Re: [ccp4bb] Occupany refinement protocol amd best practices

[Pavel Afonine]

Hi Matt,

Im wondering about occupancy refinements - both what's going on under the
> hood and what are best practices.
>

since you are quoting Phenix I suggest this bit of reading that is somewhat
relevant to your questions:
https://phenix-online.org/phenixwebsite_static/mainsite/files/newsletter/CCN_2015_07.pdf#page=12
This documents 13 typical occupancy refinement scenarios and how they can
be handled in Phenix.


> In the example I have, there is a ligand found in two distinct, partially
> overlapping sites that can be modeled is some confidence, but likely there
> are very low occupancy additional poses that blurs the electron density.
> The modeled poses are known from prior work, so even though there is
> smearing we know the ligand is in the modeled conformation. After
> perturbing the crystal these I am trying to decide what the best approach
> is to get some sort of numerical occupancy value to describe the
> distribution.
>

I apologize in advance for this trivial statement, but refinement +
validation are the tools to answer your question.
If you can model these poses with an atomic model and prove they match the
experimental diffraction data ("The modeled poses are known from prior
work" doesn't count in this regard), then you are all good! Depending on
the size ration ligand:whole structure, R factors may or may not be useful
quantifiers of the modeling choices. So your best bets may be local
quantities, such as refined ligand group occupancy, flatness of Fo-Fc map
(assessed as map values at atom positions), local map-model CC, etc. Try to
challenge your modeling decisions by placing similar to expected answers
but knowingly wrong models and see how that changes the fit/quality
criteria -- that may give you a way to assess uncertainty.

1.  In Phenix, how is the occupancy number determined?


Please refer to the paper I mentioned above, and let me know if you have
additional questions.


> Is there a real-space correlation between the experimental density and the
> model(weighted to occ) that is optimized?


Yes, but very indirectly through optimization of overall standard ML target
function.


>   How can this go wrong?


It can go wrong in as many ways as the refinement target function has local
minima, that is in many thousands ways!


> I fear that the smearing and heterogenous nature will through the
> refinement off (over or underfitting to periphery density rather than
> hyper-localized position of the model)
>

That is a valid concern that is good to keep in mind but that is not a
show-stopper!


> 2.  Are there errors associated with the occupancy numbers?
>

Yes, like with any model refinable parameter. For some discussion please
see:

https://www.nature.com/articles/s41467-018-06957-w


>
> 3.  For my own testing, I did 5% increments and manually observed Fo-Fc
> and 2Fo-Fc maps and selected a value that resulted in the lowest amount of
> both positive and negative Fo-Fc peaks.  This is how we submitted the work
> to journal but reviewer wants it to be automatically calculated.


Above mentioned paper is now more relevant in light of this question! Yes,
you can do manual sampling to get starting values (because the closer they
are to the true values, the better chances refinement is successful), then
do some refinement starting with these values.

Good luck!
Pavel



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[ccp4bb] Occupany refinement protocol amd best practices

[Matt McLeod]

Hi all,

Im wondering about occupancy refinements - both what's going on under the hood 
and what are best practices.

In the example I have, there is a ligand found in two distinct, partially 
overlapping sites that can be modeled is some confidence, but likely there are 
very low occupancy additional poses that blurs the electron density. The 
modeled poses are known from prior work, so even though there is smearing we 
know the ligand is in the modeled conformation. After perturbing the crystal 
these I am trying to decide what the best approach is to get some sort of 
numerical occupancy value to describe the distribution.

1.  In Phenix, how is the occupancy number determined? Is there a real-space 
correlation between the experimental density and the model(weighted to occ) 
that is optimized?  How can this go wrong?  I fear that the smearing and 
heterogenous nature will through the refinement off (over or underfitting to 
periphery density rather than hyper-localized position of the model)

2.  Are there errors associated with the occupancy numbers?

3.  For my own testing, I did 5% increments and manually observed Fo-Fc and 
2Fo-Fc maps and selected a value that resulted in the lowest amount of both 
positive and negative Fo-Fc peaks.  This is how we submitted the work to 
journal but reviewer wants it to be automatically calculated.  Is my approacg 
problematic besides the subjective nature of me determining what is a minimized 
peaks?  Is this less so reliable to automated fitting?  Will this really make a 
difference?  To be most honest, how should I let phenix approximate these 
values?  Set B-factor to average protein b?  Refine both simultaneously?  Or?

