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

[Keller, Jacob]

I would think the most information-reflecting representation for systematic 
absences (or maybe for all reflections) would be not I/sig but the reflection's 
(|log|) ratio to the expected intensity in that shell (median intensity, say). 
Thus, when the median intensity is 1000 counts, and one observes a spot of 1 
count, this would be quite information-rich even though its I/sig would be 
really small.

This approach would also reflect the lesser information contained in twinned 
data, where deviations from mean intensities are smaller, even though I/sig be 
quite large.

Regarding spots that are not systematic absence candidates, isn't it true that 
a very weak spot (e.g. 10 counts) of I/sig = 2 might contain more information 
than a strong spot (1000 counts) of I/sig = 20 in the same shell, if the median 
counts in the shell were 1000?

I used to hear rumors that maps calculated from the 1000 strongest spots were 
almost tantamount to using all reflections; maybe these flaky maps would be 
improved by using instead the 1000 reflections which deviate most from expected 
intensities? (i.e., both stronger and weaker.)

Maybe more generally, should refinement incorporate weighting for these deviant 
spots? Or maybe it already does, but my understanding was that I/sig was the 
most salient for weighting.

I guess the general idea is that the more unexpected the value is, the more it 
captures something unique about the thing being measured, thus more information.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
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-Original Message-
From: CCP4 bulletin board  On Behalf Of Kay Diederichs
Sent: Tuesday, March 10, 2020 2:48 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] [3dem] Which resolution?

I'd say that it depends on your state of knowledge, and on their I and sigma.

- if you know the space group for sure before you do the measurement of the 
systematic absences, their I and sigma don't matter to you (because they don't 
influence your mental model of the experiment), so their information content is 
(close to) zero.
- if the space group is completely unknown, some groups of reflections (e.g. 
h,k,l = 0,0,2n+1) can only be considered "potentially systematic absences". 
Then both I and sigma matter. "small" or "high" I/sigma for each member of such 
a group of reflections would indeed add quite some information in this 
situation, so an information content of up to 1 bit would be justified. 
"intermediate" I/sigma (say, 0.5 to 2) would be closer to zero bit, since it 
does not let you safely decide between "yes" or "no" (the recent paper by Randy 
Read and coworkers relates I and sigma to bits of information, but not in the 
context of decision making from potentially systematic absent reflections). 

So it is not quite straightforward, I think.

best wishes,
Kay

On Tue, 10 Mar 2020 01:26:03 +0100, James Holton  wrote:

>I'd say they are 1 bit each, since they are the answer to a yes-or-no 
>question.
>
>-James Holton
>MAD Scientist
>
>On 2/27/2020 6:32 PM, Keller, Jacob wrote:
>> How would one evaluate the information content of systematic absences?
>>
>> JPK
>>
>> On Feb 26, 2020 8:14 PM, James Holton  wrote:
>> In my opinion the threshold should be zero bits.  Yes, this is where
>> CC1/2 = 0 (or FSC = 0).  If there is correlation then there is 
>> information, and why throw out information if there is information to 
>> be had?  Yes, this information comes with noise attached, but that is 
>> why we have weights.
>>
>> It is also important to remember that zero intensity is still useful 
>> information.  Systematic absences are an excellent example.  They 
>> have no intensity at all, but they speak volumes about the structure.  
>> In a similar way, high-angle zero-intensity observations also tell us 
>> something.  Ever tried unrestrained B factor refinement at poor 
>> resolution?  It is hard to do nowadays because of all the safety 
>> catches in modern software, but you can get great R factors this way.
>> A telltale sign of this kind of "over fitting" is remarkably large 
>> Fcalc values beyond the resolution cutoff.  These don't contribute to 
>> the R fa

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

[Keller, Jacob]

How would one evaluate the information content of systematic absences?

JPK

On Feb 26, 2020 8:14 PM, James Holton  wrote:
In my opinion the threshold should be zero bits.  Yes, this is where CC1/2 = 0 
(or FSC = 0).  If there is correlation then there is information, and why throw 
out information if there is information to be had?  Yes, this information comes 
with noise attached, but that is why we have weights.

It is also important to remember that zero intensity is still useful 
information.  Systematic absences are an excellent example.  They have no 
intensity at all, but they speak volumes about the structure.  In a similar 
way, high-angle zero-intensity observations also tell us something.  Ever tried 
unrestrained B factor refinement at poor resolution?  It is hard to do nowadays 
because of all the safety catches in modern software, but you can get great R 
factors this way.  A telltale sign of this kind of "over fitting" is remarkably 
large Fcalc values beyond the resolution cutoff.  These don't contribute to the 
R factor, however, because Fobs is missing for these hkls. So, including 
zero-intensity data suppresses at least some types of over-fitting.

The thing I like most about the zero-information resolution cutoff is that it 
forces us to address the real problem: what do you mean by "resolution" ?  Not 
long ago, claiming your resolution was 3.0 A meant that after discarding all 
spots with individual I/sigI < 3 you still have 80% completeness in the 3.0 A 
bin.  Now we are saying we have a 3.0 A data set when we can prove 
statistically that a few non-background counts fell into the sum of all spot 
areas at 3.0 A.  These are not the same thing.

Don't get me wrong, including the weak high-resolution information makes the 
model better, and indeed I am even advocating including all the noisy zeroes.  
However, weak data at 3.0 A is never going to be as good as having strong data 
at 3.0 A.  So, how do we decide?  I personally think that the resolution 
assigned to the PDB deposition should remain the classical I/sigI > 3 at 80% 
rule.  This is really the only way to have meaningful comparison of resolution 
between very old and very new structures.  One should, of course, deposit all 
the data, but don't claim that cut-off as your "resolution".  That is just 
plain unfair to those who came before.

Oh yeah, and I also have a session on "interpreting low-resolution maps" at the 
GRC this year.  
https://www.grc.org/diffraction-methods-in-structural-biology-conference/2020/

So, please, let the discussion continue!

-James Holton
MAD Scientist

On 2/22/2020 11:06 AM, Nave, Colin (DLSLtd,RAL,LSCI) wrote:
Alexis
This is a very useful summary.

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

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

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

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

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

Regards
Colin



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

Hi all,

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

Notwithstanding notational problems (e.g. strict equations as opposed to 
approximation 

[ccp4bb] Sodium Ion Binding?

[Keller, Jacob]

Dear Crystallographers,

Does anyone know of a good biophysical way to identify or quantify sodium ion 
binding to a protein, besides crystallography and ITC? Is this possible with 
SPR, perhaps? Mass spec? Gel shifts? Examples would be greatly appreciated!

All the best,

Jacob Keller


+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
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Re: [ccp4bb] Figure of merit in refinement

[Keller, Jacob]

>>And as we often end our beer-discussions - may be all protein space groups 
>>are actually true P1, just close enough to satisfy the high symmetry rules .. 
>>but this is getting a bit philosophical I know ..

Could we add that all crystals are twinned, just some are in such a way as to 
be a problem?

JPK


On Wed, Oct 16, 2019 at 6:24 PM Randy Read 
mailto:rj...@cam.ac.uk>> wrote:
James,

Where we diverge is with your interpretation that big differences lead to small 
FOMs.  The size of the FOM depends on the product of Fo and Fc, not their 
difference.  The FOM for a reflection where Fo=1000 and Fc=10 is very different 
from the FOM for a reflection with Fo=5000 and Fc=4010, even though the 
difference is the same.

Expanding on this:

1. The FOM actually depends more on the E values, i.e. reflections smaller than 
average get lower FOM values than ones bigger than average.  In the resolution 
bin from 5.12 to 5.64Å of 2vb1, the mean observed intensity is 20687 and the 
mean calculated intensity is 20022, which means that 
Eobs=Sqrt(145.83/20687)=0.084 and Ecalc=Sqrt(7264/20022)=0.602.  This 
reflection gets a low FOM because the product (0.050) is such a small number, 
not because the difference is big.

2. You have to consider the role of the model error in the difference, because 
for precisely-measured data most of the difference comes from model error.  In 
this resolution shell, the correlation coefficient between Iobs and Fcalc^2 is 
about 0.88, which means that sigmaA is about Sqrt(0.88) = 0.94.  The variance 
of both the real and imaginary components of Ec (as an estimate of the phased 
true E) will be (1-0.94^2)/2 = 0.058, so the standard deviations of the real 
and imaginary components of Ec will be about 0.24.  In that context, the 
difference between Eobs and Ecalc is nothing like a 2000-sigma outlier.

Looking at this another way, the reason why the FOM is low for this reflection 
is that the conditional probability distribution of Eo given Ec has significant 
values on the other side of the origin of the complex plane. That means that 
the *phase* of the complex Eo is very uncertain.  The figures in this web page 
(https://www-structmed.cimr.cam.ac.uk/Course/Statistics/statistics.html)
 should help to explain that idea.

Best wishes,

Randy


On 16 Oct 2019, at 16:02, James Holton 
mailto:jmhol...@lbl.gov>> wrote:


All very true Randy,

But nevertheless every hkl has an FOM assigned to it, and that is used to 
calculate the map.  Statistical distribution or not, the trend is that hkls 
with big amplitude differences get smaller FOMs, so that means large 
model-to-data discrepancies are down-weighted.  I wonder sometimes at what 
point this becomes a self-fulfilling prophecy?  If you look in detail and the 
Fo-Fc differences in pretty much any refined structure in the PDB you will find 
huge outliers.  Some are hundreds of sigmas, and they can go in either 
direction.

Take for example reflection -5,2,2 in the highest-resolution lysozyme structure 
in the PDB: 2vb1.  Iobs(-5,2,2) was recorded as 145.83 ± 3.62 (at 5.4 Ang) with 
Fcalc^2(-5,2,2) = 7264.  A 2000-sigma outlier!  What are the odds?   On the 
other hand, Iobs(4,-6,2) = 1611.21 ± 30.67 vs Fcalc^2(4,-6,2) = 73, which is in 
the opposite direction.  One can always suppose "experimental errors", but ZD 
sent me these images and I have looked at all the spots involved in these hkls. 
 I don't see anything wrong with any of them.  The average multiplicity of this 
data set was 7.1 and involved 3 different kappa angles, so I don't think these 
are "zingers" or other weird measurement problems.

I'm not just picking on 2vb1 here.  EVERY PDB entry has this problem.  Not sure 
where it comes from, but the FOM assigned to these huge differences is always 
small, so whatever is causing them won't show up in an FOM-weighted map.

Is there a way to "change up" the statistical distribution that assigns FOMs to 
hkls?  Or are we stuck with this systematic error?

-James Holton
MAD Scientist
On 10/4/2019 9:31 AM, Randy Read wrote:
Hi James,

I'm sure you realise this, but it's important for other readers to remember 
that the FOM is a statistical quantity: we have a probability distribution for 
the true phase, we pick one phase (the "centroid" phase that should minimise 
the RMS error in the density map), and then the FOM is the expected value of 
the phase error, obtained by taking the cosines of all possible phase 
differences and weighting by the probability of that phase difference.  Because 
it's a statistical quantity from a random distribution, you really can't expect 
this to agree reflection by reflection!  It's a good start to see that the 
overall values are good, but if you want to look more closely you have to look 
a groups of reflections, e.g. 

Re: [ccp4bb] ITC question -dimer vs monomer

[Keller, Jacob]

I don't understand what you are trying to do-are you trying to show, by the 
difference in ITC response, that the predictions you made about the 
oligomerization are true?

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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From: CCP4 bulletin board  On Behalf Of Bernhard Rupp
Sent: Thursday, October 3, 2019 11:06 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] ITC question -dimer vs monomer

Hi Fellows,

please let me ask the respective experts an ITC question: I have 2 proteins, 
stable and dialyzed in identical buffer.
A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer 
will form.

Intuitively, it should make a difference whether I titrate the dimer with the 
monomer or vice versa.
In the first case, a momomer would initially meet a lot of free dimers, and I 
would expect that randomly, a AB2 complex
is more likely to form than a A2B2 (let's disregard any more complex 
colligative/cooperative effects).

If I drip the dimer into the monomer pool, it is quite likely that the B dimer 
meets 2 free As, and I get right away a higher population of A2B2s.

Maybe at dilutions of ITC and with sufficient equilibration that is not an 
issue at all (again, absent any cooperative effects that might alter the first 
Kd vs. the second, despite the sites on the dimer are at least initially 
equivalent).

Can someone guide me towards literature about this or perhaps share some 
first-hand experience?

Many thanks, BR

--
Bernhard Rupp
http://www.hofkristallamt.org/
b...@hofkristallamt.org
+1 925 209 7429
+43 676 571 0536
--
Many plausible ideas vanish
at the presence of thought
--




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Re: [ccp4bb] SeMet data

[Keller, Jacob]

Why do you think you are rejecting anomalous data? What do the normal 
tell-tales reveal, like anom CC?

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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-Original Message-
From: CCP4 bulletin board  On Behalf Of L. Doyle
Sent: Monday, August 26, 2019 6:45 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] SeMet data

I have some Seleno-Methionine protein crystals (12 SeMet of 211 amino acids, 
incorporation verified by Mass Spec). I've already collected several datasets 
(ALS BL5.0.2) but I seem to be losing (rejecting?) a lot of anomalous signal 
during data processing. I'm most familiar with HKL2000, but I have tried XDS 
and DIALS auto-processing. Here is a scan: 
https://urldefense.proofpoint.com/v2/url?u=https-3A__ibb.co_LZqm33p=DwIFaQ=LU6cRtx0xgB8s29tIz9Olw=eLCg9eJ4Rs_LnxfUWsp7FSxhIEcZYmTSU4Uyq1bRYPI=IY2dWiiG1i1JgjTXPAPNw3-KHrUr53w37DPc7mNTmQk=eA1o_iIowAlvk0KtH7k81LUBeSWfBfCsL7yvZK7KWvM=
  and here is an example of a frame: 
https://urldefense.proofpoint.com/v2/url?u=https-3A__ibb.co_gR3ZR47=DwIFaQ=LU6cRtx0xgB8s29tIz9Olw=eLCg9eJ4Rs_LnxfUWsp7FSxhIEcZYmTSU4Uyq1bRYPI=IY2dWiiG1i1JgjTXPAPNw3-KHrUr53w37DPc7mNTmQk=0Vn8zTPkITbHZuSG2lgAXAZZ5rpwTmNDfRvUjYPvYXs=
 . Each frame is 0.25° and I'm using inverse beam with wedge size 1°. Maybe I 
need to adjust my collection strategy? All previous datasets have been in space 
group P 21 with dimensions of approx. 24.5Å, 85Å, 40Å, 90°, 96.5°, 90°. I'm 
sure there are additional things I can be doing in HKL but I've run out of 
ideas. Any advice or recommendations would be appreciated. Please let me know 
if you need additional information. 

Thank you,
Lindsey



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[ccp4bb] Resonant Scattering Directionality

[Keller, Jacob]

Dear Crystallographers,

It seems to be a usual assumption that anomalous scattering is essentially 
angularly-independent, e.g.:

http://pd.chem.ucl.ac.uk/pdnn/diff1/anomscat.htm

But why the can't we see anomalous-only spots at e.g. 1 Ang resolution in a 2 
Ang data set?

This actually has some repercussions for a non-x-ray (but still resonance) 
idea, so it would be helpful to know...

All the best,

Jacob

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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Re: [ccp4bb] challenges in structural biology

[Keller, Jacob]

What about developing a theory of how crystallization happens, i.e., what does 
the microscopic “picture” look like when crystals are forming, then predicting 
based on that picture? I remember looking into these things about ten years 
ago, and there were some cool things being done with various scattering methods 
and with AFM, but am not sure now what is the state of that art.

It would seem to me that crystallization is the search for intermolecular 
docking sites of sufficiently good (albeit presumably weak) affinity and 
consistent with the formation of a 3D lattice. I wonder what the affinity of 
these sites is, actually—I guess somewhere in the micromolar range, based on 
usual protein concentrations under crystallization conditions (10 mg/ml of a 40 
kD protein is 250 uM).

Presumably the various docking sites would change affinity based on the 
crystallization conditions, which would explain why some crystallization 
conditions work, others don’t?

Maybe a systematic look at all crystallization contacts in the PDB might yield 
some insight into crystallization? Maybe it’s already been done?

JPK



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Re: [ccp4bb] Density questionable?

[Keller, Jacob]

This is the old question of what a structural model represents. One perspective 
is that it represents the things one is certain about above some threshold, 
from the crystallographic data and maps alone. The other perspective is that it 
represents the most likely guess of what is actually there. I prefer the second 
"most likely" approach since it incorporates more information, namely the 
knowledge of what's in the solutions, the way proteins pack, the protein's 
sequence, etc. And everyone agrees to the use of such priors when it comes to 
bond lengths and similar. I guess it boils down to the weighting of prior 
information.

A problem arises, however, when users from one camp look at models from the 
other camp, and I do not really have a solution to this, especially as the two 
camps don't tend to see both sides of the question. Could there be two versions 
of each model: a "robustly-observed" and a "most-likely" version?

Maybe this debate could also be raised at the Holton conference?

Jacob

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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-Original Message-
From: CCP4 bulletin board  On Behalf Of Peer Mittl
Sent: Monday, July 22, 2019 6:05 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Density questionable?

Dear Colleagues,

We are working on a structure where the density for a whole protein chain (>200 
aa) is questionable, since the B-factors exceed 200 Å2 (2.3 Ang resolution). 
However, the initial difference density map and the feature enhanced map 
(normal 2fo-fc map to a minor extend) support the presence of this chain. 
Putting the chain seems equally wrong as not putting it. Putting it reduces 
Rfree by 0.3%. As a conservative researcher I feel tempted to deposit the 
structure without this highly mobile/weakly occupied chain, but other 
researchers may say "he has missed something". Handling this chain like a 
weakly occupied water is probably wrong, but what is the optimal/correct way? 
Is there a general opinion on how the escape this dilemma?

All the best,
Peer



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Re: [ccp4bb] challenges in structural biology

[Keller, Jacob]

I like both of these points! I would comment/add the following:

1) What are the tools we can use for metals in structural biology? Note, I am 
biased here.

-Including validation of solute ions like Na/K/Cl etc
-Some metric of identity confidence?

2) Micro electron diffraction methods - ability to use on small crystals (which 
speaks to Tom's point) and a potential bridge between cryo-EM and X-ray 
diffraction techniques?

-What about an XFEL equivalent for electrons with their greater scattering? I 
looked into this a while back, and there seemed to be some pretty tough things 
about it, but is there hope? Maybe any purifiable protein could be solved in 
this way? Would it have to be done by diffraction, or could focusing work 
somehow?

JPK




Sarah EJ Bowman, PhD

Associate Research Scientist, Hauptman-Woodward Medical Research Institute 
Director, High-Throughput Crystallization Screening Center

https://urldefense.proofpoint.com/v2/url?u=http-3A__www.getacrystal.org=DwIF-g=LU6cRtx0xgB8s29tIz9Olw=eLCg9eJ4Rs_LnxfUWsp7FSxhIEcZYmTSU4Uyq1bRYPI=HLRpGMGxTyZSjqW-aeakIElqVXKo3CvO_O36dUmNgKY=7uXULBobMzHYWnHc-YO4fFjHH7PbGQXufIRarsr4dQE=
 

sbow...@hwi.buffalo.edu


From: CCP4 bulletin board  on behalf of Peat, Tom 
(Manufacturing, Parkville) 
Sent: Monday, July 15, 2019 8:07 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: challenges in structural biology

Hello Tim,

I'm not sure this question is specific to crystallography- I believe the same 
can be asked of any experiment in any field?
And if one wants to get into true costs- was it worth it to build the Large 
Hadron Collider to statistically prove that the Higgs boson exists?
I'm guessing it was worth it to the folks that got their name on the paper...
Cheers, tom

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tim Grüne
Sent: Tuesday, 16 July 2019 6:09 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] challenges in structural biology

Dear James,

10) are the biological questions that you can answer with a (crystal) structure 
sufficiently relevant to justify the resources?

Best,
Tim



Am 15.07.2019 21:44, schrieb Holton, James M:
> Hello folks,
>
> I have the distinct honor of chairing the next Gordon Research 
> Conference on Diffraction Methods in Structural Biology (July 26-31 
> 2020).  This meeting will focus on the biggest challenges currently 
> faced by structural biologists, and I mean actual real-world 
> challenges.  As much as possible, these challenges will take the form 
> of friendly competitions with defined parameters, data, a scoring 
> system, and "winners", to be established along with other unpublished 
> results only at the meeting, as is tradition at GRCs.
>
> But what are the principle challenges in biological structure 
> determination today?  I of course have my own ideas, but I feel like 
> I'm forgetting something.  Obvious choices are:
> 1) getting crystals to diffract better
> 2) building models into low-resolution maps (after failing at #1)
> 3) telling if a ligand is really there or not
> 4) the phase problem (dealing with weak signal, twinning and
> pseudotranslation)
> 5) what does "resolution" really mean?
> 6) why are macromolecular R factors so much higher than small-molecule 
> ones?
> 7) what is the best way to process serial crystallography data?
> 8) how should one deal with non-isomorphism in multi-crystal methods?
> 9) what is the "structure" of something that won't sit still?
>
> What am I missing?  Is industry facing different problems than 
> academics?  Are there specific challenges facing electron-based 
> techniques?  If so, could the combined strength of all the world's 
> methods developers solve them?  I'm interested in hearing the voice of 
> this community.  On or off-list is fine.
>
> -James Holton
> MAD Scientist
>
>
> ##
> ##
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.u
> k_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1=DwIF-g=LU6cRtx0xgB8
> s29tIz9Olw=eLCg9eJ4Rs_LnxfUWsp7FSxhIEcZYmTSU4Uyq1bRYPI=HLRpGMGxTyZ
> SjqW-aeakIElqVXKo3CvO_O36dUmNgKY=Q23TMeWBhuH2_xEAYk3-wjMz65NwxYH-I3E
> fSr0dcAs=

--
--
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] Fo-Fc density close to cysteine residue

[Keller, Jacob]

How about radiation-damaged/smashed Sulphur? You could test this by refining 
occupancy of the cys S.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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From: CCP4 bulletin board  On Behalf Of Lumbini Yadav
Sent: Wednesday, July 10, 2019 7:12 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Fo-Fc density close to cysteine residue

Thank you for the valuable input

On Wed, Jul 10, 2019 at 4:25 PM Eleanor Dodson 
mailto:eleanor.dod...@york.ac.uk>> wrote:
Well - that more or less proves the residue is a CYS - there is a peak in the 
PHAN map right on the S.
And that the extra density does not contain an anomalous scatterer so is 
probably not a metal or a sulphate or...

