tried to buy one, my recollection was that it was available from 
Hellma, but I couldn't (and still can't) find my Hellma paper catalog, 
and for some reason I did not find the Hellma web site when I wanted to 
buy a cuvette. I have since learned to spell Google.

I have the starnacells dot com paper catalog in front of me. What you 
want is almost catalog number 16.12F-Q-1.5/Z15. The Z parameter should 
probably be closer to 14 than 15mm.

That catalog number from Starna is probably the one that I bought some 
years ago, and here is my recollection of what happened: The cell 
arrived, and I found that light did not go through unless I pushed it 
sideways. I measured the old Dynapro cuvette, and it was 12.45x12.45mm. 
The cell from Starna was 12.25x12.35mm. I returned it and Starna sent me 
a replacement that was 12.35x12.25mm. When this comedy of rectangular 
cuvettes tired me I returned the nth cell that they sent me and gave up 
trying to buy one.

One more detail: the above recollections are about a 13,714-year old 
DynaPro with the microcuvette device added as post-translational 
modification. It's so old that the serial number is probably negative. I 
don't know if a more recent instrument has the same cuvette holder geometry.

I hope that partial information helps,
   Dan




Gloria Borgstahl wrote:
 Does any one know of a source of these cuvettes?
 Protein Solution doesn't exist anymore
 and Wyatt no longer has these.

[ccp4bb] Disrupt beta sheet formation

[Reza Khayat]

Hi,

I have a protein that is prone to aggregation. I think the problem may be 
caused by a stretch of six-residues at the N-terminus that is predicted to form 
a beta-strand. I think this beta-strand is open ended and may form beta-
sheets (similar to amyloid fibers) leading to the aggregation I observe. Are 
there any chemicals that would disrupt short beta-sheet formation? Thanks.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070
www.khayatlab.org

[ccp4bb] Solvent channels

[Reza Khayat]

Hi,

I'd like to do some soaking experiments with a relatively large molecule. Can 
someone suggest a program/method to display the solvent channels of a 
crystal? We have the crystal structure. I'd like to see if the channels are 
large 
enough to allow the molecule to travel to the hypothesized binding site. 
Thanks.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070
www.khayatlab.org

Re: [ccp4bb] negative density around disulfide bond

[Reza Khayat]

What does an omit map look like, if you omit both cysteines?

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070
www.khayatlab.org


 Original message 
Date: Mon, 2 Jun 2014 09:31:28 -0700
From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf 
of Pavel Afonine pafon...@gmail.com)
Subject: Re: [ccp4bb] negative density around disulfide bond  
To: CCP4BB@JISCMAIL.AC.UK

   Ezequiel,
   since you mentioned you tried Phenix too:
   in Phenix you can remove a particular disulfide bond
   by using a parameter, for**example:

   disulfide_bond_exclusions_selection_string=(chain A
   and resseq 1 and name SG) or (chain B and resseq 10
   and name SG)

   This works in the command line and GUI. Please let
   me know (off-list) if you any help with this.

   Pavel

   On Sun, Jun 1, 2014 at 10:08 PM, Eze Chivi
   ezech...@outlook.com.ar wrote:

 Hello, when I refine my structure, I see negative
 density around the disulfide bond. I have 7 copies
 per ASU, and I can see this density in many of
 them. In some cases, I see positive density also
 (negative in the center of the straight line
 linking S atoms, and positive in both sides). What
 can I try to solve it? Is it due to radiation
 damage? Alternative conformation (partial
 oxidation)? Incorrect disulfide geometry
 parameters? My resolution is 2.1 A, R/Rfree are
 around 0.220/0.243, and similar results with
 refmac5, phenix and PDBREDO. Please find two
 example pictures in attachment.
 Thanks for your help!

 Ezequiel

[ccp4bb] Imaging Facilities Manager - Research Assistant Professor

[Reza Khayat]

 
package covering health insurance, pension and retirement 
benefits, paid parental leave, and savings programs.  We also 
provide mentoring and support for research, scholarship, and 
publication as part of our commitment to ongoing faculty 
professional development.

HOW TO APPLY

From our job posting system, select Apply Now, create or log 
in to a user account, and provide the requested information.  
If you are viewing this posting from outside our system, 
access the employment page on our web site and search for this 
vacancy using the Job ID or Title.  

