Re: [ccp4bb] what happens when freezer goes down

[Artem Evdokimov]

Hi,

My condolences. This is not a fun experience!

Your plasmids will be OK :)

Glycerol stocks may be rescued as long as after going to room temperature
they did NOT get frozen again. Re-freeze will greatly reduce viability -
but even after one re-freeze you should be able to streak the glycerol
stocks onto appropriate plates and get a few (or more) colonies. Notably
this does not mean that expression from these rescued glycerols will be
good; furthermore there are quite a few protein expression constructs in
our labs that do not store well as glycerol stocks - fresh DNA
transformation is the only way to get consistent expression for them.

Proteins in frozen cell pellets: that's iffy. The outcome greatly depends
on the protein itself - for instance I had several proteins that had
activity loss issues even when stored in LN2. In a -80 freezer they would
lose 40-50% activity in a week, and a single freeze/thaw/freeze would kill
them instantly (even in cell pellets). Then again, other proteins are fine
'no matter what'. In cases like yours it really helps to have some sort of
parameter you can follow (activity, or something).

Frozen protein samples - can be even more iffy than pellets.

Pellets are generally cheap to make, though. Unless they are
isotope-labeled or suchlike.

Artem

- Cosmic Cats approve of this message


On Fri, Apr 25, 2014 at 11:43 AM, Jacqueline Vitali  wrote:

> Dear colleagues,
>
> I know this is not related to ccp4 but I am in need of an answer and many
> of you work with cells etc.
>
> My building had a major malfunction of electricity and the power backup
> did not kick in.  My -80C freezer was without power for over 24 hours until
> I found out.  Because it is small, it goes fast to room temp.  I had many
> glycerol stocks in it with cells, cells with plasmids etc. as well as cell
> pellets.  I am trying to rescue things.
>
> My question is what happens to cell pellets.  I had many as I like to
> start purification at the cell pellet level.  Are these destroyed when they
> go to room temp for 24 hours or are they ok?
>
> Thanks.
>
> Jackie Vitali
> Cleveland State University
>
>
>
>
>

Re: [ccp4bb] AW: [ccp4bb] Twinning VS. Disorder

[Yale]

Thank you all for your comments/references and now I have a better 
understanding of what could be actually happening. But I have a feeling that 
disorder, where the twin domains can interfere with each other, is not actually 
so unusual. And in some cases MR might be possible to reveal a partial 
structure. (I ran into papers like this before but I couldn't think of one at 
the top of my head.) Why there is not a package out there to allow refinement 
against such data? I feel that if you have phase information it is possible to 
model the translation between different micro-domains...

Sincerely,
Chen


> On Apr 25, 2014, at 3:21 PM, "Keller, Jacob"  wrote:
> 
> Thanks to all—I’ve got the paper now
>  
> JPK
>  
> From: Keller, Jacob 
> Sent: Friday, April 25, 2014 1:58 PM
> To: 'Oliver Zeldin'
> Cc: CCP4BB@jiscmail.ac.uk
> Subject: RE: [ccp4bb] AW: [ccp4bb] Twinning VS. Disorder
>  
> Does anyone know of a place where one can obtain this reference for free? I 
> would contact Darwin himself, but I suspect he wouldn’t write back. I think 
> this is the original paper proposing the mosaic block model, and I’d really 
> like to see his reasoning.
>  
> Darwin, C. G. (1922). Philos. Mag. 43, 800±829. The reflexion of X-rays from 
> imperfect crystals
> 
> JPK
>  
> From: oliver.zel...@gmail.com [mailto:oliver.zel...@gmail.com] On Behalf Of 
> Oliver Zeldin
> Sent: Friday, April 25, 2014 1:03 PM
> To: Keller, Jacob
> Cc: CCP4BB@jiscmail.ac.uk
> Subject: Re: [ccp4bb] AW: [ccp4bb] Twinning VS. Disorder
>  
> Dear Jacob,
> 
> In terms of the effect of crystal (lattice) defects on diffraction spot 
> profiles, there are two great papers by Colin Nave that discuss this: 
> http://journals.iucr.org/d/issues/1998/05/00/issconts.html and 
> http://journals.iucr.org/d/issues/1998/05/00/issconts.html . There is also 
> this paper on the nature of mosaic micro-domains: 
> http://journals.iucr.org/d/issues/2000/08/00/en0024/en0024.pdf. 
> 
> I am sure there must be other references for the 'nature' of lattice 
> disorder, and it anyone can point to them, I would be grateful.
> 
> Cheers,
> 
> Oliver
>  
> 
> On Fri, Apr 25, 2014 at 6:20 AM, Keller, Jacob  
> wrote:
> >your Gedankenexperiment on powder diffraction is not correct. You would 
> >record a powder diffraction pattern if you rotated a single crystal around 
> >the beam axis and record the result on a single image.
> 
> If you wanted to do it with a single crystal, you would have to rotate the 
> crystal through all possible rotations in 3d, not just around the axis of the 
> beam, because you would then miss all the reflections which were not in the 
> diffraction condition at that phi angle. I agree that it could be done this 
> way (not sure why this is important though.)
> 
> >This rotation does not affect the mosaicity and the mosaicity of a powder 
> >sample related only to the mosaicity of the micro crystals present in the 
> >powder. You also do not get arcs when reducing the powderness but you start 
> >seeing single spots. This can often be observed in the presence of ice rings.
> 
> You are talking about "powderness," which I would guess is a measure of the 
> completeness of the sampling of all possible orientations of the constituent 
> crystals, and I agree with what you say would happen. I said, however, 
> mosaicity, which is a  measure of the breadth of the distribution of the 
> orientation angles of the microdomains/microcrystals. By decreasing 
> "powderness," one would do nothing to mosaicity. If one could arrange the 
> microdomains into some range of orientation angles, one would reduce the 
> mosaicity, and get arcs. I wish I had a picture of an arc-containing 
> diffraction pattern--I've seen them from time to time, and they're always of 
> course bad news. Anyone on the list have such a diffraction pattern handy?
> 
> JPK
> 
> 
> 
> 
> On 04/25/2014 09:32 AM, Keller, Jacob wrote:
> > Is the following being neglected?
> >
> > In a crystal with these putative mosaic microdomains, there will be
> > interference between microdomains at their edges/borders (at least),
> > but since most microdomains are probably way smaller than the
> > coherence length of 3-10 microns, presumably all unit cells in domain
> > A interfere with all unit cells in domains B, C, etc, which are in the
> > same coherence volume. In fact, as I said too unclearly in a previous
> > post, as the putative microdomains become smaller and smaller to the
> > limit of one unit cell, they become indistinguishable from unit cell
> > parameter variation. So I am becoming increasingly suspicious about
> > the existence of microdomains, and wonder what hard evidence there is
> > for their existence?
> >
> > As a thought experiment, one can consider the microdomain theory taken
> > to its limit: a powder diffraction image. In powder diffraction, there
> > are so many crystals (read: microdomains) that each spot is manifested
> > at its Bragg angle at every possible radial positi

Re: [ccp4bb] Systematically shifted peak in 2Fo-Fc and BDF maps

[Petr Leiman]

Thanks to all who replied to my message.

