Re: [ccp4bb] happy/sad maps

[Sindelar, Charles]

Hi James!  I’m not sure exactly what cpp4, coot and phenix do for their FFT’s, 
but I’m assuming they must mask off the ‘corners’ of the 3D FFT data cube 
transform (where the frequency is greater than the Nyquist) during the forwards 
transform.

If you take your Cheshire map and do the FFT in matlab or octave, this is not 
done and I can confirm you get the smiley back. The cryo-EM software I am 
familiar with does filter away the FFT corners during the first 3D 
reconstruction step, so bypassing this issue.

Fun demo- this would be a great exercise for students!

Chuck


From: CCP4 bulletin board  on behalf of James Holton 

Date: Friday, April 28, 2023 at 11:49 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] happy/sad maps
Its still April, but this one isn't a joke.

The smiley-face electron density in the left panel of the attached image
has the remarkable property that any attempt to sharpen or blur the map
turns it into the frowny-face on the right.  If you'd like to try this
yourself, the hidden_frown.map file is available in this tarball:
https://nam12.safelinks.protection.outlook.com/?url=https%3A%2F%2Fbl831.als.lbl.gov%2F~jamesh%2Fbugreports%2Ffft_042423.tgz=05%7C01%7Ccharles.sindelar%40YALE.EDU%7Cc059178a4b774611f6ec08db48001b68%7Cdd8cbebb21394df8b4114e3e87abeb5c%7C0%7C0%7C638182937669477928%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C=jl16hGjaqmChVOHWb%2FAvgtvPTVFOt06wBqBYoxPk%2FZw%3D=0

In fact, any use of an FFT, even with the sharpening B set to zero,
turns the smiley into a frowny face. There is no way to get the smiley
face back (except opening the file again).  Yes, that's right, even just
a simple back-and-forth FFT: turning this hidden_frown.map into
structure factors and then back into a map again, gives you a frowny
face.  This happens using coot, ccp4 and phenix.

Wait, what!?  Isn't a Fourier transform supposed to preserve
information? As in: you can jump back and forth between real and
reciprocal space with impunity? Without introducing error?  Well, yes,
it is SUPPOSED to work like that, but the 3D FFT algorithms of
structural biology have a ... quirk. If you start with structure factors
and make a map out of them, you can convert it back-and-forth as often
as you want with 100% preservation of information.  However, if you
start with a real-space map (such as from cryoEM), a back-and-forth
conversion gives you a different map. This new map can then be
transformed back-and-forth all you want and be 100% preserved. It has
been "christened" by the FFT, but it is not the same as the starting
map, which is impossible to recover from the FFT-transformed data.
Information has been lost.  It is fine for crystallography (which starts
with structure factors), but for techniques such as cryoEM that start
with maps, using an FFT changes the data.

What information is being lost?  Sharp edges. These turn into ripples
covering all of real and reciprocal space. Do real-world data have sharp
edges?  Well, the all-or-nothing masks we use to model bulk solvent are
one example. Also, if you "mask off" otherwise smooth density with an
all-or-nothing mask, you will get similar ripples.  Another example of a
sharp edge might be the large changes between adjacent pixels you see
when a single electron hits a detector. For example, if you make a map
with just one non-zero voxel and run it back-and-forth through FFT you
will find that voxel loses from 50% to 99% of its value (depending on
the size of the map).  How much does this actually impact cryo-EM data?
That is my question.

What evil magic did I wield to make this map?  Well, I drew a smiley and
frowny face by hand, converted them to maps, and then I generated random
noise within the boundaries of the smiley face. I ran this noisy map
back-and-forth through FFT, and then subtracted the map that survives
the FFT from the pre-FFT map.  This cheshire_smile.map has the
interesting property that all of the structure factors calculated from
it are zero. It has an RMSD of 1.4, but after a back-and-forth FFT this
RMSD drops to 1e-7.  I generated the hidden_frown.map by simply summing
the frowny.map with cheshire_smile.map.

