Re: [ccp4bb] powdery residue on pucks?

2020-02-22 Thread Zachary A. Wood 
We only notice the dust when we let pucks thaw while still assembled. We had 
always assumed that the dust was due to oxidation (different types of metals in 
contact with the water from condensation). We have not seen the dust since we 
began taking apart pucks and removing pins prior to thawing them. Since doing 
this, we have also noticed that the pin bases are less prone to oxidation, 
which is nice (and supports our electrochemistry hypothesis).  Maybe give that 
a try?

Best regards,

Z

***
Zachary A. Wood, Ph.D.
Associate Professor and Graduate Coordinator
Department of Biochemistry & Molecular Biology
University of Georgia
Life Sciences Building, Rm A426B
120 Green Street
Athens, GA  30602-7229
Office: 706-583-0304
Lab:706-583-0303
FAX: 706-542-1738
***




On Feb 22, 2020, at 3:42 AM, Steiner, Roberto 
<2497b6493202-dmarc-requ...@jiscmail.ac.uk>
 wrote:

[EXTERNAL SENDER - PROCEED CAUTIOUSLY]

Fully agree with what others have said. When ’the dust’ happened to us we had 
to replace that particular shipping dewar as T was not holding for very long.

Best wishes
Roberto

On 22 Feb 2020, at 00:33, Emilia C. Arturo (Emily) 
mailto:ecgart...@gmail.com>> wrote:

Hi All,

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

Regards,
Emily.

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

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




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Professor Roberto Steiner
Randall Centre for Cell and Molecular Biophysics
Faculty of Life Sciences and Medicine
King's College London

roberto.stei...@kcl.ac.uk
Phone 0044 20 78488216
Fax0044 20 78486435

Room 3.10A
New Hunt's House
Guy's Campus
SE1 1UL
London






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Re: [ccp4bb] Contradictory result between ITC and cocrystal structure

2020-02-22 Thread Petri Kursula 
Hi,
a likely scenario is that your mutant crystallises in the same 
conformation/packing as the wild-type protein, and this conformation is good 
for ligand binding. In solution, your mutant protein may be more flexible/open 
than the wild-type and affinity hence lower. We see this quite often. Various 
solution structure techniques will shed light on this.

Another possibility, assuming that you did ITC only at one temperature, is that 
you are “lucky” enough to be at the temperature where enthalpy for binding is 
zero for the mutant (but not wild-type). This you can find out by carrying out 
ITC at different temperatures.
Petri
 
Petri Kursula
--
Professor 
--
Department of Biomedicine
University of Bergen, Norway
http://www.uib.no/en/rg/petrikursula
petri.kurs...@uib.no
--
Faculty of Biochemistry and Molecular Medicine
Biocenter Oulu
University of Oulu, Finland
--





> On 22 Feb 2020, at 07:31, monika chandravanshi 
>  wrote:
> 
> Dear All,
> 
>   I have a situation, where a mutant protein does not exhibit any 
> heat change upon titration with cognate ligand in the ITC experiment. 
> However, it co-crystallizes with the respective cognate ligand. Also, the 
> cocrystal structure reveals the conservation of the hydrogen bonding networks 
> except for the mutated residues. I would like to know the possible reason for 
> the no heat change in the ITC experiment. 
> 
> Looking forward to hearing from you.
> 
> -- 
> -
> 
> With Kind Regards
> 
> Monika Chandravanshi
> PhD Scholar, 
> Department of Biosciences and Bioengineering
> Indian Institute of Technology Guwahati, Guwahati India
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
> 



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Re: [ccp4bb] Contradictory result between ITC and cocrystal structure

2020-02-22 Thread Srivastava, Dhiraj 
As other said, having co-crystal doesn’t mean you will have measurable 
affinity. Further no heat in ITC experiments doesn’t mean no binding. Which 
buffer you used during previous ITC experiment? If heat in the previous 
experiment (wild type) was due to heat of ionization and you removed the 
residue involved in protonation/deprotonation event, you will not see any heat.
If there is 10-20 fold decrease in affinity by mutation, you may not be able to 
see significant heat during ITC experiment as well.


Dhiraj



From: CCP4 bulletin board  on behalf of vipul panchal 

Sent: Saturday, February 22, 2020 3:25 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Contradictory result between ITC and cocrystal structure

Hi Monika,

If the protein cocrystalize with ligand doesn't mean it interacts with protein.
You have provided insufficient information here. Does the ligand is bound to 
mutant protein with atleast 2 or 3 hydrogen bonds (how many residues are 
mutated?) ? If no that means, it is just cocrystalizing but not interacting 
with mutant protein.