The most logical explanation to the observed phenomenon is likely the 
following. During crystallization, Sr ions from the crystallization solution 
kick out a fraction of the Mg(OH)6 ions from the binding site in question. The 
cavity of the binding site is big and the Sr ion binds at a slightly different 
position compared to the Mg(OH)6 ion. The BDF map shows the shifted position of 
the Sr ion, whereas the 2Fo-Fc map shows the position of the Mg(OH)6 ion. This 
is possible because the occupancy of the Sr ion at that site is low - the 
height of the Sr peak in the BDF map is less than 15% of the maximal peak 
height - and the contribution of the Sr ion to the 2Fo-Fc map is negligibly 
small. The “normal” (2Fo-Fc) scattering of the Sr ion is essentially completely 
masked by the Mg(OH)6 ion scattering because the two overlap (they are 0.47 A 
apart) and the latter has a 5-6 times higher occupancy.

Eleanor’s and Phoebe Rice’s comments were extremely useful in solving this 
puzzle (at least for me - we will see if the referees agree that this is a 
solution). This particular comment by Phoebe was really insightful considering 
she did not see the actual data but only read my somewhat incomplete 
description: “Could it be a mix of species - partly Sr and partly some 
non-anomalous-scattering entity that sits in a slightly shifted position?”

CCP4BB is an incredible resource full of really clever people! Thanks to all 
again!

Petr


On Apr 24, 2014, at 11:39, Petr Leiman 
mailto:petr.lei...@epfl.ch>> wrote:

Dear All,

We are looking for an explanation for a very strange observation.

Problem:
We have two fully independent data sets (two different crystals), in which the 
Bijvoet Difference Fourier map peak of one particular metal site is shifted by 
0.47 A from its position in the 2Fo-Fc map.

Relevant information:
The resolution of both data sets is 1.5-1.6 A.
The 2Fo-Fc and BDP maps are calculated using the same phases.
The metal ion is water hydrated and all the details are crystal clear in both 
2Fo-Fc maps.
The crystals are grown in the presence of Sr and the data sets are collected at 
the Sr K-edge.
There are many other Sr sites and all strong peaks in the BDF map overlap with 
2Fo-Fc map peaks perfectly. The Sr site in question is not the strongest, but 
it is well above the noise level of the BDF map.

Additional information:
The weird site is actually fully buried inside an internal cavity (it is 
surrounded by protein atoms from all sides), but a Sr atom is able to diffuse 
into this cavity somehow. All other Sr sites are on the surface of the protein.

Any thoughts about why a non-noise BDF map peak would not overlap with a 2Fo-Fc 
map peak are welcome!

Thank you very much,

Petr

--
Petr Leiman
EPFL
BSP 415
CH-1015 Lausanne
Switzerland
Office: +41 21 69 30 441
Mobile: +41 79 538 7647
Fax: +41 21 69 30 422

Re: [ccp4bb] anomalous signal

[Jim Pflugrath]

 should be above 0.80

There seems to be plenty of signal there with all values above 1.02.  We have 
solved structures with less multiplicity and lower .

There is a different criteria of "signal" for when you know the positions of 
the anomalous substructure atoms and when you need to find the positions of the 
anomalous substructure atoms.

As for "no signal", I think I am on record that there is always an anomalous 
signal. :)  But can you detect it?

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Faisal Tarique 
[faisaltari...@gmail.com]
Sent: Friday, April 25, 2014 4:06 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] anomalous signal

Dear all

sorry about my previous mail where i forgot to mention that the data was 
collected on home source at Cuk alpha and at 1.54A.

written below is the log file of an anomalous data processed through SHELXC..my 
question is ..what is the strength of anomalous signal ?? as it is said "For 
zero signal  and  should be about 0.80". Then in the present 
case is there really a signal or can be assumed no signal..we are expecting one 
Ca atom bound to the protein at its active site..the redundancy of the data is 
11.6..with this signal strength can we assume Ca to be present there or 
whatever little anomalous if present is due to something elseor there is no 
signal at all ??...

Resl.   Inf - 8.0 - 6.0 - 5.0 - 4.0 - 3.8 - 3.6 - 3.4 - 3.2 - 3.0 - 2.8 - 2.60
 N(data) 375   493   580  1319   450   538   679   866  1081  1414  1709
 58.8  38.6  32.6  38.3  27.7  27.2  21.9  18.4  12.6   9.5   6.1
 %Complete  94.7  99.0  99.3  99.5 100.0  99.6  99.7  99.8  99.6  99.6  90.9
1.65  1.27  1.18  1.25  1.19  1.12  1.11  1.11  0.97  1.02  1.05

--
Regards

Faisal
School of Life Sciences
JNU

[ccp4bb] anomalous signal

[Faisal Tarique]

Dear all

sorry about my previous mail where i forgot to mention that the data was
collected on home source at Cuk alpha and at 1.54A.

written below is the log file of an anomalous data processed through
SHELXC..my question is ..what is the strength of anomalous signal ?? as it
is said "For zero signal  and  should be about 0.80". Then
in the present case is there really a signal or can be assumed no
signal..we are expecting one Ca atom bound to the protein at its active
site..the redundancy of the data is 11.6..with this signal strength can we
assume Ca to be present there or whatever little anomalous if present is
due to something elseor there is no signal at all ??...

Resl.   Inf - 8.0 - 6.0 - 5.0 - 4.0 - 3.8 - 3.6 - 3.4 - 3.2 - 3.0 - 2.8 -
2.60
 N(data) 375   493   580  1319   450   538   679   866  1081  1414  1709
 58.8  38.6  32.6  38.3  27.7  27.2  21.9  18.4  12.6   9.5   6.1
 %Complete  94.7  99.0  99.3  99.5 100.0  99.6  99.7  99.8  99.6  99.6  90.9
1.65  1.27  1.18  1.25  1.19  1.12  1.11  1.11  0.97  1.02  1.05

-- 
Regards

Faisal
School of Life Sciences
JNU

[ccp4bb] anomalous signal

[Faisal Tarique]

Dear all

written below is the log file of an anomalous data processed through
SHELXC..my question is ..what is the strength of anomalous signal ?? as it
is said "For zero signal  and  should be about 0.80". Then
in the present case is there really a signal or can be assumed no
signal..we are expecting one Ca atom bound to the protein at its active
site..the redundancy of the data is 11.6..with this signal strength can we
assume Ca to be present there or whatever little anomalous if present is
due to something elseor there is no signal at all ??...

Resl.   Inf - 8.0 - 6.0 - 5.0 - 4.0 - 3.8 - 3.6 - 3.4 - 3.2 - 3.0 - 2.8 -
2.60
 N(data) 375   493   580  1319   450   538   679   866  1081  1414  1709
 58.8  38.6  32.6  38.3  27.7  27.2  21.9  18.4  12.6   9.5   6.1
 %Complete  94.7  99.0  99.3  99.5 100.0  99.6  99.7  99.8  99.6  99.6  90.9
1.65  1.27  1.18  1.25  1.19  1.12  1.11  1.11  0.97  1.02  1.05



-- 
Regards

Faisal
School of Life Sciences
JNU

Re: [ccp4bb] "NEW! Try depositing X-ray data using the wwPDB deposition system"

[Andreas Förster]

I was getting very excited here for a second.  Deposition of X-ray data? 
 Is this finally possible?  Raw diffraction data, in other words? 
Image files?


Sadly, the text of the email doesn't support that conclusion.


Andreas



On 25/04/2014 8:16, Rachel Kramer Green wrote:

Depositing a new X-ray crystal structure to the PDB archive?Try the new
and improved wwPDB Deposition system at
http://deposit.wwpdb.org/deposition/.

Since the new system went live on January 27^th , 2014, ~700 structures
have been deposited and promptly annotated.Detailed information on the
system can be found at http://www.wwpdb.org/system_info.html.The new
system was developed to allow the wwPDB partners to meet the evolving
needs of the scientific community over the next decade, including
support for very large systems, complex chemistry, and joint use of
multiple experimental methods. The system replaces all current
deposition and annotation systems in use at the wwPDB deposition
centers, and will lead to improved efficiency and consistency.

