Re: [ccp4bb] Water

[Shekhar Mande]

Welljust to add, it has been our contention that many of the metal ions
have been modelled as waters in several structures- due perhaps to the lack
of sufficiently high resolution data.  We published some of the potential
metal binding sites in many structures a few years ago:

Proteins. 2008 Mar;70(4):1206-18.

Shekhar

On Thu, Mar 8, 2012 at 9:42 AM, Parthasarathy Sampathkumar <
spart...@gmail.com> wrote:

> Dear Uma,
>
> The water pictured in W12-1.jpg: could this be a potential metal ion? If
> you flip the side chain on Asn at 3.08Angstrom, then this has 3 or 4
> coordination with oxygen atoms. So, provided your crystallization condition
> or buffer contains metal ion(s), you could attempt to see if it fits better
> with a refinement cycle.
>
> May be a similar situation with the water described in W11-1.jpg as well?
> Difficult to say from these figures.
>
> COOT within the "validate" wizard has an option to search for
> "hihgly-coordinated waters" like the one you have pictured.
>
> Hope this helps,
> Partha
>
> On Wed, Mar 7, 2012 at 4:21 PM, Uma Ratu  wrote:
>
>> Dear Roger:
>>
>> Thank you very much for your comments. I use them as guideline and remove
>> many 'false waters".
>>
>> Still, I am not clear of some of these 'waters' are real or not. I have
>> the pic attached.
>>
>> In Pic-W11-1, the 'water' is connected to the adjust residues with 4
>> contacts, which are 'N' or 'O' atoms. I would consider this 'water' is
>> false. My question is: if these 4 contacts include "C" from residues, will
>> it be a polar contact or not?
>>
>> In Pic-W12-1, the 'water' is connected to the adjust residues with 3
>> contacts. The 4th is to another 'water'.
>> Will this 'water' is true or not? Similar case is seen in Pic-W190-1
>>
>> In Pic-W109-1, some 'waters' are connected to adjust residues, some not.
>> Are these 'water' true or not?
>>
>> Further more,
>> > and the b-factors are not way out of line,
>>
>> I am not clear on how to define "out of line".
>> How to find b-factor of individual residue in Coot? I search the web, but
>> find no answer.
>>
>> Thank you for advice
>>
>> Uma
>>
>> On Wed, Mar 7, 2012 at 11:44 AM, Roger Rowlett wrote:
>>
>>> Uma,
>>>
>>> Remember that your structure, ultimately, is a model. A model is your
>>> best judgment of the true representation of the protein structure in your
>>> crystal. Your model should make chemical sense. Coot is pretty good at
>>> placing waters, but it cannot substitute entirely for the experimentalist.
>>> Coot will miss some waters, and mis-assign others into weak, unmodeled or
>>> alternate side- or main-chain density, or into density that might be
>>> attributable to cations and anions or other crystallization materials. Your
>>> waters should be subjected to inspection and verification. It is really
>>> helpful to turn on environment distances in Coot when you do this. Even in
>>> a large protein model, it is possible to inspect all waters for
>>> reasonableness pretty quickly. If you have no significant positive or
>>> negative difference density, and the b-factors are not way out of line, and
>>> hydrogen bonding partners are reasonable, then modeling a water is probably
>>> a good call.
>>>
>>> Waters should have hydrogen bonding partners with side chains or
>>> main-chain polar atoms, within reasonable distances, or be withing hydrogen
>>> bonding distance of other waters that are (chains of waters). If a "water"
>>> has strong electron density and more than 4 polar contacts, you might
>>> consider anion or cation occupancy. Most anions and cations will have
>>> higher electron density, and appropriately different types of polar
>>> contacts. (e.g. you might find sulfates near a cluster of basic residues).
>>> Low occupancy anions can often look a lot like water. PEGs can create ugly
>>> "snakes" of variable density that may be challenging to model. Modeling
>>> non-protein structural bits is endlessly entertaining for the protein
>>> crystallographer. ;)
>>>
>>> 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 3/7/2012 11:20 AM, Uma Ratu wrote:
>>>
>>> Dear All:
>>>
>>> I try to add water to my model.
>>>
>>> Here is how I did:
>>> Coot: Find Wates
>>>  Map: FWT PHWT;  1.8 rmsd; Distances to protein atoms:
>>> 2.4 min/3.2 max
>>>
>>> Coot found 270 water molecules.
>>>
>>> I then examed these waters. Most of them had ball shape. Some had two or
>>> more balls together. Some had irregular shape (not glabol shape).
>>>
>>> I run Water Check. The program did not find any mis-matched water.
>>>
>>> Here is my question: how could I tell the waters are real? Or something
>>> else?
>>>
>>> Thank you for advice
>>>
>>> Ros
>>>
>>>
>>>
>>>
>>>
>>
>


-- 
Sh

Re: [ccp4bb] off topic: fluorescence polarization positive control

[KAMESH Narasimhan (GIS)]

If you have fluorescein (10-100nM) in your lab, you could measure the effect of 
varying the amount of glycerol (0 to 100%) on the fluorescence anisotropy of 
fluorescein in a phosphate buffer. The anisotropy should increase with 
increasing concentration of glycerol. Fluorescein by itself in a buffer should 
have very very low anisotropy.

Or you could do a fluorescence quenching experiment by titrating potassium 
iodide (0 to 500mM KI) into a fluorescein solution. The anisotropy should 
increase with increasing KI. (because of decrease in fluorescence lifetime)

I have never done the above experiments, as I always had a macromolecular 
binding assay that worked. But you could give the above recipe a try. It should 
work.

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Huiming Li
Sent: Thursday, March 08, 2012 2:21 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] off topic: fluorescence polarization positive control

Hi all,

  Sorry for the off topic question.  Could someone suggest a positive control 
for FP assay? I am developing this assay and would like to use it for trouble 
shooting an instrument.

Thanks,

Huiming Li

Re: [ccp4bb] Water

[Parthasarathy Sampathkumar]

Dear Uma,

The water pictured in W12-1.jpg: could this be a potential metal ion? If
you flip the side chain on Asn at 3.08Angstrom, then this has 3 or 4
coordination with oxygen atoms. So, provided your crystallization condition
or buffer contains metal ion(s), you could attempt to see if it fits better
with a refinement cycle.

May be a similar situation with the water described in W11-1.jpg as well?
Difficult to say from these figures.

COOT within the "validate" wizard has an option to search for
"hihgly-coordinated waters" like the one you have pictured.