But that still doesnt explain WHAT it is . Sorry not to be more help..

Eleanor

On Wed, 10 Jul 2019 at 11:32, Lumbini Yadav 
mailto:lumbin...@gmail.com>> wrote:
No I am using ccp4i. I tried doing SAD refinement in refmac and the output 
image is attached below .
I do not seen density near cysteine that was visible in Fo-Fc map

On Wed, Jul 10, 2019 at 3:40 PM Eleanor Dodson 
mailto:eleanor.dod...@york.ac.uk>> wrote:
The key word for refmac is ANOM MAPONLY
Are you using GUI2?
Eleanor

On Wed, 10 Jul 2019 at 09:32, Lumbini Yadav 
mailto:lumbin...@gmail.com>> wrote:
I have soaked my crystals in  sodium dithionite a reducing agent. I have not 
done mass spec but sequence is confirmed

On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel 
mailto:dbon...@som.umaryland.edu>> wrote:
Have you mass-speced the protein before crystallization to make sure it wasn’t 
derivatized during expression and/or purification, or compared the mass spec of 
the crystals verses purified protein? Any fancy reagents or other reductants 
used during purification?
What about S-Acetyl-cysteine (3-letter code: SCY).

Best,

Dan

Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Lumbini Yadav
Sent: Tuesday, July 09, 2019 5:22 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Fo-Fc density close to cysteine residue

Dear all,

We have found a huge Fo-Fc density close to cysteine residue (see attached 
image) in the structure with resolution of 1.2A. In the crystallization 
condition, we have PEG 3350, Potassium phosphate monobasic, glycerol and 
protein was in Tris and NaCl. Before freezing the crystals were soaked in 
mother liquor containing sodium dithionite.

I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD, 
CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in all the 
screenings we do see some part of Fo-Fc density unaddressed at 3 sigma.

Does anyone have an idea about what this density could be? Covalent 
modification?

Thanks.


Kind regards,
Lumbini



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Re: [ccp4bb] DNA or RNA

[Keller, Jacob]

Hi Reza,

What about seeing whether RNAse and DNAse incubations kill the complex?

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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From: CCP4 bulletin board  On Behalf Of Reza Khayat
Sent: Thursday, June 20, 2019 6:16 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] DNA or RNA


Hi,



Sorry for the non-crystallography question. We have a protein complex with 
nucleic acid and would like to know if the nucleic acid is DNA or RNA. Is there 
a sensitive method for doing this where we don't need buckets of the sample? 
Thanks.



Best wishes,
Reza
​
Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031



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Re: [ccp4bb] beryllium chloride

[Keller, Jacob]

ct: Re: [ccp4bb] beryllium chloride

No, that should read



Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu<mailto:diana.tomch...@utsouthwestern.edu>
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Apr 1, 2019, at 5:54 PM, Keller, Jacob 
mailto:kell...@janelia.hhmi.org>> wrote:

Is that 4+ an April fools’ joke? Pretty crazy if not…can’t think of another ion 
with such a charge, well except things like DNA and proteins, but not single 
atoms.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Aaron Finke
Sent: Monday, April 1, 2019 6:45 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] beryllium chloride

American Elements sells BeCl2 but you’d have to check with them on what scale 
they sell it at. They tend to do custom manufacturing.

https://www.americanelements.com/beryllium-chloride-7787-47-5

BeCl2 dissociates in aqueous solution to form Be(H2O)4+ 2Cl-.


Aaron
--
Aaron Finke
Staff Scientist, MacCHESS
Cornell University
e-mail: af...@cornell.edu<mailto:af...@cornell.edu>

On Apr 1, 2019, at 17:07, Alexandra Deaconescu 
mailto:alexandra_deacone...@brown.edu>> wrote:
Hello,

Is anyone aware of a company that sells Beryllium chloride in the US? Sigma 
does not carry it any longer, and a quick Google search failed to reveal 
alternatives.

Thank you very much,

Alexandra


--
Alexandra Deaconescu, B.E., Ph.D.
Assistant Professor
Brown University

Office: (401) 863-3215
Wet Lab: (401) 863-6729
Computational Lab: (401) 863-7031

For Mail:
Laboratories of Molecular Medicine
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Providence, RI 02903

For Courier:
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Brown University
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Website: www.deaconesculab.com<http://www.deaconesculab.com/>

Admin
Ms. Christina Fournier
Email: christina_fournier[at]brown.edu<http://brown.edu/>
Mailing Address:
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Confidentiality Notice:
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UT Southwestern

Medical Center


The future of medicine, today.



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Re: [ccp4bb] beryllium chloride

[Keller, Jacob]

Is that 4+ an April fools’ joke? Pretty crazy if not…can’t think of another ion 
with such a charge, well except things like DNA and proteins, but not single 
atoms.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
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received this message by mistake, please reply to this message and follow with 
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From: CCP4 bulletin board  On Behalf Of Aaron Finke
Sent: Monday, April 1, 2019 6:45 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] beryllium chloride

American Elements sells BeCl2 but you’d have to check with them on what scale 
they sell it at. They tend to do custom manufacturing.

https://www.americanelements.com/beryllium-chloride-7787-47-5

BeCl2 dissociates in aqueous solution to form Be(H2O)4+ 2Cl-.

Aaron
--
Aaron Finke
Staff Scientist, MacCHESS
Cornell University
e-mail: af...@cornell.edu

On Apr 1, 2019, at 17:07, Alexandra Deaconescu 
mailto:alexandra_deacone...@brown.edu>> wrote:
Hello,

Is anyone aware of a company that sells Beryllium chloride in the US? Sigma 
does not carry it any longer, and a quick Google search failed to reveal 
alternatives.

Thank you very much,

Alexandra


--
Alexandra Deaconescu, B.E., Ph.D.
Assistant Professor
Brown University

Office: (401) 863-3215
Wet Lab: (401) 863-6729
Computational Lab: (401) 863-7031

For Mail:
Laboratories of Molecular Medicine
70 Ship St. GE-4
Providence, RI 02903

For Courier:
Laboratories of Molecular Medicine
Brown University
70 Ship St., Chestnut St. Loading Dock
Providence, RI 02903

Website: www.deaconesculab.com

Admin
Ms. Christina Fournier
Email: christina_fournier[at]brown.edu
Mailing Address:
Box G-E, Brown University,
Providence, RI 02912-G
Telephone: 401-863-2782

Confidentiality Notice:
This e-mail message, including any attachments, is for the sole use of the 
intended recipient(s) and may contain confidential, proprietary and privileged 
information. Any unauthorized review, use, disclosure or distribution is 
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immediately and destroy or permanently delete all copies of the original 
message.



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Re: [ccp4bb] Interesting pattern on a crystallization drop

[Keller, Jacob]

It's a crystal with a very large lattice.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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From: CCP4 bulletin board  On Behalf Of Beatriz Gomes 
Guimaraes
Sent: Wednesday, March 27, 2019 2:44 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Interesting pattern on a crystallization drop


Dear all,



I would like to share with you a surprising pattern I found when examining some 
crystallization plates (attached figures).



It is less obvious looking the photos, but apparently the "lines" are formed by 
precipitated protein and there are some "bubbles" with small drops inside. I 
wish they were microcrystals but I do not think this is the case.

I was suprised by the symmetry !



And it is not completely random because for the same condition the difference 
between the two drops are : protein alone ("hexagon") and protein + ligand 
("rhombus")


crystallization condition is:

0.01 M Cobalt(II) chloride hexahydrate

0.1 M Tris pH 8.5

20% w/v Polyvinylpyrrolidone K 15



Have you seen anything similar before?



Thank you for your comments!

Beatriz




--
Beatriz Guimarães
Laboratory of Structural Biology and Protein Engineering
Instituto Carlos Chagas - ICC / FIOCRUZ Paraná
Rua Prof. Algacyr Munhoz Mader, 3775   Bloco C
CIC 81350-010
Curitiba - PR, Brasil
Tel.:+55(41)3316-3225/2104-3438



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[ccp4bb] KCl in SDS-PAGE workarounds?

[Keller, Jacob]

Dear crystallographers,

It has been my experience that KCl does nasty things when loading SDS-PAGE 
gels. Does anyone have an easy workaround, perhaps TCA precipitation? Ideally 
this would be something nicely quantitative yet quick and easy….

Any suggestions appreciated.

All the best,

Jacob Keller

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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From: CCP4 bulletin board  On Behalf Of Artem Evdokimov
Sent: Saturday, March 2, 2019 8:32 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ionic strength for extraction buffer of membrane proteins

Hi Alex,

In my experience you just have to experiment with these parameters (in however 
of a limited space that is afforded by your experimental set-up). If you have 
the right tools you can slog through semifactorial or DoE-based scouting of 
various parameters with relative ease. These parameters typically include pH, 
salinity, detergent(s), and other variables (e.g. the nature of the buffer and 
salt(s) - many cases exist where the 'standard' choices do not work well).

Literature-based analogies do help on occasion. For example, membrane-spanning 
p450-type proteins often prefer high salt, and often would do better in sodium 
acetate as opposed to chloride. I've worked with ion channels that preferred 3M 
NaCl, or 2M MgCl2 and other fairly weird conditions...

Artem
- Cosmic Cats approve of this message


On Fri, Mar 1, 2019 at 7:27 AM Alex Perálvarez Marín 
mailto:aperalva...@gmail.com>> wrote:
Dear all,

any reference as a guide for selecting the appropriate salt and
concentration for membrane proteins extraction buffer?

Best,

Alex

--
Alex Perálvarez-Marín, Ph.D.
Centre d'Estudis en Biofísica / Unitat de Biofísica
Edifici M
Universitat Autònoma de Barcelona
08193 Cerdanyola del Vallés
Barcelona
Spain
Phone: +34 93 581 4504
FAX:  +34 93 581 1907
e-mail: aperalva...@gmail.com<mailto:aperalva...@gmail.com>
LinkedIn: 
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Re: [ccp4bb] SO4 or PO4

[Keller, Jacob]

I see what you’re saying—you’re probably right.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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From: Goldman, Adrian 
Sent: Sunday, February 17, 2019 10:48 PM
To: Keller, Jacob 
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] SO4 or PO4

The total number of electrons in the two systems is identical, so you are 
looking for that extra fraction located closer to S than to P due to the change 
in atomic number.
I haven’t checked but I doubt if this is easy even in ccdb, and I would be 
stunned if it is possible in the pdb. There might be a small chance by 
classifying h-bonding and implied lone pairs and angles, but I think even this 
can be difficult.
Sent from my iPhone

On 18 Feb 2019, at 03:39, Keller, Jacob 
mailto:kell...@janelia.hhmi.org>> wrote:
Let me clarify. I was thinking not of the anomalous but of the regular 2FO-FC 
density, which should be easy enough to quantify and compare to the relative 
heights in thousands of high-resolution PO4’s and S04’s in the PDB. One could 
even get a pretty good estimate of the likelihood the unknown’s being each. I 
would think this could really nail it.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

From: Eleanor Dodson 
mailto:eleanor.dod...@york.ac.uk>>
Sent: Sunday, February 17, 2019 6:12 PM
To: Keller, Jacob mailto:kell...@janelia.hhmi.org>>
Cc: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] SO4 or PO4

Well - I try to quantify the relative anom peak heights by checking those over  
MET or CYS S sites with similar B values v the disputed one.. The expected 
difference between P and S isnt very big, but it might give you a 
crystallographic clue.

Eleanor

On Sun, 17 Feb 2019 at 17:31, Keller, Jacob 
mailto:kell...@janelia.hhmi.org>> wrote:
Shouldn’t it be possible to look at the ratio of peak heights of O’s versus S 
or P to figure out which is more likely? The must be thousands of examples in 
the pdb with which to determine the ratio, even if one restricts the analysis 
to “high resolution” structures.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
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From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Yu Qiu
Sent: Sunday, February 17, 2019 9:11 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] SO4 or PO4

Hi Shijun,

I had a similar issue a while ago that a acetylated lysine binding site was 
occupied by acetate from buffer. In this case, I would suggest trying 
crystallize in a conditions without SO4, e.g. using NH4Cl instead, if it is 
reproducible.

Best,
Yu

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ???
Sent: Sunday, February 17, 2019 8:57 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [EXTERNAL] Re: [ccp4bb] SO4 or PO4


Dear all

Thanks for all your suggestions. PO4 is from my Ligand  PI3P, but I cannot see 
the electron density of the PI3P tail, and the binding site is exactly there. 
While my crystal buffer salt is 50mM (NH4)2SO4, so I am afraid the SO4 occupied 
the PO4 position.BTW, I can see the other SO4 electron density in the other 
places, but is small than 

Re: [ccp4bb] SO4 or PO4

[Keller, Jacob]

Let me clarify. I was thinking not of the anomalous but of the regular 2FO-FC 
density, which should be easy enough to quantify and compare to the relative 
heights in thousands of high-resolution PO4’s and S04’s in the PDB. One could 
even get a pretty good estimate of the likelihood the unknown’s being each. I 
would think this could really nail it.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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From: Eleanor Dodson 
Sent: Sunday, February 17, 2019 6:12 PM
To: Keller, Jacob 
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] SO4 or PO4

Well - I try to quantify the relative anom peak heights by checking those over  
MET or CYS S sites with similar B values v the disputed one.. The expected 
difference between P and S isnt very big, but it might give you a 
crystallographic clue.

Eleanor

On Sun, 17 Feb 2019 at 17:31, Keller, Jacob 
mailto:kell...@janelia.hhmi.org>> wrote:
Shouldn’t it be possible to look at the ratio of peak heights of O’s versus S 
or P to figure out which is more likely? The must be thousands of examples in 
the pdb with which to determine the ratio, even if one restricts the analysis 
to “high resolution” structures.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Yu Qiu
Sent: Sunday, February 17, 2019 9:11 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] SO4 or PO4

Hi Shijun,

I had a similar issue a while ago that a acetylated lysine binding site was 
occupied by acetate from buffer. In this case, I would suggest trying 
crystallize in a conditions without SO4, e.g. using NH4Cl instead, if it is 
reproducible.

Best,
Yu

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ???
Sent: Sunday, February 17, 2019 8:57 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [EXTERNAL] Re: [ccp4bb] SO4 or PO4


Dear all

Thanks for all your suggestions. PO4 is from my Ligand  PI3P, but I cannot see 
the electron density of the PI3P tail, and the binding site is exactly there. 
While my crystal buffer salt is 50mM (NH4)2SO4, so I am afraid the SO4 occupied 
the PO4 position.BTW, I can see the other SO4 electron density in the other 
places, but is small than this one, and the diffraction resolution is 1.6A.

Best Regards

Shijun

-Original Messages-
From:"Roger Rowlett" mailto:rrowl...@colgate.edu>>
Sent Time:2019-02-17 00:47:55 (Sunday)
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Cc:
Subject: Re: [ccp4bb] SO4 or PO4
Two things to look at that could provide a clue:

Examine the anomalous map for some density over the central atom. Sulfur will 
often, but not always have significant anomalous density depending on the 
wavelength and quality of data set.

Phosphate is normally HPO4= or H2PO4-. Look for phosphate donor to acceptor 
hydrogen bonding contacts. Sulfate rarely has donor to acceptor hydrogen 
bonding contacts, as it is SO4= at any reasonable pH.

Roger Rowlett

On Sat, Feb 16, 2019, 4:06 AM 张士军 
<21620150150...@stu.xmu.edu.cn<mailto:21620150150...@stu.xmu.edu.cn> wrote:

Dear all

I have got a crystal grown at the condition both have ion of SO4 and PO4, and 
the diffraction resolution is very well, but the problem is coming: how to tell 
which is which just from electron density? I think they are exactly same. 
Thanks a lot !!!

Beat Regards

Shijun



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Re: [ccp4bb] SO4 or PO4

[Keller, Jacob]

Shouldn’t it be possible to look at the ratio of peak heights of O’s versus S 
or P to figure out which is more likely? The must be thousands of examples in 
the pdb with which to determine the ratio, even if one restricts the analysis 
to “high resolution” structures.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
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message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

From: CCP4 bulletin board  On Behalf Of Yu Qiu
Sent: Sunday, February 17, 2019 9:11 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] SO4 or PO4

Hi Shijun,

I had a similar issue a while ago that a acetylated lysine binding site was 
occupied by acetate from buffer. In this case, I would suggest trying 
crystallize in a conditions without SO4, e.g. using NH4Cl instead, if it is 
reproducible.

Best,
Yu

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ???
Sent: Sunday, February 17, 2019 8:57 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] Re: [ccp4bb] SO4 or PO4


Dear all

Thanks for all your suggestions. PO4 is from my Ligand  PI3P, but I cannot see 
the electron density of the PI3P tail, and the binding site is exactly there. 
While my crystal buffer salt is 50mM (NH4)2SO4, so I am afraid the SO4 occupied 
the PO4 position.BTW, I can see the other SO4 electron density in the other 
places, but is small than this one, and the diffraction resolution is 1.6A.

Best Regards

Shijun


-Original Messages-
From:"Roger Rowlett" mailto:rrowl...@colgate.edu>>
Sent Time:2019-02-17 00:47:55 (Sunday)
To: CCP4BB@JISCMAIL.AC.UK
Cc:
Subject: Re: [ccp4bb] SO4 or PO4
Two things to look at that could provide a clue:

Examine the anomalous map for some density over the central atom. Sulfur will 
often, but not always have significant anomalous density depending on the 
wavelength and quality of data set.

Phosphate is normally HPO4= or H2PO4-. Look for phosphate donor to acceptor 
hydrogen bonding contacts. Sulfate rarely has donor to acceptor hydrogen 
bonding contacts, as it is SO4= at any reasonable pH.

Roger Rowlett

On Sat, Feb 16, 2019, 4:06 AM 张士军 
<21620150150...@stu.xmu.edu.cn wrote:

Dear all

I have got a crystal grown at the condition both have ion of SO4 and PO4, and 
the diffraction resolution is very well, but the problem is coming: how to tell 
which is which just from electron density? I think they are exactly same. 
Thanks a lot !!!

Beat Regards

Shijun



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Re: [ccp4bb] hybrid photon counter in the home lab

[Keller, Jacob]

Wow, that’s pretty impressive. I hadn’t realized the high levels of DQE you had 
achieved. If you could make one for visible photons, you’d make a lot of 
microscopists really happy.

I also had not realized that QE of other detectors is intensity dependent—seems 
counter-intuitive.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

From: Marcus Winter 
Sent: Wednesday, January 16, 2019 11:25 AM
To: Keller, Jacob ; CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] hybrid photon counter in the home lab



Dear Jacob,


Thank you for your reply.  You’re correct, of course.  As shown below and in 
the attachment, with Si sensors and at a photon energy of the 8 keV, the DQE of 
both the HyPix-6000HE and Pilatus3 R 200K HPC detectors are both considerably 
above 90%.


[cid:image001.png@01D4AD9E.60DA3C20]


Most importantly, this high DQE is maintained throughout the operating regime: 
from the highest to the lowest count rates.  As can be seen, this is in 
contrast to the highest performing CCD detector (the Atlas S2) and CMOS type 
detectors.  The high DQE even at the lowest count rates / lowest reflection 
intensities is the reason for the best quality highest resolution data to be 
collectable using the HPC technology detectors.


Many Thanks, Yours sincerely,

Marcus Winter
Rigaku.


From: Keller, Jacob [mailto:kell...@janelia.hhmi.org]
Sent: Wednesday, January 16, 2019 2:13 PM
To: Marcus Winter mailto:marcus.win...@rigaku.com>>; 
CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: RE: [ccp4bb] hybrid photon counter in the home lab

Doesn’t the phrase “each-and-every single photon counting capability” imply 
that quantum efficiency is 100%? I don’t think this is possible—what is the 
quantum efficiency of these detectors?

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Marcus Winter
Sent: Wednesday, January 16, 2019 3:28 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] hybrid photon counter in the home lab



Dear Wolfram,


For the past several years, Rigaku has been installing only HPC (Hybrid Photon 
Counting) X-ray area detector -based diffractometer systems: in conjunction 
with microfocus rotating anode, microfocus sealed-tube and other X-ray sources. 
 (Most recently, we have been deploying the Rigaku HyPix-6000HE HPC detector.)  
The advantages of the HPC technology over any of the phosphor-based detector 
types (CCD, CMOS and CPAD, etc…) are notable: most particularly the discrete 
each-and-every single photon counting capability, extremely high dynamic range, 
small pixel size and the single pixel point function and very rapid read-out.  
With these advantages, significantly higher resolution data can be achieved, - 
and more speedily, compared to the other detector types.  (We have a number of 
established ‘application note’ examples.)  In order to prove the advantages for 
your purposes, the best might be to collect data from a range of your own 
crystal samples.  – There is an open invitation for any seriously interested 
groups to collect data from their crystals in our applications labs.  Please 
contact your local good Rigaku salesperson.