New or enhanced features of the deposition system include the generation
of X-ray validation reports (following the recommendations of the wwPDB
X-ray Validation Task Force), improved capture and review of ligand
information, the ability to replace coordinate and/or experimental data
files pre- and post-submission, improved communication process between
depositors and wwPDB curators, and the ability to preview and download
the PDBx/mmCIF entry file prior to submission.

Depositors will have the option to use the new system or one of the
legacy deposition tools (ADIT, AutoDep) for most of 2014. After the
transition to the new system, the legacy tools will be available for a
limited period of time to complete any unfinished deposition sessions.

The wwPDB continues to improve the deposition interface and supporting
functionality as it receives more submissions and input.Questions,
comments, and suggestions should be sent to:
http://deposit-feedback.wwpdb.org_._

--



Rachel Kramer Green, Ph.D.

RCSB PDB

kra...@rcsb.rutgers.edu

*New!*Deposit X-ray data with the wwPDB at:

http://deposit.wwpdb.org/deposition (NMR and 3DEM coming soon).

___

Twitter: https://twitter.com/#!/buildmodels

Facebook: http://www.facebook.com/RCSBPDB



--
  Andreas Förster
 Crystallization and X-ray Facility Manager
   Centre for Structural Biology
  Imperial College London

Re: [ccp4bb] AW: [ccp4bb] Twinning VS. Disorder

[Keller, Jacob]

Thanks to all—I’ve got the paper now

JPK

From: Keller, Jacob
Sent: Friday, April 25, 2014 1:58 PM
To: 'Oliver Zeldin'
Cc: CCP4BB@jiscmail.ac.uk
Subject: RE: [ccp4bb] AW: [ccp4bb] Twinning VS. Disorder

Does anyone know of a place where one can obtain this reference for free? I 
would contact Darwin himself, but I suspect he wouldn’t write back. I think 
this is the original paper proposing the mosaic block model, and I’d really 
like to see his reasoning.

Darwin, C. G. (1922). Philos. Mag. 43, 800±829. The reflexion of X-rays from 
imperfect crystals
JPK

From: oliver.zel...@gmail.com 
[mailto:oliver.zel...@gmail.com] On Behalf Of Oliver Zeldin
Sent: Friday, April 25, 2014 1:03 PM
To: Keller, Jacob
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] AW: [ccp4bb] Twinning VS. Disorder

Dear Jacob,

In terms of the effect of crystal (lattice) defects on diffraction spot 
profiles, there are two great papers by Colin Nave that discuss this: 
http://journals.iucr.org/d/issues/1998/05/00/issconts.html and 
http://journals.iucr.org/d/issues/1998/05/00/issconts.html . There is also this 
paper on the nature of mosaic micro-domains: 
http://journals.iucr.org/d/issues/2000/08/00/en0024/en0024.pdf.

I am sure there must be other references for the 'nature' of lattice disorder, 
and it anyone can point to them, I would be grateful.
Cheers,
Oliver

On Fri, Apr 25, 2014 at 6:20 AM, Keller, Jacob 
mailto:kell...@janelia.hhmi.org>> wrote:
>your Gedankenexperiment on powder diffraction is not correct. You would record 
>a powder diffraction pattern if you rotated a single crystal around the beam 
>axis and record the result on a single image.
If you wanted to do it with a single crystal, you would have to rotate the 
crystal through all possible rotations in 3d, not just around the axis of the 
beam, because you would then miss all the reflections which were not in the 
diffraction condition at that phi angle. I agree that it could be done this way 
(not sure why this is important though.)

>This rotation does not affect the mosaicity and the mosaicity of a powder 
>sample related only to the mosaicity of the micro crystals present in the 
>powder. You also do not get arcs when reducing the powderness but you start 
>seeing single spots. This can often be observed in the presence of ice rings.
You are talking about "powderness," which I would guess is a measure of the 
completeness of the sampling of all possible orientations of the constituent 
crystals, and I agree with what you say would happen. I said, however, 
mosaicity, which is a  measure of the breadth of the distribution of the 
orientation angles of the microdomains/microcrystals. By decreasing 
"powderness," one would do nothing to mosaicity. If one could arrange the 
microdomains into some range of orientation angles, one would reduce the 
mosaicity, and get arcs. I wish I had a picture of an arc-containing 
diffraction pattern--I've seen them from time to time, and they're always of 
course bad news. Anyone on the list have such a diffraction pattern handy?

JPK




On 04/25/2014 09:32 AM, Keller, Jacob wrote:
> Is the following being neglected?
>
> In a crystal with these putative mosaic microdomains, there will be
> interference between microdomains at their edges/borders (at least),
> but since most microdomains are probably way smaller than the
> coherence length of 3-10 microns, presumably all unit cells in domain
> A interfere with all unit cells in domains B, C, etc, which are in the
> same coherence volume. In fact, as I said too unclearly in a previous
> post, as the putative microdomains become smaller and smaller to the
> limit of one unit cell, they become indistinguishable from unit cell
> parameter variation. So I am becoming increasingly suspicious about
> the existence of microdomains, and wonder what hard evidence there is
> for their existence?
>
> As a thought experiment, one can consider the microdomain theory taken
> to its limit: a powder diffraction image. In powder diffraction, there
> are so many crystals (read: microdomains) that each spot is manifested
> at its Bragg angle at every possible radial position on the detector.
> Mosaicity would be, what, 360 degrees?
> So, now imagine decreasing the mosaicity to lower values, and one gets
> progressively shorter arcs which at lower values become spots.
> Doesn’t this mean that the contribution from microdomain mosaicity
> should be to make the spots more like arcs, as we sometimes see in
> terrible diffraction patterns, and not just general broadening of
> spots? Put another way: mosaicity should broaden spots in the radial
> direction (arcs), and unit cell parameter variation should produce
> straight broadening in the direction of the unit cell variation of
> magnitude proportional to the degree of variation in that direction.
>
> JPK
>
>
> From: CCP4 bulletin board 
> [mailto:CCP4BB@JISCMAIL.AC.UK

[ccp4bb] "NEW! Try depositing X-ray data using the wwPDB deposition system"

[Rachel Kramer Green]

Depositing a new X-ray crystal structure to the PDB archive?Try the new 
and improved wwPDB Deposition system at 
http://deposit.wwpdb.org/deposition/.


Since the new system went live on January 27^th , 2014, ~700 structures 
have been deposited and promptly annotated.Detailed information on the 
system can be found at http://www.wwpdb.org/system_info.html.The new 
system was developed to allow the wwPDB partners to meet the evolving 
needs of the scientific community over the next decade, including 
support for very large systems, complex chemistry, and joint use of 
multiple experimental methods. The system replaces all current 
deposition and annotation systems in use at the wwPDB deposition 
centers, and will lead to improved efficiency and consistency.


New or enhanced features of the deposition system include the generation 
of X-ray validation reports (following the recommendations of the wwPDB 
X-ray Validation Task Force), improved capture and review of ligand 
information, the ability to replace coordinate and/or experimental data 
files pre- and post-submission, improved communication process between 
depositors and wwPDB curators, and the ability to preview and download 
the PDBx/mmCIF entry file prior to submission.


Depositors will have the option to use the new system or one of the 
legacy deposition tools (ADIT, AutoDep) for most of 2014. After the 
transition to the new system, the legacy tools will be available for a 
limited period of time to complete any unfinished deposition sessions.