Any insight would be appreciated.
Matt



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Re: [ccp4bb] Fragile Crystals

[Hough, Michael (DLSLtd,RAL,LSCI)]

Hi Elizabeth,

As suggested VMXi at Diamond may be able to help with in situ data collection 
without harvesting. I'd be happy to discuss if useful?

cheers

Mike

Sent from Outlook for Android

From: CCP4 bulletin board  on behalf of CCP4BB 
<193323b1e616-dmarc-requ...@jiscmail.ac.uk>
Sent: Thursday, November 23, 2023 7:04:45 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Fragile Crystals

Hi

Sorry if someone else has mentioned this already, but how about shooting your 
crystals in situ (i.e. while sitting in the plate)? I know that VMXi at Diamond 
can do this (in fact it's designed for his purpose) but haven't collected data 
in long enough not to know if it can be done elsewhere.

Harry
--
Dr Harry Powell

On 22 Nov 2023, at 20:04, Phil Jeffrey 
mailto:pjeff...@princeton.edu>> wrote:

Hello Morgan

In addition to the other good suggestions, I have a few observations of my own.

If your crystals crack without handling or adding anything to the drop, then 
they are extremely environment-sensitive.  If that's the case, testing at room 
temperature will be problematic because that tends to be somewhat stressful on 
the crystal either mechanically (ye olde capillary mount method) or via 
dehydration (loop mounts with the sleeve).

Growing in the presence of at least a little cryoprotectant as per Vaheh would 
be less stressful than multi-step processes like Tao-Hsin's advice unless your 
crystals can re-anneal after stress.  Mounting directly from the drop is 
probably essential, and mounting under oil is a good thing to try in addition - 
apart from anything else oil on the drop slows down the environmental changes.  
Using Mitegen mounts might be less stressful on some crystals than standard 
nylon loops if they are mechanically sensitive.  Spending some time optimizing 
the mechanics of your freezing technique might help significantly in reducing 
the amount of time your crystal dehydrates while moving through air.
(Jim Pflugrath's article is full of useful information:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4461322/ )

Small crystals often freeze more smoothly than large ones - even for robust 
crystals like tetragonal lysozyme.  Try a lot of crystals - I've had projects 
were two different crystals in the same loop from the same drop showed 
radically different diffraction.  Also I've encountered several cases where the 
appearance of disorder varies within a crystal when using a microfocus 
synchrotron beam line (I mostly use FMX and AMX at NSLS2).

Lastly, really cranky crystals rings a distant bell of something we encountered 
in the p19(INK4d)-Cdk6 structure back in the 1990's.  I think it was Jie-Oh Lee 
that did the hard work on this, but in many instances crystals cracked in situ 
when merely opening the drop, and the fix was by adding a cross-linker to the 
well, resealing the drop and waiting for the cross-linker to diffuse:

"The crystals were pretreated with glutaraldehyde (diffused into the drop from 
a reservoir of 30% glutaraldehyde) to reduce their tendency to crack and lose 
diffraction along b* and c*."
https://www.nature.com/articles/26155#Sec9

Most crystals don't love being cross-linked, and I would call this a successful 
instance of a desperation maneuver.

Good luck.
Phil Jeffrey
Princeton

On 11/22/23 11:44 AM, Blake, Morgan Elizabeth wrote:
Hello
I am a PhD student working on a crystallography project to wrap up my 
dissertation research. I have purified a complex of two proteins, and I can 
consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane pH 
7.5. These crystals have sharp edges and can grow to a large size (greater than 
0.5 mm), but the crystals seem to be very fragile. When we open the drops to 
harvest the crystals, we have little time to harvest the crystals before they 
crack. When we move the crystals to a cryoprotectant, over time they start 
fracturing. We've tried using different percentages of glycerol, ethylene 
glycol, PEG400, and oil for cryoprotectants with no success. Needless to say, 
the crystals do not diffract well, with spot patterns that look very 
streaky/mosaic, which I presume is due to the defects that we see in 
harvesting/handling. We have screened for alternate crystallization conditions, 
but we seem to get the same morphology in other conditions. Does anyone have 
suggestions for additives we could use post-crystallization to help stabilize 
our crystals?
Thanks for your advice!

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Re: [ccp4bb] Quaternary Structure concept

[Jon Cooper]

Hello, it sounds like a monomeric protein which has been cleaved in 4 places so 
that would not give it 'quaternary' structure. If the nicked monomers assembled 
into dimers or timer, etc, then it becomes quaternary.