But isn't this map getting less noisy? Yes it is, but the interpretation
clearly changes as well.

Why does this happen?  It is because of a finite resolution cutoff. Oh!
What a relief! You don't have super-high resolution, do you? Well, no,
almost nobody has signal out beyond 1.0 A, but we do have noise.  In
diffraction data this noise is removed by simply not measuring it.  For
map data, however, the problem is that noise at very high frequencies
(small-number resolutions) is hard to avoid. This is because of another
phenomenon NMR spectroscopists are very familiar with: aliasing, or
"folding".  If any high-spatial frequency noise exists above half the
sampling rate (or "Nyquist frequency") it still gets 

[ccp4bb] Isopeptide and shelxl topology file

[StrBio]

Hello,

Can you suggest how to make isopeptide bond in coot and generate .cif file
to refine the structure in phenix?

Also need suggestions to create shelxl topology file for non standard amino
acids.
I used PRODGR before but seems like the sever is down last couple of
months. Any alternatives?

Thank you
Amit



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Re: [ccp4bb] happy/sad maps

[Lijun Liu]

what about if you run a 2-D FFT rather than 2-D FFT, if your hand drawing map 
in on a 2-D paper?

> On Apr 28, 2023, at 10:48 AM, James Holton  wrote:
> 
> Its still April, but this one isn't a joke.
> 
> The smiley-face electron density in the left panel of the attached image has 
> the remarkable property that any attempt to sharpen or blur the map turns it 
> into the frowny-face on the right.  If you'd like to try this yourself, the 
> hidden_frown.map file is available in this tarball:
> https://bl831.als.lbl.gov/~jamesh/bugreports/fft_042423.tgz
> 
> In fact, any use of an FFT, even with the sharpening B set to zero, turns the 
> smiley into a frowny face. There is no way to get the smiley face back 
> (except opening the file again).  Yes, that's right, even just a simple 
> back-and-forth FFT: turning this hidden_frown.map into structure factors and 
> then back into a map again, gives you a frowny face.  This happens using 
> coot, ccp4 and phenix.
> 
> Wait, what!?  Isn't a Fourier transform supposed to preserve information? As 
> in: you can jump back and forth between real and reciprocal space with 
> impunity? Without introducing error?  Well, yes, it is SUPPOSED to work like 
> that, but the 3D FFT algorithms of structural biology have a ... quirk. If 
> you start with structure factors and make a map out of them, you can convert 
> it back-and-forth as often as you want with 100% preservation of information. 
>  However, if you start with a real-space map (such as from cryoEM), a 
> back-and-forth conversion gives you a different map. This new map can then be 
> transformed back-and-forth all you want and be 100% preserved. It has been 
> "christened" by the FFT, but it is not the same as the starting map, which is 
> impossible to recover from the FFT-transformed data. Information has been 
> lost.  It is fine for crystallography (which starts with structure factors), 
> but for techniques such as cryoEM that start with maps, using an FFT changes 
> the data.
> 
> What information is being lost?  Sharp edges. These turn into ripples 
> covering all of real and reciprocal space. Do real-world data have sharp 
> edges?  Well, the all-or-nothing masks we use to model bulk solvent are one 
> example. Also, if you "mask off" otherwise smooth density with an 
> all-or-nothing mask, you will get similar ripples.  Another example of a 
> sharp edge might be the large changes between adjacent pixels you see when a 
> single electron hits a detector. For example, if you make a map with just one 
> non-zero voxel and run it back-and-forth through FFT you will find that voxel 
> loses from 50% to 99% of its value (depending on the size of the map).  How 
> much does this actually impact cryo-EM data?  That is my question.
> 
> What evil magic did I wield to make this map?  Well, I drew a smiley and 
> frowny face by hand, converted them to maps, and then I generated random 
> noise within the boundaries of the smiley face. I ran this noisy map 
> back-and-forth through FFT, and then subtracted the map that survives the FFT 
> from the pre-FFT map.  This cheshire_smile.map has the interesting property 
> that all of the structure factors calculated from it are zero. It has an RMSD 
> of 1.4, but after a back-and-forth FFT this RMSD drops to 1e-7.  I generated 
> the hidden_frown.map by simply summing the frowny.map with cheshire_smile.map.
> 
> But isn't this map getting less noisy? Yes it is, but the interpretation 
> clearly changes as well.
> 
> Why does this happen?  It is because of a finite resolution cutoff. Oh! What 
> a relief! You don't have super-high resolution, do you? Well, no, almost 
> nobody has signal out beyond 1.0 A, but we do have noise.  In diffraction 
> data this noise is removed by simply not measuring it.  For map data, 
> however, the problem is that noise at very high frequencies (small-number 
> resolutions) is hard to avoid. This is because of another phenomenon NMR 
> spectroscopists are very familiar with: aliasing, or "folding".  If any 
> high-spatial frequency noise exists above half the sampling rate (or "Nyquist 
> frequency") it still gets recorded, but shows up in a lower-frequency Fourier 
> coefficient. It is not possible to remove such aliasing noise after 
> digitization. Upon discretization of the signal (FFT or no) all these high 
> frequencies join with lower frequency terms, and so survive any low-pass 
> filtering. Darn.
> 
> Why am I bringing this up?  Because if there is noise out beyond the FFT 
> resolution limit it implies there is also noise out beyond the 
> Nyquist-Shannon limit as well. If that is the case, direct-space imaging data 
> may be a lot noisier than it needs to be. In general, in digital signal 
> processing of things like sound an analog low-pass filter is always installed 
> at the input of any digitizer.  Perhaps this is why de-focusing works better 
> than being at focus?
> 
> What is the solution? Well, for things like the bulk 