The wwPDB continues to improve the deposition interface and supporting 
functionality as it receives more submissions and input.Questions, 
comments, and suggestions should be sent to: 
http://deposit-feedback.wwpdb.org_._


--



Rachel Kramer Green, Ph.D.

RCSB PDB

kra...@rcsb.rutgers.edu

*New!*Deposit X-ray data with the wwPDB at:

http://deposit.wwpdb.org/deposition (NMR and 3DEM coming soon).

___

Twitter: https://twitter.com/#!/buildmodels

Facebook: http://www.facebook.com/RCSBPDB

Re: [ccp4bb] AW: [ccp4bb] Twinning VS. Disorder

[Keller, Jacob]

Does anyone know of a place where one can obtain this reference for free? I 
would contact Darwin himself, but I suspect he wouldn’t write back. I think 
this is the original paper proposing the mosaic block model, and I’d really 
like to see his reasoning.

Darwin, C. G. (1922). Philos. Mag. 43, 800±829. The reflexion of X-rays from 
imperfect crystals
JPK
From: oliver.zel...@gmail.com [mailto:oliver.zel...@gmail.com] On Behalf Of 
Oliver Zeldin
Sent: Friday, April 25, 2014 1:03 PM
To: Keller, Jacob
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] AW: [ccp4bb] Twinning VS. Disorder

Dear Jacob,

In terms of the effect of crystal (lattice) defects on diffraction spot 
profiles, there are two great papers by Colin Nave that discuss this: 
http://journals.iucr.org/d/issues/1998/05/00/issconts.html and 
http://journals.iucr.org/d/issues/1998/05/00/issconts.html . There is also this 
paper on the nature of mosaic micro-domains: 
http://journals.iucr.org/d/issues/2000/08/00/en0024/en0024.pdf.

I am sure there must be other references for the 'nature' of lattice disorder, 
and it anyone can point to them, I would be grateful.
Cheers,
Oliver

On Fri, Apr 25, 2014 at 6:20 AM, Keller, Jacob 
mailto:kell...@janelia.hhmi.org>> wrote:
>your Gedankenexperiment on powder diffraction is not correct. You would record 
>a powder diffraction pattern if you rotated a single crystal around the beam 
>axis and record the result on a single image.
If you wanted to do it with a single crystal, you would have to rotate the 
crystal through all possible rotations in 3d, not just around the axis of the 
beam, because you would then miss all the reflections which were not in the 
diffraction condition at that phi angle. I agree that it could be done this way 
(not sure why this is important though.)

>This rotation does not affect the mosaicity and the mosaicity of a powder 
>sample related only to the mosaicity of the micro crystals present in the 
>powder. You also do not get arcs when reducing the powderness but you start 
>seeing single spots. This can often be observed in the presence of ice rings.
You are talking about "powderness," which I would guess is a measure of the 
completeness of the sampling of all possible orientations of the constituent 
crystals, and I agree with what you say would happen. I said, however, 
mosaicity, which is a  measure of the breadth of the distribution of the 
orientation angles of the microdomains/microcrystals. By decreasing 
"powderness," one would do nothing to mosaicity. If one could arrange the 
microdomains into some range of orientation angles, one would reduce the 
mosaicity, and get arcs. I wish I had a picture of an arc-containing 
diffraction pattern--I've seen them from time to time, and they're always of 
course bad news. Anyone on the list have such a diffraction pattern handy?

JPK




On 04/25/2014 09:32 AM, Keller, Jacob wrote:
> Is the following being neglected?
>
> In a crystal with these putative mosaic microdomains, there will be
> interference between microdomains at their edges/borders (at least),
> but since most microdomains are probably way smaller than the
> coherence length of 3-10 microns, presumably all unit cells in domain
> A interfere with all unit cells in domains B, C, etc, which are in the
> same coherence volume. In fact, as I said too unclearly in a previous
> post, as the putative microdomains become smaller and smaller to the
> limit of one unit cell, they become indistinguishable from unit cell
> parameter variation. So I am becoming increasingly suspicious about
> the existence of microdomains, and wonder what hard evidence there is
> for their existence?
>
> As a thought experiment, one can consider the microdomain theory taken
> to its limit: a powder diffraction image. In powder diffraction, there
> are so many crystals (read: microdomains) that each spot is manifested
> at its Bragg angle at every possible radial position on the detector.
> Mosaicity would be, what, 360 degrees?
> So, now imagine decreasing the mosaicity to lower values, and one gets
> progressively shorter arcs which at lower values become spots.
> Doesn’t this mean that the contribution from microdomain mosaicity
> should be to make the spots more like arcs, as we sometimes see in
> terrible diffraction patterns, and not just general broadening of
> spots? Put another way: mosaicity should broaden spots in the radial
> direction (arcs), and unit cell parameter variation should produce
> straight broadening in the direction of the unit cell variation of
> magnitude proportional to the degree of variation in that direction.
>
> JPK
>
>
> From: CCP4 bulletin board 
> [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
> Ian Tickle Sent: Thursday, April 24, 2014 7:01 PM To:
> CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] AW: 
> [ccp4bb] Twinning VS.
> Disorder
>
>
> Dear Herman On 24 April 2014 22:32,
> mailto:herman.schreu...@sanofi.c

Re: [ccp4bb] what happens when freezer goes down

[David Blum]

Jackie,

We grow cells routinely and freeze pellets after fermentation.  In general,
proteins are fairly stable until you break cells so you are probably ok
unless your protein is very heat labile and it sat at room temperature for
hours.  However as I mentioned, there is a kind of buffering from the cell
that can stabilize the protein until you are ready to use it.  The glycerol
stocks are probably ok as well since you need just a small inoculum to get
your culture growing.  The plasmids may be the most affected, but you can
sequence if you have aberrant expression.

Best,

David

-- 

David L. Blum, Ph.D.

Director, Bioexpression and Fermentation Facility

Department of Biochemistry and Molecular Biology

University of Georgia

120 Green Street room A414A

Athens, GA 30602

http://bff.uga.edu/

b...@uga.edu


On Fri, Apr 25, 2014 at 12:43 PM, Jacqueline Vitali  wrote:

> Dear colleagues,
>
> I know this is not related to ccp4 but I am in need of an answer and many
> of you work with cells etc.
>
> My building had a major malfunction of electricity and the power backup
> did not kick in.  My -80C freezer was without power for over 24 hours until
> I found out.  Because it is small, it goes fast to room temp.  I had many
> glycerol stocks in it with cells, cells with plasmids etc. as well as cell
> pellets.  I am trying to rescue things.
>
> My question is what happens to cell pellets.  I had many as I like to
> start purification at the cell pellet level.  Are these destroyed when they
> go to room temp for 24 hours or are they ok?
>
> Thanks.
>
> Jackie Vitali
> Cleveland State University
>
>
>
>
>

[ccp4bb] CCP4-6.4.0 Update 013

[Charles Ballard]

Dear CCP4 Users

An update for the CCP4-6.4.0 series has just been released, consisting
of the following changes

* qtpisa and pisa
 - New features
 + concentration dependence of predicted oligomeric states to aid the 
identification of biological units in protein's working conditions
 + multi-parameter interaction radar for more consistent discrimination 
between biologically-relevant interfaces and crystal contacts
 + help support (qtpisa only)

Note that auto-updates work only with CCP4 6.4.0 series, therefore
please upgrade if necessary.  The Update Manager is now included in the
package so you do not need to install it separately.  In addition, all
available updates will be installed automatically if you are using Setup
Manager for CCP4 installation.