Hope this helps,
Partha

On Wed, Mar 7, 2012 at 4:21 PM, Uma Ratu  wrote:

> Dear Roger:
>
> Thank you very much for your comments. I use them as guideline and remove
> many 'false waters".
>
> Still, I am not clear of some of these 'waters' are real or not. I have
> the pic attached.
>
> In Pic-W11-1, the 'water' is connected to the adjust residues with 4
> contacts, which are 'N' or 'O' atoms. I would consider this 'water' is
> false. My question is: if these 4 contacts include "C" from residues, will
> it be a polar contact or not?
>
> In Pic-W12-1, the 'water' is connected to the adjust residues with 3
> contacts. The 4th is to another 'water'.
> Will this 'water' is true or not? Similar case is seen in Pic-W190-1
>
> In Pic-W109-1, some 'waters' are connected to adjust residues, some not.
> Are these 'water' true or not?
>
> Further more,
> > and the b-factors are not way out of line,
>
> I am not clear on how to define "out of line".
> How to find b-factor of individual residue in Coot? I search the web, but
> find no answer.
>
> Thank you for advice
>
> Uma
>
> On Wed, Mar 7, 2012 at 11:44 AM, Roger Rowlett wrote:
>
>> Uma,
>>
>> Remember that your structure, ultimately, is a model. A model is your
>> best judgment of the true representation of the protein structure in your
>> crystal. Your model should make chemical sense. Coot is pretty good at
>> placing waters, but it cannot substitute entirely for the experimentalist.
>> Coot will miss some waters, and mis-assign others into weak, unmodeled or
>> alternate side- or main-chain density, or into density that might be
>> attributable to cations and anions or other crystallization materials. Your
>> waters should be subjected to inspection and verification. It is really
>> helpful to turn on environment distances in Coot when you do this. Even in
>> a large protein model, it is possible to inspect all waters for
>> reasonableness pretty quickly. If you have no significant positive or
>> negative difference density, and the b-factors are not way out of line, and
>> hydrogen bonding partners are reasonable, then modeling a water is probably
>> a good call.
>>
>> Waters should have hydrogen bonding partners with side chains or
>> main-chain polar atoms, within reasonable distances, or be withing hydrogen
>> bonding distance of other waters that are (chains of waters). If a "water"
>> has strong electron density and more than 4 polar contacts, you might
>> consider anion or cation occupancy. Most anions and cations will have
>> higher electron density, and appropriately different types of polar
>> contacts. (e.g. you might find sulfates near a cluster of basic residues).
>> Low occupancy anions can often look a lot like water. PEGs can create ugly
>> "snakes" of variable density that may be challenging to model. Modeling
>> non-protein structural bits is endlessly entertaining for the protein
>> crystallographer. ;)
>>
>> 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 3/7/2012 11:20 AM, Uma Ratu wrote:
>>
>> Dear All:
>>
>> I try to add water to my model.
>>
>> Here is how I did:
>> Coot: Find Wates
>>  Map: FWT PHWT;  1.8 rmsd; Distances to protein atoms:
>> 2.4 min/3.2 max
>>
>> Coot found 270 water molecules.
>>
>> I then examed these waters. Most of them had ball shape. Some had two or
>> more balls together. Some had irregular shape (not glabol shape).
>>
>> I run Water Check. The program did not find any mis-matched water.
>>
>> Here is my question: how could I tell the waters are real? Or something
>> else?
>>
>> Thank you for advice
>>
>> Ros
>>
>>
>>
>>
>>
>

Re: [ccp4bb] Water

[Eleanor Dodson]

I guess we would all like to be able to do that!

High resolution structures show that a) there are well defined H2Os with 
tidy H bonds. b) there are multiple networks where the waters (and many 
side chains) have partial occupancy c) there is a soup of other "stuff" 
which was in the crystallisation medium and is very difficult to model..


There are some lessons - never forget what was in the crystallisation ..
remember that the solvent structure will never be complete

good luck
eleanor 




On Mar 7 2012, Uma Ratu wrote:


Dear All:

I try to add water to my model.

Here is how I did:
Coot: Find Wates
Map: FWT PHWT;  1.8 rmsd; Distances to protein atoms: 2.4
min/3.2 max

Coot found 270 water molecules.

I then examed these waters. Most of them had ball shape. Some had two or
more balls together. Some had irregular shape (not glabol shape).

I run Water Check. The program did not find any mis-matched water.

Here is my question: how could I tell the waters are real? Or something
else?

Thank you for advice

Ros



--
Professor Eleanor Dodson
YSNL, Dept of Chemistry
University of York
Heslington YO10 5YW
tel: 00 44 1904 328259
Fax: 00 44 1904 328266

Re: [ccp4bb] Water

[Bernhard Rupp (Hofkristallrat a.D.)]

Ø  Some of these 'water' have more than 4 contacts, I would consider them as
'false'. 

 

How about bifurcated hydrogen bonds?

 

BR

Re: [ccp4bb] REFMAC Riding Hydrogens

[Garib N Murshudov]

Hi

1) refmac's default option has been "generate all" starting from version 5 (it 
is around 14 years). The reason is for most tests generating hydrogens gave 
good R or geometry. And if you look at the original paper by Ramachandran (I 
think it was pulished in 1963) you see using hard ball approximation and 
including all atoms generated plot is very similar to what we know as 
Ramachandran plot. A little bit more work on included hydrogen plus phi/psi 
torsion angle restraints (in free form, not from Ramachandran plot) should 
generate faithfully Ramchandran plot. It needs to be tested properly and 
carefully. refmac version 4 did not have hydrogen generation option.
2) ccp4i's option has always been use hydrogens if present in file (I 
personally do not like this option: this option should be used with care)
3) If there is no non-bondedn ... others then it is very likely that hydrogens 
were not used. 
4) I am not sure that there is a fullproof way to know if hydrogens were added 
or not.

Regards
Garib


On 7 Mar 2012, at 15:00, Paul Smith wrote:

> Hello CCP4bb,
> 
> Firstly, thanks to all for your comments.  However, I'm still unsure how to 
> sort all of this riding hydrogen business out.
> 
> Robbie's comments seem particularly apt:
> 
> "Because there were some reporting errors in the past 
> (http://proteincrystallography.org/ccp4bb/message18808.html) it is hard to 
> tell from the PDB when refinement with hydrogens became hip."
> 
> Is there any foolproof way to know if a recently deposited file was refined 
> with riding hydrogens in REFMAC, especially since some such structures do not 
> yet have publications associated with them? How about the value of NON-BONDED 
> CONTACTS OTHERS? If that is other than NULL, does that mean riding hydrogens 
> were present?
> 
> Also, how about refmac version numbers?  Is there a clear demarcation when 
> riding hydrogens a) became available, b) became default, or c) became default 
> in the CCP4i GUI?
> 
> Thanks again,
> 
> --Paul
> 
> From: Robbie Joosten 
> To: CCP4BB@JISCMAIL.AC.UK 
> Sent: Tuesday, March 6, 2012 4:26 AM
> Subject: Re: [ccp4bb] REFMAC Riding Hydrogens
> 
> Hi Everyone,
>  
> Pavel’s statement is likely a bit of an exaggeration, but he has a valid (yet 
> hard to prove point). The default in CCP4i was (and is?) to use hydrogens 
> only if present in the input file. This is IMO not a safe default.
> Because there were some reporting errors in the past 
> (http://proteincrystallography.org/ccp4bb/message18808.html) it is hard to 
> tell from the PDB when refinement with hydrogens became hip. Discussions on 
> this BB show that at the use of riding hydrogens is still not fully accepted, 
> especially at low resolution (where they actually help most).
>  
> Cheers,
> Robbie
>  
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Pavel 
> Afonine
> Sent: Monday, March 05, 2012 21:53
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] REFMAC Riding Hydrogens
>  
> Dear Tim,
>  
> good catch, thanks; I could craft that phrase more carefully! Although often 
> it may not be quite fair to take phrases out of context: this newsletter 
> article was written in the context of macromolecular refinement. And yes, 
> "recently" may be a broad term -:)
>  
> All the best,
> Pavel
> On Mon, Mar 5, 2012 at 12:45 PM, Tim Gruene  wrote:
> -BEGIN PGP SIGNED MESSAGE-
> Hash: SHA1
> 
> Dear Pavel,
> 
> you may want to add to the structures mentioned in [1] one or two
> organic structures present in the Cambridge Database.
> 
> "Until recently it was customary to ignore hydrogen atoms throughout the
> process of crystallographic X-­‐ray structure determination." [1]
> 
> 'recently' as in 1997 [2]? Even though 1997 is probably a poor
> estimation of the corresponding year...
> 
> Cheers,
> Tim
> 
> 
> [1] "On contribution of hydrogen atoms to X-ray scattering"
> http://www.phenix-online.org/newsletter/
> [2] http://shelx.uni-ac.gwdg.de/SHELX/shelx.pdf
> 
> On 03/05/2012 09:14 PM, Pavel Afonine wrote:
> > Hi,
> >
> > On Mon, Mar 5, 2012 at 11:52 AM, Matthew Franklin 
> > wrote:
> >
> >> Adding the riding hydrogens generally gives you some improvement in R
> >> factors even with a good quality (i.e. stereochemically correct) model.
> >>
> >
> > and here are the results of more or less systematic test that prove this:
> >
> > see "On contribution of hydrogen atoms to X-ray scattering"
> > here:
> > http://www.phenix-online.org/newsletter/
> >
> > Pavel
> >
> - --
> Dr Tim Gruene
> Institut fuer anorganische Chemie
> Tammannstr. 4
> D-37077 Goettingen
> 
> GPG Key ID = A46BEE1A
> -BEGIN PGP SIGNATURE-
> Version: GnuPG v1.4.11 (GNU/Linux)
> Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/
> 
> iD8DBQFPVSXkUxlJ7aRr7hoRAm1TAJ9Hyfhkl3yhD5QSKw9I4RSK58m0fACgmlxk
> YGILzeMam/3gQVmCeh0vQ8k=
> =3m2J
> -END PGP SIGNATURE-
>  
> 
> 

Garib N Murshudov 
Structural Studies Division
MRC Laboratory of Molecular Biology
H

Re: [ccp4bb] Water

[Uma Ratu]

Hi, Joel:

Thank you for your comments.

Some of these waters have 4 contacts all with "O" from adjacent residues.
As "O" can be doner as well as acceptor, I would consider them as 'real'
water. Some of these 'water' have more than 4 contacts, I
would consider them as 'false'.

I lower the H-bond seeting to 2 - 3.2. This helps to reduce the noise.

>The B-factor is displayed in Coot along the bottom (left) when you middle
click on an atom. You can also see the B-factor when  you read the pdb file
as text



I found the B-factor using both ways.



Thank you very much!



Uma

On Wed, Mar 7, 2012 at 5:39 PM, Joel Tyndall wrote:

>  Hi Uma,
>
> ** **
>
> Water has the capability of making 4 h-bonds, 2 from the two non-bonding
> pairs of electrons (h-bond acceptors - expect an N-H from an amide for
> example) as well as the two hydrogens (h-bond donors). I would refine all
> those waters and assume they are waters. If the distance to the other atoms
> is between 2.5-3.2 then you can assume the water to be correct. In many
> cases waters will h-bond (only)  to other water molecules.
>
> ** **
>
> The B-factor is displayed in Coot along the bottom (left) when you middle
> click on an atom. You can also see the B-factor when you read the pdb file
> as text
>
> ** **
>
> Hope this helps.
>
> ** **
>
> _
>
> Joel Tyndall, PhD
>
> Senior Lecturer in Medicinal Chemistry
> National School of Pharmacy
> University of Otago
> PO Box 56 Dunedin 9054
> New Zealand   
>
> Skype: jtyndall
> http://www.researcherid.com/rid/C-2803-2008
>
> Pukeka Matua
> Te Kura Taiwhanga Putaiao
> Te Whare Wananga o Otago
> Pouaka Poutapeta 56 Otepoti 9054
> Aotearoa
>
> Ph / Waea   +64 3 4797293
> Fax / Waeawhakaahua +64 3 4797034
>
> ** **
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Uma
> Ratu
> *Sent:* Thursday, 8 March 2012 10:22 a.m.
>
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] Water
>
> ** **
>
> Dear Roger:
>
>  
>
> Thank you very much for your comments. I use them as guideline and remove
> many 'false waters". 
>
>  
>
> Still, I am not clear of some of these 'waters' are real or not. I have
> the pic attached.
>
>  
>
> In Pic-W11-1, the 'water' is connected to the adjust residues with 4
> contacts, which are 'N' or 'O' atoms. I would consider this 'water' is
> false. My question is: if these 4 contacts include "C" from residues, will
> it be a polar contact or not?
>
>  
>
> In Pic-W12-1, the 'water' is connected to the adjust residues with 3
> contacts. The 4th is to another 'water'. 
>
> Will this 'water' is true or not? Similar case is seen in Pic-W190-1
>
>  
>
> In Pic-W109-1, some 'waters' are connected to adjust residues, some not.
> Are these 'water' true or not?
>
>  
>
> Further more, 
>
> > and the b-factors are not way out of line, 
>
>  
>
> I am not clear on how to define "out of line". 
>
> How to find b-factor of individual residue in Coot? I search the web, but
> find no answer. 
>
>  
>
> Thank you for advice
>
>  
>
> Uma
>
> On Wed, Mar 7, 2012 at 11:44 AM, Roger Rowlett 
> wrote:
>
> Uma,
>
> Remember that your structure, ultimately, is a model. A model is your best
> judgment of the true representation of the protein structure in your
> crystal. Your model should make chemical sense. Coot is pretty good at
> placing waters, but it cannot substitute entirely for the experimentalist.
> Coot will miss some waters, and mis-assign others into weak, unmodeled or
> alternate side- or main-chain density, or into density that might be
> attributable to cations and anions or other crystallization materials. Your
> waters should be subjected to inspection and verification. It is really
> helpful to turn on environment distances in Coot when you do this. Even in
> a large protein model, it is possible to inspect all waters for
> reasonableness pretty quickly. If you have no significant positive or
> negative difference density, and the b-factors are not way out of line, and
> hydrogen bonding partners are reasonable, then modeling a water is probably
> a good call.
>
> Waters should have hydrogen bonding partners with side chains or
> main-chain polar atoms, within reasonable distances, or be withing hydrogen
> bonding distance of other waters that are (chains of waters). If a "water"
> has strong electron density and more than 4 polar contacts, you might
> consider anion or cation occupancy. Most anions and cations will have
> higher electron density, and appropriately different types of polar
> contacts. (e.g. you might find sulfates near a cluster of basic residues).
> Low occupancy anions can often look a lot like water. PEGs can create ugly
> "snakes" of variable density that may be challenging to model. Modeling
> non-protein structural bits is endlessly entertaining for the protein
> crystallog

Re: [ccp4bb] Water

[Joel Tyndall]

Hi Uma,

Water has the capability of making 4 h-bonds, 2 from the two non-bonding pairs 
of electrons (h-bond acceptors - expect an N-H from an amide for example) as 
well as the two hydrogens (h-bond donors). I would refine all those waters and 
assume they are waters. If the distance to the other atoms is between 2.5-3.2 
then you can assume the water to be correct. In many cases waters will h-bond 
(only)  to other water molecules.