Another possibility is, during cocrystalization ligand concentration is very 
high so as to force the interaction which otherwise is not possible as you 
observed with ITC. In other terms, affinity is too low to be detected by ITC.

Cheers,
Vipul

On Sat, 22 Feb, 2020, 12:01 PM monika chandravanshi, 
mailto:chandravanshi.monik...@gmail.com>> 
wrote:
Dear All,

  I have a situation, where a mutant protein does not exhibit any 
heat change upon titration with cognate ligand in the ITC experiment. However, 
it co-crystallizes with the respective cognate ligand. Also, the cocrystal 
structure reveals the conservation of the hydrogen bonding networks except for 
the mutated residues. I would like to know the possible reason for the no heat 
change in the ITC experiment.

Looking forward to hearing from you.

--
-

With Kind Regards

Monika Chandravanshi
PhD Scholar,
Department of Biosciences and Bioengineering
Indian Institute of Technology Guwahati, Guwahati India



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Re: [ccp4bb] Contradictory result between ITC and cocrystal structure

2020-02-22 Thread Srivastava, Dhiraj 
As other said, having co-crystal doesn’t mean you will have measurable 
affinity. Further no heat in ITC experiments doesn’t mean no binding. Which 
buffer you used during previous ITC experiment? If heat in the previous 
experiment (wild type) was due to heat of ionization and you removed the 
residue involved in protonation/deprotonation event, you will not see any heat.
If there is 10-20 fold decrease in affinity by mutation, you may not be able to 
see significant heat during ITC experiment as well.


Dhiraj



From: CCP4 bulletin board  on behalf of vipul panchal 

Sent: Saturday, February 22, 2020 3:25 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Contradictory result between ITC and cocrystal structure

Hi Monika,

If the protein cocrystalize with ligand doesn't mean it interacts with protein.
You have provided insufficient information here. Does the ligand is bound to 
mutant protein with atleast 2 or 3 hydrogen bonds (how many residues are 
mutated?) ? If no that means, it is just cocrystalizing but not interacting 
with mutant protein.

Another possibility is, during cocrystalization ligand concentration is very 
high so as to force the interaction which otherwise is not possible as you 
observed with ITC. In other terms, affinity is too low to be detected by ITC.

Cheers,
Vipul

On Sat, 22 Feb, 2020, 12:01 PM monika chandravanshi, 
mailto:chandravanshi.monik...@gmail.com>> 
wrote:
Dear All,

  I have a situation, where a mutant protein does not exhibit any 
heat change upon titration with cognate ligand in the ITC experiment. However, 
it co-crystallizes with the respective cognate ligand. Also, the cocrystal 
structure reveals the conservation of the hydrogen bonding networks except for 
the mutated residues. I would like to know the possible reason for the no heat 
change in the ITC experiment.

Looking forward to hearing from you.

--
-

With Kind Regards

Monika Chandravanshi
PhD Scholar,
Department of Biosciences and Bioengineering
Indian Institute of Technology Guwahati, Guwahati India



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Re: [ccp4bb] Contradictory result between ITC and cocrystal structure

2020-02-22 Thread Patra, Dhabaleswar 
Dear Monika,

If your protein-ligand interaction is at millimolar, you need to increase 
injection volume in order to see heat change. Also, you can do the experiment 
at low temperature such as 10 degree Celsius. You can follow this paper where I 
have crystallized weak binding ligand and complemented with ITC.
https://pubmed.ncbi.nlm.nih.gov/24957055-structure-interactions-and-evolutionary-implications-of-a-domain-swapped-lectin-dimer-from-mycobacterium-smegmatis/?from_term=Dhabaleswar+Patra_pos=7
Structure, Interactions and Evolutionary Implications of a Domain-Swapped 
Lectin Dimer From Mycobacterium Smegmatis - 
PubMed
Crystal structure determination of the lectin domain of MSMEG_3662 from 
Mycobacterium smegmatis and its complexes with mannose and methyl-α-mannose, 
the first effort of its kind on a mycobacterial lectin, reveals a structure 
very similar to β-prism II fold lectins from plant sources, but with extens …
pubmed.ncbi.nlm.nih.gov
Hope this help.