Many Thanks, Yours sincerely,

Marcus Winter
Rigaku.


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of wtempel
Sent: Tuesday, January 15, 2019 6:20 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] hybrid photon counter in the home lab


Hi,
I would value your opinions in this equipment-related question.
Allé et al have compared detector types with a molybdemon source for a small 
molecule application<https://dx.doi.org/10.1088/0031-8949/91/6/063001>. Are 
there simil

Re: [ccp4bb] hybrid photon counter in the home lab

[Keller, Jacob]

Doesn’t the phrase “each-and-every single photon counting capability” imply 
that quantum efficiency is 100%? I don’t think this is possible—what is the 
quantum efficiency of these detectors?

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

From: CCP4 bulletin board  On Behalf Of Marcus Winter
Sent: Wednesday, January 16, 2019 3:28 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] hybrid photon counter in the home lab



Dear Wolfram,


For the past several years, Rigaku has been installing only HPC (Hybrid Photon 
Counting) X-ray area detector -based diffractometer systems: in conjunction 
with microfocus rotating anode, microfocus sealed-tube and other X-ray sources. 
 (Most recently, we have been deploying the Rigaku HyPix-6000HE HPC detector.)  
The advantages of the HPC technology over any of the phosphor-based detector 
types (CCD, CMOS and CPAD, etc…) are notable: most particularly the discrete 
each-and-every single photon counting capability, extremely high dynamic range, 
small pixel size and the single pixel point function and very rapid read-out.  
With these advantages, significantly higher resolution data can be achieved, - 
and more speedily, compared to the other detector types.  (We have a number of 
established ‘application note’ examples.)  In order to prove the advantages for 
your purposes, the best might be to collect data from a range of your own 
crystal samples.  – There is an open invitation for any seriously interested 
groups to collect data from their crystals in our applications labs.  Please 
contact your local good Rigaku salesperson.

Many Thanks, Yours sincerely,

Marcus Winter
Rigaku.


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of wtempel
Sent: Tuesday, January 15, 2019 6:20 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] hybrid photon counter in the home lab


Hi,
I would value your opinions in this equipment-related question.
Allé et al have compared detector types with a molybdemon source for a small 
molecule application. Are 
there similar published comparisons for protein crystallography? What benefits 
can I expect from replacing a CCD detector with a hybrid photon counter at an 
energy of 8 keV and in the absence of the flux that a modern synchrotron 
provides?

Thank you in advance.
Wolfram Tempel



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Re: [ccp4bb] translational NCS & twinning

[Keller, Jacob]

>>I feel you went ahead with right strategy.

I agree with this part regarding lowering symmetry.

>>For 2.1 A datasrt, the appropriate drop in Rfree/ Rwork is a strong 
>>indicator, i believe.

This is not true—even non-twinned data will improve in R values with twinning 
operators added as parameters. And twin-refined structures always have lower R 
values.
JPK


On Thu 10 Jan, 2019, 3:52 PM Donghyuk Shin 
mailto:sdh...@gmail.com> wrote:
Dear all,

I am having tough time with my Xtal data sets those seem to be twinned or have 
translational NCS, and it will be greatly appreciated if you can give me some 
advices or comments!

Data was initially processed with XDS and scaled with aimless without 
specifying certain space group (SG).
Aimless picked the P 63 2 2 for the best SG, but the xtriage indicates there is 
non-origin peak after patterson analysis. (attached log)
And, I could not get the proper MR-solution from this data sets.

Because I read that twinning and tNCS cannot be properly distinguished at high 
SG, I went down to subgroup either P32 or P6 assuming that there is twinning 
which make data set seems to have apparently high SG. (procedure was same 
XDS->aimless but I specified the SG to keep them)
Then, xtriage still indicates there is non-origin peak as before, but found 
twin laws for the data sets (attached log).
However, I still could not get the right MR-solution.
Then, I went even further down to P3 or C2, and xtriage found more twin laws 
which is make sense because of the lower SG. (attached log)
Again, I could not get the MR-solution.
For all the MR running above, I assumed that phaser(ccp4 module) automatically 
applied tNCS if they present. or should I have to tick on button in the expert 
parameters?

Then, I went back to the image and processed the datasets with mosflm by 
checking the indexed spots.
During this step, I played with the threshold for indexing to follow the strong 
spot for get correct SG.
I am not sure whether this is correct or not, but by putting high threshold for 
indexing (e.g. ~15) I could index the data with C2 which has half dimension for 
a,b axes (116.348,  67.218, 182.861,  90, 90, 90) to the original unit cell 
(232.533, 134.202, 182.67, 90, 90, 90).
With this, I could put 3 molecules in ASU by MR. During refinement, I felt that 
the R values were not dropping, and I applied twin refinement.
without twin refinement the R values were (0.39/0.44, work/free), and applying 
twin refinement gave me significantly better values (0.23/0.26).
Because there were 6 twin operators which may cause this huge R value drop, I 
speculate whether this is true or not.

Your comments will be greatly helpful!

With you all the best,
Donghyuk





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Re: [ccp4bb] 2019 New Year’s resolutions of a cryo-EM newbie

[Keller, Jacob]

6) I most sincerely hope that, if I stick to my five New Year’s resolutions 
and stop wasting the cryo-EM community’s time, my fellow Canadians will not 
extradite me to the fake-news country at our southern border.


I know this is a joke, but we in the USA are not a fake news people, and please 
don’t punish us for the sins of our leadership--it’s painful for many of us.

JPK



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Re: [ccp4bb] Experimental phasing vs molecular replacement

[Keller, Jacob]

>>That said, model phases are not so bad.  In fact, in all my experiments with 
>>fake data the model-phased 2mFo-DFc map always has the best correlation to 
>>the "true" map.  If you substitute the "true" phases and use the 2mFo-DFc 
>>coefficients you actually make things worse. Counter-intuitive, but true.

I don't understand what you mean by true and fake here--can you clarify? How 
are the true map and phases generated (from an original true model, I assume?), 
and how are the fake data generated? (Also from the true model?) I am wondering 
whether there is some circular reasoning?

JPK



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Re: [ccp4bb] Long term storage for raw images/ crystallographic data sets

[Keller, Jacob]

I saw explicitly that it is not limited to EU.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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-Original Message-
From: CCP4 bulletin board  On Behalf Of 
graeme.win...@diamond.ac.uk
Sent: Thursday, November 29, 2018 4:22 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Long term storage for raw images/ crystallographic data 
sets

Dear Tim,

I do not think Zenodo is limited to Europeans - at least I could not find this 
on their policy page:

http://about.zenodo.org/policies/

I know of plenty of uploads from Japan for example

Best wishes Graeme

On 29 Nov 2018, at 21:16, Tim Gruene 
mailto:tim.gru...@psi.ch>> wrote:

Dear Raquel,

when they are associated with a publication, you can publish them on 
data.sbgrid.org in the US or at 
zenodo.org in Europe.

Best regards,
Tim

On Thursday, November 29, 2018 9:54:02 PM CET Lieberman, Raquel L wrote:
Dear All,

How do your labs handle long-term raw data backups? My lab is maxing out our 
6TB RAID backup (with two off-site mirrors) so I am investigating our next long 
term solution. The vast majority of the data sets are published structures 
(i.e. processed data deposited in PDB) or redundant/unusable so immediate 
access is not anticipated, but the size of data sets is increasing quickly with 
time, so I am looking for a scalable-yet-affordable solution.

Would be grateful for input into various options, e.g. bigger HD/RAIDs, cloud 
backup, tape, anything else.

I will compile.

Thank you,

Raquel
--
Raquel L. Lieberman, Ph.D.
Professor
School of Chemistry and Biochemistry
Georgia Institute of Technology





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Tim Gruene
- persoenlich -
OSUA/204
Forschungsstrasse 111
CH-5232 Villigen PSI
phone: +41 (0)56 310 5297

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Re: [ccp4bb] VERY old mtz file..

[Keller, Jacob]

>>  ah, nostalgia

Ah, "mantissa!" Haven't heard "mantissa" in decades...

Is there such a thing as a "praying mantissa?" Seems like there could be a good 
geek joke about it.

JPK



> However all procedures I have seen use a division of 4, which is quite 
> puzzling to me. A real data file containing meaningful numbers (eg., HKL 
> indices) would be very helpful. Thanks in advance.
> 
> Zhijie
> 
> > On Nov 13, 2018, at 2:21 PM, Johan Hattne  wrote:
> > 
> > Related by not exactly on topic: would anybody on the list be able to share 
> > old map files (not MTZ:s) with Convex, Cray, Fujitsu, or VAX reals/strings? 
> >  I’d be interested to see what those files actually look(ed) like.
> > 
> > // Best wishes; Johan
> > 
> >> On Nov 9, 2018, at 18:38, Zhijie Li  wrote:
> >> 
> >> Hi all,
> >> 
> >> On linux there are a few good GUI HEX editors. Here I’d like to 
> >> recommend BLESS, which conveniently displays all possible numerical 
> >> interpretations of the four bytes under cursor. It also allows the 
> >> user to switch between big endian or little endian through a 
> >> checkbox. Unfortunately all floats are assumed to be IEEE754, 
> >> therefore VAX floats won’t be interpreted correctly.  ( The 
> >> simplest way to convert vax to ieee float would be to write a 
> >> little program to do some bit operations. I’d be happy to take that 
> >> as my weekend project)
> >> 
> >> 
> >> BTW, along the line of space efficiency, I can’t help noticing that the 
> >> miller indices are saved as float32 in mtz, as all other numbers in mtz. 
> >> This certainly have made mtz format a beautiful homogeneous data format 
> >> ;).  In this particular case, if we have doubts about the reliability of 
> >> the machine stamp, trying to restore the miller indices would be a good 
> >> way to test hypotheses.
> >> 
> >> Zhijie
> >> 
> >>> On Nov 9, 2018, at 9:04 PM, James Holton 
> >>> <270165b9f4cf-dmarc-requ...@jiscmail.ac.uk> wrote:
> >>> 
> >>> As a beamline scientist I must say I am glad that diffraction image data 
> >>> is not usually stored as ASCII text.  In fact, I am slowly warming to the 
> >>> idea of storing it as not just binary, but compressed formats.  Problem, 
> >>> I'm sure will be that it won't be  long before we forget how to 
> >>> decompress them, as most of the algorithms we are using aren't all that 
> >>> widespread.  Probably around the same time future generations will curse 
> >>> us for using ASCII instead of unicode, which is a 16-bit standard. I'm 
> >>> sure we will be reviled for limiting ourselves so, just to save a factor 
> >>> of two in disk space.
> >>> In situations like this I always use the unix "od" command.  It makes 
> >>> everything "human readable" by converting the bytes into strings you can 
> >>> read.  Then it is just a matter of figuring out what the bytes are.
> >>> Unfortunately, "od" only decodes floats on the native platform, so if the 
> >>> mtz is from another platform (Windows vs Linux, for example), then you 
> >>> might need to do some swapping.  Thus far, I have encountered files that 
> >>> require one of a few swapping strategies in order to make them work:
> >>> 
> >>> 1 2 3 4 - no swapping
> >>> 
> >>> 4 3 2 1 - reverse all bytes
> >>> 
> >>> 3 4 1 2 - swap words and swap bytes within the words
> >>> 2 1 4 3 - reverse of previous
> >>> 
> >>> 2-1 1 4 3 - same as last, but if not all zero, decrement byte #2 
> >>> before swapping
> >>> 3 4 1 2+1 - same as 3412, but if not all zero increment byte #2 
> >>> before swapping I'm sure there are other combinations, but the oldest MTZ 
> >>> I have is only from 1996.
> >>> 
> >>> -James Holton
> >>> MAD Scientist
> >>> 
> >>> 
>  On 11/9/2018 4:47 AM, Eleanor Dodson wrote:
>  Anyone any idea what to do about this?? Created in 1992!!
>  Seems unreadable..
>  
>  No CTYP lines input for file:  1
> Indices output even if all data items flagged "missing"
>  Warning, NOT all LABOUT data lines given
>  Warning: Machine stamp corrupted? Assuming native format. 
> >> CCP4 library signal library_file:End of File (Error)
>  
>  
>  To unsubscribe from the CCP4BB list, click the following link:
>  https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>  
> >>> 
> >>> 
> >>> To unsubscribe from the CCP4BB list, click the following link:
> >>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> >>> 
> >> 
> >> To unsubscribe from the CCP4BB list, click the following link:
> >> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> >> 
> > 
> >  Research Specialist @ Gonen Lab 
> > 
> >  UCLA * 615 Charles E. Young Drive South
> > BSRB #347 * Los Angeles, CA 90095
> > 
> > 
> > 
> > 
> > To unsubscribe from the CCP4BB list, click the following link:
> > 

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

[Keller, Jacob]

I deposited a dataset on SBGrid from a calmodulin-peptide complex which has 
some "nice" features: merohedral twinning (with variable twin fraction in the 
same dataset) and unavoidable detector cutoffs. It's relatively easy to 
integrate, but solving is somewhat harder. There's a paper on it too which 
gives the "answers" I came up with.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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-Original Message-
From: CCP4 bulletin board  On Behalf Of Whitley, Matthew 
J
Sent: Wednesday, September 19, 2018 8:16 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

Dear colleagues,

For teaching purposes, I am looking for a small number (< 5) of macromolecular 
diffraction datasets (raw images) that might be considered 'difficult' for a 
beginning crystallography student to process.  By 'difficult' I generally mean 
not able to be processed automatically by a common processing package (XDS, 
Mosflm, DIALS, etc) using default settings, i.e., no black box "click and done" 
processing. The datasets I am looking for would have some stumbling block such 
as incorrect experimental parameters recorded in the image headers, multiple 
lattices that cause indexing to fail, datasets for which determining the 
correct space group is tricky, datasets for experiments in which the crystal 
slipped or moved in the beam, or anything else you can think of.  The idea is 
for these beginning students to examine several datasets that highlight various 
phenomena that can lead one astray during processing.

A good candidate dataset would also ideally comprise a modest number of images 
so as to keep integration time to a minimum.  Factors that are mostly 
irrelevant for my purpose: resolution (as long as better than
~3.5 Å), source (home vs synchrotron), presence/absence of anomalous 
scattering,  presence/absence of ligands, monomeric vs oligomeric structures, 
etc.  Also, to be clear, I am not looking for datasets that have so many 
pathologies that they would require many long hours of work for an expert to 
process correctly.

I have checked public repositories such as proteindiffraction.org and SBGrid 
databank, but all of the datasets I acquired from these sources process 
satisfactorily with little effort, and in any event I know of no way to search 
for 'challenging' datasets.  (I also wonder whether anybody is in the habit of 
depositing, shall we say, less-than-pristine images to public repositories?)

If you know of such a dataset that is already publicly available, or if you 
have such a dataset that you are willing to share for solely educational 
purposes, I would appreciate hearing from you, either on- or off-list.

Thank you in advance for your suggestions.

Matthew

--
Matthew J. Whitley, Ph.D.
Department of Pharmacology & Chemical Biology University of Pittsburgh School 
of Medicine




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Re: [ccp4bb] cell discrepancies and stuck refinement using different XDS-versions

[Keller, Jacob]

>>But there is no rule without exception,

Well, occasionally there is.

JPK




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Re: [ccp4bb] Some sort of "closure"?

[Keller, Jacob]

Wow, something really happened! I wonder how many citations of those 
articles/structures are out there? 1000? I hadn't realized it was more than 
just that Nature paper. I hope nothing important is built on them.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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From: CCP4 bulletin board  On Behalf Of Mark J van Raaij
Sent: Thursday, July 19, 2018 7:59 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Some sort of "closure"?

Not sure if this has been posted here yet, but I don't remember it and only 
just saw this:
https://www.federalregister.gov/documents/2018/04/16/2018-07782/findings-of-research-misconduct
[it's about the famous case of fabricated structures and structure factors]

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij
Editor of Acta Crystallographica F, Structural Biology Communications
http://journals.iucr.org/f/




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Re: [ccp4bb] [ccp4bb] Oxford University Press

[Keller, Jacob]

I don't fully understand the cynicism in the response, unless it is simply the 
outpouring from years of painstaking review work for no monetary reward in a 
very broken publication system. Everybody I have talked to thinks the system is 
terrible, and various solutions have been proposed. One of these is, believe it 
or not, what I suggested, which is to pay reviewers. It is a huge amount of 
work, if one has a conscience about it, and the number of people with the 
expertise and experience required for this type of work is exceedingly small. 
Further, a real review of a paper takes significantly more than 2 hours of 
work, depending on the type of paper (I think you were joking about 2 h, but 
not totally sure). What really kills me is that the publishing houses are 
making money hand over fist, and this because they know that they can get very 
cooperative scientists (G-d bless them) to do huge amounts of work for free. It 
is really scandalous, and I am not sure why we scientists go along with it. To 
put it bluntly: we are being exploited by the journals; why not do something 
about it? I am sure that the journals will not be overjoyed to release their 
grip on the profits, but that should not stop us.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hughes, 
Jon
Sent: Saturday, June 30, 2018 6:13 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] AW: [ccp4bb] [ccp4bb] Oxford University Press

great idea! 2 hours at €200 per hour makes €1000 - sounds like an eminently 
reasonable starting point for negotiations. if the publishers don't like our 
price, they can do the reviewing themselves - and after a while no one will 
bother to buy the resulting rubbish anyhow! in the mean time we put our stuff 
online directly (without wasting our time, for example, still formatting 
reference lists in the 21st century!). we have them over a barrel. the only 
problem i see is how to get grants without papers in Nature. anyone have a 
solution to that one?
one final aspect is: who gets the money? surely the universities etc. should 
get it, not us: the taxpayer pays us already. 
best,
jon

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Keller, 
Jacob
Gesendet: Samstag, 30. Juni 2018 00:00
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] [ccp4bb] Oxford University Press

The one I don't get is why not pay reviewers? $1000 per review? If you look at 
publishers' profit margins, you will see that they can afford it. I actually 
think the scientific community should go on a "review strike" until reviewers 
get paid.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Petr 
Leiman
Sent: Friday, June 29, 2018 4:47 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] [ccp4bb] Oxford University Press

Indeed! Scientists in the Soviet Bloc got paid for publishing their scientific 
papers (and maybe for citations as well - not sure about that one)! We need to 
change the current system! Although these changes could be accompanied by many 
other pleasant virtues of the Soviet regime. 

Petr


> On Jun 29, 2018, at 8:11 AM, Hughes, Jon  
> wrote:
> 
> whose paper? our universities pay subscriptions for these journals and we 
> even pay on top of that for the pages of our publications (even when they're 
> not actually printed!), whilst we review papers for free! sounds like a 
> well-validated way to use taxpayers' money to keep the expensive company cars 
> etc. nice and shiny. why don't universities just require reimbursement for 
> the time we 

Re: [ccp4bb] [ccp4bb] Oxford University Press

[Keller, Jacob]

The one I don't get is why not pay reviewers? $1000 per review? If you look at 
publishers' profit margins, you will see that they can afford it. I actually 
think the scientific community should go on a "review strike" until reviewers 
get paid.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Petr 
Leiman
Sent: Friday, June 29, 2018 4:47 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] [ccp4bb] Oxford University Press

Indeed! Scientists in the Soviet Bloc got paid for publishing their scientific 
papers (and maybe for citations as well - not sure about that one)! We need to 
change the current system! Although these changes could be accompanied by many 
other pleasant virtues of the Soviet regime. 

Petr


> On Jun 29, 2018, at 8:11 AM, Hughes, Jon  
> wrote:
> 
> whose paper? our universities pay subscriptions for these journals and we 
> even pay on top of that for the pages of our publications (even when they're 
> not actually printed!), whilst we review papers for free! sounds like a 
> well-validated way to use taxpayers' money to keep the expensive company cars 
> etc. nice and shiny. why don't universities just require reimbursement for 
> the time we invest to insure that the merchandise is up to standard? €100 per 
> hour would be cheap. seems to me as though some capitalists need to add a few 
> lines to their balance sheets
> best, jon
> 
> -Ursprüngliche Nachricht-
> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
> Robbie Joosten
> Gesendet: Freitag, 29. Juni 2018 13:42
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: Re: [ccp4bb] Oxford University Press
> 
> Yes, but think of all the money they miss due to your pirating of their paper 
> ;) It's the typical discussion about whether piracy of copyrighted material 
> leads to loss or gain of revenue. There are a lot of models here, but not 
> necessarily well-validated.
> 
> Anyway, if people want to read your papers and cannot get them from 
> ResearchGate, I'm sure they can find them on another online 
> collection, a hub of some sort ;)
> 
> Cheers,
> Robbie
> 
>> -Original Message-
>> From: Bernhard Rupp [mailto:hofkristall...@gmail.com]
>> Sent: Friday, June 29, 2018 13:23
>> To: 'Robbie Joosten'; CCP4BB@JISCMAIL.AC.UK
>> Subject: RE: [ccp4bb] Oxford University Press
>> 
>> Agreed, but for 10 years old papers this seems a bit of overkill
>> 
>> 
>> 
>> From: CCP4 bulletin board  On Behalf Of Robbie 
>> Joosten
>> Sent: Friday, June 29, 2018 12:11
>> To: CCP4BB@JISCMAIL.AC.UK
>> Subject: Re: [ccp4bb] Oxford University Press
>> 
>> 
>> 
>> Were they open access papers? If they were, than OUP is being too 
>> aggressive (IMO), but otherwise it makes sense. I also find the 
>> ResearchGate is rather aggressive in bugging you to upload papers 
>> that are readily available from the publisher. The whole business bit 
>> in scientific publishing is a necessary (?) evil, but I guess if 
>> given the choice one should publish somewhere where you as an author retain 
>> copyright.
>> 
>> 
>> 
>> Cheers,
>> 
>> Robbie
>> 
>> 
>> 
>> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
>> Bernhard Rupp
>> Sent: Friday, June 29, 2018 11:42
>> To: CCP4BB@JISCMAIL.AC.UK
>> Subject: [ccp4bb] Oxford University Press
>> 
>> 
>> 
>> Hi Fellows,
>> 
>> 
>> 
>> just an advisory that Oxford University Press is pretty aggressive in
>> 
>> enforcing copyright - I had to remove 2 Bioinformatics papers
>> 
>> from ResearchGate.
>> 
>> 
>> 
>> Fortunately, authors have choices, too
>> 
>> 
>> 
>> Cheers, BR
>> 
>> --
>> 
>> Bernhard Rupp
>> 
>> http://www.hofkristallamt.org/
>> 
>> b...@hofkristallamt.org
>> 
>> +1 925 209 7429
>> 
>> +43 676 571 0536
>> 
>> --
>> 
>> Many plausible ideas vanish
>> 
>> at the presence of thought
>> 
>> --
>> 
>> 
>> 
>> 
>> 
>> 
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>> 
>> 
>> 
>> 
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> 

Re: [ccp4bb] Electron density

[Keller, Jacob]

I know you mentioned trying buffer components, but it does look a lot like TRIS 
to me, maybe a different conformation than you’re modelling?