Best wishes, Jon Cooper. jon.b.coo...@protonmail.com

Sent from Proton Mail mobile

 Original Message 
On 22 Nov 2023, 20:58, BRUNO DI GERONIMO QUINTERO wrote:

> Dear CCP4BB comunnity;
>
> I am not a structural biologist thus far.
>
> I would like to inquire about the concept of quaternary structure and its 
> applicability to the system I am currently investigating. The protein under 
> study is a human lysosomal protein (Gene MAN2B1) that initially presents as a 
> single chain. Upon entry into the lysosome (facilitated by the signal 
> peptide), it undergoes cleavage four times in four external loops, resulting 
> in the formation of five different "chains." The tertiary structure remains 
> unchanged, and only after denaturation and purification, it becomes possible 
> to observe the five distinct peptides. If the protein remains in the 
> endoplasmic reticulum (ER), it exists as a single chain, as there is no 
> cleavage due to the higher pH in the ER.
>
> My question is as follows: can we consider the protein inside the lysosome as 
> having a quaternary structure? It originates from a monomer, and the subunits 
> exhibit different sequences while interacting with each other. But indeed, 
> there are 5 different peptides.
>
> I would appreciate your expertise and consideration of this perhaps 
> unconventional question, which is causing me significant concern.
>
> Best regards, Bruno Di Geronimo.
>
> ---
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



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Re: [ccp4bb] Fragile Crystals

[CCP4BB]

Hi

Sorry if someone else has mentioned this already, but how about shooting your 
crystals in situ (i.e. while sitting in the plate)? I know that VMXi at Diamond 
can do this (in fact it's designed for his purpose) but haven't collected data 
in long enough not to know if it can be done elsewhere.

Harry
--
Dr Harry Powell

> On 22 Nov 2023, at 20:04, Phil Jeffrey  wrote:
> 
> Hello Morgan
> 
> In addition to the other good suggestions, I have a few observations of my 
> own.
> 
> If your crystals crack without handling or adding anything to the drop, then 
> they are extremely environment-sensitive.  If that's the case, testing at 
> room temperature will be problematic because that tends to be somewhat 
> stressful on the crystal either mechanically (ye olde capillary mount method) 
> or via dehydration (loop mounts with the sleeve).
> 
> Growing in the presence of at least a little cryoprotectant as per Vaheh 
> would be less stressful than multi-step processes like Tao-Hsin's advice 
> unless your crystals can re-anneal after stress.  Mounting directly from the 
> drop is probably essential, and mounting under oil is a good thing to try in 
> addition - apart from anything else oil on the drop slows down the 
> environmental changes.  Using Mitegen mounts might be less stressful on some 
> crystals than standard nylon loops if they are mechanically sensitive.  
> Spending some time optimizing the mechanics of your freezing technique might 
> help significantly in reducing the amount of time your crystal dehydrates 
> while moving through air.
> (Jim Pflugrath's article is full of useful information:
> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4461322/ )
> 
> Small crystals often freeze more smoothly than large ones - even for robust 
> crystals like tetragonal lysozyme.  Try a lot of crystals - I've had projects 
> were two different crystals in the same loop from the same drop showed 
> radically different diffraction.  Also I've encountered several cases where 
> the appearance of disorder varies within a crystal when using a microfocus 
> synchrotron beam line (I mostly use FMX and AMX at NSLS2).
> 
> Lastly, really cranky crystals rings a distant bell of something we 
> encountered in the p19(INK4d)-Cdk6 structure back in the 1990's.  I think it 
> was Jie-Oh Lee that did the hard work on this, but in many instances crystals 
> cracked in situ when merely opening the drop, and the fix was by adding a 
> cross-linker to the well, resealing the drop and waiting for the cross-linker 
> to diffuse:
> 
> "The crystals were pretreated with glutaraldehyde (diffused into the drop 
> from a reservoir of 30% glutaraldehyde) to reduce their tendency to crack and 
> lose diffraction along b* and c*."
> https://www.nature.com/articles/26155#Sec9
> 
> Most crystals don't love being cross-linked, and I would call this a 
> successful instance of a desperation maneuver.
> 
> Good luck.
> Phil Jeffrey
> Princeton
> 
>> On 11/22/23 11:44 AM, Blake, Morgan Elizabeth wrote:
>> Hello
>> I am a PhD student working on a crystallography project to wrap up my 
>> dissertation research. I have purified a complex of two proteins, and I can 
>> consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane 
>> pH 7.5. These crystals have sharp edges and can grow to a large size 
>> (greater than 0.5 mm), but the crystals seem to be very fragile. When we 
>> open the drops to harvest the crystals, we have little time to harvest the 
>> crystals before they crack. When we move the crystals to a cryoprotectant, 
>> over time they start fracturing. We've tried using different percentages of 
>> glycerol, ethylene glycol, PEG400, and oil for cryoprotectants with no 
>> success. Needless to say, the crystals do not diffract well, with spot 
>> patterns that look very streaky/mosaic, which I presume is due to the 
>> defects that we see in harvesting/handling. We have screened for alternate 
>> crystallization conditions, but we seem to get the same morphology in other 
>> conditions. Does anyone have suggestions for additives we could use 
>> post-crystallization to help stabilize our crystals?
>> Thanks for your advice!
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
>> 
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> 