Regards,
Dhabaleswar Patra
Purdue University, IN



From: CCP4 bulletin board  on behalf of vipul panchal 

Sent: Saturday, February 22, 2020 4:25 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Contradictory result between ITC and cocrystal structure

Hi Monika,

If the protein cocrystalize with ligand doesn't mean it interacts with protein.
You have provided insufficient information here. Does the ligand is bound to 
mutant protein with atleast 2 or 3 hydrogen bonds (how many residues are 
mutated?) ? If no that means, it is just cocrystalizing but not interacting 
with mutant protein.

Another possibility is, during cocrystalization ligand concentration is very 
high so as to force the interaction which otherwise is not possible as you 
observed with ITC. In other terms, affinity is too low to be detected by ITC.

Cheers,
Vipul

On Sat, 22 Feb, 2020, 12:01 PM monika chandravanshi, 
mailto:chandravanshi.monik...@gmail.com>> 
wrote:
Dear All,

  I have a situation, where a mutant protein does not exhibit any 
heat change upon titration with cognate ligand in the ITC experiment. However, 
it co-crystallizes with the respective cognate ligand. Also, the cocrystal 
structure reveals the conservation of the hydrogen bonding networks except for 
the mutated residues. I would like to know the possible reason for the no heat 
change in the ITC experiment.

Looking forward to hearing from you.

--
-

With Kind Regards

Monika Chandravanshi
PhD Scholar,
Department of Biosciences and Bioengineering
Indian Institute of Technology Guwahati, Guwahati India



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Re: [ccp4bb] [3dem] Which resolution?

2020-02-22 Thread Nave, Colin (DLSLtd,RAL,LSCI) 
Alexis
This is a very useful summary.

You say you were not convinced by Marin's derivation in 2005. Are you convinced 
now and, if not, why?

My interest in this is that the FSC with half bit thresholds have the danger of 
being adopted elsewhere because they are becoming standard for protein 
structure determination (by EM or MX). If it is used for these mature 
techniques it must be right!

It is the adoption of the ½ bit threshold I worry about. I gave a rather weak 
example for MX which consisted of partial occupancy of side chains, substrates 
etc. For x-ray imaging a wide range of contrasts can occur and, if you want to 
see features with only a small contrast above the surroundings then I think the 
half bit threshold would be inappropriate.

It would be good to see a clear message from the MX and EM communities as to 
why an information content threshold of ½ a bit is generally appropriate for 
these techniques and an acknowledgement that this threshold is 
technique/problem dependent.

We might then progress from the bronze age to the iron age.

Regards
Colin



From: CCP4 bulletin board  On Behalf Of Alexis Rohou
Sent: 21 February 2020 16:35
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] [3dem] Which resolution?

Hi all,

For those bewildered by Marin's insistence that everyone's been messing up 
their stats since the bronze age, I'd like to offer what my understanding of 
the situation. More details in this thread from a few years ago on the exact 
same topic:
https://mail.ncmir.ucsd.edu/pipermail/3dem/2015-August/003939.html
https://mail.ncmir.ucsd.edu/pipermail/3dem/2015-August/003944.html

Notwithstanding notational problems (e.g. strict equations as opposed to 
approximation symbols, or omission of symbols to denote estimation), I believe 
Frank & Al-Ali and "descendent" papers (e.g. appendix of Rosenthal & Henderson 
2003) are fine. The cross terms that Marin is agitated about indeed do in fact 
have an expectation value of 0.0 (in the ensemble; if the experiment were 
performed an infinite number of times with different realizations of noise). I 
don't believe Pawel or Jose Maria or any of the other authors really believe 
that the cross-terms are orthogonal.

When N (the number of independent Fouier voxels in a shell) is large enough, 
mean(Signal x Noise) ~ 0.0 is only an approximation, but a pretty good one, 
even for a single FSC experiment. This is why, in my book, derivations that 
depend on Frank & Al-Ali are OK, under the strict assumption that N is large. 
Numerically, this becomes apparent when Marin's half-bit criterion is plotted - 
asymptotically it has the same behavior as a constant threshold.

So, is Marin wrong to worry about this? No, I don't think so. There are indeed 
cases where the assumption of large N is broken. And under those circumstances, 
any fixed threshold (0.143, 0.5, whatever) is dangerous. This is illustrated in 
figures of van Heel & Schatz (2005). Small boxes, high-symmetry, small objects 
in large boxes, and a number of other conditions can make fixed thresholds 
dangerous.