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Daniel 
Garcia
Sent: Sunday, May 13, 2018 8:40 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Electron density

Dear all,

I am currently refining a structure and found a intriguing electron density at 
the protein surface (pictures attached, the Fo-Fc map is contoured at >3.5 
sigma). My first candidates were molecules from my protein prep or 
crystallisation buffer, but none of them seem to fit well. I can observe that 
the ligand is nearby the side chains of a tyrosine, a lysine, a threonine and a 
glutamate residue, and it is close to the carbonyl oxygens of the protein 
backbone of a nearby loop. The shape of this density is not pyramidal, but it 
is not planar either.

Do you have any suggestions to solve this density based on your own experience? 
My crystallisation buffer contains tartrate, ammonium sulphate, and CHES, and 
my protein is in Tris buffer containing DTT and sodium chloride.

Best regards,

--
Mario

Re: [ccp4bb] According correct space group assignment...

[Keller, Jacob]

Why not try direct methods on both SG options, and maybe P1 as well? Depending 
on the wavelength and multiplicity, you might also have some good anomalous 
signal from the P's.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Rafal 
Dolot
Sent: Friday, April 20, 2018 9:31 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] According correct space group assignment...

Dear CCP4BB,

I've recently collected data for 11mer build of DNA (9xG, 2xT). XDS, and DIALS 
gave me similar solution - SG: I2(1)2(1)2(1) or I222, with cell dimension 
20.65, 22.96, 43.37, 90, 90, 90, what is too small for this size of the 
molecule. 11mer is rich in G, so we expect the G-tetraplex formation. Data were 
collected to almost 1 A, so it should be enough for trials with direct 
methods/ab initio solution. What I should do first to find correct SG and/or 
cell parameters?

Best regards,

Rafal
-- 
|--|
|Rafal Dolot, Ph.D.|
|  |
|Polish Academy of Sciences|
|Centre of Molecular and Macromolecular Studies|
|Department of Bioorganic Chemistry|
|Macromolecular Crystallography Team   |
|Sienkiewicza 112  |
|90-363 Lodz, Poland   |
|Phone: +48(42)6803215 |
|Cell:  +48 502897781  |
|--|

Re: [ccp4bb] point group...321

[Keller, Jacob]

Yes, several, but it's only important when merging multiple data sets. And it's 
not really ambiguities but alternatives.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
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message with any third party, without a written consent of the sender. If you 
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-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Gihan 
Ketawala
Sent: Thursday, March 29, 2018 4:27 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] point group...321

Hi, 
Can the point group 321 show indexing ambiguities? if not since I'm dealing 
with non-anomalous dataset can I use 3m1 instead?

Thank you,
Best,
Gihan

Re: [ccp4bb] A new capability on the STARANISO server: "PDBpeep"

[Keller, Jacob]

>>>These simple things are easily forgotten if there isn't direct visual 
>>>evidence for their existence.

Maybe someday we scientists will collectively realize the huge importance of 
intuitive data presentation and focus more on it, scorn it less as "mere 
presentation"? When one thinks of the most powerful breakthroughs in the 
history of science, one is struck by the advances of simply presenting data (or 
collecting data) in a way that speaks directly to the intuition, e.g., 
heliocentrism, the DNA double helix, structural bio in general, cartesian 
graphing. What about things like molecular graphics, or the ribbon diagrams and 
other representations which underlie them? All of these are "mere 
presentations" but have driven science forward incredibly. Shouldn't we focus 
on making more data-to-intuition translators?

For example, what there was a VR interface in which the user could actually 
feel physical forces in protein molecules, get a touch-intuitive sense of what 
proteins are like? We would already "know" so much more about proteins!

Jacob

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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Re: [ccp4bb] A new capability on the STARANISO server: "PDBpeep"

[Keller, Jacob]

>> I am not sure that there is any such thing as an up-to-date estimate of 
>> the prevalence of anisotropy in the PDB - but now you can get a feel for it 
>> yourself by looking at any entries you want. However please do not submit 
>> the whole PDB to the server - yet ;-) .

It would be great to have a figure showing distributions of anisotropy across 
the pdb, perhaps filtering for various parameters like resolution, lattice 
size, membrane proteins, etc. And this should be helpful for assaying the 
importance of dealing with anisotropy using STARANISO and others.

>> From looking at anisotropy as visible through the overall scaling 
>> Debye-Waller factor, I would say that whenever anisotropy is allowed by the 
>> Laue group, it will be present, even if mild. Symmetry lower than cubic 
>> means that intermolecular contacts along directions that are not 
>> symmetry-equivalent will be different, and there is no reason why different 
>> contacts should create identical degrees of long-range order.

Yes, that makes all the sense in the world. I wonder how one would evaluate in 
a given case whether the usual isotropic approximation is good enough? Or 
perhaps there's no reason to use the isotropic assumption at all, now that 
there are ways of dealing with anisotropy?

JPK

Re: [ccp4bb] A new capability on the STARANISO server: "PDBpeep"

[Keller, Jacob]

Wow, this is really cool--just tried a quick look at a recent membrane protein 
(5eqi) and you can see right away that there is anisotropy.

I would guess this can be found in the literature, but how prevalent is 
anisotropy in the PDB?

Jacob Keller

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
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-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Gerard 
Bricogne
Sent: Wednesday, March 28, 2018 11:56 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] A new capability on the STARANISO server: "PDBpeep"

Dear all,

 Ever since the WebGL viewer became available on the STARANISO server, we 
have found ourselves using it with increasing frequency to take a quick look (a 
"peep") at the diffraction data deposited with various PDB entries - for 
example, to try and identify a root cause for some sub-optimal refinement 
results, or, quite often, just out of sheer curiosity!

 This involved a totally straightforward procedure whereby the diffraction 
data file associated with a given PDB entry was downloaded from the PDB and 
subsequently uploaded to the STARANISO server.

 Gradually, however, this operation became so popular among some of us that 
we thought it would be useful to implement this simple procedure as an 
autonomous capability - and thus was born "PDBpeep" !

 You can access this new feature by connecting to 

   http://staraniso.globalphasing.org/cgi-bin/PDBpeep.cgi 

and enter a PDB code into the box. As indicated on that page, this provides 
only a cursory look at the overall quality of each dataset, and any further 
analysis or output can only be obtained by submitting the datafile to the 
STARANISO server. Better results would clearly be obtainable if the raw images 
for these datasets had been deposited and could be reprocessed, with the 
untruncated output of that processing then being submitted to the STARANISO 
server (reprocessing the images with autoPROC would combine those two steps 
into a single one).

 Most of the deposited datasets have been isotropically truncated, and 
their 3D view in WebGL often suggests that this truncation was too drastic. A 
number of entries will show infelicities - such as cusps and/or missing angular 
ranges, or even stripes caused by gaps between the modules of pixel detectors 
if the beam centre is at a position symmetric relative to those gaps - all 
marked up in blue.


 Our purpose in sharing this capability with the community is to bring a 
further contribution to the process of making everyone more "data quality 
aware" and keen to scrutinise more closely the protocols by which they collect 
diffraction data, or have such data collected on their behalf.


 We will be grateful to receive feedback about PDBpeep, just as we have 
been about the STARANISO server itself.


 With best wishes,

 The STARANISO developers.

Re: [ccp4bb] protein quasicrystals?

[Keller, Jacob]

I would say definitely not a quasicrystal, just multiple crystals, but the 
spots look like salt spots to me, being very sharp and intense. The puzzling 
thing is that the low-resolution zone is so populated, which would argue a bit 
against salt.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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From: yu@sanofi.com [mailto:yu@sanofi.com]
Sent: Tuesday, February 13, 2018 4:03 PM
To: Keller, Jacob <kell...@janelia.hhmi.org>; CCP4BB@JISCMAIL.AC.UK
Subject: RE: [ccp4bb] protein quasicrystals?

As asked by a few people, here are the images of crystals and diffraction.

Thanks,
Yu

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, 
Jacob
Sent: Tuesday, February 13, 2018 4:00 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [EXTERNAL] Re: [ccp4bb] protein quasicrystals?

I also would love to see an image….

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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message with any third party, without a written consent of the sender. If you 
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From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of James 
Phillips
Sent: Tuesday, February 13, 2018 3:03 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] protein quasicrystals?

There are programs which are good at indexing patterns from multiply twinned 
crystals. Bruker AXS has one, to my knowledge. There may be other sources. I 
suggest you try that first before you invoke a quasicrystal explanation.




James Phillips

On Tue, Feb 13, 2018 at 12:07 PM, Takanori Nakane 
<tnak...@mrc-lmb.cam.ac.uk<mailto:tnak...@mrc-lmb.cam.ac.uk>> wrote:
Hi,

"dials.reciprocal_space_viewer" is very useful to identify multiple lattices.
For quasicrystal and modulated crystals, "dials.rs_mapper" is also very
useful.

Best regards,

Takanori Nakane

> Have you tried microseeding of these sphere crystals? It may help to get
> better crystals.
>
>
> Burak
>
> 
> From: CCP4 bulletin board 
> <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Yu Qiu
> <yu@sanofi.com<mailto:yu@sanofi.com>>
> Sent: 13 February 2018 15:09:43
> To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
> Subject: [ccp4bb] protein quasicrystals?
>
>
> Hi,
>
>
>
> I have been trying to crystallize a protein complex and keep getting
> sphere shape crystals. The diffraction is around 3 angstrom, but looks
> like multiple lattices. I am wondering if it could be a quasi crystal? Is
> there anyone has such experience?
>
>
>
> Thanks,
>
> Yu
>

Re: [ccp4bb] protein quasicrystals?

[Keller, Jacob]

I also would love to see an image….

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of James 
Phillips
Sent: Tuesday, February 13, 2018 3:03 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] protein quasicrystals?

There are programs which are good at indexing patterns from multiply twinned 
crystals. Bruker AXS has one, to my knowledge. There may be other sources. I 
suggest you try that first before you invoke a quasicrystal explanation.




James Phillips

On Tue, Feb 13, 2018 at 12:07 PM, Takanori Nakane 
> wrote:
Hi,

"dials.reciprocal_space_viewer" is very useful to identify multiple lattices.
For quasicrystal and modulated crystals, "dials.rs_mapper" is also very
useful.

Best regards,

Takanori Nakane

> Have you tried microseeding of these sphere crystals? It may help to get
> better crystals.
>
>
> Burak
>
> 
> From: CCP4 bulletin board 
> > on behalf of Yu Qiu
> >
> Sent: 13 February 2018 15:09:43
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] protein quasicrystals?
>
>
> Hi,
>
>
>
> I have been trying to crystallize a protein complex and keep getting
> sphere shape crystals. The diffraction is around 3 angstrom, but looks
> like multiple lattices. I am wondering if it could be a quasi crystal? Is
> there anyone has such experience?
>
>
>
> Thanks,
>
> Yu
>

Re: [ccp4bb] Might be of interest to this group

[Keller, Jacob]

I hope everyone realizes that I am very happy with off-topics, and love the 
praying mantis thing. It didn’t come off as cynical, did it? I just thought it 
funny to post to the crystallography list, and somewhat humorous to picture 
praying mantises running COOT etc.

We should really find an organism which sees in reciprocal space, figure out 
how it solves the phase problem, then implement it in software. Or train 
ourselves to do it by wearing Fourier glasses—then we could just do all of our 
modelling in reciprocal space, save a lot of time.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

Re: [ccp4bb] Might be of interest to this group

[Keller, Jacob]

Or for the Praying Mantis Crystallographers’ association?

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Andreas 
Forster
Sent: Friday, February 9, 2018 11:13 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Might be of interest to this group

Wouldn’t that be something for the PyMOL mailing list?

All best - Andreas
On Fri, 9 Feb 2018 at 20:38, David Schuller 
> wrote:

http://www.cell.com/current-biology/fulltext/S0960-9822(18)30014-9

A Novel Form of Stereo Vision in the Praying Mantis
Vivek Nityananda,Ghaith Tarawneh, Sid Henriksen,Diana Umeton,Adam Simmons, 
Jenny C.A. Read

DOI: https://doi.org/10.1016/j.cub.2018.01.012

Current Biology

--

===

All Things Serve the Beam

===

   David J. Schuller

   modern man in a post-modern world

   MacCHESS, Cornell University

   schul...@cornell.edu

Re: [ccp4bb] Issues with latest XDS (20171218)

[Keller, Jacob]

How about including all of this stuff in refinement? Only adds another ~10-100 
[restrained] parameters…

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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message with any third party, without a written consent of the sender. If you 
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From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of wtempel
Sent: Wednesday, January 24, 2018 12:19 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Issues with latest XDS (20171218)

I agree that accurate and complete image header information is useful, but not 
universal. How about a routine similar to 
dials.discover_better_experimental_model?
W.

On Wed, Jan 24, 2018 at 10:12 AM, Engin Özkan 
> wrote:
Dear all,

I have to agree with all sentiments stated, and I have definitely seen the 
value of restraining distance values in weak and anisotropic data, which is 
great for those of us who deal with such data on a regular basis. However, I 
also just went through a search of all refined DISTANCE values on all (~20) 
CORRECT.LP files on my hard drive. Mismatches between input and refined values 
of up to 0.25% (1 mm in 400 mm) is not uncommon for data collected over several 
different beam lines. Many of us collect data at synchrotrons remotely and/or 
without ever talking to a beamline scientists, and expecting users to end up 
with perfect distance values in their auto-scripted XDS.INP files might be 
expecting the perfect in an imperfect, fast-moving data-collection world. A 
solution that accommodates both viewpoints, if at all possible, would indeed be 
great.

Best,

Engin


On 1/24/18 8:06 AM, "Weiergräber, Oliver H." wrote:
Dear Clemens, dear Kay,

I absolutely agree with your statement regarding responsibilities. 
Unfortunately, for a user of a synchrotron beamline, it is hardly possible to 
_know_ that the distance (or any beam or camera parameter) provided by the 
beamline software is in error. In the end indications can only be derived from 
the behaviour of software used for processing. Of course developers are not 
responsible for hardware issues, but since such issues do occur in real life, 
the software may still help spotting them ;-)
Given that IDXREF runs very fast, why not have it probe the shifts of 
parameters for a full refinement scope and issue a warning if things look 
suspicious, giving the user options how to proceed (similar to the 
not-so-infrequent case of "insufficient percentage of indexed reflections").

Best regards
Oliver


   PD Dr. Oliver H. Weiergräber
   Institute of Complex Systems
   ICS-6: Structural Biochemistry
   Tel.: +49 2461 61-2028
   Fax: +49 2461 61-9540





From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] 
on behalf of Clemens Vonrhein 
[vonrh...@globalphasing.com]
Sent: Wednesday, January 24, 2018 2:39 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Issues with latest XDS (20171218)

Dear Oliver,

yes, there are other changes to the parameter refinement procedure
within XDS as far as I understand. These were introduced to robustify
XDS processing (and parameter refinement) when encountering very poor
datasets, cases where the crystal moves out of the beam in certain
orientations or empty loops. That works very well it seems - at least
for us (and we were involved in discussing those cases with both
Wolfgang Kabsch and Kay Diederichs).

I'm sure you agree that if the crystal to detector distance in the
header is wrong by such a large amount (over 1 mm), the (proper)
solution is to fix this at the point of collecting the data and
writing the header. As Kay says: there should be no reason for an
instrument/beamline to not know that distance very accurately and to
write the correct value in the header. It has the same importance as
e.g. wavelength/energy or oscillation range.

Of course, if a user already has images with an inaccurate value and
wants to process those, the old behaviour of XDS might be able to fix
that particular problem for you. But this does feel like patching up
simple-to-fix problems at the wrong stage, namely processing instead
of at the beamline side ... with the "danger" that they will never get
fixed properly because 

Re: [ccp4bb] "Atomic resolution"

[Keller, Jacob]

>I tell people it is when your resolution is less than the bond length that 
>connects the two atoms.

I thought this was sort of a pitfall, since the Bragg spacings don't 
necessarily map on to conventional resolution. But it would fit the 1.5 Ang 
estimate.

Also, resolution would depend a lot on phase accuracy/precision, no?

JPK


On Thu, Jan 11, 2018 at 1:59 PM, Thomas Edwards <t.a.edwa...@leeds.ac.uk> wrote:
> Dear Jacob,
>
> Ah... this old chestnut!
>
> Current EM people say that they are at atomic resolution because they 
> are building atomic models (naive??).
>
> I have been criticised in the past for using the term with say 2.2A 
> diffraction data. By co-authors and reviewers alike. When I was young 
> and naive.
>
> My (current) definition would be yours - visible with data.
> I think 1.5A is about right for X-ray. Maybe higher res?
>
> I’m sure there are lots of rigorous ways to think. I probably haven’t 
> taken that route. However, I think it is a semantic problem that might 
> benefit from some disambiguation rather than rigour.
>
> It depends why you are asking the question...
>
> Sorry..!
>
> Ed is: Out and about...
> Sent from iPhone6sPlus.
>
> On 11 Jan 2018, at 19:31, Keller, Jacob <kell...@janelia.hhmi.org> wrote:
>
> Dear Crystallographers,
>
>
>
> Has there been a consensus as to what is meant by “atomic resolution?” 
> Seems like the term is taken by various practitioners to mean different 
> things.
>
>
>
> A related question: at what resolution are atoms “visible” using only 
> the data? I have an empirical feeling that this would be around 1.5 
> Ang Bragg spacings, but on the other hand, one can contour up most 
> maps and see individual atom peaks. I would be interested to hear a 
> more rigorous way to think about this.
>
>
>
> All the best,
>
>
>
> Jacob Keller
>
>
>
> +
>
> Jacob Pearson Keller
>
> Research Scientist / Looger Lab
>
> HHMI Janelia Research Campus
>
> 19700 Helix Dr, Ashburn, VA 20147
>
> (571)209-4000 x3159
>
> +
>
>
>
> The content of this email is confidential and intended for the 
> recipient specified in message only. It is strictly forbidden to share 
> any part of this message with any third party, without a written consent of 
> the sender.
> If you received this message by mistake, please reply to this message 
> and follow with its deletion, so that we can ensure such a mistake 
> does not occur in the future.
>
>

Re: [ccp4bb] "Atomic resolution"

[Keller, Jacob]

The reason behind this query is that I want to illustrate the power of prior 
knowledge in data analysis. I want to say something like “even though atoms 
cannot be directly observed at worse than X resolution, which represents Y% of 
the PDB, all of these data sets have been fit correctly with atomic models. 
This is due entirely to the power of the excellent priors which exist in 
crystallography.”

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
(571)209-4000 x3159
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

From: Thomas Edwards [mailto:t.a.edwa...@leeds.ac.uk]
Sent: Thursday, January 11, 2018 2:59 PM
To: Keller, Jacob <kell...@janelia.hhmi.org>
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] "Atomic resolution"

Dear Jacob,

Ah... this old chestnut!

Current EM people say that they are at atomic resolution because they are 
building atomic models (naive??).

I have been criticised in the past for using the term with say 2.2A diffraction 
data. By co-authors and reviewers alike. When I was young and naive.

My (current) definition would be yours - visible with data.
I think 1.5A is about right for X-ray. Maybe higher res?

I’m sure there are lots of rigorous ways to think. I probably haven’t taken 
that route. However, I think it is a semantic problem that might benefit from 
some disambiguation rather than rigour.

It depends why you are asking the question...

Sorry..!
Ed is: Out and about...
Sent from iPhone6sPlus.

On 11 Jan 2018, at 19:31, Keller, Jacob 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> wrote:
Dear Crystallographers,

Has there been a consensus as to what is meant by “atomic resolution?” Seems 
like the term is taken by various practitioners to mean different things.

A related question: at what resolution are atoms “visible” using only the data? 
I have an empirical feeling that this would be around 1.5 Ang Bragg spacings, 
but on the other hand, one can contour up most maps and see individual atom 
peaks. I would be interested to hear a more rigorous way to think about this.

All the best,

Jacob Keller

+++++
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
(571)209-4000 x3159
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

[ccp4bb] "Atomic resolution"

[Keller, Jacob]

Dear Crystallographers,

Has there been a consensus as to what is meant by "atomic resolution?" Seems 
like the term is taken by various practitioners to mean different things.