Please report any bugs to c...@stfc.ac.uk.

Many thanks for using CCP4.

The CCP4 Core Team--
Scanned by iCritical.

Re: [ccp4bb] AW: [ccp4bb] Twinning VS. Disorder

[Oliver Zeldin]

Dear Jacob,

In terms of the effect of crystal (lattice) defects on diffraction spot
profiles, there are two great papers by Colin Nave that discuss this:
http://journals.iucr.org/d/issues/1998/05/00/issconts.html and
http://journals.iucr.org/d/issues/1998/05/00/issconts.html . There is also
this paper on the nature of mosaic micro-domains:
http://journals.iucr.org/d/issues/2000/08/00/en0024/en0024.pdf.

I am sure there must be other references for the 'nature' of lattice
disorder, and it anyone can point to them, I would be grateful.

Cheers,

Oliver


On Fri, Apr 25, 2014 at 6:20 AM, Keller, Jacob wrote:

> >your Gedankenexperiment on powder diffraction is not correct. You would
> record a powder diffraction pattern if you rotated a single crystal around
> the beam axis and record the result on a single image.
>
> If you wanted to do it with a single crystal, you would have to rotate the
> crystal through all possible rotations in 3d, not just around the axis of
> the beam, because you would then miss all the reflections which were not in
> the diffraction condition at that phi angle. I agree that it could be done
> this way (not sure why this is important though.)
>
> >This rotation does not affect the mosaicity and the mosaicity of a powder
> sample related only to the mosaicity of the micro crystals present in the
> powder. You also do not get arcs when reducing the powderness but you start
> seeing single spots. This can often be observed in the presence of ice
> rings.
>
> You are talking about "powderness," which I would guess is a measure of
> the completeness of the sampling of all possible orientations of the
> constituent crystals, and I agree with what you say would happen. I said,
> however, mosaicity, which is a  measure of the breadth of the distribution
> of the orientation angles of the microdomains/microcrystals. By decreasing
> "powderness," one would do nothing to mosaicity. If one could arrange the
> microdomains into some range of orientation angles, one would reduce the
> mosaicity, and get arcs. I wish I had a picture of an arc-containing
> diffraction pattern--I've seen them from time to time, and they're always
> of course bad news. Anyone on the list have such a diffraction pattern
> handy?
>
> JPK
>
>
>
>
> On 04/25/2014 09:32 AM, Keller, Jacob wrote:
> > Is the following being neglected?
> >
> > In a crystal with these putative mosaic microdomains, there will be
> > interference between microdomains at their edges/borders (at least),
> > but since most microdomains are probably way smaller than the
> > coherence length of 3-10 microns, presumably all unit cells in domain
> > A interfere with all unit cells in domains B, C, etc, which are in the
> > same coherence volume. In fact, as I said too unclearly in a previous
> > post, as the putative microdomains become smaller and smaller to the
> > limit of one unit cell, they become indistinguishable from unit cell
> > parameter variation. So I am becoming increasingly suspicious about
> > the existence of microdomains, and wonder what hard evidence there is
> > for their existence?
> >
> > As a thought experiment, one can consider the microdomain theory taken
> > to its limit: a powder diffraction image. In powder diffraction, there
> > are so many crystals (read: microdomains) that each spot is manifested
> > at its Bragg angle at every possible radial position on the detector.
> > Mosaicity would be, what, 360 degrees?
> > So, now imagine decreasing the mosaicity to lower values, and one gets
> > progressively shorter arcs which at lower values become spots.
> > Doesn’t this mean that the contribution from microdomain mosaicity
> > should be to make the spots more like arcs, as we sometimes see in
> > terrible diffraction patterns, and not just general broadening of
> > spots? Put another way: mosaicity should broaden spots in the radial
> > direction (arcs), and unit cell parameter variation should produce
> > straight broadening in the direction of the unit cell variation of
> > magnitude proportional to the degree of variation in that direction.
> >
> > JPK
> >
> >
> > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
> > Ian Tickle Sent: Thursday, April 24, 2014 7:01 PM To:
> > CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] AW: [ccp4bb] Twinning VS.
> > Disorder
> >
> >
> > Dear Herman On 24 April 2014 22:32,
> > mailto:herman.schreu...@sanofi.com>>
> > wrote:
> >
> > The X-ray coherent length is depending on the crystal, not the
> > synchrotron and my gut feeling is that it is at least several hundred
> > unit cells, but here other experts may correct me.
> >
> >
> > I assume you meant that the coherence length is a property of the beam
> > (e.g. for a Cu target source it's related to the lifetime of the
> > excited Cu K-alpha state), not the crystal, e,g, see
> > http://www.aps.anl.gov/Users/Meeting/2010/Presentations/WK2talk_Vartan
> > iants.pdf (slides 8-11).  The relevant property of the crystal is the

[ccp4bb] what happens when freezer goes down

[Jacqueline Vitali]

Dear colleagues,

I know this is not related to ccp4 but I am in need of an answer and many
of you work with cells etc.

My building had a major malfunction of electricity and the power backup did
not kick in.  My -80C freezer was without power for over 24 hours until I
found out.  Because it is small, it goes fast to room temp.  I had many
glycerol stocks in it with cells, cells with plasmids etc. as well as cell
pellets.  I am trying to rescue things.

My question is what happens to cell pellets.  I had many as I like to start
purification at the cell pellet level.  Are these destroyed when they go to
room temp for 24 hours or are they ok?

Thanks.

Jackie Vitali
Cleveland State University

Re: [ccp4bb] coot problems to decrease R FREE

[Pavel Afonine]

Hi Robbie,


>   I agree that you bias R-free after the real-space refinement
> >
> >
> > well, ok, isn't it enough to realize that this is bad and should be
> avoided ? (I
> > guess we all know we should never bias Rfree!)
> >
> >
> >   My point was that we normally do not calculate R-free after real-
> > space refinement,
> >
> >
> > It's not about whether you compute something or not. It's about whether
> > you expose free-r reflections to refinement and to current model, given
>  that
> > they should never ever see any refinement or model or optimization under
> > any circumstances. Otherwise they immediately become non-free.
> Yet we do optimise weights against R-free, both in phenix.refine (from the
> manual: "phenix.refine uses automatic procedure to determine the weights
> between X-ray target and stereochemistry or ADP restraints. To optimize
> these weights (that is to find those resulting in lowest Rfree factors)")
> and in PDB_REDO (well against LL-free, but that is the same test set). And
> we do not go the distance to use something like an R-sleep set to keep a
> set of reflections really independent. Here we are introducing bias as
> well, but we seem to have accepted that this is not a big problem.  Or we
> chose to ignore it.



This is all true. There is a subtle line, however, between actively
refining your model against free reflections (and therefore against Rfree)
in real or reciprocal spaces, and using Rfree at discrete points of time to
make certain decisions, like visually inspect the numbers and say I like
this model more because it has lower Rfree, or choose weight because one
yields a lower Rfree than the other. Technically, I agree, it is also using
test reflections, and it would be the best if "sleep" set of reflections is
used for this instead!

>From practical side, it's easy to demonstrate that if you start with a
model and data set composed of work and free reflections, and having
Rfree>Rwork with some gap, then compute two maps: one using only work
reflections, and another using all work+test. Then refine model against the
two maps.

In case of refinement against the first map (calculated using work
reflections only), both Rfree and Rwork will change, Rfree>Rwork will
remain true and the gap will be maintained.

In case of refinement against the second map (calculated using all,
work+test, reflections), both Rfree and Rwork will change, and Rfree will
*very* quickly approach Rwork, so Rwork~Rfree.