It would indeed be better to use a non-fixed threshold. So why am I not using 
the 1/2-bit criterion in my own work? While numerically it behaves well at most 
resolution ranges, I was not convinced by Marin's derivation in 2005. 
Philosophically though, I think he's right - we should aim for FSC thresholds 
that are more robust to the kinds of edge cases mentioned above. It would be 
the right thing to do.

Hope this helps,
Alexis



On Sun, Feb 16, 2020 at 9:00 AM Penczek, Pawel A 
mailto:pawel.a.penc...@uth.tmc.edu>> wrote:
Marin,

The statistics in 2010 review is fine. You may disagree with assumptions, but I 
can assure you the “statistics” (as you call it) is fine. Careful reading of 
the paper would reveal to you this much.
Regards,
Pawel


On Feb 16, 2020, at 10:38 AM, Marin van Heel 
mailto:marin.vanh...@googlemail.com>> wrote:


 EXTERNAL EMAIL 
Dear Pawel and All others 
This 2010 review is - unfortunately - largely based on the flawed statistics I 
mentioned before, namely on the a priori assumption that the inner product of a 
signal vector and a noise vector are ZERO (an orthogonality assumption).  The 
(Frank & Al-Ali 1975) paper we have refuted on a number of occasions (for 
example in 2005, and most recently in our BioRxiv paper) but you still take 
that as the correct relation between SNR and FRC (and you never cite the 
criticism...).
Sorry
Marin

On Thu, Feb 13, 2020 at 10:42 AM Penczek, Pawel A 
mailto:pawel.a.penc...@uth.tmc.edu>> wrote:
Dear Teige,

I am wondering whether you are familiar with

Resolution measures in molecular electron microscopy.
Penczek PA. Methods Enzymol. 2010.
Citation

Methods Enzymol. 2010;482:73-100. doi: 10.1016/S0076-6879(10)82003-8.

You will find there answers to all questions you asked and much more.

Regards,
Pawel Penczek

Regards,
Pawel
___
3dem mailing list

Re: [ccp4bb] Contradictory result between ITC and cocrystal structure

2020-02-22 Thread vipul panchal 
Hi Monika,

If the protein cocrystalize with ligand doesn't mean it interacts with
protein.
You have provided insufficient information here. Does the ligand is bound
to mutant protein with atleast 2 or 3 hydrogen bonds (how many residues are
mutated?) ? If no that means, it is just cocrystalizing but not interacting
with mutant protein.

Another possibility is, during cocrystalization ligand concentration is
very high so as to force the interaction which otherwise is not possible as
you observed with ITC. In other terms, affinity is too low to be detected
by ITC.

Cheers,
Vipul

On Sat, 22 Feb, 2020, 12:01 PM monika chandravanshi, <
chandravanshi.monik...@gmail.com> wrote:

> Dear All,
>
>   I have a situation, where a mutant protein does not exhibit
> any heat change upon titration with cognate ligand in the ITC experiment.
> However, it co-crystallizes with the respective cognate ligand. Also, the
> cocrystal structure reveals the conservation of the hydrogen bonding
> networks except for the mutated residues. I would like to know the possible
> reason for the no heat change in the ITC experiment.
>
> Looking forward to hearing from you.
>
> --
> *-*
>
> *With Kind Regards*
>
> *Monika Chandravanshi*
> *PhD Scholar, *
> *Department of Biosciences and Bioengineering*
> *Indian Institute of Technology Guwahati, Guwahati India*
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] powdery residue on pucks?

2020-02-22 Thread Steiner, Roberto 
Fully agree with what others have said. When ’the dust’ happened to us we had 
to replace that particular shipping dewar as T was not holding for very long.

Best wishes
Roberto

On 22 Feb 2020, at 00:33, Emilia C. Arturo (Emily) 
mailto:ecgart...@gmail.com>> wrote:

Hi All,

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

Regards,
Emily.

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

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




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Professor Roberto Steiner
Randall Centre for Cell and Molecular Biophysics
Faculty of Life Sciences and Medicine
King's College London

roberto.stei...@kcl.ac.uk
Phone 0044 20 78488216
Fax0044 20 78486435

Room 3.10A
New Hunt's House
Guy's Campus
SE1 1UL
London






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