[ccp4bb] Quaternary Structure concept

[BRUNO DI GERONIMO QUINTERO]

Dear CCP4BB comunnity;

I am not a structural biologist thus far.

I would like to inquire about the concept of quaternary structure and its
applicability to the system I am currently investigating. The protein under
study is a human lysosomal protein (Gene MAN2B1) that initially presents as
a single chain. Upon entry into the lysosome (facilitated by the signal
peptide), it undergoes cleavage four times in four external loops,
resulting in the formation of five different "chains." The tertiary
structure remains unchanged, and only after denaturation and purification,
it becomes possible to observe the five distinct peptides. If the protein
remains in the endoplasmic reticulum (ER), it exists as a single chain, as
there is no cleavage due to the higher pH in the ER.

My question is as follows: can we consider the protein inside the lysosome
as having a quaternary structure? It originates from a monomer, and the
subunits exhibit different sequences while interacting with each other. But
indeed, there are 5 different peptides.

I would appreciate your expertise and consideration of this perhaps
unconventional question, which is causing me significant concern.

Best regards, Bruno Di Geronimo.



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Re: [ccp4bb] Fragile Crystals

[Phil Jeffrey]

Hello Morgan

In addition to the other good suggestions, I have a few observations of 
my own.


If your crystals crack without handling or adding anything to the drop, 
then they are extremely environment-sensitive.  If that's the case, 
testing at room temperature will be problematic because that tends to be 
somewhat stressful on the crystal either mechanically (ye olde capillary 
mount method) or via dehydration (loop mounts with the sleeve).


Growing in the presence of at least a little cryoprotectant as per Vaheh 
would be less stressful than multi-step processes like Tao-Hsin's advice 
unless your crystals can re-anneal after stress.  Mounting directly from 
the drop is probably essential, and mounting under oil is a good thing 
to try in addition - apart from anything else oil on the drop slows down 
the environmental changes.  Using Mitegen mounts might be less stressful 
on some crystals than standard nylon loops if they are mechanically 
sensitive.  Spending some time optimizing the mechanics of your freezing 
technique might help significantly in reducing the amount of time your 
crystal dehydrates while moving through air.

(Jim Pflugrath's article is full of useful information:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4461322/ )

Small crystals often freeze more smoothly than large ones - even for 
robust crystals like tetragonal lysozyme.  Try a lot of crystals - I've 
had projects were two different crystals in the same loop from the same 
drop showed radically different diffraction.  Also I've encountered 
several cases where the appearance of disorder varies within a crystal 
when using a microfocus synchrotron beam line (I mostly use FMX and AMX 
at NSLS2).


Lastly, really cranky crystals rings a distant bell of something we 
encountered in the p19(INK4d)-Cdk6 structure back in the 1990's.  I 
think it was Jie-Oh Lee that did the hard work on this, but in many 
instances crystals cracked in situ when merely opening the drop, and the 
fix was by adding a cross-linker to the well, resealing the drop and 
waiting for the cross-linker to diffuse:


"The crystals were pretreated with glutaraldehyde (diffused into the 
drop from a reservoir of 30% glutaraldehyde) to reduce their tendency to 
crack and lose diffraction along b* and c*."

https://www.nature.com/articles/26155#Sec9

Most crystals don't love being cross-linked, and I would call this a 
successful instance of a desperation maneuver.


Good luck.
Phil Jeffrey
Princeton

On 11/22/23 11:44 AM, Blake, Morgan Elizabeth wrote:

Hello

I am a PhD student working on a crystallography project to wrap up my 
dissertation research. I have purified a complex of two proteins, and I 
can consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS 
propane pH 7.5. These crystals have sharp edges and can grow to a large 
size (greater than 0.5 mm), but the crystals seem to be very fragile. 
When we open the drops to harvest the crystals, we have little time to 
harvest the crystals before they crack. When we move the crystals to a 
cryoprotectant, over time they start fracturing. We've tried using 
different percentages of glycerol, ethylene glycol, PEG400, and oil for 
cryoprotectants with no success. Needless to say, the crystals do not 
diffract well, with spot patterns that look very streaky/mosaic, which I 
presume is due to the defects that we see in harvesting/handling. We 
have screened for alternate crystallization conditions, but we seem to 
get the same morphology in other conditions. Does anyone have 
suggestions for additives we could use post-crystallization to help 
stabilize our crystals?