I've seen it numerous times when developing real-space refinement in
Phenix, and I think I can still find the script that demonstrates
specifically this scenario.

All the best,
Pavel

[ccp4bb] AW: [ccp4bb] Twinning VS. Disorder

[Robert Sweet]

Dear James,

I have a slightly different way to think about transverse coherence.  I 
heard Mark Sutton give a talk about this at the APS a few years ago, and 
here are graphics from a similar talk given by Alec Sandy at BNL: 
http://www.bnl.gov/nsls2/workshops/docs/XPCS/XPCS_Sandy.ppt


The eqn. he gives on figure 8 is this:

  L(coherence) =
Lambda*(source-to-observation-point-distance) / 2*pi*sigma(source)

This is the wavelength divided by the angle subtended by the source viewed 
by the observer, with a 2.pi in there for some reason.  The way I explain 
it (though I cannot derive it this way) is that as you view the source, 
consider that your eye sees a ray coming from the top of the source, and 
one coming from the bottom.  As you move your eye up and down, the two 
rays will slide back and forth against one another. The coherence length 
is how far you move your eye to have them slip about 1/4 of a wave.  In 
Mark's APS example you see the horizontal is 7 microns, the vertical is 
200.  This reflects the fact that the typical synchrotron source is much 
wider than it is high.


Bob



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of James Holton 
[jmhol...@lbl.gov]
Sent: Thursday, April 24, 2014 6:59 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] AW: [ccp4bb] Twinning VS. Disorder

There are two kinds of "coherence length": transverse and longitudinal.  
Longitudinal coherence is often quoted as delta-lambda/lambda, which is easy to calculate 
but unfortunately completely irrelevant for diffraction from crystals.  If it weren't 
then Laue diffraction wouldn't produce spots.

Transverse coherence tends to be around 3-10 microns with 1 A x-rays, depending on the 
detector distance.  Yes, that's right, the detector distance.  Longer detector distances 
give you a bigger coherence length, especially when the source is "very far 
away", like it is at a synchrotron.

How this happens is easiest to picture if you consider the simplest possible diffraction situation: a 
"point" source of x-rays, two atoms, and a detector.  As long as the atoms are very close together 
relative to the distances from the sample to the source and the detector, then you have the "far 
field" diffraction situation.  This is where both atoms are within the "coherence length", 
Bragg's diagram for Bragg's Law holds: parallel incoming rays and parallel outgoing rays.

But what if the atoms are very far apart?  Obviously, the scattering from two atoms on different 
sides of the room will just add as intensities.  And if they are very close together, then Bragg's 
Law holds and they scatter "coherently".  What most people think of as the 
"coherence length" is the point of transition between these two kinds of scattering.

This point is rather conveniently defined as the distance between two atoms when the path from the source to one atom 
to a given detector pixel becomes 0.5 wavelengths longer than the same path through the other atom.  As long as both 
atoms lie in the "Bragg plane" (that's the plane perpendicular to the "s" vector, which is the 
vector difference between the incoming and outgoing beam directions), the far-field approximation tells us they should 
also be "in phase", but if they are far enough apart the 0.5 A change in total path length is enough to 
change the scattering completely from constructive to destructive interference.  In ordinary optics, this is called the 
edge of the first "Fresnel zone".

So, if your source is "very far away", emitting 1 A x-rays, and your detector is 1 meter away, then 
moving one atom 10 microns away from the centerline of the beam makes the path from that atom to the detector 
1-sqrt(1^2+10e-6^2) = 0.5 A longer.  So that implies the "coherence length" is 10 microns.  But if 
the detector is only 100 mm away, that gives you 0.1-sqrt(0.1^2+3e-6^2) = 0.5 A, so 3 um is the 
"coherence length".

Of course, this is for the ideal case of a point source very far away.  In reality finite beam divergence will mess up 
the "coherence" inasmuch as a divergent source looks like an array of sources all viewing the sample through 
a pinhole.  What you then get on the detector is the sum of the patterns from all those sources, so the 
"coherence" is not as clean.  That is, you don't see the Fourier transform of the crystal shape in every 
spot.  Mosaic spread also messes up "coherence" in this way.  Some might even define the mosaic "domain 
size" as the inverse of the effective coherence length.

But, the long and short of all this is that as long as your detector pixels are bigger 
than the "coherence length" the coherence doesn't really matter.

Hope that makes sense,

-James Holton
MAD Scientist



On Thu, Apr 24, 2014 at 2:32 PM, 
mailto:herman.schreu...@sanofi.com>> wrote:
Dear Chen,

Twinning can be thought of as of two or more macro-crystals glued or grown 
together. The reason that the reflections often overlap is t

Re: [ccp4bb] PyMol and Schrodinger

[Eugene Krissinel]

Thank you Piotr, this is very useful to know.

Cheers,

Eugene

On 24 Apr 2014, at 22:18, Piotr Sliz wrote:

> Eugene,
> 
> SBGrid Consortium supports certain paid programs such as Pymol, Schrodinger, 
> and Geneious, and academic laboratories have access to a limited pool of 
> tokens. For more serious computations it might be easier to get an individual 
> license, yet for starters the SBGrid shared-token library is certainly a 
> viable option. 
> 
> Access to the commercial tokens is included in SBGrid membership, though 
> limited depending on your geographic location and affiliation. Please email 
> h...@sbgrid.org for a more detailed description of this program.
> 
> Kind Regards,
> 
> Piotr
> 


-- 
Scanned by iCritical.

Re: [ccp4bb] AW: [ccp4bb] Twinning VS. Disorder

[Keller, Jacob]

>your Gedankenexperiment on powder diffraction is not correct. You would record 
>a powder diffraction pattern if you rotated a single crystal around the beam 
>axis and record the result on a single image.

If you wanted to do it with a single crystal, you would have to rotate the 
crystal through all possible rotations in 3d, not just around the axis of the 
beam, because you would then miss all the reflections which were not in the 
diffraction condition at that phi angle. I agree that it could be done this way 
(not sure why this is important though.)

>This rotation does not affect the mosaicity and the mosaicity of a powder 
>sample related only to the mosaicity of the micro crystals present in the 
>powder. You also do not get arcs when reducing the powderness but you start 
>seeing single spots. This can often be observed in the presence of ice rings.

You are talking about "powderness," which I would guess is a measure of the 
completeness of the sampling of all possible orientations of the constituent 
crystals, and I agree with what you say would happen. I said, however, 
mosaicity, which is a  measure of the breadth of the distribution of the 
orientation angles of the microdomains/microcrystals. By decreasing 
"powderness," one would do nothing to mosaicity. If one could arrange the 
microdomains into some range of orientation angles, one would reduce the 
mosaicity, and get arcs. I wish I had a picture of an arc-containing 
diffraction pattern--I've seen them from time to time, and they're always of 
course bad news. Anyone on the list have such a diffraction pattern handy?