[ccp4bb] happy/sad maps

[James Holton]

Its still April, but this one isn't a joke.

The smiley-face electron density in the left panel of the attached image 
has the remarkable property that any attempt to sharpen or blur the map 
turns it into the frowny-face on the right.  If you'd like to try this 
yourself, the hidden_frown.map file is available in this tarball:

https://bl831.als.lbl.gov/~jamesh/bugreports/fft_042423.tgz

In fact, any use of an FFT, even with the sharpening B set to zero, 
turns the smiley into a frowny face. There is no way to get the smiley 
face back (except opening the file again).  Yes, that's right, even just 
a simple back-and-forth FFT: turning this hidden_frown.map into 
structure factors and then back into a map again, gives you a frowny 
face.  This happens using coot, ccp4 and phenix.


Wait, what!?  Isn't a Fourier transform supposed to preserve 
information? As in: you can jump back and forth between real and 
reciprocal space with impunity? Without introducing error?  Well, yes, 
it is SUPPOSED to work like that, but the 3D FFT algorithms of 
structural biology have a ... quirk. If you start with structure factors 
and make a map out of them, you can convert it back-and-forth as often 
as you want with 100% preservation of information.  However, if you 
start with a real-space map (such as from cryoEM), a back-and-forth 
conversion gives you a different map. This new map can then be 
transformed back-and-forth all you want and be 100% preserved. It has 
been "christened" by the FFT, but it is not the same as the starting 
map, which is impossible to recover from the FFT-transformed data. 
Information has been lost.  It is fine for crystallography (which starts 
with structure factors), but for techniques such as cryoEM that start 
with maps, using an FFT changes the data.


What information is being lost?  Sharp edges. These turn into ripples 
covering all of real and reciprocal space. Do real-world data have sharp 
edges?  Well, the all-or-nothing masks we use to model bulk solvent are 
one example. Also, if you "mask off" otherwise smooth density with an 
all-or-nothing mask, you will get similar ripples.  Another example of a 
sharp edge might be the large changes between adjacent pixels you see 
when a single electron hits a detector. For example, if you make a map 
with just one non-zero voxel and run it back-and-forth through FFT you 
will find that voxel loses from 50% to 99% of its value (depending on 
the size of the map).  How much does this actually impact cryo-EM data?  
That is my question.