The B-factor is displayed in Coot along the bottom (left) when you middle click 
on an atom. You can also see the B-factor when you read the pdb file as text

Hope this helps.

_
Joel Tyndall, PhD

Senior Lecturer in Medicinal Chemistry
National School of Pharmacy
University of Otago
PO Box 56 Dunedin 9054
New Zealand
Skype: jtyndall
http://www.researcherid.com/rid/C-2803-2008
Pukeka Matua
Te Kura Taiwhanga Putaiao
Te Whare Wananga o Otago
Pouaka Poutapeta 56 Otepoti 9054
Aotearoa

Ph / Waea   +64 3 4797293
Fax / Waeawhakaahua +64 3 4797034

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Uma Ratu
Sent: Thursday, 8 March 2012 10:22 a.m.
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Water

Dear Roger:

Thank you very much for your comments. I use them as guideline and remove many 
'false waters".

Still, I am not clear of some of these 'waters' are real or not. I have the pic 
attached.

In Pic-W11-1, the 'water' is connected to the adjust residues with 4 contacts, 
which are 'N' or 'O' atoms. I would consider this 'water' is false. My question 
is: if these 4 contacts include "C" from residues, will it be a polar contact 
or not?

In Pic-W12-1, the 'water' is connected to the adjust residues with 3 contacts. 
The 4th is to another 'water'.
Will this 'water' is true or not? Similar case is seen in Pic-W190-1

In Pic-W109-1, some 'waters' are connected to adjust residues, some not. Are 
these 'water' true or not?

Further more,
> and the b-factors are not way out of line,

I am not clear on how to define "out of line".
How to find b-factor of individual residue in Coot? I search the web, but find 
no answer.

Thank you for advice

Uma
On Wed, Mar 7, 2012 at 11:44 AM, Roger Rowlett 
mailto:rrowl...@colgate.edu>> wrote:
Uma,

Remember that your structure, ultimately, is a model. A model is your best 
judgment of the true representation of the protein structure in your crystal. 
Your model should make chemical sense. Coot is pretty good at placing waters, 
but it cannot substitute entirely for the experimentalist. Coot will miss some 
waters, and mis-assign others into weak, unmodeled or alternate side- or 
main-chain density, or into density that might be attributable to cations and 
anions or other crystallization materials. Your waters should be subjected to 
inspection and verification. It is really helpful to turn on environment 
distances in Coot when you do this. Even in a large protein model, it is 
possible to inspect all waters for reasonableness pretty quickly. If you have 
no significant positive or negative difference density, and the b-factors are 
not way out of line, and hydrogen bonding partners are reasonable, then 
modeling a water is probably a good call.

Waters should have hydrogen bonding partners with side chains or main-chain 
polar atoms, within reasonable distances, or be withing hydrogen bonding 
distance of other waters that are (chains of waters). If a "water" has strong 
electron density and more than 4 polar contacts, you might consider anion or 
cation occupancy. Most anions and cations will have higher electron density, 
and appropriately different types of polar contacts. (e.g. you might find 
sulfates near a cluster of basic residues). Low occupancy anions can often look 
a lot like water. PEGs can create ugly "snakes" of variable density that may be 
challenging to model. Modeling non-protein structural bits is endlessly 
entertaining for the protein crystallographer. ;)

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 3/7/2012 11:20 AM, Uma Ratu wrote:
Dear All:

I try to add water to my model.

Here is how I did:
Coot: Find Wates
 Map: FWT PHWT;  1.8 rmsd; Distances to protein atoms: 2.4 
min/3.2 max

Coot found 270 water molecules.

I then examed these waters. Most of them had ball shape. Some had two or more 
balls together. Some had irregular shape (not glabol shape).

I run Water Check. The program did not find any mis-matched water.

Here is my question: how could I tell the waters are real? Or something else?

Thank you for advice

Ros

[ccp4bb] Directories & Projects Spring Cleaning

[mark Mayer]

I'd appreciate suggestions about how to reorganize my My Directories&ProjectDir 
listing.
Its currently got several years worth of work and is getting hard to work with.
I'd like to archive old projects (say by year) but keep all the CCP4i files for 
each project intact, so that I can easily go back to them in the future without 
having to wade through the current multi year list.

Thanks

[ccp4bb] off topic: fluorescence polarization positive control

[Huiming Li]

Hi all,
 
  Sorry for the off topic question.  Could someone suggest a positive control 
for FP assay? I am developing this assay and would like to use it for trouble 
shooting an instrument.
 
Thanks,
 
Huiming Li
  

[ccp4bb] PhD position Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, University of Oslo

[jens Preben Morth]

*Dear CCP4
*

*Please forward this advertisement to suitable candidates*

*best Preben
*

*
Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, 
University of Oslo*


**

*1 PhD research fellowship*

*Background:*

The Centre for Molecular Medicine Norway (NCMM, www.ncmm.uio.no) was 
established in 2008 as a national partnership institution with the 
European Molecular Biology Laboratory (www.embl.org). NCMM investigates 
and focuses on disease mechanisms in cancer, immune and hematological 
disorders as well as cardiovascular diseases.


A PhD position is available in the laboratory of Dr. Preben Morth. 
Theposition is funded by an NCMM grant and is available immediately. The 
Focus of the research will include membrane protein involved with 
osmoregulation and anion transport in bacteria and higher eukaryotes. 
Projects focus on bicarbonate transport through the plasma membrane and 
the intra cellular complexes involved with their tight control.


**

*The candidate:*

We search for highly motivated individuals holding an MSc, M.D. or 
equivalent degree with an interest in doing a PhD in experimental 
science working in an interactive international environment with 
techniques as outlined above. Applicants with X-ray structural biology 
background will be prioritized. Knowledge on membrane protein 
characterization using biophysical methods will be an advantage.


The University of Oslo has an agreement for all employees, aiming to 
secure rights to research results and intellectual property.


*The application:*

**The application should include:

1)A one-page statement of motivation and research interests

2)CV

3)Copies of Graduation Certificates

4)Names and contact details of 2-3 references (name, relation to 
candidate, e-mail and telephone number)


The position is for three years with a preferred start in May or June 2012.