JPK




On 04/25/2014 09:32 AM, Keller, Jacob wrote:
> Is the following being neglected?
> 
> In a crystal with these putative mosaic microdomains, there will be 
> interference between microdomains at their edges/borders (at least), 
> but since most microdomains are probably way smaller than the 
> coherence length of 3-10 microns, presumably all unit cells in domain 
> A interfere with all unit cells in domains B, C, etc, which are in the 
> same coherence volume. In fact, as I said too unclearly in a previous 
> post, as the putative microdomains become smaller and smaller to the 
> limit of one unit cell, they become indistinguishable from unit cell 
> parameter variation. So I am becoming increasingly suspicious about 
> the existence of microdomains, and wonder what hard evidence there is 
> for their existence?
> 
> As a thought experiment, one can consider the microdomain theory taken 
> to its limit: a powder diffraction image. In powder diffraction, there 
> are so many crystals (read: microdomains) that each spot is manifested 
> at its Bragg angle at every possible radial position on the detector. 
> Mosaicity would be, what, 360 degrees?
> So, now imagine decreasing the mosaicity to lower values, and one gets 
> progressively shorter arcs which at lower values become spots.
> Doesn’t this mean that the contribution from microdomain mosaicity 
> should be to make the spots more like arcs, as we sometimes see in 
> terrible diffraction patterns, and not just general broadening of 
> spots? Put another way: mosaicity should broaden spots in the radial 
> direction (arcs), and unit cell parameter variation should produce 
> straight broadening in the direction of the unit cell variation of 
> magnitude proportional to the degree of variation in that direction.
> 
> JPK
> 
> 
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
> Ian Tickle Sent: Thursday, April 24, 2014 7:01 PM To:
> CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] AW: [ccp4bb] Twinning VS. 
> Disorder
> 
> 
> Dear Herman On 24 April 2014 22:32,
> mailto:herman.schreu...@sanofi.com>>
> wrote:
> 
> The X-ray coherent length is depending on the crystal, not the 
> synchrotron and my gut feeling is that it is at least several hundred 
> unit cells, but here other experts may correct me.
> 
> 
> I assume you meant that the coherence length is a property of the beam 
> (e.g. for a Cu target source it's related to the lifetime of the 
> excited Cu K-alpha state), not the crystal, e,g, see 
> http://www.aps.anl.gov/Users/Meeting/2010/Presentations/WK2talk_Vartan
> iants.pdf (slides 8-11).  The relevant property of the crystal is the 
> size of the microdomains.  You don't get interference because 
> coherence length << domain size, i.e. the beam is not coherent over 
> more than
> 1 domain.  This is true for in-house sources & synchrotrons, I guess 
> for FELs it's different, i.e. much greater coherence length?
> This relates to a question I asked on the BB some time ago: if the 
> coherence length is long enough would you start to see the effects of 
> interference in twinned crystals, i.e. would the summation of 
> intensities break down? I defer to the experts on synchrotrons & FELs! 
> Cheers -- Ian
> 

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

GPG Key ID = A46BEE1A

-BEGIN PGP SI

[ccp4bb] Postdoctoral Position Available at Rutgers University

[Matthew Neiditch]

A postdoctoral position is available in the Department of Microbiology and 
Molecular Genetics at Rutgers University - New Jersey Medical School to study 
structural mechanisms regulating bacterial signaling and perform SAR studies of 
antimicrobial compounds.  In addition to a recent Ph.D., the ideal candidate 
will have a strong background in macromolecular X-ray crystallography, 
biochemistry, protein purification, and molecular biology. A working knowledge 
of bacterial genetics and computational modeling of protein-ligand interactions 
is a plus. Candidates with demonstrated success in another field and a strong 
commitment to learning the crystallographic process will also be considered. 

Please submit: CV and at least 2 letters of reference by email to 
matthew.neidi...@rutgers.edu with the subject line “Postdoctoral Position 
Applicant”


Matthew B. Neiditch, Ph.D.
Associate Professor
Dept. of Microbiology and Molecular Genetics
New Jersey Medical School
Rutgers, The State University of New Jersey
225 Warren St., Room E450U
Newark, NJ 07103

Re: [ccp4bb] coot problems to decrease R FREE

[Robbie Joosten]

Hi Pavel,

 
>   I agree that you bias R-free after the real-space refinement
> 
> 
> well, ok, isn't it enough to realize that this is bad and should be avoided ? 
> (I
> guess we all know we should never bias Rfree!)
> 
> 
>   My point was that we normally do not calculate R-free after real-
> space refinement,
> 
> 
> It's not about whether you compute something or not. It's about whether
> you expose free-r reflections to refinement and to current model, given  that
> they should never ever see any refinement or model or optimization under
> any circumstances. Otherwise they immediately become non-free.
Yet we do optimise weights against R-free, both in phenix.refine (from the 
manual: "phenix.refine uses automatic procedure to determine the weights 
between X-ray target and stereochemistry or ADP restraints. To optimize these 
weights (that is to find those resulting in lowest Rfree factors)") and in 
PDB_REDO (well against LL-free, but that is the same test set). And we do not 
go the distance to use something like an R-sleep set to keep a set of 
reflections really independent. Here we are introducing bias as well, but we 
seem to have accepted that this is not a big problem.  Or we chose to ignore it.

 
>   but after reciprocal space refinement. Here the bias is removed
> again.
> 
> 
> Hm.. How you know this? I guess it requires a great deal of effort to prove
> this!.
We use this whenever we are forced to use a new test set. E.g. when doing 
molecular replacement into an isomorphous cell without access to the original 
test set (this has been discussed on the BB a few times) or when doing k-fold 
cross validation. Whenever a new test set in chosen, enough model tuning (i.e. 
refinement) is needed to make the model independent of the test set (you may 
need to perturb the model somewhat by resetting the B-factors or by giving the 
coordinates a nudge). At refinement convergence the maximum amount of tuning is 
attained. And at true convergence, the starting point doesn't matter (although 
local minima complicate matters).  

With k-fold cross validation we can actually show that there is no obvious 
bias: Say we build and refine a model with test set 0 left out, then any 
further refinement with k different test sets left out should start out with 
R-free substantially biased for any set i!=0, that is R-free(i) < R-free(0). 
This is indeed what we see. 
Now if the refinements converge and there still is bias then we would still get 
R-free(i) < R-free(0) for all sets i!=0. This is not what we see (at least not 
for most PDB_REDO test cases). Of course, these are examples of the worst sort 
of test set bias. The bias introduced in real-space refinement is more subtle. 
At least that is what I assume, but this is something you said you could 
quantify. A proper estimate of the problem (rather than a principle we don't 
quite adhere to all the time anyway), would really help.

Cheers,
Robbie  

> 
> 
>   In practical terms we have to choose the best option:
>   1) Refine against maps with missing reflections and the possibility of
> artefacts that lead to suboptimal results. This keeps R-free unbiased, even
> directly after real-space refinement.
>   2) Refine against maps with test reflections set to Fcalc. This biases
> against the current model, but should have less artefacts. This too keeps R-
> free unbiased directly after real-space refinement.
>   3) Refine against maps with all reflections. This should give the best
> fitting results, but does introduce bias to the test set. However, this bias
> 
> 
> These are all valid points. However they do not advocate for biasing Rfree.
> Yuo can always fill in missing reflections with something else (different from
> your genuine free-r set of reelections) and thus address two issues at once :
> eliminate missing terms and not use free-r reflections for this!
> 
> Pavel

Re: [ccp4bb] AW: [ccp4bb] Twinning VS. Disorder

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Jakob,

your Gedankenexperiment on powder diffraction is not correct. You
would record a powder diffraction pattern if you rotated a single
crystal around the beam axis and record the result on a single image.
This rotation does not affect the mosaicity and the mosaicity of a
powder sample related only to the mosaicity of the micro crystals
present in the powder. You also do not get arcs when reducing the
powderness but you start seeing single spots. This can often be
observed in the presence of ice rings.