A related question: at what resolution are atoms "visible" using only the data? 
I have an empirical feeling that this would be around 1.5 Ang Bragg spacings, 
but on the other hand, one can contour up most maps and see individual atom 
peaks. I would be interested to hear a more rigorous way to think about this.

All the best,

Jacob Keller

+++++
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
(571)209-4000 x3159
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

[ccp4bb] Na-Binding Protein?

[Keller, Jacob]

Dear Crystallographers,

Is anyone aware of a soluble protein which changes large-scale conformation +/- 
Na+, and is specific for Na+ per se, or at least ignores K+ and Ca++? E.g., Rb+ 
or Li+ might be okay. Structural info would be a plus, but not a sine qua non.

Similarly, what about with K+ or Cl- specificities, but oblivious to similar 
common ions?

All the best,

Jacob Keller

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
(571)209-4000 x3159
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

[ccp4bb] Finding Homologs with Specific Residues Conserved

[Keller, Jacob]

Dear Crystallographer-Bioinformaticians,

Is anyone aware of a way to tweak BLAST or similar software to be able to 
specify certain residues to be absolutely required, e.g., active site residues? 
I guess one can winnow broad-scale resuts with scripts, but it would seem to be 
a pretty common type of thing to do, and might change results slightly a la 
psi-BLAST.

All the best,

Jacob Keller

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
(571)209-4000 x3159
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

Re: [ccp4bb] Crystal structure of an unknown protein

[Keller, Jacob]

Wow, pretty cool—you must have solved it to very high resolution to know the 
sequence from the structure. I cannot imagine, however, how you got this 
contaminant—maybe phage infection of your bacterial culture? Anyway, I agree 
with BLAST-ing the sequence, seeing what you get that is closest.

JPK


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jiyong Su
Sent: Thursday, December 14, 2017 7:09 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Crystal structure of an unknown protein

Dear CCP4bb,

In 2014, I collected a high quality data set from a crystal. But I could not 
solve the structure of that crystal because this protein is a contaminate.
Recently, I used StruBE's Contaminer and fortunately got the solution. Thanks 
ContaMiner!!!  This protein is a contaminate protein.

However, I found this protein is an unknown protein (about 180 residues) whose 
amino acid sequence is not totally same as E.coli. There are about 20 point 
mutation sites comparing to the E.coli protein. This means this protein may be 
from an unknown bacteria.

The space group of this crystal is new. There is also a new ligand in this 
protein.

My question is how could I found the primary structure of this protein and how 
to deposit this protein in PDB.

Best regards,

Jiyong

Re: [ccp4bb] coordinate transformation

[Keller, Jacob]

Wouldn't the PISA server do something like this?

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kajander, 
Tommi A
Sent: Wednesday, December 13, 2017 8:50 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] coordinate transformation


Hello,

If someone could point this out would be very helpful... Wasnt there a simple 
script somewhere that would transfer coordinates close to origin - if they for 
some reason are not? Just cant find anything right away. Sure i have done this 
before...



Thanks,

Tommi

Re: [ccp4bb] P212121 twinning

[Keller, Jacob]

Try P2 SG’s.

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Carmela 
Garcia
Sent: Sunday, December 3, 2017 12:30 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] P212121 twinning

Hi,

The dimensions for a native are 58 103 220, with small differences for the 
derivatives.

Best,

Carmela.

On 3 Dec 2017, at 18:18, Sridhar Prasad 
> wrote:

Hello,
  Can you please share the unit cell dimensions.

Cheers,
Sridhar

On Dec 3, 2017 9:13 AM, "Carmela Garcia" 
> wrote:
Dear all,

I know that some years ago a similar situation was discussed here, and I wonder 
if someone has new insights about these problems.

My protein is a dimer in solution. I tried several derivatives for SAD, and all 
my datasets seem to have the same problem, including the native crystals. I 
processed the data with XDS and the space group determination was done with 
Pointless, being a P212121.

Checking the quality of the data, I found several problematic results:

- Translational NCS is detected. There is a peak at (0.50, 0.40, 0.50)
- The L test suggests twinning (L statistic = 0.41)
- The mean acentric moments I from input data have the following values:
/^2 : 4.396
/^3: 34.478
/^4: 361.084
All these values are way higher than the expected ones for non-twinned data.
- The twinning fraction from L-test is 0.22

This would all suggest that my space group is wrong, and that I should proceed 
in a lower symmetry group, but I don’t know how should I continue. I know about 
cases where a P43212 or a P41212 were suggested but in fact the correct space 
group was P212121, but I would not know how to continue going down. Checking my 
images, I can see some streaky spots, and the crystals grow first as needles 
that eventually become plates and rarely crystals. All these would be a clear 
indicative of twinning, but I would not expect to have the same results with 
different derivatives. We thought about some kind of reticular twinning, and I 
wonder if there is a way to solve it.

Any idea about how to proceed or to identify the problem would be welcome.

Thanks,

Carmela.



Carmela Garcia-Doval
University of Zurich
Department of Biochemistry
Winterthurerstrasse 
190
CH-8057 Zurich
Switzerland
E-mail: c.gar...@bioc.uzh.ch

Re: [ccp4bb] Regarding Patents

[Keller, Jacob]

>>Isn’t that exactly the idea of a patent? Instead of keeping the invention
a trade secret (occasionally a viable alternative) you publish the invention,
and the inventor (and in general, the supporting institutions) can get
rewarded if someone plans to use the idea commercially.

I agree with this especially because someone else is, after all, going to 
commercialize it and charge money for it.







Best, BR

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Abhishek 
Anan
Sent: Saturday, November 4, 2017 05:31
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Regarding Patents

I second Gert's thoughts
Best,
Abhishek

On Sat, Nov 4, 2017 at 10:21 AM, Gert Vriend 
> wrote:
A related question. If you have a crystal structure and found a novel ligand 
binding site that can be used to regulate protein activity, could you patent 
such "binding site"? If not, how to make the best use of such findings?

I would say that the best one can do with important novel 
data/information/knowledge/insights is to publish it so the world can benefit 
from it.

Gert

Re: [ccp4bb] AW: Re: [ccp4bb] Basic Crystallography/Imaging Conundrum

[Keller, Jacob]

>>My understanding is that EM people will routinely switch to diffraction mode 
>>when they want accurate measurements.  You lose the phase information but, 
>>since EM lenses tend to have imperfections, you get better measurements of 
>>the intensities.

Only to my knowledge in the case of crystalline samples like 2D crystals.

>>Of course the loss of phases is a serious problem when you don't have a model 
>>of the object as precise as our atomic models.

From where does this precision arise, I wonder? I guess priors for atom-based 
models are pretty invariant. On the other hand, who says that such priors, 
albeit of many more varieties, don't exist for larger biological samples, such 
as zebrafish brains and drosophila embryos/larvae? Anyway, right now, the state 
of the art of modelling in these fluorescence data sets is hand-drawing circles 
around things that look interesting, hoping the sample does not shift too much, 
or perhaps using some tracking. But it could be so much better!

JPK

Re: [ccp4bb] AW: Re: [ccp4bb] Basic Crystallography/Imaging Conundrum

[Keller, Jacob]

"Quality of image" has a lot of parameters, including resolution, noise, 
systematic errors, etc. I am not aware of a global "quality of image" metric.

One other consideration, not related to your comment: imagine if we had an 
x-ray lens through which we could take confocal images of a protein molecule or 
crystal, output as a voxel array. Would we really still prefer to measure 
diffraction patterns rather than the equivalent real space image, even assuming 
we had some perfect way to solve the phase problem? Or conversely, should we 
try to do fluorescence imaging in diffraction mode, due to its purported 
information efficiency?

JPK

-Original Message-
From: herman.schreu...@sanofi.com [mailto:herman.schreu...@sanofi.com] 
Sent: Friday, November 10, 2017 10:22 AM
To: Keller, Jacob <kell...@janelia.hhmi.org>; CCP4BB@JISCMAIL.AC.UK
Subject: AW: [ccp4bb] AW: Re: [ccp4bb] Basic Crystallography/Imaging Conundrum

At the bottom line, it is the quality of the image, not only the amount of 
pixels that counts. Adding more megapixels to a digital camera with a poor lens 
(as some manufacturers did), did not result in any sharper or better images.
Herman


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Keller, 
Jacob
Gesendet: Freitag, 10. November 2017 15:48
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] AW: Re: [ccp4bb] Basic Crystallography/Imaging 
Conundrum

It seems, then, to be generally agreed that the conversion between voxels and 
Fourier terms was valid, each containing the same amount of information, but 
the problem was in the representation, and there was just trickery of the eye. 
I was thinking and hoping this would be so, since it allows a pretty direct 
comparison of crystal data to microscopic imaging data. I guess a litmus test 
would be to decide whether a voxel version of the electron density map would 
work equivalently well in crystallographic software, which I suspect it would. 
If so, then the same techniques--so effective in extracting information for the 
relatively information-poor crystal structures--could be used on fluorescence 
imaging data, which come in voxels.

Regarding information-wealth, in Dale's example, the whole hkl set was 4.1 MB. 
One frame in a garden-variety XYZT fluorescence image, however, contains about 
2000 x 2000 x 100 voxels at 16-bit, i.e., 400 million bits or 50 MB. In some 
data sets, these frames come at 10 Hz or more. I suspect that the I/sigma is 
also much better in the latter. So, with these data, and keeping a 
data:parameters ratio of ~4, one could model about 100 million parameters. This 
type of modelling, or any type of modelling for that matter, remains almost 
completely absent in the imaging world, perhaps because the data size is 
currently so unwieldy, perhaps also because sometimes people get nervous about 
model biases, perhaps also because people are still improving the imaging 
techniques. But just imagine what could be done with some crystallography-style 
modelling!

Jacob Keller



-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tristan 
Croll
Sent: Friday, November 10, 2017 8:36 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] AW: Re: [ccp4bb] Basic Crystallography/Imaging Conundrum

Or a nice familiar 2D example: the Ramachandran plot with 7.5 degree binning, 
as a grid (left) or with bicubic smoothing (right). Different visualisations of 
the same data, but the right-hand image uses it better.

On 2017-11-10 08:24, herman.schreu...@sanofi.com wrote:
> In line with Dale's suggestions, I would suggest that you reformat 
> your voxel map into the format of an electron density map and look at 
> it with coot. I am sure it will look much better and much more like 
> the electron density we are used to look at. Alternatively, you could 
> display an bona fide electron density map as voxel blocks and I am 
> sure it will look similar to the voxel map you showed in your first 
> email.
> 
> Best,
> Herman
> 
> -Ursprüngliche Nachricht-
> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
> Dale Tronrud
> Gesendet: Freitag, 10. November 2017 08:08
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: [EXTERNAL] Re: [ccp4bb] Basic Crystallography/Imaging 
> Conundrum
> 
>Ethan and I apparently agree that anomalous scattering is "normal"
> and Friedel's Law is just an approximation.  I'll presume that your 
> "unique" is assuming otherwise and your 62,500 reflections only 
> include half of reciprocal space.  The full sphere of data would 
> include 125,000 reflections.  Since the cube root of 125,000 is 50 you 
> get a range of indices from -25 to +25 which would give you 2 A 
> resolution, which is still far from your hope of 1 A.
> 
>For your test case of 

Re: [ccp4bb] AW: Re: [ccp4bb] Basic Crystallography/Imaging Conundrum

[Keller, Jacob]

ction.  (e.q. a 
> sampling rate of 0.5 A for 1 A resolution data)  This is, of course, 
> the Nyquist Theorem which states that you have to sample at twice the 
> frequency of the highest resolution Fourier coefficient.
> 
>   This is exactly how an FFT works.  It allocates the memory required 
> to store the structure factors and it returns the map in that same 
> array - The number of bytes is unchanged.  It also guarantees that the 
> calculation is reversible as no information is lost in either 
> direction.
> 
>So, why does your blocky image look so bad?  First you have sampled 
> too coarsely.  You should have twice the sampling rate in each 
> direction.
> 
>The next point is more subtle.  You are displaying each voxel as a 
> block.  This is not correct.  The sharp lines that occur at the 
> boundaries between the blocks is a high frequency feature which is not 
> consistent with a 1 A resolution image.  Your sample points should be 
> displayed at discrete points since they are not the average density 
> within a block but the value of the density at one specific point.
> 
>What is the density of the map between the sampled points?  The 
> Fourier series provides all the information needed to calculate them 
> and you can calculate values for as fine a sampling rate as you like, 
> just remember that you are not adding any more information because 
> these new points are correlated with each other.
> 
>If you have only the samples of a map and want to calculate Fourier 
> coefficients there are many sets of Fourier coefficients that will 
> reproduce the sampled points equally well.  We specify a unique 
> solution in the FFT by defining that all reflections of resolution 
> higher than 1 A must be identically equal to zero.  When you calculate 
> a map from a set of coefficients that only go to 1 A resolution this 
> is guaranteed.
> 
>When you are calculating coefficients from any old map you had 
> better ensure that the map you are sampling does not contain 
> information of a higher resolution than twice your sampling rate.
> This is a problem when calculating Fcalc from an atomic model.  You 
> calculate a map from the model and FFT it, but you can't sample that 
> map at 1/2 the resolution of your interest.  You must sample that map 
> much more finely because an atomic model implies Fourier coefficients 
> of very high resolution.
> (Otherwise phase extension would be impossible)  This problem was 
> discussed in detail in Lynn Ten Eyck's 1976 paper on Fcalc FFT's but 
> is often forgotten.  Gerard Bricogne's papers on NCS averaging from 
> the 1970's also discusses these matters in great depth.
> 
>In summary, your blocky picture (even with double sampling) is not 
> a valid representation because it is not blurry like a 1 A resolution 
> map should be.  To create an accurate image you need to oversample the 
> map sufficiently to prevent the human eye from detecting aliasing 
> artifacts such as the straight lines visible in your blocky picture.
> This requires very fine sampling because the eye is very sensitive to 
> straight lines.  When using a map for any purpose other than FFTing 
> you will need to oversample the map by some amount to prevent aliasing 
> artifacts and the amount of oversampling will depend on what you are 
> doing to the map (Again, see Gerard's papers.)  Such an oversampled 
> map will have many more voxels but no more information because the 
> density values are correlated.
> 
> Dale Tronrud
> 
> On 11/9/2017 4:10 PM, Keller, Jacob wrote:
>> Dear Crystallographers,
>> 
>>  
>> 
>> I have been considering a thought-experiment of sorts for a while, 
>> and wonder what you will think about it:
>> 
>>  
>> 
>> Consider a diffraction data set which contains 62,500 unique 
>> reflections from a 50 x 50 x 50 Angstrom unit cell, with each 
>> intensity measured perfectly with 16-bit depth. (I am not sure what 
>> resolution this corresponds to, but it would be quite high even in 
>> p1, I think--probably beyond 1.0 Angstrom?). Thus, there are 62,500 x 
>> 16 bits (125 KB) of information in this alone, and there is an HKL 
>> index associated with each intensity, so that I suppose contains 
>> information as well. One could throw in phases at 16-bit as well, and 
>> get a total of 250 KB for this dataset.
>> 
>>  
>> 
>> Now consider an parallel (equivalent?) data set, but this time 
>> instead of reflection intensities you have a real space voxel map of 
>> the same
>> 50 x 50 x 50 unit cell consisting of 125,000 voxels, each of which 
>> has a 16-bit electron density value, and an associated xyz inde

Re: [ccp4bb] Basic Crystallography/Imaging Conundrum

[Keller, Jacob]

>>62500 is < 40^3, so ±20 indices on each axis.
50Å / 20 = 2.5Å,  so not quite 2.5Å resolution

Nice--thanks for calculating that. Couldn't remember how to do it off-hand, and 
I guess my over-estimate comes from most protein crystals having some symmetry. 
I don't really think it affects the question though--do you?

>>All that proves is that assigning each 1x1x1 voxel a separate density value 
>>is a very inefficient use of information.  Adjacent voxels are not 
>>independent, and no possible assignment of values will get around the 
>>inherent blockiness of the representation.

Not sure what this means--what is the precise definition or measure of 
"efficient use of information?" Like a compression algorithm? Are diffraction 
data sets like compressed data?

Also, the "blockiness" of representation is totally ancillary--you can do all 
of the smoothing you want, I think, and the voxel map will still be basically 
lousy. No?

>>I know!  Let's instead of assigning a magnitude per voxel, let's assign a 
>>magnitude per something-resolution-sensitive, like a sin wave.   Then for 
>>each hkl measurement we get one sin wave term.   Add up all the sine waves 
>>and what do you get?  Ta da.  A nice map.

It was good of proto-crystallographers to invent diffraction as a way to apply 
Fourier Series. I don't know--it seems funny to me that somehow diffraction is 
able to harness "efficient information use," whereas the voxel map is not. I am 
looking for more insight into this.

>>Aren't Fourier series marvelous?

Well, I have always liked FTs, but your explanations are not particularly 
enlightening to me yet.

I will re-iterate that the reason I brought this up is that the imaging world 
might learn a lot from crystallography's incredible extraction of all possible 
information through the use of priors and modelling.

Also, I hope you noticed that all of the parameters about the crystallographic 
data set were extremely optimistic, and in reality the information content 
would be far less.

One could compare the information content of the derived structure to that of 
the measurements to get a metric for "information extraction," perhaps, and 
this could be applied across many types of experiments in different fields. I 
nominate crystallography for the best ratio.

JPK



 
> Assuming that it is apt, however: is this a possible way to see the power of 
> all of our Bayesian modelling? Could one use our modelling tools on such a 
> grainy picture and arrive at similar results?
>
> Are our data sets really this poor in information, and we just model the heck 
> out of them, as perhaps evidenced by our scarily low data:parameters ratios?
> 
> My underlying motivation in this thought experiment is to illustrate the 
> richness in information (and poorness of modelling) that one achieves in 
> fluorescence microscopic imaging. If crystallography is any measure of the 
> power of modelling, one could really go to town on some of these terabyte 5D 
> functional data sets we see around here at Janelia (and on YouTube).
> 
> What do you think?
> 
> Jacob Keller
> 
> +
> Jacob Pearson Keller
> Research Scientist / Looger Lab
> HHMI Janelia Research Campus
> 19700 Helix Dr, Ashburn, VA 20147
> (571)209-4000 x3159
> +
> 

--
Ethan A Merritt, Dept of Biochemistry
Biomolecular Structure Center,  K-428 Health Sciences Bldg
MS 357742,   University of Washington, Seattle 98195-7742

Re: [ccp4bb] Yet another "what's my blob" thread

[Keller, Jacob]

Looks like it's at a symmetry/NCS axis, so that complicates appearances...

JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Lucas
Sent: Monday, October 02, 2017 6:06 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Yet another "what's my blob" thread

I'm in the later stages of solving a structure which contains two tetramers in 
the asymetric unit. I found these two blobs (in equivalent positions on each 
tetramer) with positively charged residues around it. Crystallization condition 
is Magnesium chloride, Bis-tris and PEG3350. While the second blob looks like a 
metal, the first one has a weird shape even though they are expected to have 
the same thing. Any ideas?

Lucas

Re: [ccp4bb] include corners in mosflm

[Keller, Jacob]

You’ve got a point about including data, but on the other hand, I would assume 
one would (almost always) set the collection parameters so as not to require 
use of the corners. And “swung out” mode is pretty atypical, so would be 
strange to set a default for it.

JPK

From: herman.schreu...@sanofi.com [mailto:herman.schreu...@sanofi.com]
Sent: Wednesday, September 27, 2017 2:44 AM
To: Keller, Jacob <kell...@janelia.hhmi.org>; CCP4BB@JISCMAIL.AC.UK
Subject: AW: [ccp4bb] include corners in mosflm

With a detector in swing-out position, one has to include the corners. Also, 
why should one discard potential data during processing? Based on the 
statistics, one can always discard data afterwards if it is not good or too 
incomplete.

HS

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Keller, 
Jacob
Gesendet: Dienstag, 26. September 2017 22:14
An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Betreff: Re: [ccp4bb] include corners in mosflm

Why on earth would one want that to be the *default*? I understand that there 
may be the odd unrepeatable dataset collected too close, or there may be 
occasionally be hardward limitations, but I cannot understand how this would be 
a recurring problem….

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of CCP4BB
Sent: Tuesday, September 26, 2017 4:11 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] include corners in mosflm

Hi Ed

I'm afraid not; that's one thing that can't be changed to a different default.
Harry
--
Dr Harry Powell
Chairman of European Crystallographic Association SIG9 (Crystallographic 
Computing)

On 26 Sep 2017, at 20:34, Edwin Pozharski 
<pozharsk...@gmail.com<mailto:pozharsk...@gmail.com>> wrote:
By default, iMosflm excludes corners from processing.  Is there a simple way to 
make it the default to go all the way to the corner instead of detector edge?  
I could of course set the max resolution for processing to some outrageous 
value that is guaranteed to be outside of the image, but perhaps I am missing a 
more intelligent option in the gui.  (I vaguely recall HKL2000 having a 
Edge/Corner/Other) radiobutton).

There is a whole separate question as to wisdom of including corners, of 
course.  Yes, adding a resolution shell with robust data will improve model 
quality even if such shell is woefully incomplete. On the other hand, it's 
possible that fill-in option for missing reflections in map calculation may 
make maps more biased. A reasonable solution to this would be to use 2 
different resolution limits in refinement and map calculation - not hard to 
script for that yet I don't know if any refinement software provides such 
option natively.
Ed.