Candidates should provide a CV/resume and statement of 
scholarly interests.

CLOSING DATE

The posting will close on June 20, 2014.

JOB SEARCH CATEGORY

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pluralistic community and continue to seek excellence through 
diversity and inclusion. EO/AA Employer.


Go to the following link to apply:

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HRAM.HRS_CE.GBL?
Page=HRS_CE_JOB_DTLAction=AJobOpeningId=10784SiteId=1Posti
ngSeq=1

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070
www.khayatlab.org

Re: [ccp4bb] HisTrap Trap

[Reza Khayat]

I think the new versions of GE's HisTrap columns can address 
these problems. Try contacting someone at GE. 

I think a number of labs have had such problems in the past 
and the culprit has been believed to be a chelator in the 
media but never confirmed because the manufacturer of the 
media is not willing to disclose the contents for the media. 


Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070
www.khayatlab.org


 Original message 
Date: Mon, 19 May 2014 14:38:12 +
From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf 
of Keller, Jacob kell...@janelia.hhmi.org)
Subject: Re: [ccp4bb] HisTrap Trap  
To: CCP4BB@JISCMAIL.AC.UK



   Well, this is of only possible relevance, but in a
   previous lab, we used sf9 cells/media quite a bit,
   and there was always an issue similar to this, due
   to [we thought] ferritin being secreted into the
   medium, and sucking up the metals. Many, in fact,
   crystallized ferritin this way by mistake! Is the
   concentrated flow-through colored, indicating
   protein-bound metals, or do the metals go through a
   concentrator, indicating free or
   small-molecule-complexed metals? What about pH?



   What about Gibco's formulation--is it available? The
   mysterious dispersant could easily be either some
   chelator like EDTA. You could try ITC with Gibco in
   the cuvette, titrate with metals if you have a
   machine handy--it's just an hour or so.



   JPK









   From: CCP4 bulletin board
   [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bernhard
   Rupp
   Sent: Monday, May 19, 2014 10:14 AM
   To: CCP4BB@JISCMAIL.AC.UK
   Subject: [ccp4bb] HisTrap Trap



   Hi Fellows,



   my lab mates successfully expressed a glycoprotein
   in CHO cells in serum free medium, and

   the protein captures nicely on HisTrap Excel 1ml
   columns (obviously, high yield is not my
   problem...).

   We load ca 1L supernatant at 0.5 ml/min, and eluate
   with a steep imidazole gradient. 20mM Imidazole
   buffer for regeneration.

   Works fine (and often...see yield remark).



   Overcome by common crystallographers' greed (nor
   creed), we switched to stable xfected HEK293, and
   cell free medium Gibco CD 293.

   The first run gave high final yields  cheers.

   The second run less of either, because the small
   HisTrap column essentially dissolved - the medium
   collapsed,

   Ni leaches out, kaput as kaput goes.

   A 3rd run on a similar previously working column
   lead to the same result.



   Only thing changed was the cells and medium. Same
   buffers, same gradients, same Akta equipment, same
   lab techs.



   Before I improve the statistics by ruining further
   columns, has anybody experienced such a calamity
   that might

   be blamable on secret media components or similar?
   There is a mysterious `proprietary dispersant'
   preventing

   cell adhesion quoted



   Best wishes, BR



   --
--

   Bernhard Rupp

   b...@ruppweb.org

   b...@hofkristallamt.org

   http://www.ruppweb.org/

   --
-

[ccp4bb] Seeking postdoctoral candidate

[Reza Khayat]

Hi,

The research group of Dr. Reza Khayat at the City College of 
New York (www.khayatlab.org) is seeking candidate postdoctoral 
fellows to study host pathogen interaction using a combination 
of cryo-electron microscopy (cryo-EM), X-ray crystallography, 
biophysics and biochemistry. The research group is 
particularly interested in elucidating the structural 
mechanism by which enveloped and nonenveloped viruses and 
bacteriophages interact with their hosts to: acquire cellular 
entry, hijack cellular machinery, disassemble, replicate, 
assemble and egress. The group is also interested in 
determining the mechanism by which host antibodies and 
synthetic DNA aptamers neutralize the viruses.