*For further information:*

http://www.ncmm.uio.no/research/groups/membrane-transport/

http://www.ncmm.uio.no/research/groups/membrane-transport/morth-bio/

*Deadline for applications: *

*April9, 2012 *labeled with *Ref.no.**2012/3375-- PhD research fellow*

Inquiries regarding the positions can be directed to Dr. Preben Morth 
(j.p.mo...@ncmm.uio.no ).


Inquiries regarding the application can be directed to Nina Modahl 
(nina.mod...@ncmm.uio.no )


--
J. Preben Morth, Ph.D
Group Leader
Membrane Transport Group
Nordic EMBL Partnership
Centre for Molecular Medicine Norway (NCMM)
University of Oslo
P.O.Box 1137 Blindern
0318 Oslo, Norway

Email: j.p.mo...@ncmm.uio.no
Tel: +47 2284 0794

http://www.jpmorth.dk

Re: [ccp4bb] FW: endotoxin removal

[Kevin Jin]

I tried to use 250mM Tris pH 7.6, 1M NaCl and 5~10% 2-propanol for
dialysis. It works,



On Wed, Mar 7, 2012 at 6:50 AM, Jerry McCully
 wrote:
>
>
> Dear All;
>
>     We purified a His tag protein by Ni-NTA and gel-filtration from
> E.coli.
>
>  We tried two endotoxin removal resins from Pierce. However, it is
> hard to remove the endotoxins in the purified protein because the protein
> bound to the resin as well.
>
>  This protein contains quite a few aromatic residues and has a pI
> around 6.
>
>  Any ideas to remove the endotoxins will be highly appreciated.
>
>     best regards,
>
> Jerry McCully
>
>

Re: [ccp4bb] Water

[Roger Rowlett]

  
  
Uma,
  
  Remember that your structure, ultimately, is a model. A model is
  your best judgment of the true representation of the protein
  structure in your crystal. Your model should make chemical sense.
  Coot is pretty good at placing waters, but it cannot substitute
  entirely for the experimentalist. Coot will miss some waters, and
  mis-assign others into weak, unmodeled or alternate side- or
  main-chain density, or into density that might be
attributable to cations and anions or other crystallization
materials. Your waters should be subjected to inspection and
verification. It is really helpful to turn on environment distances
in Coot when you do this. Even in a large protein model, it is
possible to inspect all waters for reasonableness pretty quickly. If
you have no significant positive or negative difference density, and
the b-factors are not way out of line, and hydrogen bonding partners
are reasonable, then modeling a water is probably a good call.

Waters should have hydrogen bonding partners with side chains or
main-chain polar atoms, within reasonable distances, or be withing
hydrogen bonding distance of other waters that are (chains of
waters). If a "water" has strong electron density and more than 4
polar contacts, you might consider anion or cation occupancy. Most
anions and cations will have higher electron density, and
appropriately different types of polar contacts. (e.g. you might
find sulfates near a cluster of basic residues). Low occupancy
anions can often look a lot like water. PEGs can create ugly
"snakes" of variable density that may be challenging to model.
Modeling non-protein structural bits is endlessly entertaining for
the protein crystallographer. ;)

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 3/7/2012 11:20 AM, Uma Ratu wrote:

  Dear All:
   
  I try to add water to my model. 
   
  Here is how I did:
  Coot: Find Wates
   Map: FWT PHWT;  1.8 rmsd; Distances to
protein atoms: 2.4 min/3.2 max
   
  Coot found 270 water molecules. 
   
  I then examed these waters. Most of them had ball shape. Some
had two or more balls together. Some had irregular shape (not
glabol shape).
   
  I run Water Check. The program did not find any mis-matched
water.
   
  Here is my question: how could I tell the waters are real? Or
something else?
   
  Thank you for advice
   
  Ros
   
   
   

  

[ccp4bb] Water

[Uma Ratu]

Dear All:

I try to add water to my model.

Here is how I did:
Coot: Find Wates
 Map: FWT PHWT;  1.8 rmsd; Distances to protein atoms: 2.4
min/3.2 max

Coot found 270 water molecules.

I then examed these waters. Most of them had ball shape. Some had two or
more balls together. Some had irregular shape (not glabol shape).

I run Water Check. The program did not find any mis-matched water.

Here is my question: how could I tell the waters are real? Or something
else?

Thank you for advice

Ros

Re: [ccp4bb] FW: endotoxin removal

[lieh low]

Hi Jerry,
We have used 0.05% Triton X-100 in our wash buffer to wash the protein
on column (Ni-IMAC for His-tagged proteins and MabSelect for
antibodies). 20 column-volumes wash followed by 20 column-volume wash
without detergent is typically enough to reduce the endotoxin
significantly.
I would also suggest checking your FPLC system, and decontaminate with
2N NaOH if neccessary.
Hope that helps
Regards,
ray

On Wed, Mar 7, 2012 at 9:50 AM, Jerry McCully
 wrote:
>
>
> Dear All;
>
>     We purified a His tag protein by Ni-NTA and gel-filtration from
> E.coli.
>
>  We tried two endotoxin removal resins from Pierce. However, it is
> hard to remove the endotoxins in the purified protein because the protein
> bound to the resin as well.
>
>  This protein contains quite a few aromatic residues and has a pI
> around 6.
>
>  Any ideas to remove the endotoxins will be highly appreciated.
>
>     best regards,
>
> Jerry McCully
>
>

Re: [ccp4bb] REFMAC Riding Hydrogens

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Paul,

I doubt there is a foolproof way - if the authors did not describe the
refinement procedure properly in their publication and the PDB file
lacks this information in its header, I guess you cannot tell.