Re: [ccp4bb] include corners in mosflm

[Keller, Jacob]

Why on earth would one want that to be the *default*? I understand that there 
may be the odd unrepeatable dataset collected too close, or there may be 
occasionally be hardward limitations, but I cannot understand how this would be 
a recurring problem….

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of CCP4BB
Sent: Tuesday, September 26, 2017 4:11 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] include corners in mosflm

Hi Ed

I'm afraid not; that's one thing that can't be changed to a different default.
Harry
--
Dr Harry Powell
Chairman of European Crystallographic Association SIG9 (Crystallographic 
Computing)

On 26 Sep 2017, at 20:34, Edwin Pozharski 
> wrote:
By default, iMosflm excludes corners from processing.  Is there a simple way to 
make it the default to go all the way to the corner instead of detector edge?  
I could of course set the max resolution for processing to some outrageous 
value that is guaranteed to be outside of the image, but perhaps I am missing a 
more intelligent option in the gui.  (I vaguely recall HKL2000 having a 
Edge/Corner/Other) radiobutton).

There is a whole separate question as to wisdom of including corners, of 
course.  Yes, adding a resolution shell with robust data will improve model 
quality even if such shell is woefully incomplete. On the other hand, it's 
possible that fill-in option for missing reflections in map calculation may 
make maps more biased. A reasonable solution to this would be to use 2 
different resolution limits in refinement and map calculation - not hard to 
script for that yet I don't know if any refinement software provides such 
option natively.
Ed.

Re: [ccp4bb] Risk assessment for heavy atom soaking - examples?

[Keller, Jacob]

Well, you can add to your list Silver sulfadiazine, mercurochrome, and 
merthiolate, all OTC antiseptics. The mercurochrome, since it contains Br, 
might be used as another standard for the Br edge.

JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of James 
Holton
Sent: Tuesday, September 12, 2017 1:46 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Risk assessment for heavy atom soaking - examples?

One more correction,

It seems brominated vegetable oil (BMO) really does contain Br atoms!  I could 
have sworn I read in some reputable source long ago that the process of 
"bromination" was an old term for general reduction of double bonds and did not 
necessarily involve bromine. Usually hydrogen.  I remembered this because I 
thought it was hugely counter-intuitive.  Now, of course, I cannot find that 
reference. So, who am I to pit the validity of my memory against Wikipedia and 
a long list of links to health-nut web blogs?  Guess I was wrong about that.

The Mountain Dew I am drinking right now has a very faint X-ray fluorescence 
peak that could be Br.  Hard to be sure above background. So I will have to get 
a sample of neat BMO to sit next to my shampoo, pepto and sunscreen on my shelf 
of heavy atom compounds that are on the FDA's GRAS list:
https://www.fda.gov/food/ingredientspackaginglabeling/gras/

Remarkably, the MSDS for BMO is less scary than that of ordinary vegetable oil. 
 This raises more than one interesting topic, but the most relevant here I 
think is "bio-availability".  Selenomethionine is much much more bioavailable 
than selenium sulfide, which is the active ingredient in my dandruff shampoo.  
Apparently, humans don't absorb it, but microorganisms can mistake it for a 
source of sulfur.

I expect the bio-availability of Hg in pizza is pretty high considering how it 
bio-amplifies in fish, so I stand by my APE.  But it is always prudent to read 
the MSDS before you open a bottle, and then read the MSDS of something similar 
just to put it in perspective.

-James Holton
MAD Scientist

On 9/6/2017 12:59 PM, James Holton wrote:
> Was just pointed out to me off-list that my anchovy data was off, so I 
> just double-checked the FDA website:
>
> https://www.fda.gov/food/foodborneillnesscontaminants/metals/ucm115644
> .htm
>
>
> Turns out the latest number for anchovies is 0.016 ppm, or 0.5 ug per 
> ounce (28g).
>
> So, if you use a whole 2 oz can, that's still ~ 1 microgram Hg as the 
> Anchovie Pizza Equivalent.
>
> And it looks like one piece of bigeye tuna sushi could be as much as 
> ~14g*1.816ppm = 25 APEs
>
> -James Holton
> MAD Scientist
>
> On 9/6/2017 11:44 AM, James Holton wrote:
>> Something that could perhaps be of use here is what I like to call 
>> the "Anchovie Pizza Equivalent" (APE), which is about 1 microgram of 
>> mercury.  According to the Food and Drug Administration website here 
>> in the USA the average mercury content of anchovies is 0.34 ppm, 
>> which is about 1 microgram per ounce of fish.  Tuna can be higher, 
>> but varies a lot from fish to fish.  My point here is that most 
>> institutions regard the amount of mercury you bring onsite for 
>> purposes of eating for lunch, be it sushi or pizza, as small enough 
>> to be negligible.  I tend to agree.  So, one could argue that 1 
>> microgram of Hg per day is a "safe amount".  Especially if you don't 
>> eat it.
>>
>> In terms of protein crystals, a 100 micron wide cube has a volume of
>> 1 nanoliter, and if it were soaked to a final concentration of 50 mM 
>> Hg that is 1e-9 L * 50e-3 mol/L *200 g/mol = 10 ng.  So, 100 protein 
>> crystals soaked with Hg add up to roughly 1 APE.  Please note that I 
>> am in no way encouraging you to eat your protein crystals, and 
>> especially not the solutions you soak them in.  You should do your 
>> own APE calculations for those.  But I do think it important to note 
>> just how tiny the amount of metal in our crystals really is.
>>
>> Now, mercury is purportedly the second-most-toxic metal after 
>> Plutonium.  But Pu derivatives are uncommon.  In fact, until recently
>> (4zhd) Pu derivatives were unheard of. The authors I'm sure will tell 
>> you 4zhd involved no small amount of paperwork. But as long as you 
>> are not working with Pu, you can regard every other metal as less 
>> toxic than Hg.
>>
>> Another good example is selenium; by far the most common metal 
>> derivative.  Although toxic, Se is also a dietary requirement. I 
>> suppose this is an excellent demonstration of what "moderation"
>> really means.  The Recommended Daily Allowance (RDA) of selenium in 
>> the USA for adult men and pregnant women is 55-60 micrograms per day.  
>> In crystals, one Se atom per 100 amino acids at 50% solvent comes to 
>> an overall concentration of 50 mM.  So, a 100 micron crystal contains 
>> about 4 ng of Se.  It would take 15,000 such crystals to add up to 
>> the US RDA.  The synchrotrons I work at don't go 

Re: [ccp4bb] Unknown positive electron density

[Keller, Jacob]

Fourier truncation ripples?

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of CRAIG A 
BINGMAN
Sent: Monday, August 21, 2017 6:20 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Unknown positive electron density

Betty,

I think that f’’ for Ba at this wavelength is around 3.5 electrons, and f'' for 
As is expected to be about 3 electrons. Is there a nearby crystallographic 
symmetry axis?

Craig

On Aug 21, 2017, at 5:05 PM, Betty Chu 
> wrote:

Hi Craig,
The data collection wavelength was 0.92 Angstroms. Since we observe anomalous 
signal for Ba at this wavelength, we would expect greater anomalous signal if 
As were present. There is a possibility for weak anomalous signal in this 
positive density, but the weak anomalous signal only shows up if I try to model 
a Ba in the density. Without modelling anything, there is no anomalous signal.

This is what the map looks like after one round of refinement with the Ba in 
the density. But since there are waters that are 1.6 Angstroms, 1.9 Angstroms, 
and 2.2 Angstroms away from the Ba, which is smaller than the coordination 
distance between Ba and water, we are skeptical of the Ba being there.

https://cbsostorage.chem.umd.edu/owncloud/index.php/s/O4lI2iKvRsQUoRo

Thank you,
Betty

On Mon, Aug 21, 2017 at 5:21 PM, CRAIG A BINGMAN 
> wrote:
What is the data collection wavelength/energy? Would you expect significant 
anomalous diffraction from As at this wavelength?

On Aug 21, 2017, at 11:37 AM, Betty Chu 
> wrote:

Hi Shailesh,
When I modelled in the Barium ion with octahedrally coordinated waters and ran 
the refinement, the distances from the barium to some of the waters ended up 
being too close (<2.2 Angstroms). Also, the positive electron density is 
connected. If the density indicated barium with coordinated waters, would that 
mean there are multiple ones present in the positive density?
Here are more views of the connected positive density.

https://cbsostorage.chem.umd.edu/owncloud/index.php/s/j5CgZIJe6NKgUlZ

https://cbsostorage.chem.umd.edu/owncloud/index.php/s/YbiNFbT8AW1iHUQ

On Mon, Aug 21, 2017 at 12:00 PM, Shailesh Tripathi 
> wrote:
Looks like Ba2+. Since it exist with coordination number 6 or above check what 
geometry water is following there (trigonal bipiramidal or so on). Water might 
also be shared by symmetry related Ba cation.



Shailesh Kumar Tripathi,
Phone: 9686289668


On Mon, Aug 21, 2017 at 9:17 PM, Betty Chu 
> wrote:
Yes, I have. The cacodylate ion does not fit well into the density.

On Mon, Aug 21, 2017 at 11:44 AM, Pradeep Pallan 
> wrote:
Did you try modelling in a cacodylate ion (CH3)2AsO2-?

On Mon, Aug 21, 2017 at 10:19 AM, Betty Chu 
> wrote:
Dear ccp4bb,
I am refining a 1.40 Angstrom data set for a DNA oligonucleotide. While the 
model for the DNA fits very well into the density, there is a patch of positive 
electron density in the solvent space that we are having trouble with.

The screenshot can be viewed through this link:
https://cbsostorage.chem.umd.edu/owncloud/index.php/s/J5cKnOpCC4vb1VC

In the screenshot, the yellow color is the anomalous map and a barium ion is 
fitted into density near the positive green electron density.

The oligonucleotide was purchased from IDT. The crystallization condition is 
15% MPD, 120 mM BaCl2, and 30 mM NaCaC pH 6.4. I have tried modelling Ba2+ with 
coordinated waters, MPD, and sodium cacodylate into the electron density, but 
none of those fit well.
Any suggestions regarding the identity of this electron density is much 
appreciated. Thank you!

Sincerely,
Betty Chu
Paukstelis Research Group
Department of Chemistry and Biochemistry
University of Maryland, College Park



--

---
Pradeep Pallan

Re: [ccp4bb] Primer design

[Keller, Jacob]

>No need of the whole exome. Sequencing The second PCR product will do the job 
>I guess. Second PCR (from the cDNA pool) with specific forward primer and and 
>oligodA reverse primer. Surely a matter of less than $3 

$3 is a major understimation, but I see your point. On the other hand, it is 
important to consider new technologies, and it would be a service to the 
scientific community to publish the exome somewhere, so other researchers would 
not have to spend their $3.

JPK





Best,

DKG


- Original Message -
From: "Jacob Keller" 
To: "Debasish Kumar Ghosh" , CCP4BB@JISCMAIL.AC.UK
Sent: Monday, July 24, 2017 6:48:25 PM
Subject: RE: Primer design

Or sequence the whole exome for what, $500-1000?

JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Debasish 
Kumar Ghosh
Sent: Monday, July 24, 2017 7:43 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Primer design

Dear Syed,

The process is very trivial to clone your gene of interest. Assuming your gene 
is transcribed as mono-cistronic mRNA, take the oligodT primer as reverse 
primer. First isolate the total RNA from the tissue or cells and do the cDNA 
synthesis using oligodT primer followed by gene specific PCR with forward 
primer and oligodA primer from the cDNA pool. Sequence the PCR product to get 
to know the first stop codon in the ORF. Making the specific reverse primer 
from the sequence is then just matter of time.  
Hope this helps.

Best wishes,

Debasish

CSIR- Senior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group Centre for DNA Fingerprinting and 
Diagnostics Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab) Lab URL: 
http://www.cdfd.org.in/labpages/computational_functional_genomics.html



- Original Message -
From: "syed ibrahim" <048c02cac012-dmarc-requ...@jiscmail.ac.uk>
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, July 24, 2017 4:56:00 PM
Subject: [ccp4bb] Primer design

Hello All

I am interested in cloning a gene from a plant. I searched the database only 
partial sequence is available, ie: for 160 residues only. The full length of 
the protein is around 570 residues. I designed forward primer and I have no 
clue to design reverse primer.

Any help

Thank you

Syed

Re: [ccp4bb] Primer design

[Keller, Jacob]

Or sequence the whole exome for what, $500-1000?

JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Debasish 
Kumar Ghosh
Sent: Monday, July 24, 2017 7:43 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Primer design

Dear Syed,

The process is very trivial to clone your gene of interest. Assuming your gene 
is transcribed as mono-cistronic mRNA, take the oligodT primer as reverse 
primer. First isolate the total RNA from the tissue or cells and do the cDNA 
synthesis using oligodT primer followed by gene specific PCR with forward 
primer and oligodT primer from the cDNA pool. Sequence the PCR product to get 
to know the first stop codon in the ORF. Making the specific reverse primer 
from the sequence is then just matter of time.  
Hope this helps.

Best wishes,

Debasish

CSIR- Senior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group Centre for DNA Fingerprinting and 
Diagnostics Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab) Lab URL: 
http://www.cdfd.org.in/labpages/computational_functional_genomics.html



- Original Message -
From: "syed ibrahim" <048c02cac012-dmarc-requ...@jiscmail.ac.uk>
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, July 24, 2017 4:56:00 PM
Subject: [ccp4bb] Primer design

Hello All

I am interested in cloning a gene from a plant. I searched the database only 
partial sequence is available, ie: for 160 residues only. The full length of 
the protein is around 570 residues. I designed forward primer and I have no 
clue to design reverse primer.

Any help

Thank you

Syed

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Keller, Jacob]

>Ideally you should convert polarization to anisotropy. Simple enough – but 
>some referees can get picky…

What is the argument for anisotropy being better?

JPK

Re: [ccp4bb] Fine Phi Slicing

[Keller, Jacob]

Based on this, a vision for the future:

A warehouse filled with sealed-tube, top-hat-profiled sources, super-accurate 
goniostats, and Dectris detectors, a robot running back and forth from a 
central dewar to place the crystals, all images 1-bit, intensities measured as 
probabilities; a day when crystal-frying will finally come to an end, or will 
be used routinely (as RIP) as the sure-fire way to solve crystal structures.

JPK



-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of James 
Holton
Sent: Thursday, July 20, 2017 2:07 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Fine Phi Slicing

An important aspect of fine phi slicing that has not been mentioned yet (and 
took me a long time to figure out) is the impact of read-out time.  
Traditionally, read-out time is simply a delay that makes the overall data 
collection take longer, but with so-called "shutterless" data collection the 
read-out time can have a surprising impact on data quality.  It's 2 ms on my 
Pilatus3 S 6M.  This doesn't sound like much, and indeed 2 ms is also the 
timing jitter of my x-ray shutter, which had not been a problem with CCD 
detectors for 15 years.  The difference is that with so-called "shutterless" 
data collection not only can appreciable intensity fall into this 2 ms hole, 
but none of the data processing programs have a way to "correct" for it.  What 
you end up with is Rmerge/Rmeas values of 15-30% in the lowest-angle bin, and 
correspondingly low overall I/sigma.  At first,  I couldn't even solve lysozyme 
by S-SAD!  This had been an easy task with the Q315r I had just replaced.  The 
difference turned out to be "noise" coming from this read-out gap.

The 2 ms gap between images is only important if it is comparable to the time 
it takes a relp to transit the Ewald sphere.  At 1 deg/s and mosaic spread of 
0.5 deg this is 0.002 deg of missing data, or about 1% error in integrated 
intensity.  This is fine for most applications.  But if you are turning at 25 
deg/s with a room-temperature crystal of mosaicity
0.05 deg, then you could loose the spot entirely in a 2 ms read-out gap (100% 
error).  This is one of several arguments for fine phi slicing, where you make 
sure that every spot is not just observed, but split over
2-3 images.  This also helps the pile-up correction Gerd already mentioned.  
What is often overlooked, however, is that the error due to read-out gap is 
only relevant to partials.  Fulls don't experience it at all, so wide phi 
slicing is practically immune to it.  But with fine phi slicing everything is a 
partial, and 100% of the spots are going to take on read-out-gap error. So, 
what is the solution?  Slow down.

The problem with slowing down the spindle, of course, is radiation damage.  If 
you've got a flux of 1e12 photons/s into a 100x100 micron beam spot and 1 A 
wavelength you are dosing metal-free protein crystals at about 50 kGy/s.  Most 
room-temperature crystals can endure no more than 200 kGy, so they will live 
for about 4 seconds in this beam.  A detector framing at 25 Hz will only get 
100 images, no matter what the spindle speed. The decision then: is it better 
to get 100 deg at 1 
deg/image?  or 2.5 deg with fine phi slicing?   That is, if the mosaic 
spread is 0.05 deg, you can do no more than 0.025 deg/image and still barely 
qualify as "fine phi slicing". The 25 Hz framing rate dictates no less than 40 
ms exposures, and that means turning the spindle at (0.025 deg/ 0.04 s) = 0.625 
deg/s. Thus, we cover 2.5 deg in the 4 seconds before the crystal dies.  That's 
just algebra.  The pragmatic consequence is the difference between getting a 
complete dataset from one crystal and needing to merge 40 crystals.

Of course, you can attenuate 40x and get 100 fine-sliced degrees, but that will 
take 40x more beam time.  The images will also be 40x weaker.  
In my experience you need at least an average of 1 photon/pixel/image before 
even the best data processing algorithms start to fall over.  You can actually 
calculate photons/pixel/image beforehand if you know your flux and how thick 
your sample is:

photons/pixel = 1.2e-5*flux*exposure*thickness/pixels

where flux is in photons/s, exposure in seconds, thickness in microns and 
1.2e-5 comes from the NIST elastic scattering cross section of light atoms (C, 
N, O are all ~ 0.2 cm^2/g), the rough density of protein crystals (1.2 g/cm^3), 
and the fact that about half of all scattered photons land on a flat detector 
at typical distance from the sample, or: 
0.2*1.2*(1e-4 cm/um)/2 = 1.155e-5

So, if your flux is 1e12 photons/s and your sample is 100 um thick you will get 
~1 photon/pixel on a 6M in about 5 ms.  That corresponds to a framing rate of 
200 Hz.  If the detector can't go that fast, you need to attenuate.  Note this 
is the total sample thickness, including the stuff around the crystal.  Air 
scatter counts as 1 micron of sample thickness for every mm of air between 

Re: [ccp4bb] Pilatus Issues

[Keller, Jacob]

Regarding daftness, it seems that the detector is wider than tall, which should 
improve the ratio of Lorentz-problematic reflections to good, fast-moving ones. 
So I assume it was a choice between that and excluding some spots in the 
gap--an appropriate calculation could be done to see whether this is 
appropriate.

I was curious about the polarization issue you mentioned: how does horizontal 
orientation speak to polarization? I don't understand the connection.

All the best,

Jacob Keller



-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Gerard 
Bricogne
Sent: Saturday, July 15, 2017 5:31 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Pilatus Issues

Dear John,

 Having just seen Andreas's message regarding the best source of support to 
address your enquiry, I have a further remark to make about your instrument.

 As this is a lab instrument, the Omega axis would be vertical, and indeed 
the beam stop shadow (vertical on the top module) and the diffuse shadow of the 
sample holder (vertical on the bottom module) would confirm this. This being 
the case, it is quite simply *daft* to have the gap between the two modules 
being horizontal. That is done on purpose on synchrotron beamlines because of 
the polarisation of the beam (which is why Omega is horizontal on such 
beamlines), but in a lab system the gap should be in the vertical direction. As 
currently placed in your system, this gap is cutting into perfectly good data, 
whereas if it were vertical instead, it would only cut out data that are 
getting perilouly close to the cusp anyway.

 You should ask the manufacturer of your diffractometer to rotate your 
detector by 90 degrees! Someone in the OEM world forgot about the Lorentz 
factor ;-) .


 With best wishes,
 
  Gerard.

--
On Fri, Jul 14, 2017 at 05:14:03PM +0100, John Hardin wrote:
> Hi,
> 
> We have recently noticed an issue with our Pilatus (biased pixels/vertical 
> lines).
> I was curious as to whether anyone else has seen this or might know what 
> could have caused it?
> 
> Best,
> John
> 

Re: [ccp4bb] Fine Phi Slicing

[Keller, Jacob]

Hi Graeme,

I see your point about the blind region and also the tile lines. But 2-theta 
would have the advantage of also shifting the low-res spots to entirely new 
pixels, which would be harder through rotation. Also, wouldn't rotating about 
the beam axis shift the spots to variable degrees across rotation space, with 
some angles (+/- 90 deg) negligibly shifted?

Further, does it give anyone pause: Graeme makes a subtle implication that most 
samples die before collecting 360 degrees, which I think may be true. What can 
be done about this tragic lack of attenuation? One possibility is to model the 
radiation damage in refinement, but wouldn't it make a lot more sense to have a 
lot of good attenuators installed by default (or use sealed-tube sources!).

JPK



-Original Message-
From: graeme.win...@diamond.ac.uk [mailto:graeme.win...@diamond.ac.uk] 
Sent: Friday, July 14, 2017 1:37 AM
To: Keller, Jacob <kell...@janelia.hhmi.org>
Cc: ccp4bb@jiscmail.ac.uk
Subject: Re: [ccp4bb] Fine Phi Slicing

Jacob

If you have a complete 360 deg data set and your sample is still alive, and you 
have a multi-axis gonio, I would recommend rotating the crystal about the beam 
(ideally by ~ maximum scattering 2-theta angle) and collecting again. This 
would record your blind region as well as moving the reflections to different 
pixels, and (as a bonus) also will move reflections out from the tile join 
regions into somewhere they can be measured, which would not happen for small 
2-theta shift.