Thanks for your advice!



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Re: [ccp4bb] Fragile Crystals

[Tao-Hsin Chang]

Hi Morgan Elizabeth,

Just following up on Vaheh’s suggestion, you can also add 2-4% of 
cryoprotectant, which includes your mother liquor (and your protein if crystals 
have dissolved), into the crystallization drop daily or weekly after crystals 
appear. Repeat this step until reaching the desired concentration of 
cryoprotectant. Additionally, I will harvest smaller crystals in your case.

If you can test the diffraction at room temperature, it seems to be the first 
priority for me to determine whether it is worthwhile to spend time on this 
crystallization condition and construct.

Good luck!

Best wishes,
Tao-Hsin

> On Nov 22, 2023, at 12:26 PM, Oganesyan, Vaheh 
>  wrote:
> 
> Hi Morgan Elizabeth,
>  
> In some cases adding 1-2% of cryoprotectant into crystallization drop during 
> setting those drops up helps to introduce 25-30% of the same cryoprotectant 
> during harvest, provided you still can get those crystals to grow. Worked for 
> me in several cases.
>  
> Vaheh
>  
> From: CCP4 bulletin board  > On Behalf Of Blake, Morgan Elizabeth
> Sent: Wednesday, November 22, 2023 11:44 AM
> To: CCP4BB@JISCMAIL.AC.UK 
> Subject: [ccp4bb] Fragile Crystals
>  
> Hello!
>  
> I am a PhD student working on a crystallography project to wrap up my 
> dissertation research. I have purified a complex of two proteins, and I can 
> consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane 
> pH 7.5. These crystals have sharp edges and can grow to a large size (greater 
> than 0.5 mm), but the crystals seem to be very fragile. When we open the 
> drops to harvest the crystals, we have little time to harvest the crystals 
> before they crack. When we move the crystals to a cryoprotectant, over time 
> they start fracturing. We've tried using different percentages of glycerol, 
> ethylene glycol, PEG400, and oil for cryoprotectants with no success. 
> Needless to say, the crystals do not diffract well, with spot patterns that 
> look very streaky/mosaic, which I presume is due to the defects that we see 
> in harvesting/handling. We have screened for alternate crystallization 
> conditions, but we seem to get the same morphology in other conditions. Does 
> anyone have suggestions for additives we could use post-crystallization to 
> help stabilize our crystals?
>  
> Thanks for your advice!
>  
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
> 



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Re: [ccp4bb] Fragile Crystals

[Oganesyan, Vaheh]

Hi Morgan Elizabeth,

In some cases adding 1-2% of cryoprotectant into crystallization drop during 
setting those drops up helps to introduce 25-30% of the same cryoprotectant 
during harvest, provided you still can get those crystals to grow. Worked for 
me in several cases.

Vaheh

From: CCP4 bulletin board  On Behalf Of Blake, Morgan 
Elizabeth
Sent: Wednesday, November 22, 2023 11:44 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Fragile Crystals

Hello!

I am a PhD student working on a crystallography project to wrap up my 
dissertation research. I have purified a complex of two proteins, and I can 
consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane pH 
7.5. These crystals have sharp edges and can grow to a large size (greater than 
0.5 mm), but the crystals seem to be very fragile. When we open the drops to 
harvest the crystals, we have little time to harvest the crystals before they 
crack. When we move the crystals to a cryoprotectant, over time they start 
fracturing. We've tried using different percentages of glycerol, ethylene 
glycol, PEG400, and oil for cryoprotectants with no success. Needless to say, 
the crystals do not diffract well, with spot patterns that look very 
streaky/mosaic, which I presume is due to the defects that we see in 
harvesting/handling. We have screened for alternate crystallization conditions, 
but we seem to get the same morphology in other conditions. Does anyone have 
suggestions for additives we could use post-crystallization to help stabilize 
our crystals?

Thanks for your advice!