Tim

On 03/07/2012 04:00 PM, Paul Smith wrote:
> Hello CCP4bb,
> 
> Firstly, thanks to all for your comments.  However, I'm still unsure how to 
> sort all of this riding hydrogen business out.
> 
> Robbie's comments seem particularly apt:
> 
> "Because there were some reporting errors in the past 
> (http://proteincrystallography.org/ccp4bb/message18808.html) it is hard 
> to tell from the PDB when refinement with hydrogens became hip."
> 
> Is there any foolproof way to know if a recently deposited file was 
> refined with riding hydrogens in REFMAC, especially since some such 
> structures do not yet have publications associated with them? How about 
> the value of NON-BONDED CONTACTS OTHERS? If that is other than NULL, 
> does that mean riding hydrogens were present?
> 
> Also, how about refmac version numbers?  Is there a clear demarcation when 
> riding hydrogens a) became available, b) became default, or c) became default 
> in the CCP4i GUI?
> 
> Thanks again,
> 
> --Paul
> 
> 
> 
> 
>  From: Robbie Joosten 
> To: CCP4BB@JISCMAIL.AC.UK 
> Sent: Tuesday, March 6, 2012 4:26 AM
> Subject: Re: [ccp4bb] REFMAC Riding Hydrogens
>  
> 
> Hi Everyone, 
>  
> Pavel’s statement is likely a bit of an exaggeration, but he has a valid (yet 
> hard to prove point). The default in CCP4i was (and is?) to use hydrogens 
> only if present in the input file. This is IMO not a safe default. 
> Because there were some reporting errors in the past 
> (http://proteincrystallography.org/ccp4bb/message18808.html) it is hard to 
> tell from the PDB when refinement with hydrogens became hip. Discussions on 
> this BB show that at the use of riding hydrogens is still not fully accepted, 
> especially at low resolution (where they actually help most). 
>  
> Cheers,
> Robbie
>  
> From:CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Pavel 
> Afonine
> Sent: Monday, March 05, 2012 21:53
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] REFMAC Riding Hydrogens
>  
> Dear Tim,
>  
> good catch, thanks; I could craft that phrase more carefully! Although often 
> it may not be quite fair to take phrases out of context: this newsletter 
> article was written in the context of macromolecular refinement. And yes, 
> "recently" may be a broad term -:)
>  
> All the best,
> Pavel
> On Mon, Mar 5, 2012 at 12:45 PM, Tim Gruene  wrote:
> Dear Pavel,
> 
> you may want to add to the structures mentioned in [1] one or two
> organic structures present in the Cambridge Database.
> 
> "Until recently it was customary to ignore hydrogen atoms throughout the
> process of crystallographic X-­ray structure determination." [1]
> 
> 'recently' as in 1997 [2]? Even though 1997 is probably a poor
> estimation of the corresponding year...
> 
> Cheers,
> Tim
> 
> 
> [1] "On contribution of hydrogen atoms to X-ray scattering"
> http://www.phenix-online.org/newsletter/
> [2] http://shelx.uni-ac.gwdg.de/SHELX/shelx.pdf
> 
> On 03/05/2012 09:14 PM, Pavel Afonine wrote:
>> Hi,
> 
>> On Mon, Mar 5, 2012 at 11:52 AM, Matthew Franklin wrote:
> 
>>> Adding the riding hydrogens generally gives you some improvement in R
>>> factors even with a good quality (i.e. stereochemically correct) model.
>>>
> 
>> and here are the results of more or less systematic test that prove this:
> 
>> see "On contribution of hydrogen atoms to X-ray scattering"
>> here:
>> http://www.phenix-online.org/newsletter/
> 
>> Pavel
> 

- -- 
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A

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=zini
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Re: [ccp4bb] setting occupancy for disordered water

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear ARKO,

you can edit the PDB file by doubling the line of the respective water,
assign the the "Alternate location indicator" (column 17) to 'A' and 'B'
(see e.g. http://www.wwpdb.org/documentation/format33/sect9.html#ATOM),
and change the coordinates from 'B' to the second peak.

Make sure that the water is not near a symmetry axis - there are often
artefact of density near symmetry elements.

Tim

On 03/07/2012 02:20 PM, arka chakraborty wrote:
> Hi all,
> 
> I am having a structure where there is a disordered water .I have fixed one
> water into the density, yet there is a positive density about 1.6 ang away.
> I want to put in another water and attribute 0.50 occupancy to each of the
> the waters while attributing them to  one water in reality. I would like to
> know how to modify the pdb so that phenix.refine accepts it.
> 
> Thanks in advance,
> 
> Regards,
> 
> ARKO
> 

- -- 
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A

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Version: GnuPG v1.4.10 (GNU/Linux)
Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

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IVxnxkZNnSxXiIF2LNdr8AU=
=E2g7
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Re: [ccp4bb] REFMAC Riding Hydrogens

[Paul Smith]

Hello CCP4bb,

Firstly, thanks to all for your comments.  However, I'm still unsure how to 
sort all of this riding hydrogen business out.

Robbie's comments seem particularly apt:

"Because there were some reporting errors in the past 
(http://proteincrystallography.org/ccp4bb/message18808.html) it is hard 
to tell from the PDB when refinement with hydrogens became hip."

Is there any foolproof way to know if a recently deposited file was 
refined with riding hydrogens in REFMAC, especially since some such 
structures do not yet have publications associated with them? How about 
the value of NON-BONDED CONTACTS OTHERS? If that is other than NULL, 
does that mean riding hydrogens were present?

Also, how about refmac version numbers?  Is there a clear demarcation when 
riding hydrogens a) became available, b) became default, or c) became default 
in the CCP4i GUI?

Thanks again,

--Paul




 From: Robbie Joosten 
To: CCP4BB@JISCMAIL.AC.UK 
Sent: Tuesday, March 6, 2012 4:26 AM
Subject: Re: [ccp4bb] REFMAC Riding Hydrogens
 

Hi Everyone, 
 
Pavel’s statement is likely a bit of an exaggeration, but he has a valid (yet 
hard to prove point). The default in CCP4i was (and is?) to use hydrogens only 
if present in the input file. This is IMO not a safe default. 
Because there were some reporting errors in the past 
(http://proteincrystallography.org/ccp4bb/message18808.html) it is hard to tell 
from the PDB when refinement with hydrogens became hip. Discussions on this BB 
show that at the use of riding hydrogens is still not fully accepted, 
especially at low resolution (where they actually help most). 
 
Cheers,
Robbie
 
From:CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Pavel 
Afonine
Sent: Monday, March 05, 2012 21:53
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] REFMAC Riding Hydrogens
 
Dear Tim,
 
good catch, thanks; I could craft that phrase more carefully! Although often it 
may not be quite fair to take phrases out of context: this newsletter article 
was written in the context of macromolecular refinement. And yes, "recently" 
may be a broad term -:)
 
All the best,
Pavel
On Mon, Mar 5, 2012 at 12:45 PM, Tim Gruene  wrote:
-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Pavel,

you may want to add to the structures mentioned in [1] one or two
organic structures present in the Cambridge Database.

"Until recently it was customary to ignore hydrogen atoms throughout the
process of crystallographic X-­‐ray structure determination." [1]

'recently' as in 1997 [2]? Even though 1997 is probably a poor
estimation of the corresponding year...

Cheers,
Tim


[1] "On contribution of hydrogen atoms to X-ray scattering"
http://www.phenix-online.org/newsletter/
[2] http://shelx.uni-ac.gwdg.de/SHELX/shelx.pdf

On 03/05/2012 09:14 PM, Pavel Afonine wrote:
> Hi,
>
> On Mon, Mar 5, 2012 at 11:52 AM, Matthew Franklin wrote:
>
>> Adding the riding hydrogens generally gives you some improvement in R
>> factors even with a good quality (i.e. stereochemically correct) model.
>>
>
> and here are the results of more or less systematic test that prove this:
>
> see "On contribution of hydrogen atoms to X-ray scattering"
> here:
> http://www.phenix-online.org/newsletter/
>
> Pavel
>
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A
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=3m2J
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Re: [ccp4bb] How to reduce no. of overlaps

[Edward A. Berry]

Jim Pflugrath wrote:

In addition to reducing the beam divergence, you may wish to use a
smaller beam size by using a smaller collimator or making the slits
smaller. A smaller crystal can also help to spatially separate the Bragg
spots as can moving the detector closer to the crystal. Yes, closer to
the crystal. This is not intuitive, but arises since modern homelab
beams are not parallel but are diverging from a focal point near the
sample position. It is just something else you may wish to try.