Best,
Tim

On 04/25/2014 09:32 AM, Keller, Jacob wrote:
> Is the following being neglected?
> 
> In a crystal with these putative mosaic microdomains, there will be
> interference between microdomains at their edges/borders (at
> least), but since most microdomains are probably way smaller than
> the coherence length of 3-10 microns, presumably all unit cells in
> domain A interfere with all unit cells in domains B, C, etc, which
> are in the same coherence volume. In fact, as I said too unclearly
> in a previous post, as the putative microdomains become smaller and
> smaller to the limit of one unit cell, they become
> indistinguishable from unit cell parameter variation. So I am
> becoming increasingly suspicious about the existence of
> microdomains, and wonder what hard evidence there is for their
> existence?
> 
> As a thought experiment, one can consider the microdomain theory
> taken to its limit: a powder diffraction image. In powder
> diffraction, there are so many crystals (read: microdomains) that
> each spot is manifested at its Bragg angle at every possible radial
> position on the detector. Mosaicity would be, what, 360 degrees?
> So, now imagine decreasing the mosaicity to lower values, and one
> gets progressively shorter arcs which at lower values become spots.
> Doesn’t this mean that the contribution from microdomain mosaicity
> should be to make the spots more like arcs, as we sometimes see in
> terrible diffraction patterns, and not just general broadening of
> spots? Put another way: mosaicity should broaden spots in the
> radial direction (arcs), and unit cell parameter variation should
> produce straight broadening in the direction of the unit cell
> variation of magnitude proportional to the degree of variation in
> that direction.
> 
> JPK
> 
> 
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf
> Of Ian Tickle Sent: Thursday, April 24, 2014 7:01 PM To:
> CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] AW: [ccp4bb] Twinning
> VS. Disorder
> 
> 
> Dear Herman On 24 April 2014 22:32,
> mailto:herman.schreu...@sanofi.com>>
> wrote:
> 
> The X-ray coherent length is depending on the crystal, not the
> synchrotron and my gut feeling is that it is at least several
> hundred unit cells, but here other experts may correct me.
> 
> 
> I assume you meant that the coherence length is a property of the
> beam (e.g. for a Cu target source it's related to the lifetime of
> the excited Cu K-alpha state), not the crystal, e,g, see
> http://www.aps.anl.gov/Users/Meeting/2010/Presentations/WK2talk_Vartaniants.pdf
> (slides 8-11).  The relevant property of the crystal is the size of
> the microdomains.  You don't get interference because coherence
> length << domain size, i.e. the beam is not coherent over more than
> 1 domain.  This is true for in-house sources & synchrotrons, I
> guess for FELs it's different, i.e. much greater coherence length?
> This relates to a question I asked on the BB some time ago: if the
> coherence length is long enough would you start to see the effects
> of interference in twinned crystals, i.e. would the summation of
> intensities break down? I defer to the experts on synchrotrons &
> FELs! Cheers -- Ian
> 

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

GPG Key ID = A46BEE1A

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Re: [ccp4bb] AW: [ccp4bb] Twinning VS. Disorder

[Keller, Jacob]

Is the following being neglected?

In a crystal with these putative mosaic microdomains, there will be 
interference between microdomains at their edges/borders (at least), but since 
most microdomains are probably way smaller than the coherence length of 3-10 
microns, presumably all unit cells in domain A interfere with all unit cells in 
domains B, C, etc, which are in the same coherence volume. In fact, as I said 
too unclearly in a previous post, as the putative microdomains become smaller 
and smaller to the limit of one unit cell, they become indistinguishable from 
unit cell parameter variation. So I am becoming increasingly suspicious about 
the existence of microdomains, and wonder what hard evidence there is for their 
existence?

As a thought experiment, one can consider the microdomain theory taken to its 
limit: a powder diffraction image. In powder diffraction, there are so many 
crystals (read: microdomains) that each spot is manifested at its Bragg angle 
at every possible radial position on the detector. Mosaicity would be, what, 
360 degrees? So, now imagine decreasing the mosaicity to lower values, and one 
gets progressively shorter arcs which at lower values become spots. Doesn’t 
this mean that the contribution from microdomain mosaicity should be to make 
the spots more like arcs, as we sometimes see in terrible diffraction patterns, 
and not just general broadening of spots? Put another way: mosaicity should 
broaden spots in the radial direction (arcs), and unit cell parameter variation 
should produce straight broadening in the direction of the unit cell variation 
of magnitude proportional to the degree of variation in that direction.

JPK


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ian Tickle
Sent: Thursday, April 24, 2014 7:01 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] AW: [ccp4bb] Twinning VS. Disorder


Dear Herman
On 24 April 2014 22:32, 
mailto:herman.schreu...@sanofi.com>> wrote:

The X-ray coherent length is depending on the crystal, not the synchrotron and 
my gut feeling is that it is at least several hundred unit cells, but here 
other experts may correct me.


I assume you meant that the coherence length is a property of the beam (e.g. 
for a Cu target source it's related to the lifetime of the excited Cu K-alpha 
state), not the crystal, e,g, see 
http://www.aps.anl.gov/Users/Meeting/2010/Presentations/WK2talk_Vartaniants.pdf 
(slides 8-11).  The relevant property of the crystal is the size of the 
microdomains.  You don't get interference because coherence length << domain 
size, i.e. the beam is not coherent over more than 1 domain.  This is true for 
in-house sources & synchrotrons, I guess for FELs it's different, i.e. much 
greater coherence length?  This relates to a question I asked on the BB some 
time ago: if the coherence length is long enough would you start to see the 
effects of interference in twinned crystals, i.e. would the summation of 
intensities break down?
I defer to the experts on synchrotrons & FELs!
Cheers
-- Ian

[ccp4bb] AW: [ccp4bb] AW: [ccp4bb] Twinning VS. Disorder

[Herman . Schreuder]

Dear Ian and James,
Here I learned something new. I assumed that coherence length would be limited 
by crystal quality, e.g. mosaicity and microdomains etc. which apparently is 
not the case. For me, one of the characteristics of twinning is that there is 
no interference between the twin domains. If the twin domains get so small that 
there is interference, these phenomenon are usually referred to as lattice 
translocation disorder, but that might be my personal interpretation.
So maybe I focused too narrowly on the word twinning and should have mentioned 
the lattice translocation disorder. However, in this case the usual twinning 
options in refinement programs cannot be used, since these assume summation of 
intensities.

Cheers,
Herman

Von: Ian Tickle [mailto:ianj...@gmail.com]
Gesendet: Freitag, 25. April 2014 01:01
An: Schreuder, Herman R&D/DE
Cc: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] AW: [ccp4bb] Twinning VS. Disorder


Dear Herman
On 24 April 2014 22:32, 
mailto:herman.schreu...@sanofi.com>> wrote:

The X-ray coherent length is depending on the crystal, not the synchrotron and 
my gut feeling is that it is at least several hundred unit cells, but here 
other experts may correct me.


I assume you meant that the coherence length is a property of the beam (e.g. 
for a Cu target source it's related to the lifetime of the excited Cu K-alpha 
state), not the crystal, e,g, see 
http://www.aps.anl.gov/Users/Meeting/2010/Presentations/WK2talk_Vartaniants.pdf 
(slides 8-11).  The relevant property of the crystal is the size of the 
microdomains.  You don't get interference because coherence length << domain 
size, i.e. the beam is not coherent over more than 1 domain.  This is true for 
in-house sources & synchrotrons, I guess for FELs it's different, i.e. much 
greater coherence length?  This relates to a question I asked on the BB some 
time ago: if the coherence length is long enough would you start to see the 
effects of interference in twinned crystals, i.e. would the summation of 
intensities break down?
I defer to the experts on synchrotrons & FELs!
Cheers
-- Ian