The group has dedicated access to a JEOL 2100 and Zeiss EM902 
for grid preparation and screening, and to the resources at 
the New York Structural Biology Center (NYSBC) that include 
facilities for cryo-EM grid and sample preparation, JEOL 
3200FSC, Tecnai F20, JEOL 2100F, JEOL 1230 and a FEI Helios 
650. The group also has dedicated access to state-of-art 
instrumentation to be housed at the Advanced Science and 
Research Center (ASRC). Both NYSBC and ASRC are located in 
adjacent buildings and within five minute walking distance 
from the laboratory (GPS: 40.815927, -73.951201).

The group has open access to multiple Gryphon LCP robots for 
conducting crystallization experiments at multiple 
temperatures, Alchemist DT for screen optimization, and 
Minstrel HT for crystal imaging and data storage. The group 
shares the following instrumentation: microplate readers, ITC, 
DLS, CD, and bioreactors.

Candidates must have a Ph.D., be highly motivated, exhibit 
independence, and have experience with protein purification. 
Salary will be competitive for the NYC area and be 
commensurate with experience and prior success. Please send a 
cover letter, CV, and the names with contact information for 
three references to rkha...@ccny.cuny.edu.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070
www.khayatlab.org

Re: [ccp4bb] First stucture of FCFV

[Reza Khayat]

I think fungus dependent crystallization has occurred for some 
labs. A paper that pops into mind is from my graduate 
laboratory (not my work though):

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2225192/

Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070
www.khayatlab.org


 Original message 
Date: Thu, 3 Apr 2014 06:36:37 -0700
From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf 
of Chad Brautigam cabrautc...@yahoo.com)
Subject: Re: [ccp4bb] First stucture of FCFV  
To: CCP4BB@JISCMAIL.AC.UK

   I once encountered mold-dependent crystallization of
   a protein.  Wouldn't that have made for a lively
   Methods section?
   Luckily, we determined the structure from crystals
   derived from a different, non-moldy condition.
   Whew.
   Chad
   From: Artem Evdokimov artem.evdoki...@gmail.com
   To: CCP4BB@JISCMAIL.AC.UK
   Sent: Thursday, April 3, 2014 7:55 AM
   Subject: Re: [ccp4bb] First stucture of FCFV
   Common molds like aspergillus or penicillium. After
   a while you sometimes get sporangia, then you can
   tell with more certainty. ..
   A.
   On Apr 3, 2014 3:50 AM, Bernhard Rupp
   hofkristall...@gmail.com wrote:

 Several people were asking what this FCFV
 tentacles actually might be. I think it is some
 fungus/yeast growing out of nutritious drops. Does
 resemble fungus/mushroom mycelium. I have also
 some that look like huge bacteriophages with nice
 heads on them, probably yeast buds. There is also
 a yeast lab next to the Xtallization facility :-/
 *** feel free to speculate.

 Best, BR

Re: [ccp4bb] ITC with unfolded proteins

[Reza Khayat]

Hi,

I think the experiment is doable, but how would you decouple 
protein-protein interaction from folding of the unfolded 
protein due to protein interaction?

Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070


 Original message 
Date: Fri, 14 Mar 2014 18:07:48 +0530
From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf 
of Anita P crystals...@gmail.com)
Subject: [ccp4bb] ITC with unfolded proteins  
To: CCP4BB@JISCMAIL.AC.UK

   Hello everyone,
   I have a query for the scientists working on
   protein-protein interaction.
   It is known that some proteins exist in unfolded or
   molten globule state and attain structure on
   interaction with other folded proteins.
   Many a times, it is difficult to obtain the
   structure of these complexes.
   Is it possible to quantitatively determine the
   thermodynamics of interaction between an unfolded
   protein and a folded protein using ITC? Later may be
   perform an alascan to determine the residues of the
   unfolded partner involved in the interaction.
   Please share your ideas
   cheers**
   Anita

Re: [ccp4bb] saving map isovalue surface to file?

[Reza Khayat]

USCF Chimera can save maps as a VRML and other formats. You 
can use 3D animation software to change formats from VRML to 
anything else.


Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070


 Original message 
Date: Tue, 4 Mar 2014 10:38:13 -0800
From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf 
of Alastair Fyfe af...@ucsc.edu)
Subject: [ccp4bb] saving map isovalue surface to file?  
To: CCP4BB@JISCMAIL.AC.UK

Is there an easy way to save a map isovalue surface to a file 
in a 
standard triangulated mesh format such as ply? This could 
be done with 
vtk or similar libraries, but, if there's already a script-
accessible 
coot or pymol command, it would be helpful. Pymol can do this 
for 
molecular surfaces, but I haven't found a reference for maps.
thanks,
Alastair Fyfe

[ccp4bb] Se-Met and disulfides

[Reza Khayat]

Hi,

I'm sure this question has been addressed before and I 
apologize for asking it again. I'm working on a protein 
predicted to have multiple disulfide bonds. The protein is 
expressed in an E. coli strain with an oxidizing cytoplasm, 
and is purified under nonreducing conditions (no DTT, b-
ME...). I'd like to produce some Se-Met incorporated protein 
for phasing. Given that Se-Met can oxidize, its anomalous 
scattering is dependent on its oxidation state, and a mixture 
of oxidized and reduced Se-Met makes phasing difficult, what 
is the suggested protocol to follow in my situation? Here's 
hoping that the solution is not dumping a bunch of Se-Met into 
all the purification buffers...  

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070


 Original message 
Date: Thu, 27 Feb 2014 11:22:10 +
From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf 
of Vicky Tsirkoni vicky.tsirk...@pharm.kuleuven.be)
Subject: [ccp4bb]  
To: CCP4BB@JISCMAIL.AC.UK

   Hi Prerana,
   You could try 4°C as well (dont forget to use
   lower protein concentration) in my case this worked
   better.
   Cheers,
   Vicky G. Tsirkone, MSc
   Laboratory for Biocrystallography
   Department of Pharmaceutical and Pharmacological
   Sciences
   KU Leuven
   ON II Herestraat 49 - box 822
   3000 Leuven | Belgium
   Tel.: +32 16 3 23419
   e-mail: vicky.tsirk...@pharm.kuleuven.be

   

   From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on
   behalf of Prerana G. [tracy...@gmail.com]
   Sent: Monday, February 24, 2014 5:23 PM
   To: CCP4BB@JISCMAIL.AC.UK
   Subject: [ccp4bb]
   Hi Vicky,
   I tried to change the ratio of paraffin:silicon oil
   to 1:2, but still the protein precipitated. So I
   tried to do it other way round, I kept
   paraffin:silicon oil ratio as 2:1 and so far it has
   not precipitated. Apart from that, I separately used
   the two oils.In silicon oil there was immediate
   precipitation, but none in parafin oil.
   Prerana

Re: [ccp4bb] off-topic: bug busting

[Reza Khayat]

A lot of ageing sonicators are not really ageing. Have you tried cleaning the 
tip of the sonicator? If not, remove the tip from the sonicator and use a 800 
Grit wet sandpaper to make it as shiny as it was when you first bought it. I do 
this every three times I use the sonicator. A lot of crap deposits on it with 
every use. 

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070


 Original message 
Date: Tue, 4 Feb 2014 12:40:51 -0500
From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Roger Rowlett 
rrowl...@colgate.edu)
Subject: Re: [ccp4bb] off-topic: bug busting  
To: CCP4BB@JISCMAIL.AC.UK

   BeadBeater. http://biospec.com/. Gentle,
   aerosol-free way to break 15-350 mL of cell paste
   (2-150 g wet packed cells).

   Cheers,

   ___
   Roger S. Rowlett
   Gordon  Dorothy Kline Professor
   Department of Chemistry
   Colgate University
   13 Oak Drive
   Hamilton, NY 13346

   tel: (315)-228-7245
   ofc: (315)-228-7395
   fax: (315)-228-7935
   email: rrowl...@colgate.edu

   On 2/4/2014 11:49 AM, Phoebe A. Rice wrote:

 Some time ago, there was a nice discussion of
 cost-effective, wimpy protein-friendly ways to
 break open E. coli.  We're thinking about
 replacing an aging sonicator.  If people have a
 favorite gizmo, could they repeat that advice?
 thank you,
   Phoebe Rice

 ++

 Phoebe A. Rice
 Dept. of Biochemistry  Molecular Biology
 The University of Chicago

 773 834 1723; pr...@uchicago.edu
 http://bmb.bsd.uchicago.edu/Faculty_and_Research/

 http://www.rsc.org/shop/books/2008/9780854042722.asp