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



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Re: [ccp4bb] Fragile Crystals

[srikannathasan velupillai]

Hi Blake,
 You can cross seed and get new condition with good stable crystal
system. Also you could try VMXi beamline, you do not need to use cryo and
use the plate directly.. You have to use Greiner Crystal QuickX ™ or
MiTeGen In-Situ-1 ™ plates.

Thanks Kannan
Show quoted text

On Wed, 22 Nov 2023, 16:54 Blake, Morgan Elizabeth,  wrote:

> Hello!
>
> I am a PhD student working on a crystallography project to wrap up my
> dissertation research. I have purified a complex of two proteins, and I can
> consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane
> pH 7.5. These crystals have sharp edges and can grow to a large size
> (greater than 0.5 mm), but the crystals seem to be very fragile. When we
> open the drops to harvest the crystals, we have little time to harvest the
> crystals before they crack. When we move the crystals to a cryoprotectant,
> over time they start fracturing. We've tried using different percentages of
> glycerol, ethylene glycol, PEG400, and oil for cryoprotectants with no
> success. Needless to say, the crystals do not diffract well, with spot
> patterns that look very streaky/mosaic, which I presume is due to the
> defects that we see in harvesting/handling. We have screened for alternate
> crystallization conditions, but we seem to get the same morphology in other
> conditions. Does anyone have suggestions for additives we could use
> post-crystallization to help stabilize our crystals?
>
> Thanks for your advice!
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
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Re: [ccp4bb] Fragile Crystals

[Sarah Bowman]

Hi Morgan,

Some of the things that you could try with the conditions that you have already 
found:

1) Lower the temperature for incubation, which might slow down crystal 
formation and decrease potential defects as the crystals form

2) Harvest the crystals using a humidity control device like the Watershed from 
MiTeGen

3) Try adding oil to the top of the well and harvest through the oil as 
cryoprotectant

4) Try different cryos and different methods of adding cryoprotectant

5) Try collecting at room temperature – that may help identify if the issue is 
the crystals or the cryo

Finally, you may need to find other conditions in which your complex forms 
crystals.  Feel free to reach out to the National Crystallization Center at HWI 
if you need assistance with finding new conditions (www.getacrystal.org).

Good luck!
Sarah

Sarah EJ Bowman PhD
Associate Investigator | Hauptman-Woodward Medical Research Institute
Director | National High-Throughput Crystallization Center

p: +1 716 898 8623
e: sbowman at hwi.buffalo.edu

Research Webpage
Crystallization Center Webpage

Hauptman-Woodward Medical Research Institute
700 Ellicott Street | Buffalo, NY 14203

From: CCP4 bulletin board  on behalf of "Blake, Morgan 
Elizabeth" 
Reply-To: "Blake, Morgan Elizabeth" 
Date: Wednesday, November 22, 2023 at 11:54 AM
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: [ccp4bb] Fragile Crystals

Hello! I am a PhD student working on a crystallography project to wrap up my 
dissertation research. I have purified a complex of two proteins, and I can 
consistently grow crystals in 10% PEG3350,
Warning! This message was sent from outside your organization and we were 
unable to verify the sender.

sophospsmartbannerend
Hello!

I am a PhD student working on a crystallography project to wrap up my 
dissertation research. I have purified a complex of two proteins, and I can 
consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane pH 
7.5. These crystals have sharp edges and can grow to a large size (greater than 
0.5 mm), but the crystals seem to be very fragile. When we open the drops to 
harvest the crystals, we have little time to harvest the crystals before they 
crack. When we move the crystals to a cryoprotectant, over time they start 
fracturing. We've tried using different percentages of glycerol, ethylene 
glycol, PEG400, and oil for cryoprotectants with no success. Needless to say, 
the crystals do not diffract well, with spot patterns that look very 
streaky/mosaic, which I presume is due to the defects that we see in 
harvesting/handling. We have screened for alternate crystallization conditions, 
but we seem to get the same morphology in other conditions. Does anyone have 
suggestions for additives we could use post-crystallization to help stabilize 
our crystals?

Thanks for your advice!



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Re: [ccp4bb] Fragile Crystals

[David J. Schuller]

There are multiple avenues you could explore.

If you think handling is an issue, you could look into growing and collecting 
with "in situ"  plates.

You could test for diffraction at room temp, or even get a full data set at 
room temp by combining data from multiple crystals. If diffraction is not good 
at room temp, it is probably not going to be better under cryo conditions.