But the pattern is also diverging from a point at the sample?

I'm guessing the focus point is somewhere between the crystal
and the detector, so by moving the detector closer you are better
approximating "focus on the detector" rather than "focus on the crystal"?

With a home source one probably has room for a Huber goniometer with
arcs, or better yet one of those goniometers that allows rotation
up to 90* about a point at the crystal, so the crystal doesn't move
out of the cold stream as you rotate.

One can also cheat on the mosaicity during integration by fixing
it at a small fraction of the true mosaicity. This is called
"cutting off the wings" or more euphemistically "peak height
sampling". The accuracy will suffer, but not as much as you
might expect- probably because if spot profiles are pretty
similar, the the height at  peak is a good measure of peak
volume.

eab

[ccp4bb] FW: endotoxin removal

[Jerry McCully]

Dear All;

We purified a His tag protein by Ni-NTA and gel-filtration from E.coli. 
 

 We tried two endotoxin removal resins from Pierce. However, it is hard 
to remove the endotoxins in the purified protein because the protein bound to 
the resin as well.

 This protein contains quite a few aromatic residues and has a pI 
around 6.

 Any ideas to remove the endotoxins will be highly appreciated.

best regards,

Jerry McCully



  

Re: [ccp4bb] How to reduce no. of overlaps

[Bosch, Juergen]

In addition to all the excellent suggestions if you can you can also move your 
detector away from the center of the beam aka increase the detector size in one 
dimension. Not sure if you can do that at your home source though. By moving 
the center of the beam say to the lower 9/10 of the detector you will still 
have the beam position on the detector but you will also have increased the 
total area. Needless to say you will need to collect over a larger wedge to get 
a complete dataset.

Regarding your overlap issues, it does help to spend some time thinking about 
the experiment before collecting the data, I would like to point toward the 
direction of Mosflm & strategy in conjunction with the Testgen option.

Depending how badly your overlapped data is, you might want to run it through 
XDS and see how much it can rescue, assuming you used Mosflm or HKL2000. You 
can also give the Separation Close command a try in Mosflm with Postref Width 
and Profile Optimize (RTFM for more details).

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: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://web.mac.com/bosch_lab/

Re: [ccp4bb] setting occupancy for disordered water

[Herman . Schreuder]

I would use in coot: calculate - model/fit/refine - add alt conf. This
should generate a correctly defined alternative water.
 
Good luck!
Herman




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On
Behalf Of arka chakraborty
Sent: Wednesday, March 07, 2012 2:21 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] setting occupancy for disordered water


Hi all,

I am having a structure where there is a disordered water .I
have fixed one water into the density, yet there is a positive density
about 1.6 ang away. I want to put in another water and attribute 0.50
occupancy to each of the the waters while attributing them to  one water
in reality. I would like to know how to modify the pdb so that
phenix.refine accepts it.

Thanks in advance,

Regards,

ARKO 

-- 


ARKA CHAKRABORTY
CAS in Crystallography and Biophysics
University of Madras
Chennai,India

[ccp4bb] setting occupancy for disordered water

[arka chakraborty]

Hi all,

I am having a structure where there is a disordered water .I have fixed one
water into the density, yet there is a positive density about 1.6 ang away.
I want to put in another water and attribute 0.50 occupancy to each of the
the waters while attributing them to  one water in reality. I would like to
know how to modify the pdb so that phenix.refine accepts it.

Thanks in advance,

Regards,

ARKO

-- 

*ARKA CHAKRABORTY*
*CAS in Crystallography and Biophysics*
*University of Madras*
*Chennai,India*

Re: [ccp4bb] How to reduce no. of overlaps

[Mark J van Raaij]

my experiences:
C2 with a long axis parallel to the shortest crystal dimension, crystals were 
plates. Used prebent loops to fish the crystals. Personally I haven't tried to 
bend loops in mounted crystals as Frank does, but it sounds very useful.
bar-shaped P321 crystals with hexagonal cross-sections, these naturally "fell" 
into loops in the "right" orientations. Too "right" usually, because you also 
don't want the long axis too parallel to the rotation axis and have a large 
blind cone compromising completeness, 10-20º from parallel is great, here a 
minikappa or the bendable loop mounts are very useful.

Mark J van Raaij
Laboratorio M-4
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 7 Mar 2012, at 08:44, Frank von Delft wrote:

> Yes... quite.  Thanks!
> 
> (Beam, rotation axis... how can you expect me to keep all these new-fangled 
> inventions apart?!)
> 
> 
> 
> On 07/03/2012 07:33, VAN RAAIJ , MARK JOHAN wrote:
>> typo correction, you'll want the long axis parallel to the rotation
>> axis, not to the beam.
>> Mark
>> 
>> Quoting Frank von Delft:
>> 
>>> You probably have to tilt your crystal, so that the long axis is
>>> parallel to the beam.  We do this routinely:  cut a plastic pipette
>>> tip to have a sharp point, then push the loop where it attaches to
>>> the pin, to bend the crystal itself.
>>> 
>>> You have to identify from your diffraction whether the long axis is
>>> pointing through the face or the edge of the loop.  As it's P6,
>>> chances are it's through the face, because long-axis P6 tends to
>>> make flat hexagons which lie flush with the face.  So you have to
>>> bend so the face of the loop upwards.
>>> 
>>> You'll have to practice this first, though, so put up an empty loop.
>>>  Top tips:
>>> * Don't breathe!  You'll blow the cryostream away.
>>> * Bend the loop towards (rather than away from) the rim edge of
>>> the pin to which it's glued.
>>> * Don't breathe!
>>> * Practise practise practise.
>>> 
>>> 
>>> Another thing:  most in-house sources allow you to reduce divergence
>>> of the beam.  You lose intensity, but no matter, just expose longer.
>>>  That also improves overlap.
>>> 
>>> Cheers
>>> phx
>>> 
>>> 
>>> 
>>> On 07/03/2012 04:56, Dipankar Manna wrote:
 Dear Crystallographers,
 
 I am working on a protein having SG P6, the cell parameters are a=
 79, b= 79, c= 325. The crystals are forming in big size and with
 very good shape. It also diffracting very well in Home source
 facility both in terms of resolution and intensity. But the only
 problem is the number of overlaps. Its showing much more than the
 good spots. As a result the completeness is showing maximum up to
 65% even after collecting 180 degrees. I am unable to get a
 complete data. I tried with reducing the oscillation angel to 0.3
 degree/0.5 degree but it did not improve that much. Please give me
 some suggestions.
 