See http://scripts.iucr.org/cgi-bin/paper?BA0020 Figure 16 as excellent 
illustration of this.

Biggest risk with this is getting *moving* shadows on the data on the second 
run, as an effective 45-50 degree chi shift (say) will usually be a pretty wide 
opening angle for a kappa gonio. XDS and DIALS both have mechanisms to deal 
with this, and automated processing packages are able to apply these given a 
reasonable understanding of the beamline.

Also saves building 2-theta axes which can handle 92 kg ;o)

Cheers Graeme

On 13 Jul 2017, at 21:00, Keller, Jacob 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> wrote:

I thought there was a new paper from the Pilatus people saying fine slicing is 
worth it even beyond the original 1/2 mosaicity rule?

I would think, actually, more gains would made by doing light exposures at, 
say, 1/3 mosaicity, collecting 360 deg, then shifting the detector in 2theta by 
a degree or two to shift uniformly the spots to new pixels, maybe accompanied 
by a kappa change. One would have to remember about the two-theta when 
processing, however!

JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Gerd 
Rosenbaum
Sent: Thursday, July 13, 2017 3:40 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] weird diffraction pattern

Dear Gerard,

   my "sound like a sales person" was meant as poking a little fun - nothing 
serious, of course.

I and our users like our not-so-new-anymore Pilatus3 6M. It's a great detector 
in many ways. But, there is a lot of hype that this detector solves 
all-problem, for instance fine slicing that is claimed to be only possible with 
a pixel array detector. People get carried away and use
0.01 degree slices even as the mosaicity of their sample is, say, 0.3 degree. 
Slicing beyond 1/3 of the mosaicity will gain you very little - only more 
frames, more processing time.

This discourse is already drifting away from the original topic of the thread 
so I will comment on the other arguments  you made like resolution in a private 
e-mail.

Best regards,

Gerd

On 13.07.2017 14:00, Gerard Bricogne wrote:
Dear Gerd,

 I can assure you that I have no shares in Dectris nor any commecial 
connections with them. What I do have is a lot of still vivid memories of CCD 
images, with their wooly point-spread function that was affected by 
fine-grained spatial variability as well as by irredicible inaccuracies in the 
geometric corrections required to try and undo the distortions introduced by 
the fiber-optic taper. By comparison the pixel-array detectors have a very 
regular structure, so that slight deviations from exact registering of the 
modules can be calibrated with high accuracy, making it possible to get very 
small residuals between calculated and observed spot positions. That, I 
certainly never saw with CCD images.

 I do think that asking for the image width was a highly pertinent question 
in this case, that had not been asked. As a specialist you might know how to 
use a CCD to good effect in fine-slicing mode, but it is amazing how many 
people there are still out there who are told to use 0.5 or even 1.0 degree 
image widths.

 Compensating the poor PSF of a CCD by fine slicing in the angular 
dimension is a tall order. With a Pilatus at 350mm from the crystal, the 
angular separation between 174-micron pixels is 0.5 milli

[ccp4bb] Fine Phi Slicing

[Keller, Jacob]

I thought there was a new paper from the Pilatus people saying fine slicing is 
worth it even beyond the original 1/2 mosaicity rule?

I would think, actually, more gains would made by doing light exposures at, 
say, 1/3 mosaicity, collecting 360 deg, then shifting the detector in 2theta by 
a degree or two to shift uniformly the spots to new pixels, maybe accompanied 
by a kappa change. One would have to remember about the two-theta when 
processing, however!

JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Gerd 
Rosenbaum
Sent: Thursday, July 13, 2017 3:40 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] weird diffraction pattern

Dear Gerard,

my "sound like a sales person" was meant as poking a little fun - nothing 
serious, of course.

I and our users like our not-so-new-anymore Pilatus3 6M. It's a great detector 
in many ways. But, there is a lot of hype that this detector solves 
all-problem, for instance fine slicing that is claimed to be only possible with 
a pixel array detector. People get carried away and use
0.01 degree slices even as the mosaicity of their sample is, say, 0.3 degree. 
Slicing beyond 1/3 of the mosaicity will gain you very little - only more 
frames, more processing time.

This discourse is already drifting away from the original topic of the thread 
so I will comment on the other arguments  you made like resolution in a private 
e-mail.

Best regards,

Gerd

On 13.07.2017 14:00, Gerard Bricogne wrote:
> Dear Gerd,
>
>   I can assure you that I have no shares in Dectris nor any 
> commecial connections with them. What I do have is a lot of still 
> vivid memories of CCD images, with their wooly point-spread function 
> that was affected by fine-grained spatial variability as well as by 
> irredicible inaccuracies in the geometric corrections required to try 
> and undo the distortions introduced by the fiber-optic taper. By 
> comparison the pixel-array detectors have a very regular structure, so 
> that slight deviations from exact registering of the modules can be 
> calibrated with high accuracy, making it possible to get very small 
> residuals between calculated and observed spot positions. That, I 
> certainly never saw with CCD images.
>
>   I do think that asking for the image width was a highly 
> pertinent question in this case, that had not been asked. As a 
> specialist you might know how to use a CCD to good effect in 
> fine-slicing mode, but it is amazing how many people there are still 
> out there who are told to use 0.5 or even 1.0 degree image widths.
>
>   Compensating the poor PSF of a CCD by fine slicing in the 
> angular dimension is a tall order. With a Pilatus at 350mm from the 
> crystal, the angular separation between 174-micron pixels is 0.5 milliradian.
> To achieve that separation in the angular (rotation) dimension, the 
> equivalent image width would have to be 0.03 degree. For an EIGER the 
> numbers become 75 microns, hence 0.21 milliradian i.e. 0.012 degree.
>
>   Hence my advice, untainted by any commercial agenda :-) .
>   
>   
>   With best wishes,
>   
>Gerard.
>
> --
> On Thu, Jul 13, 2017 at 01:25:08PM -0500, Gerd Rosenbaum wrote:
>> Dear Gerard,
>>
>> you sound like a sales person for Dectris. Fine slicing is perfectly 
>> fine with CCD detectors - it takes a bit longer because of the step 
>> scan instead of continuous scan. The read noise issue is often 
>> overstated compared to the sample induced scatter background. If for 
>> fine slicing at 0.05 degree or less the diffraction peaks go too 
>> close to the read noise make a longer exposure - signal goes up, 
>> ratio signal to sample-induced-BG less, as for any fine slicing, same read 
>> noise.
>>
>> It would be helpful to analyze the dense spot packing along layer 
>> lines if we knew the wavelength and the sample-to-detector distance 
>> (assuming this is a 300 mm detector) and the rotation width - as you 
>> pointed out. That would help to distinguish between multiple crystals 
>> (my guess) and lattice translocation disorder. Fine slicing is 
>> definitely needed to figure out what the diffraction pattern at 120 
>> degree could tell you in terms of strong anisotropy .
>>
>> Best regard.
>>
>> Gerd
>>
>> On 13.07.2017 08:20, Gerard Bricogne wrote:
>>> Dear Tang,
>>>
>>>   I noticed that your diffraction images seem to have been 
>>> recorded on a 3x3 CCD detector. With this type of detector, fine 
>>> slicing is often discouraged (because of the readout noise), and yet 
>>> with the two long cell axes you have, any form of thick (or only 
>>> semi-fine) slicing would result in spot overlaps.
>>>
>>>   What, then, was your image width? Would you have access to a 
>>> beamline with a Pilatus detector so that you could collect 
>>> fine-sliced data?
>>>
>>>   I would tend to agree with Herman that your crystals might be 
>>> cursed with lattice translocation 

Re: [ccp4bb] crystallization optimization

[Keller, Jacob]

I’d be curious whether anyone has ever published an empirical phase diagram 
that looks like the one posted here, since I think real experiments have a lot 
more free parameters than those included in the phase diagram.

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Oganesyan, Vaheh
Sent: Thursday, July 13, 2017 9:50 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] crystallization optimization

What I’m about to write should be referred as a question rather than an answer. 
However, it might also help to find the answer to crystallization question 
discussed here.
The good old crystallization diagram so far for me was something that I’d look 
after successful crystallization story and find in which direction my 
optimization went. Each condition in every screen is just a point at the 
diagram. Were on the diagram that point is situated you don’t know because the 
scales of X and Y axes are unknown. You can find those scales by deliberately 
setting up similar screens with diluted (or concentrated, or both) of protein 
sample (Y axis scale) and diluted (mostly) crystallization screen. This is the 
way I can make use of the crystallization diagram. Unfortunately, often we 
cannot spare enough protein to do so. In such cases going through different 
screens and looking for similar conditions sometime allows finding horizontal 
line on which your crystallization position should be. After this few 
optimization attempts at different protein concentrations may help finding 
position on the diagram and clues where to go.

I hope what I just wrote makes sense. If there is a better way of using 
crystallization diagram I’d love to hear.

Regards,

Vaheh Oganesyan
www.medimmune.com

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Philippe 
BENAS
Sent: Thursday, July 13, 2017 12:47 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] crystallization optimization

Dear all,

I fully agree with all the suggestions, but it seems that no one has raised the 
issue of the solubility curve changes on the pH. If the dilution of the protein 
or precipating agent can indeed modify starting and the equilbrium points on 
the phase diagram, I would also suggest trying various pH as they can change a 
whole lot of the net protein charge, therefore the corresponding solubility 
curve and nucleation zone and hence the entire corresponding phase diagram (for 
more info PubMed search with "Madeleine Riess-Kautt" as keywords, a great 
scientist who dedidacted her career to understanding of the so-called 
Hofmeister series).

All the best,
Philippe


Philippe BENAS, Ph.D.
Dog in the manger
"Un importun survient qui trouble l'intimité, qui arrête l'expansion, qui glace 
le plaisir, - probablement comme un étranger tombant au milieu d'enfants en 
train de danser une ronde", Alfred Delvau, Dictionnaire de la langue verte 
(1866).

Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS
Faculté de Pharmacie, Université Paris Descartes
Case 48
Av, de l'Observatoire
F-75270 PARIS cedex 06
+33.1.5373.1599
E-mails: 
philippe.be...@parisdescartes.fr, 
philippe_be...@yahoo.fr
URLs: 
http://lcrbw.pharmacie.univ-paris5.fr/
 , 
http://lcrbw.pharmacie.univ-paris5.fr/spip.php?article18




De : Patrick Shaw Stewart >
À : CCP4BB@JISCMAIL.AC.UK
Envoyé le : Mercredi 12 juillet 2017 17h28
Objet : Re: [ccp4bb] crystallization optimization


Alun

I agree Frank's point is very interesting - and he intriguingly refers us to 
the phase diagram.

Is the point that Line A is longer than Line B ?

Best wishes

Patrick




[Inline images 2]






On 12 July 

Re: [ccp4bb] weird diffraction pattern

[Keller, Jacob]

You've got multiple lattices--try seeding approaches mentioned in a 
recent/current thread.

JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ???
Sent: Thursday, July 13, 2017 3:56 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] weird diffraction pattern

hello everyone, 
I would like to seek your opinion on my crystal hits. I am working on a 
helicase 

of which the native structure is solved and the all solution statistics are 

fine. I am trying to crystallize and solve the structure of the protein/ssDNA 

complex. I recently got some hits from commercial screens using sitting drop 

vapor diffusion. After crystallization optimization, these crystals diffract 

weakly but to 3.2 Angstroms for the longer exposure time. However, when the 

crystals rotate between 120 degrees to 180 degrees, the spots become streaky

(attached), no matter the crystals are hexagonal or flaky. I have tried to 

determine the structure by molecular replacement method, but the Rwork/Rfree 

values are huge (above 0.5) and can’t be reduced further. I suspect the 

obtained crystals quality and resulting processed statistics is the reason for 

the observed high Rwork/Rfree values. Are there any suggestions?

All comments will be appreciated!

Best,
Chenjun Tang

Re: [ccp4bb] Problem with a cell content

[Keller, Jacob]

Still seems to me that the resolution could and should be pushed a little 
further at least—CC1/2 is still high, completeness is good, I/sigma also is 
good. Why not extend a little further, say to where one of these values gets 
too low? Might improve the maps a bit.

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Koromyslova, Anna
Sent: Tuesday, July 11, 2017 3:37 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Problem with a cell content

Sorry for the confusion, NCS was found only in a dataset where the cell 
dimensions were twice bigger regardless of the sp, and with a dimer as a 
solution. The solvent content was the same.
Peak Distance   Vector
73.3% 143.8Å:   FRAC +0.000 +0.000 -0.500   (ORTH   -0.00.0 -143.8).
There was no NCS in a dataset with a smaller unit cell and sp P6222.
Thank you!

Best regards,

Anna



Dr. Anna Koromyslova, Postdoctoral researcher
German Cancer Research Center (DKFZ), F150
Im Neuenheimer Feld 242
D-69120 Heidelberg
Germany


From: Eleanor Dodson 
>
Date: Tuesday, July 11, 2017 at 8:17 PM
To: "Koromyslova, Anna" 
>
Cc: "CCP4BB@JISCMAIL.AC.UK" 
>
Subject: Re: [ccp4bb] Problem with a cell content

So SG  could be P31 2 2
What is the height of NCS vector v origin?
Eleanor
Look at hklview to see hk i  sections. Obviously all l = odd will be weak.


On 11 July 2017 at 19:12, Koromyslova, Anna 
> 
wrote:
Dear Eleanor,

NCS translation vector = 0 0 -0.5 in the final sp P6222, and in P622 or C121. 
All had the same solution.
The protein tends to form a homodimer and if I use P1 space group molecular 
replacement can find several dimers, there is no translational ncs found during 
MR, but still solvent cell content was similarly high.
Within a protein monomer there are no similar domains.

Best regards,
Anna



From: CCP4 bulletin board > 
on behalf of Eleanor Dodson 
>
Reply-To: Eleanor Dodson 
>
Date: Tuesday, July 11, 2017 at 7:29 PM
To: "CCP4BB@JISCMAIL.AC.UK" 
>
Subject: Re: [ccp4bb] Problem with a cell content

This is very strange . If you have a large non- crystallographic translation 
vector you would expect either to have two molecules in the asymmetric unit or 
your one molecule must have two very similar domains?
What is the n-c translation vector?

Could you have assigned too high symmetry ? SG maybe P 62? That could explain 
the packing clashes Z scores of 27 are pretty good.

Eleanor


On 11 July 2017 at 18:05, Oganesyan, Vaheh 
> wrote:
Похоже, что вы уже решили структуру. Почему вас беспокоит плотность ваших 
кристаллов? Кристаллы бывают разные.

First of all Fab by itself is already almost 50 kDa, so complex with antigen 
should be more than 50 kDa. Because you already solved the structure calculate 
the molecular mass based on your pdb file and rerun Matthews with correct mass. 
New numbers may be quite a bit different. Good indication of relatively low 
crystal density and consequently loose packing is the resolution of your data 
set. If you did not throw away data beyond 2.9A I’d suggest use them all. The 
reflections are too valuable to throw away. If data beyond some resolution is 
weak then they will have low contribution to the structure. Best if you 
calculate electron density maps at different resolutions at the end of 
refinement, compare them and use resolution that makes difference.



Regards,

Vaheh
8-5851

From: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Koromyslova, Anna
Sent: Tuesday, July 11, 2017 12:32 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Problem with a cell content

Dear CCP4 members,

I am working on a structure of a protein in complex with an antibody fragment 
(approx. 50kDa together). Molecular replacement with closely related proteins 
always comes up with one complex in the asymmetric unit, although MW of protein 
to which Matthews applies is 125kDa and corresponds to two complexes.
Phaser gives two warnings:
Large non-origin Patterson peak indicates that translational NCS is present.
Solutions with Z-scores greater than 27.2 (the threshold indicating a definite 
solution) were rejected for failing packing test

I couldn’t get a solution with two subunits although I have tried multiple 
combinations including only conserved parts of both proteins and different 
space groups including P1. Phenix Autobuild also yielded 

Re: [ccp4bb] Rmergicide Through Programming

[Keller, Jacob]

in Table 1, 
essentially for exactly the same reasons.

I also strongly support Francis Reyes comment about the usefulness of Rmerge at 
low resolution, and I would add to his list that it can also, in some 
circumstances, be more indicative of the wrong choice of symmetry (too high) 
than the statistics that come from POINTLESS (excellent though that program 
is!).

Andrew
On 5 Jul 2017, at 05:44, Graeme Winter 
<graeme.win...@gmail.com<mailto:graeme.win...@gmail.com><mailto:graeme.win...@gmail.com><mailto:graeme.win...@gmail.com>>
 wrote:

HI Jacob

Yes, I got this - and I appreciate the benefit of Rmeas for dealing with 
measuring agreement for small-multiplicity observations. Having this *as well* 
is very useful and I agree Rmeas / Rpim / CC-half should be the primary 
“quality” statistics.

However, you asked if there is any reason to *keep* rather than *eliminate* 
Rmerge, and I offered one :o)

I do not see what harm there is reporting Rmerge, even if it is just used in 
the inner shell or just used to capture a flavour of the data set overall. I 
also appreciate that Rmeas converges to the same value for large multiplicity 
i.e.:

Overall  InnerShell  OuterShell
Low resolution limit   39.02 39.02  1.39
High resolution limit   1.35  6.04  1.35

Rmerge  (within I+/I-) 0.080 0.057 2.871
Rmerge  (all I+ and I-)0.081 0.059 2.922
Rmeas (within I+/I-)   0.081 0.058 2.940
Rmeas (all I+ & I-)0.082 0.059 2.958
Rpim (within I+/I-)0.013 0.009 0.628
Rpim (all I+ & I-) 0.009 0.007 0.453
Rmerge in top intensity bin0.050- -
Total number of observations 1265512 16212 53490
Total number unique17515   224  1280
Mean((I)/sd(I)) 29.7 104.3   1.5
Mn(I) half-set correlation CC(1/2) 1.000 1.000 0.778
Completeness   100.0  99.7 100.0
Multiplicity72.3  72.4  41.8

Anomalous completeness 100.0 100.0 100.0
Anomalous multiplicity  37.2  42.7  21.0
DelAnom correlation between half-sets  0.497 0.766-0.026
Mid-Slope of Anom Normal Probability   1.039   - -

(this is a good case for Rpim & CC-half as resolution limit criteria)

If the statistics you want to use are there & some others also, what is the 
pressure to remove them? Surely we want to educate on how best to interpret the 
entire table above to get a fuller picture of the overall quality of the data? 
My 0th-order request would be to publish the three shells as above ;o)

Cheers Graeme



On 4 Jul 2017, at 22:09, Keller, Jacob 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org><mailto:kell...@janelia.hhmi.org><mailto:kell...@janelia.hhmi.org>>
 wrote:

I suggested replacing Rmerge/sym/cryst with Rmeas, not Rpim. Rmeas is simply 
(Rmerge * sqrt(n/n-1)) where n is the number of measurements of that 
reflection. It's merely a way of correcting for the multiplicity-related 
artifact of Rmerge, which is becoming even more of a problem with data sets of 
increasing variability in multiplicity. Consider the case of comparing a data 
set with a multiplicity of 2 versus one of 100: equivalent data quality would 
yield Rmerges diverging by a factor of ~1.4. But this has all been covered 
before in several papers. It can be and is reported in resolution bins, so can 
used exactly as you say. So, why not "disappear" Rmerge from the software?

The only reason I could come up with for keeping it is historical reasons or 
comparisons to previous datasets, but anyway those comparisons would be 
confounded by variabities in multiplicity and a hundred other things, so come 
on, developers, just comment it out!

JPK




-Original Message-
From: 
graeme.win...@diamond.ac.uk<mailto:graeme.win...@diamond.ac.uk><mailto:graeme.win...@diamond.ac.uk><mailto:graeme.win...@diamond.ac.uk>
 [mailto:graeme.win...@diamond.ac.uk]
Sent: Tuesday, July 04, 2017 4:37 PM
To: Keller, Jacob 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org><mailto:kell...@janelia.hhmi.org><mailto:kell...@janelia.hhmi.org>>
Cc: 
ccp4bb@jiscmail.ac.uk<mailto:ccp4bb@jiscmail.ac.uk><mailto:ccp4bb@jiscmail.ac.uk><mailto:ccp4bb@jiscmail.ac.uk>
Subject: Re: [ccp4bb] Rmergicide Through Programming

HI Jacob

Unbiased estimate of the true unmerged I/sig(I) of your data (I find this 
particularly useful at low resolution) i.e. if your inner shell Rmerge is 10% 
your data agree very poorly; if 2% says your data agre

Re: [ccp4bb] Rmergicide Through Programming

[Keller, Jacob]

>I do not see what harm there is reporting Rmerge, even if it is just used in 
>the inner shell or just used to capture a flavour of the data set overall. I 
>also appreciate that Rmeas converges to the same value for large multiplicity

Consider a callow young grad student, David, who being beleaguered by his 
distant advisor and armchair crystallographer, Dr. Murdstone, into improving 
the statistics of his data, resorts to every option, including trying to 
collect a high-multiplicity data set (100-fold!). To his chagrin, Rmerge keeps 
rising even in spite of these heroic efforts. His exceedingly humble post-doc 
friend and confidant, Uriah, pecks out with his clammy hands a script for 
improving Rmerge, to which he subjects David’s, and indeed all of the lab’s, 
data. Cheers resound at the improved statistics (and smaller mtz files), 
especially from Murdstone, although only MR seems to work now for solving 
structures, and final model stats generally worsen. With the help of some other 
corrective scripts written by Uriah based on Murthy’s Law, there is a lovely 
spate of papers published at well-known journals, and Dr. Murdstone becomes Sir 
Murdstone. Unfortunately, David’s data set requires experimental phasing, so he 
remains in a tailspin with progressively nastier emails arriving from Murdstone 
daily. Observing the situation, the reticent lab manager Agnes opens up the 
merging code, and deletes all mention of Rmerge. Uriah’s great successes 
suddenly stop, but somehow David is now able to solve his data set, and he 
marries Agnes, while all of the recent MR-based structures are retracted, 
notwithstanding the hundreds of subsequent papers based on their conclusions. 
David becomes a noted crystallographer with Agnes helping him slightly in the 
background, while humble Uriah finally finds useful and vastly remunerative 
employment in a pharma marketing and lobbying firm in the New World.