As for cryo, instead of trying cryoprotectant A or cryoprotectant B, you could 
try a mixture of multiple cryoprotectants:

https://pubs.acs.org/doi/abs/10.1021/acs.cgd.5b01692
Use of Multiple Cryoprotectants to Improve Diffraction Quality from Protein 
Crystals
Senda, et al.
https://doi.org/10.1021/acs.cgd.5b01692

Evidence of Kinetic Cooperativity in Dimeric Ketopantoate Reductase from 
Staphylococcus aureus
JE Sanchez, PG Gross, RW Goetze, RM Walsh Jr, WB Peeples, ZA Wood
Biochemistry 54 (21), 3360-3369

===
 All Things Serve the Beam
 ===
 David J. Schuller
 modern man in a post-modern world
 MacCHESS, Cornell University
 schul...@cornell.edu

From: CCP4 bulletin board  on behalf of Blake, Morgan 
Elizabeth 
Sent: Wednesday, November 22, 2023 11:44 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Fragile Crystals

Hello!

I am a PhD student working on a crystallography project to wrap up my 
dissertation research. I have purified a complex of two proteins, and I can 
consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane pH 
7.5. These crystals have sharp edges and can grow to a large size (greater than 
0.5 mm), but the crystals seem to be very fragile. When we open the drops to 
harvest the crystals, we have little time to harvest the crystals before they 
crack. When we move the crystals to a cryoprotectant, over time they start 
fracturing. We've tried using different percentages of glycerol, ethylene 
glycol, PEG400, and oil for cryoprotectants with no success. Needless to say, 
the crystals do not diffract well, with spot patterns that look very 
streaky/mosaic, which I presume is due to the defects that we see in 
harvesting/handling. We have screened for alternate crystallization conditions, 
but we seem to get the same morphology in other conditions. Does anyone have 
suggestions for additives we could use post-crystallization to help stabilize 
our crystals?

Thanks for your advice!



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https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



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Re: [ccp4bb] Fragile Crystals

[Nicholas Clark]

Hi Morgan,

Have you tried MMS?

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4157405/

If you can reproducibly grow crystals MMS might give you hits in different
conditions during screening. I’ve used this successfully multiple times for
crystals that wouldn’t optimize with standard methods. We were able to
solve the structure in each case.

Hope this helps.

Best,

Nick Clark

On Wed, Nov 22, 2023 at 11:54 AM Blake, Morgan Elizabeth 
wrote:

> Hello!
>
> I am a PhD student working on a crystallography project to wrap up my
> dissertation research. I have purified a complex of two proteins, and I can
> consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane
> pH 7.5. These crystals have sharp edges and can grow to a large size
> (greater than 0.5 mm), but the crystals seem to be very fragile. When we
> open the drops to harvest the crystals, we have little time to harvest the
> crystals before they crack. When we move the crystals to a cryoprotectant,
> over time they start fracturing. We've tried using different percentages of
> glycerol, ethylene glycol, PEG400, and oil for cryoprotectants with no
> success. Needless to say, the crystals do not diffract well, with spot
> patterns that look very streaky/mosaic, which I presume is due to the
> defects that we see in harvesting/handling. We have screened for alternate
> crystallization conditions, but we seem to get the same morphology in other
> conditions. Does anyone have suggestions for additives we could use
> post-crystallization to help stabilize our crystals?
>
> Thanks for your advice!
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
> 
>



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[ccp4bb] Fragile Crystals

[Blake, Morgan Elizabeth]

Hello!

I am a PhD student working on a crystallography project to wrap up my 
dissertation research. I have purified a complex of two proteins, and I can 
consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane pH 
7.5. These crystals have sharp edges and can grow to a large size (greater than 
0.5 mm), but the crystals seem to be very fragile. When we open the drops to 
harvest the crystals, we have little time to harvest the crystals before they 
crack. When we move the crystals to a cryoprotectant, over time they start 
fracturing. We've tried using different percentages of glycerol, ethylene 
glycol, PEG400, and oil for cryoprotectants with no success. Needless to say, 
the crystals do not diffract well, with spot patterns that look very 
streaky/mosaic, which I presume is due to the defects that we see in 
harvesting/handling. We have screened for alternate crystallization conditions, 
but we seem to get the same morphology in other conditions. Does anyone have 
suggestions for additives we could use post-crystallization to help stabilize 
our crystals?

Thanks for your advice!



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Re: [ccp4bb] low resolution data refinement

[M T]

Dear Liu,

If you are really confident in your MR solution some good tools to refine low 
resolution structures are LORESTR pipeline and iSOLDE software.

Best.

Michel.