What evil magic did I wield to make this map?  Well, I drew a smiley and 
frowny face by hand, converted them to maps, and then I generated random 
noise within the boundaries of the smiley face. I ran this noisy map 
back-and-forth through FFT, and then subtracted the map that survives 
the FFT from the pre-FFT map.  This cheshire_smile.map has the 
interesting property that all of the structure factors calculated from 
it are zero. It has an RMSD of 1.4, but after a back-and-forth FFT this 
RMSD drops to 1e-7.  I generated the hidden_frown.map by simply summing 
the frowny.map with cheshire_smile.map.


But isn't this map getting less noisy? Yes it is, but the interpretation 
clearly changes as well.


Why does this happen?  It is because of a finite resolution cutoff. Oh! 
What a relief! You don't have super-high resolution, do you? Well, no, 
almost nobody has signal out beyond 1.0 A, but we do have noise.  In 
diffraction data this noise is removed by simply not measuring it.  For 
map data, however, the problem is that noise at very high frequencies 
(small-number resolutions) is hard to avoid. This is because of another 
phenomenon NMR spectroscopists are very familiar with: aliasing, or 
"folding".  If any high-spatial frequency noise exists above half the 
sampling rate (or "Nyquist frequency") it still gets recorded, but shows 
up in a lower-frequency Fourier coefficient. It is not possible to 
remove such aliasing noise after digitization. Upon discretization of 
the signal (FFT or no) all these high frequencies join with lower 
frequency terms, and so survive any low-pass filtering. Darn.


Why am I bringing this up?  Because if there is noise out beyond the FFT 
resolution limit it implies there is also noise out beyond the 
Nyquist-Shannon limit as well. If that is the case, direct-space imaging 
data may be a lot noisier than it needs to be. In general, in digital 
signal processing of things like sound an analog low-pass filter is 
always installed at the input of any digitizer.  Perhaps this is why 
de-focusing works better than being at focus?


What is the solution? Well, for things like the bulk solvent mask I'd 
say some real-space "feathering" is called for before performing FFTs.  
Same goes for masked density like that used to compute CCmask. It may 
also be worth looking into the digitization process of "image first" 
structural biology methods?


My question for the BB:  can 

[ccp4bb] Registration open: Structure Meets Function Webinar #26 - New Technologies in Instruct

[ccp4mail]

Dear all,


Registration is 
open for 
the 26th edition of the Structure Meets Function 
webinar
 from Instruct-ERIC. The webinar will take place 16 May 2023 at 11:00 CEST 
(10:00 GMT).



This month will feature 4 speakers from across the Instruct-ERIC catalogue: two 
from CNB-CSIC in Madrid, Spain, and two from IGBMC of Instruct-FR2, in 
Strasbourg, France. Find out more about the webinar and register 
here.



Take a look at our previous webinars in the series 
here.





Register 
Here





Speaker 1: Javier Conesa

Speaker 2: David Herreros

Talk Title: Introduction to CNB-CSIC CLEM, and I2PC FlexiHub.



Speaker 3: Alexandre Durand

Speaker 4: Catherine Birck

Talk Title: Introduction to IGBMC Chameleon and BLI/FIDA.








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[ccp4bb] Academic position openings in Exeter, UK

[Harmer, Nicholas]

Dear all,

The Living Systems Institute in Exeter, south-west England, is advertising 
eight new academic positions at all levels. The details are available at 
https://www.exeter.ac.uk/research/livingsystems/joinus/lsirecruitment2023/. 
Most of the positions can be in any topic relevant to the institute's mission 
to discover, understand and control the fundamental rules of life. We would be 
very interested in particular in an appointment in integrative structural 
biology. We have good resources for crystallography and cryo-EM, and are part 
of BAGs with regular access to Diamond, eBIC, and ESRF.