 Regards,
 
 Dipankar
 
 /Dipankar Manna/
 
 */Aurigene Discovery Technologies Limited,/*
 
 /#39-40 KIADB Industrial Area, Electronic City, Phase II,/
 
 /Hosur Road, Bangalore- 560 100, India/
 
 /Cell: +91-9538631469 // | Office Ph  : +91 80-66204422 (Extn: 398)
  | Email ID: dipanka...@aurigene.com/
 
 
 
 
 This e-mail and any files transmitted with it are for the sole use
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 may be unlawful.
 
 Visit us at http://www.aurigene.com
>> 
>> 
>> Mark J van Raaij
>> Laboratorio M-4
>> Dpto de Estructura de Macromoléculas
>> Centro Nacional de Biotecnología - CSIC
>> c/Darwin 3, Campus Cantoblanco
>> 28049 Madrid
>> tel. 91 585 4616
>> email: mjvanra...@cnb.csic.es
>> 

Re: [ccp4bb] How to reduce no. of overlaps

[Zhijie Li]

Hi,

Besides aligning the long axis with the rotation axis, which is the most 
important, there are a few more things that may help:

1) Try optimizing the freezing to reduce the mosaicity (if not ideal), or shoot 
at RT if possible. With higher mosaicity, the shape of the reflections are 
elongated in the reciprocal space, increasing the chance of overlapping. 
2) Collect datesets in two runs, one with detector at far position to get more 
spacing between dots, another one at a closer position to get high res. 
Sometimes you may have to sacrifice some resolution for the completeness.
3) Shoot at a synchrotron with micro beam and a large detector.
4) Try integrating the images with both HKL2000 and mosflm. My past experience 
seems to suggest that mosflm is more tolerant to closely positioned reflections.

Zhijie


From: Dipankar Manna 
Sent: Tuesday, March 06, 2012 11:56 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] How to reduce no. of overlaps


Dear Crystallographers,



I am working on a protein having SG P6, the cell parameters are a= 79, b= 79, 
c= 325. The crystals are forming in big size and with very good shape. It also 
diffracting very well in Home source facility both in terms of resolution and 
intensity. But the only problem is the number of overlaps. Its showing much 
more than the good spots. As a result the completeness is showing maximum up to 
65% even after collecting 180 degrees. I am unable to get a complete data. I 
tried with reducing the oscillation angel to 0.3 degree/0.5 degree but it did 
not improve that much. Please give me some suggestions.



Regards,



Dipankar





Dipankar Manna

Aurigene Discovery Technologies Limited,

#39-40 KIADB Industrial Area, Electronic City, Phase II,

Hosur Road, Bangalore- 560 100, India

Cell: +91-9538631469  | Office Ph  : +91 80-66204422 (Extn: 398)  | Email ID: 
dipanka...@aurigene.com









This e-mail and any files transmitted with it are for the sole use of the 
intended recipient(s) and may contain confidential and privileged 
information.If you are not the intended recipient, please contact the sender by 
reply e-mail and destroy all copies of the original message.Any unauthorized 
review, use, disclosure, dissemination, forwarding,printing or copying of this 
email or any action taken in reliance on this e-mail is strictly prohibited and 
may be unlawful.

Visit us at http://www.aurigene.com

Re: [ccp4bb] How to reduce no. of overlaps

[Frank von Delft]

More accurately:  "adjustable pin".  Their *loops* are all adjustable 
(as opposed to Mitegen or MDL).


Those pins are not very useful if your crystal is already mounted.  And 
once you've bent that pin, it can't be stored again, because robots and 
tongs won't close around them.


They do have the advantage of not sometimes bending back, the way bent 
loops do.



On 07/03/2012 07:56, Zhijie Li wrote:

Hampton sells an adjustable mounted loop for this purpose.
http://hamptonresearch.com/product_detail.aspx?cid=24&sid=136&pid=385 
http://hamptonresearch.com/product_detail.aspx?cid=24&sid=136&pid=385>


*From:* Frank von Delft 
*Sent:* Wednesday, March 07, 2012 1:26 AM
*To:* CCP4BB@JISCMAIL.AC.UK 
*Subject:* Re: [ccp4bb] How to reduce no. of overlaps

You probably have to tilt your crystal, so that the long axis is 
parallel to the beam.  We do this routinely:  cut a plastic pipette 
tip to have a sharp point, then push the loop where it attaches to the 
pin, to bend the crystal itself.


You have to identify from your diffraction whether the long axis is 
pointing through the face or the edge of the loop.  As it's P6, 
chances are it's through the face, because long-axis P6 tends to make 
flat hexagons which lie flush with the face.  So you have to bend so 
the face of the loop upwards.


You'll have to practice this first, though, so put up an empty loop.  
Top tips:

* Don't breathe!  You'll blow the cryostream away.
* Bend the loop towards (rather than away from) the rim edge of 
the pin to which it's glued.

* Don't breathe!
* Practise practise practise.


Another thing:  most in-house sources allow you to reduce divergence 
of the beam.  You lose intensity, but no matter, just expose longer.  
That also improves overlap.


Cheers
phx



On 07/03/2012 04:56, Dipankar Manna wrote:


Dear Crystallographers,

I am working on a protein having SG P6, the cell parameters are a= 
79, b= 79, c= 325. The crystals are forming in big size and with very 
good shape. It also diffracting very well in Home source facility 
both in terms of resolution and intensity. But the only problem is 
the number of overlaps. Its showing much more than the good spots. As 
a result the completeness is showing maximum up to 65% even after 
collecting 180 degrees. I am unable to get a complete data. I tried 
with reducing the oscillation angel to 0.3 degree/0.5 degree but it 
did not improve that much. Please give me some suggestions.


Regards,

Dipankar

/Dipankar Manna/

*/Aurigene Discovery Technologies Limited,/*

/#39-40 KIADB Industrial Area, Electronic City, Phase II,/

/Hosur Road, Bangalore- 560 100, India/

/Cell: +91-9538631469 // | Office Ph  : +91 80-66204422 (Extn: 398)  
| Email ID: dipanka...@aurigene.com/





This e-mail and any files transmitted with it are for the sole use of 
the intended recipient(s) and may contain confidential and privileged 
information.If you are not the intended recipient, please contact the 
sender by reply e-mail and destroy all copies of the original 
message.Any unauthorized review, use, disclosure, dissemination, 
forwarding,printing or copying of this email or any action taken in 
reliance on this e-mail is strictly prohibited and may be unlawful.


Visit us at http://www.aurigene.com

Re: [ccp4bb] How to reduce no. of overlaps

[Jim Pflugrath]

In addition to reducing the beam divergence, you may wish to use a smaller beam 
size by using a smaller collimator or making the slits smaller.  A smaller 
crystal can also help to spatially separate the Bragg spots as can moving the 
detector closer to the crystal. Yes, closer to the crystal.  This is not 
intuitive, but arises since modern homelab beams are not parallel but are 
diverging from a focal point near the sample position.  It is just something 
else you may wish to try.

How you flashcool your sample will also have a large effect on the spot 
sizes/shapes.

Jim

>.
>Another thing:  most in-house sources allow you to reduce divergence of the 
>beam.  You lose intensity, >but no matter, just expose longer.  That also 
>improves overlap.
>
>Cheers
>phx