> Le 21 nov. 2023 à 13:03, Yahui Liu  a écrit :
> 
> 
> Dear all,
> I got a protein crystal dataset of 4.3 A and would like to some the structure 
> with MR.
> Now I am suffering with the refinement. I used both Refmac and Phenix.
> 
> Someone could give me a hand or  any suggestions?
> 
> All the best
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



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Re: [ccp4bb] low resolution data refinement

[Jon Cooper]

Not sure if it's been mentioned that NCS can help a lot at that resolution. We 
did some mutants derived from a 2 Angstrom native structure at about that 
resolution with 4-fold NCS and the maps were quite convincing, well, I thought 
anyway. That was in the days of RESTRAIN which, I think, had an implementation 
of NCS refinement that was a bit different from current progs. Ian will know ;-0

Best wishes, Jon Cooper. jon.b.coo...@protonmail.com

Sent from Proton Mail mobile

 Original Message 
On 21 Nov 2023, 20:27, Albert Castellvi Toledo wrote:

> Also forget to observe side chains at 4A! At this resolution is crucial to 
> critically assess the position of each amino acid in your model, taking into 
> account factors such as hinges in your structure, hydrophobic and hydrophilic 
> regions, and the types of contacts between secondary structure elements. 
> Paying attention to these details can enhance the accuracy and reliability of 
> your model.
>
> Good luck!
>
> Albert Castellví
>
> On 11/21/23 20:57, Albert Castellvi Toledo wrote:
>
>> Hello,
>>
>> At low resolutions, model bias becomes a significant issue, capable of 
>> yielding inaccurate MR solutions even with favorable figures of merit. In my 
>> experience, it is always necessary to validate MR solutions at low 
>> resolution. One effective approach involves systematically omitting small 
>> fragments from the model and see if the resulting maps suggest you the 
>> placement of the omitted fragment or not. This method enables the 
>> identification of whether the solution is genuinely robust or if it is 
>> highly influenced by model bias.
>>
>> Salut,
>>
>> Albert Castellví
>>
>> On 11/21/23 13:02, Yahui Liu wrote:
>>
>>> Dear all,
>>> I got a protein crystal dataset of 4.3 A and would like to some the 
>>> structure with MR.
>>> Now I am suffering with the refinement. I used both Refmac and Phenix.
>>>
>>> Someone could give me a hand or any suggestions?
>>>
>>> All the best
>>>
>>> ---
>>>
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>>
>> ---
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>
> ---
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



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[ccp4bb] ECA lunchtime webinar

[Manfred Weiss]

Dear all,

on the 23rd November 2023 at 13:00 CET Dr. Tatjana Barthel (HZB Berlin)

will present a talk on "Crystallographic Fragment Screening at the HZB -
Workflow,

Tools and Procedures" as part of the ECA Lunch webinar series.

You can register for the webinar here:

https://www.eventbrite.com/e/24th-eca-lunch-webinar-tickets-761521730997?aff=
oddtdtcreator.

Posted on behalf of the organizers.





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[ccp4bb] Postdoc CNRS Bordeaux

[Nicolas Reyes]

Dear all,

· A Postdoctoral position is available in the laboratory of Nico Reyes 
at the CNRS in Bordeaux, France. We are an international team working on novel 
functional and pharmacological mechanisms of human membrane proteins. We 
combine functional and biophysical approaches (ITC, fluorescence spectroscopy, 
and others) with single-particle cryoEM, and have regular access to excellent 
biochemistry, biophysics, and cryoEM in-house facilities. Recent articles from 
the lab can be found here (PMID: 35545671, 35710838, 34747040).

We are looking for a highly motivated candidate with a PhD in Structural 
Biology, Biophysics or related disciplines with a good publication record and 
willing to work on challenging projects involving human membrane proteins. 
Hands-on experience and knowledge in membrane protein expression and 
purification, as well as structural biology (X-ray crystallography, NMR, and/or 
Cryo-EM) are essential for the position. The working language in the lab is 
English, and the candidate should speak and write it comfortably. 

 

Interested candidates, please send your CV, the contact info at least 2 
references, as well as the tittle of the most interesting article you read in 
2023, along with a short paragraph explaining why the article is brilliant, to 
nicolas.re...@u-bordeaux.fr

 

Bordeaux hosts one of the top universities in France (www.u-bordeaux.com) with 
a large and international campus, where our lab is located. It is also a lively 
and fun city in the south of France nearby the ocean, mountains, and the border 
with Spain. Candidates from any nationality and genre are welcome to apply, and 
we are committed to maintain an international and equal-opportunity working 
environment.

 




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