We're very happy to speak informally to anyone interested!

Kind regards,

Nic Harmer

Nicholas Harmer
Associate Professor in Biochemistry
University of Exeter
Ext: 5179
www.exeter.ac.uk
Living Systems Institute, Stocker Road, Exeter, EX4 4QD
[Winner badge]
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This email and any attachment may contain information that is confidential, 
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under applicable legislation. It is intended for the addressee only. If you 
received this message in error, please let me know and delete the email and any 
attachments immediately. The University will not accept responsibility for the 
accuracy/completeness of this email and its attachments.




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Re: [ccp4bb] PDB redo R free

[Robbie Joosten]

To add to Tim's references, the algorithms that pdb-redo uses to deal with Rfree and possible bias are described here: https://journals.iucr.org/d/issues/2012/04/00/ba5174/index.htmlIf you happen to have a very small dataset (< 5000 reflections) pdb-redo does 10-fold cross validation and returns Rcomplete as well.Cheers,RobbieOn 28 Apr 2023 11:26, Tim Gruene  wrote:Dear Qixu Cai,

it  is a misconception that Rfree was not free anymore. The gap does
not close when you swap the reflections in Rfree set. The 'free'
reflections are freed when you do proper refinement, i.e., when the
value of the target function becomes stationary.
See https://doi.org/10.1107/S0907444997013875 and
https://doi.org/10.1107/S0907444999016868 for the mathematical
background, https://doi.org/10.1073/pnas.1502136112 for an experimental
treatment, and Ian Tickle's explanations on this topic here on the
CCP4bb.

Best wishes,
Tim


On Fri, 28 Apr 2023 16:54:07 +0800 Qixu Cai  wrote:

> Dear Robbie and Tim，
> 
> Thanks a lot for your reply. I just do not understand that if we can
> change the R free set during different rounds of refinement, the gap
> between Rwork and Rfree would be small, and Rfree would be
> meaningless, as R-free set is not "free".
> 
> Best regards,
> 
> Qixu Cai
> Email: caiq...@gmail.com
> 
> 
> 
> Robbie Joosten  于2023年4月28日周五
> 15:26写道：
> 
> > Hi Qixu,
> >
> > PDB-REDO tries to have a minimum number test set reflections to
> > reduce the error margin in R-free. As Tim says this is not a
> > problem but if you reach out privately we can change your
> > calculation to use your current testset. That is not recommended
> > though.
> >
> > Cheers,
> > Robbie
> >
> > On 28 Apr 2023 04:51, Qixu Cai  wrote:
> >
> > Dear all,
> >
> > I'm using the PDB-REDO server to refine my structure. I found that
> > PDB-REDO said that 5% R-free set is too small and it created a new
> > R-free set.
> >
> > Is possible for the PDB-REDO server to keep the original R-free set
> > to make final Rfree value comparable?
> >
> > Best regards,
> >
> > Qixu Cai
> > Email: caiq...@gmail.com
> >
> >
> > --
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
> >
> >
> >  



-- 
--
Tim Gruene
Head of the Centre for X-ray Structure Analysis
Faculty of Chemistry
University of Vienna

Phone: +43-1-4277-70202

GPG Key ID = A46BEE1A




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Re: [ccp4bb] PDB redo R free

[Tim Gruene]

Dear Qixu Cai,

it  is a misconception that Rfree was not free anymore. The gap does
not close when you swap the reflections in Rfree set. The 'free'
reflections are freed when you do proper refinement, i.e., when the
value of the target function becomes stationary.
See https://doi.org/10.1107/S0907444997013875 and
https://doi.org/10.1107/S0907444999016868 for the mathematical
background, https://doi.org/10.1073/pnas.1502136112 for an experimental
treatment, and Ian Tickle's explanations on this topic here on the
CCP4bb.

Best wishes,
Tim


On Fri, 28 Apr 2023 16:54:07 +0800 Qixu Cai  wrote:

> Dear Robbie and Tim，
> 
> Thanks a lot for your reply. I just do not understand that if we can
> change the R free set during different rounds of refinement, the gap
> between Rwork and Rfree would be small, and Rfree would be
> meaningless, as R-free set is not "free".
> 
> Best regards,
> 
> Qixu Cai
> Email: caiq...@gmail.com
> 
> 
> 
> Robbie Joosten  于2023年4月28日周五
> 15:26写道：
> 
> > Hi Qixu,
> >
> > PDB-REDO tries to have a minimum number test set reflections to
> > reduce the error margin in R-free. As Tim says this is not a
> > problem but if you reach out privately we can change your
> > calculation to use your current testset. That is not recommended
> > though.
> >
> > Cheers,
> > Robbie
> >
> > On 28 Apr 2023 04:51, Qixu Cai  wrote:
> >
> > Dear all,
> >
> > I'm using the PDB-REDO server to refine my structure. I found that
> > PDB-REDO said that 5% R-free set is too small and it created a new
> > R-free set.
> >
> > Is possible for the PDB-REDO server to keep the original R-free set
> > to make final Rfree value comparable?
> >
> > Best regards,
> >
> > Qixu Cai
> > Email: caiq...@gmail.com
> >
> >
> > --
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
> >
> >
> >  



-- 
--
Tim Gruene
Head of the Centre for X-ray Structure Analysis
Faculty of Chemistry
University of Vienna

Phone: +43-1-4277-70202

GPG Key ID = A46BEE1A



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pgpjj_Lr7i04I.pgp
Description: OpenPGP digital signature

Re: [ccp4bb] PDB redo R free

[Qixu Cai]

Dear Robbie and Tim，

Thanks a lot for your reply. I just do not understand that if we can change
the R free set during different rounds of refinement, the gap between Rwork
and Rfree would be small, and Rfree would be meaningless, as R-free set is
not "free".

Best regards,

Qixu Cai
Email: caiq...@gmail.com



Robbie Joosten  于2023年4月28日周五 15:26写道：

> Hi Qixu,
>
> PDB-REDO tries to have a minimum number test set reflections to reduce the
> error margin in R-free. As Tim says this is not a problem but if you reach
> out privately we can change your calculation to use your current testset.
> That is not recommended though.
>
> Cheers,
> Robbie
>
> On 28 Apr 2023 04:51, Qixu Cai  wrote:
>
> Dear all,
>
> I'm using the PDB-REDO server to refine my structure. I found that
> PDB-REDO said that 5% R-free set is too small and it created a new R-free
> set.
>
> Is possible for the PDB-REDO server to keep the original R-free set to
> make final Rfree value comparable?
>
> Best regards,
>
> Qixu Cai
> Email: caiq...@gmail.com
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>
>
>



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[ccp4bb] isopeptide bond and shelx topology file

[StrBio]

Hello,

Can you suggest how to make isopeptide bond in coot and generate .cif file
to refine the structure in phenix?

Also need suggestions to create shelxl topology file for non standard amino
acids.
I used PRODGR before but seems like the sever is down last couple of
months. Any alternatives?

Thank you
Amit



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Re: [ccp4bb] PDB redo R free

[Robbie Joosten]

Hi Qixu,PDB-REDO tries to have a minimum number test set reflections to reduce the error margin in R-free. As Tim says this is not a problem but if you reach out privately we can change your calculation to use your current testset. That is not recommended though.Cheers,RobbieOn 28 Apr 2023 04:51, Qixu Cai  wrote:Dear all,I'm using the PDB-REDO server to refine my structure. I found that PDB-REDO said that 5% R-free set is too small and it created a new R-free set.Is possible for the PDB-REDO server to keep the original R-free set to make final Rfree value comparable?Best regards,Qixu CaiEmail: caiq...@gmail.com


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