Is this happening right now somewhere? O Britannia, reck ye the perils of 
Rmerge.

Re: [ccp4bb] Rmergicide Through Programming

[Keller, Jacob]

I asked an editor at a prominent journal to change the template file, 
mentioning the consensus among crystallographers about the matter, but they did 
not seem willing. Maybe someone with more name-recognition would be more 
effective. Or a circulated letter from a number of prominent crystallographers? 
Or…it really doesn’t matter that much?

JPK

From: Eleanor Dodson [mailto:eleanor.dod...@york.ac.uk]
Sent: Tuesday, July 04, 2017 5:37 PM
To: Keller, Jacob <kell...@janelia.hhmi.org>
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Rmergicide Through Programming

Agree although i usually look to CC1/2.   Rmeas makes more sense than Rmerge 
but- most software reports both,  so cant you just provide Rmeas yourself   
Probably simpler to persuade journals to alter the Table 1 requirements than 
get all developers to comment those lines out!

Eleanor



On 4 July 2017 at 22:09, Keller, Jacob 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> wrote:
I suggested replacing Rmerge/sym/cryst with Rmeas, not Rpim. Rmeas is simply 
(Rmerge * sqrt(n/n-1)) where n is the number of measurements of that 
reflection. It's merely a way of correcting for the multiplicity-related 
artifact of Rmerge, which is becoming even more of a problem with data sets of 
increasing variability in multiplicity. Consider the case of comparing a data 
set with a multiplicity of 2 versus one of 100: equivalent data quality would 
yield Rmerges diverging by a factor of ~1.4. But this has all been covered 
before in several papers. It can be and is reported in resolution bins, so can 
used exactly as you say. So, why not "disappear" Rmerge from the software?

The only reason I could come up with for keeping it is historical reasons or 
comparisons to previous datasets, but anyway those comparisons would be 
confounded by variabities in multiplicity and a hundred other things, so come 
on, developers, just comment it out!

JPK




-Original Message-
From: graeme.win...@diamond.ac.uk<mailto:graeme.win...@diamond.ac.uk> 
[mailto:graeme.win...@diamond.ac.uk<mailto:graeme.win...@diamond.ac.uk>]
Sent: Tuesday, July 04, 2017 4:37 PM
To: Keller, Jacob <kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>>
Cc: ccp4bb@jiscmail.ac.uk<mailto:ccp4bb@jiscmail.ac.uk>
Subject: Re: [ccp4bb] Rmergicide Through Programming

HI Jacob

Unbiased estimate of the true unmerged I/sig(I) of your data (I find this 
particularly useful at low resolution) i.e. if your inner shell Rmerge is 10% 
your data agree very poorly; if 2% says your data agree very well provided you 
have sensible multiplicity… obviously depends on sensible interpretation. Rpim 
hides this (though tells you more about the quality of average measurement)

Essentially, for I/sig(I) you can (by and large) adjust your sig(I) values 
however you like if you were so inclined. You can only adjust Rmerge by 
excluding measurements.

I would therefore defend that - amongst the other stats you enumerate below - 
it still has a place

Cheers Graeme

> On 4 Jul 2017, at 14:10, Keller, Jacob 
> <kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> wrote:
>
>> Rmerge does contain information which complements the others.
>
> What information? I was trying to think of a counterargument to what I 
> proposed, but could not think of a reason in the world to keep reporting it.
>
> JPK
>
>
> On 4 Jul 2017, at 12:00, Keller, Jacob 
> <kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org><mailto:kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>>>
>  wrote:
>
> Dear Crystallographers,
>
> Having been repeatedly chagrinned about the continued use and reporting of 
> Rmerge rather than Rmeas or similar, I thought of a potential way to promote 
> the change: what if merging programs would completely omit Rmerge/cryst/sym? 
> Is there some reason to continue to report these stats, or are they just 
> grandfathered into the software? I doubt that any journal or crystallographer 
> would insist on reporting Rmerge per se. So, I wonder what developers would 
> think about commenting out a few lines of their code, seeing what happens? 
> Maybe a comment to the effect of "Rmerge is now deprecated; use Rmeas" would 
> be useful as well. Would something catastrophic happen?
>
> All the best,
>
> Jacob Keller
>
> ***
> Jacob Pearson Keller, PhD
> Research Scientist
> HHMI Janelia Research Campus / Looger lab
> Phone: (571)209-4000 x3159<tel:%28571%29209-4000%20x3159>
> Email: 
> kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org><mailto:kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>>
> ***
>
>
> --
> This e-mail and any attachments may c

Re: [ccp4bb] Rmergicide Through Programming

[Keller, Jacob]

I suggested replacing Rmerge/sym/cryst with Rmeas, not Rpim. Rmeas is simply 
(Rmerge * sqrt(n/n-1)) where n is the number of measurements of that 
reflection. It's merely a way of correcting for the multiplicity-related 
artifact of Rmerge, which is becoming even more of a problem with data sets of 
increasing variability in multiplicity. Consider the case of comparing a data 
set with a multiplicity of 2 versus one of 100: equivalent data quality would 
yield Rmerges diverging by a factor of ~1.4. But this has all been covered 
before in several papers. It can be and is reported in resolution bins, so can 
used exactly as you say. So, why not "disappear" Rmerge from the software?

The only reason I could come up with for keeping it is historical reasons or 
comparisons to previous datasets, but anyway those comparisons would be 
confounded by variabities in multiplicity and a hundred other things, so come 
on, developers, just comment it out!

JPK




-Original Message-
From: graeme.win...@diamond.ac.uk [mailto:graeme.win...@diamond.ac.uk] 
Sent: Tuesday, July 04, 2017 4:37 PM
To: Keller, Jacob <kell...@janelia.hhmi.org>
Cc: ccp4bb@jiscmail.ac.uk
Subject: Re: [ccp4bb] Rmergicide Through Programming

HI Jacob

Unbiased estimate of the true unmerged I/sig(I) of your data (I find this 
particularly useful at low resolution) i.e. if your inner shell Rmerge is 10% 
your data agree very poorly; if 2% says your data agree very well provided you 
have sensible multiplicity… obviously depends on sensible interpretation. Rpim 
hides this (though tells you more about the quality of average measurement) 

Essentially, for I/sig(I) you can (by and large) adjust your sig(I) values 
however you like if you were so inclined. You can only adjust Rmerge by 
excluding measurements.

I would therefore defend that - amongst the other stats you enumerate below - 
it still has a place 

Cheers Graeme

> On 4 Jul 2017, at 14:10, Keller, Jacob <kell...@janelia.hhmi.org> wrote:
> 
>> Rmerge does contain information which complements the others. 
> 
> What information? I was trying to think of a counterargument to what I 
> proposed, but could not think of a reason in the world to keep reporting it.
> 
> JPK
> 
> 
> On 4 Jul 2017, at 12:00, Keller, Jacob 
> <kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> wrote:
> 
> Dear Crystallographers,
> 
> Having been repeatedly chagrinned about the continued use and reporting of 
> Rmerge rather than Rmeas or similar, I thought of a potential way to promote 
> the change: what if merging programs would completely omit Rmerge/cryst/sym? 
> Is there some reason to continue to report these stats, or are they just 
> grandfathered into the software? I doubt that any journal or crystallographer 
> would insist on reporting Rmerge per se. So, I wonder what developers would 
> think about commenting out a few lines of their code, seeing what happens? 
> Maybe a comment to the effect of "Rmerge is now deprecated; use Rmeas" would 
> be useful as well. Would something catastrophic happen?
> 
> All the best,
> 
> Jacob Keller
> 
> ***
> Jacob Pearson Keller, PhD
> Research Scientist
> HHMI Janelia Research Campus / Looger lab
> Phone: (571)209-4000 x3159
> Email: kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>
> ***
> 
> 
> -- 
> This e-mail and any attachments may contain confidential, copyright and or 
> privileged material, and are for the use of the intended addressee only. If 
> you are not the intended addressee or an authorised recipient of the 
> addressee please notify us of receipt by returning the e-mail and do not use, 
> copy, retain, distribute or disclose the information in or attached to the 
> e-mail.
> Any opinions expressed within this e-mail are those of the individual and not 
> necessarily of Diamond Light Source Ltd. 
> Diamond Light Source Ltd. cannot guarantee that this e-mail or any 
> attachments are free from viruses and we cannot accept liability for any 
> damage which you may sustain as a result of software viruses which may be 
> transmitted in or with the message.
> Diamond Light Source Limited (company no. 4375679). Registered in England and 
> Wales with its registered office at Diamond House, Harwell Science and 
> Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
> 

Re: [ccp4bb] Rmergicide Through Programming

[Keller, Jacob]

>Rmerge does contain information which complements the others. 

What information? I was trying to think of a counterargument to what I 
proposed, but could not think of a reason in the world to keep reporting it.

JPK


On 4 Jul 2017, at 12:00, Keller, Jacob 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> wrote:

Dear Crystallographers,

Having been repeatedly chagrinned about the continued use and reporting of 
Rmerge rather than Rmeas or similar, I thought of a potential way to promote 
the change: what if merging programs would completely omit Rmerge/cryst/sym? Is 
there some reason to continue to report these stats, or are they just 
grandfathered into the software? I doubt that any journal or crystallographer 
would insist on reporting Rmerge per se. So, I wonder what developers would 
think about commenting out a few lines of their code, seeing what happens? 
Maybe a comment to the effect of "Rmerge is now deprecated; use Rmeas" would be 
useful as well. Would something catastrophic happen?

All the best,

Jacob Keller

*******
Jacob Pearson Keller, PhD
Research Scientist
HHMI Janelia Research Campus / Looger lab
Phone: (571)209-4000 x3159
Email: kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>
***


-- 
This e-mail and any attachments may contain confidential, copyright and or 
privileged material, and are for the use of the intended addressee only. If you 
are not the intended addressee or an authorised recipient of the addressee 
please notify us of receipt by returning the e-mail and do not use, copy, 
retain, distribute or disclose the information in or attached to the e-mail.
Any opinions expressed within this e-mail are those of the individual and not 
necessarily of Diamond Light Source Ltd. 
Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments 
are free from viruses and we cannot accept liability for any damage which you 
may sustain as a result of software viruses which may be transmitted in or with 
the message.
Diamond Light Source Limited (company no. 4375679). Registered in England and 
Wales with its registered office at Diamond House, Harwell Science and 
Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom

[ccp4bb] Rmergicide Through Programming

[Keller, Jacob]

Dear Crystallographers,

Having been repeatedly chagrinned about the continued use and reporting of 
Rmerge rather than Rmeas or similar, I thought of a potential way to promote 
the change: what if merging programs would completely omit Rmerge/cryst/sym? Is 
there some reason to continue to report these stats, or are they just 
grandfathered into the software? I doubt that any journal or crystallographer 
would insist on reporting Rmerge per se. So, I wonder what developers would 
think about commenting out a few lines of their code, seeing what happens? 
Maybe a comment to the effect of "Rmerge is now deprecated; use Rmeas" would be 
useful as well. Would something catastrophic happen?

All the best,

Jacob Keller

***
Jacob Pearson Keller, PhD
Research Scientist
HHMI Janelia Research Campus / Looger lab
Phone: (571)209-4000 x3159
Email: kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>
***

Re: [ccp4bb] Separating Monomers and Dimers

[Keller, Jacob]

Are you boiling your samples? Seems funny that under SDS PAGE there should be 
dimers, unless there is a disulfide link. If so, reducing agents (DTT, TCEP, 
BME) should take care of this.

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Smith Liu
Sent: Tuesday, June 27, 2017 8:41 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Separating Monomers and Dimers

it was not stable for frozen storage. if necessary，using protein fresh without 
frozen

发自网易邮箱大师
在2017年06月27日 20:22，jai 
mohan 写道：
Dear all,
I am working on a Red Fluorescent Protein (His-Tag) molecular weight around 
27kDa. After purification I ran a SDS page, the band at 27kDa confirms the 
monomer. The protein was stored at -20C, a week later again I ran a gel, this 
time I saw another new band between 50-60kDa, it confirms the protein solution 
contains both monomers and dimers. I would like to know, what is the best way 
to separate the monomers and dimers? One of my colleague advice me to go for 
sucrose gradient centrifugation and size exclusion chromatography.
However, I seek all your valuable suggestions and advice.

With best regards
Dr. S.M.Jaimohan

Re: [ccp4bb] Se-Met and Se-Cys double labelling

[Keller, Jacob]

Halide soaks anyone? Cs or NaI?

Jacob

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mark J 
van Raaij
Sent: Wednesday, June 21, 2017 12:08 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Se-Met and Se-Cys double labelling

If your data is good enough, your SeMets alone might well be enough.
Soaking native crystals in Hg compounds may also work, avoiding SeMet 
altogether. We have had a lot of success with methylmercury chloride binding to 
free Cys. You may have to experiment with different soaking times and protocols.
Finally, don't give up on MR too early, what matters is the structural 
similarity, not directly the sequence identity. We've had success once with 19% 
identity.
Native protein may be much easier to produce than the SeMet and SeMet/SeCys 
versions (and may differ a lot between proteins).

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij

On 21 Jun 2017, at 17:46, Vito Calderone 
> wrote:

I am working on a protein having 360 residues. In its sequence there are 3
Met and 5 free Cys.
I will need MAD to solve the structure since based on the sequence the
closest homologue has 20% identity匢 suppose MR would be very unlikely to
work卻o I would like to express a selenium derivative to exploit MAD.
Looking in the literature 1 Se-Met every 120 residues seems not to comply
the threshold to get a good anomalous signal. For this reason I would like
to exploit both Met and Cys so I would have 8 seleniums per 360 residues.
Could somenone suggest a reference to a protocol to express the double
mutant protein in NON auxotrophic strains of E. coli which you have
experienced working efficiently?
Thanks

Re: [ccp4bb] Refining a crystal structure with (very) high solvent content

[Keller, Jacob]

I would resolve this disagreement by repeating that "common sense is not so 
common." When I have seen a great scientist, or anyone with wisdom for that 
matter, I have seen the ability to demonstrate how complicated questions can be 
unravelled in a dazzlingly simple way, such that it almost seems trivial or 
common-sensical. Examples would include Richard Feynmann, the US Supreme Court 
Justices, and others. I would propose that Eleanor meant this kind of uncommon 
common sense.

JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Gerard 
Bricogne
Sent: Sunday, June 04, 2017 12:07 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Refining a crystal structure with (very) high solvent 
content

Dear Eleanor,

 I think this is too faint a praise for Dale. What he shows in his reply is 
not just common sense, but knowledge and understanding of the fundamentals. You 
can't do good science with common sense alone, and in our field common sense 
will not be of much help if you do not understand the Fourier transform well 
enough, for example.

 I would venture to guess that 95+% of crystallographers are in the 
unquestioned habit of making the same conceptual error that Dale has pointed 
out, viz. mistaking the rmsd of the map (which is a unit of contrast) for the 
standard deviation of a noise level in the map.
The latter quantity has nothing to do with the former, as has been pointed out 
many times.

 The problem is that this confusion is enshrined in the default values of 
certain parameters in display programs and scripts, that are assumed (not by 
their authors, but by almost everybody else) to embody all the common sense we 
need :-) .


 With best wishes,
 
  Gerard.

--
On Sun, Jun 04, 2017 at 03:36:29PM +0100, Eleanor Dodson wrote:
> Thank you Dale! You talk so much common sense..
> Eleanor
> 
> On 2 June 2017 at 23:30, Dale Tronrud  wrote:
> 
> > On 6/2/2017 1:42 PM, wtempel wrote:
> > > Hello all,
> > > crystals with high solvent content tend to diffract poorly, at 
> > > least according to intuition. Several years ago we solved a 
> > > structure 
> > >  that 
> > > appeared to buck that trend with a solvent content of ≈0.8 and 
> > > resolution beyond 2 Å, per merging statistics and visibility of spots on 
> > > diffraction images.
> > > I would welcome my colleagues’ opinions as to why I might observe 
> > > the
> > > following:
> > >
> > >  1. Paired refinement (similar to Fig. 1 in Karplus
> > > ) indicates that adding any
> > > higher resolution data beyond 3.4 Å, the lowest high resolution
> > > cut-off limit I tried, does not improve R-factors at the common
> > > lower resolution cutoff. Yes, diffraction is anisotropic in this
> > > case, but seemingly not to that extent. I hesitate to “throw out”
> > > all data beyond 3.4 Å, or whatever lower resolution cut-off I 
> > > might
> > try.
> > >  2. The Fo-Fc map, when countoured at ± 3 rmsd, includes many more
> > > (uninterpretable) features than I would expect after refinement to
> > > residuals in the mid-to-lower twenties. For expected map appearance,
> > > I had to crank up the coutour level to > 5 rmsd, like in the
> > > attached screenshot of the ADP·Mg^++ omit map.
> >
> >This is one of the prime examples of the failure of describing 
> > contour levels in terms of "sigma".  First, the number you are using 
> > is not a "standard deviation" or any other measure of the error 
> > level of the map but is simply the rms value of the map.  If you 
> > calculate the rms of a difference map where 80% of the unit cell is 
> > bulk solvent, and therefore flat, you will, of course, get a much 
> > smaller number than if the unit cell contained 80% protein with all 
> > the the expected difference map features that come from a model with 
> > an R value of ~20%.  Then when you contour at three times this 
> > absurdly small number you will see all sorts of features you are not 
> > used to seeing.  Selecting a contour level based on the e/A^3 is 
> > much less sensitive to the amount of solvent in the crystal is gives much 
> > more consistent results.
> >
> > Dale Tronrud
> > >
> > > Could these observations be linked to the high solvent content? 
> > > (1) A high solvent content structure has a higher-than-average 
> > > observation-to-parameter ratio, sufficiently high even when 
> > > limited to stronger, low-resolution reflections? (2) Map 
> > > normalization may not be attuned to such high solvent content?
> > > I am interested in analyzing the automated decision-making of the 
> > > PDB-REDO of this entry , 
> > > such as paired refinement results and selection of ADP model. 
> > > Should I find this information in the “All files (compressed)” 
> > > 

Re: [ccp4bb] Optimising data processing of a I432 dataset with 75% solvent content.

[Keller, Jacob]

It's radiation-damaged. Based on what did you say that it is not?

Also, do you have ice rings or diffuse scattering which might be present in the 
higher-res shells in certain rotation ranges but not others, such as from 
solvent in the loop?

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Michael 
Jarva
Sent: Thursday, May 18, 2017 7:48 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Optimising data processing of a I432 dataset with 75% solvent 
content.

Dear all,

I have a dataset that have two very interesting properties: a) It's in I432, 
and b) has a whooping 75% solvent content.
You might think that the solvent content obviously is a big red flag, and so 
did I, but I have phased this successfully with just one monomer, and the 
packing result does makes a lot of sense. The resulting maps contain no extra 
umodelled blobs, and trying to phase it with an additional molecules does not 
give a good solution.

The problem I have is that the diffraction intensity/Rmerge plummets/explodes 
around the 3.5Å mark (I assume because of the high solvent content) to such an 
extent that even though I have little radiation damage, 100% completeness in 
high resolution shells, and very high redundancy, any attempt to merge the 
dataset at a higher resolution has so far given no improvement to the maps.

I'm hoping that there might be a few tricks out there I can apply to the spot 
finding/integration/scaling steps have it merge in a even slightly higher 
resolution than I currently have been able to do.
Although I have a feeling that the only thing I can do is to grow another, much 
bigger, crystal...

many thanks for any feedback
/michael

See below for sample outputs from aimless:

   Overall  InnerShell  OuterShell
Low resolution limit   43.50 43.50  3.32
High resolution limit   3.10  8.78  3.10

Rmerge  (within I+/I-) 0.079 0.01021.891
Rmerge  (all I+ and I-)0.081 0.01122.502
Rmeas (within I+/I-)   0.084 0.01123.102
Rmeas (all I+ & I-)0.084 0.01123.169
Rpim (within I+/I-)0.027 0.004 7.335
Rpim (all I+ & I-) 0.020 0.003 5.450
Rmerge in top intensity bin0.010- -
Total number of observations   34917  1495  6448
Total number unique 2057   112   362
Mean((I)/sd(I)) 18.3 130.9   0.1
Mn(I) half-set correlation CC(1/2) 1.000 1.000 0.533
Completeness99.9  97.4 100.0
Multiplicity17.0  13.3  17.8

  Overall  InnerShell  OuterShell
Low resolution limit   43.50 43.50  3.84
High resolution limit   3.50  8.58  3.50

Rmerge  (within I+/I-) 0.052 0.011 2.422
Rmerge  (all I+ and I-)0.056 0.012 2.659
Rmeas (within I+/I-)   0.055 0.011 2.553
Rmeas (all I+ & I-)0.058 0.013 2.738
Rpim (within I+/I-)0.017 0.004 0.804
Rpim (all I+ & I-) 0.014 0.003 0.644
Rmerge in top intensity bin0.010- -
Total number of observations   24596  1690  6071
Total number unique 1462   120   343
Mean((I)/sd(I)) 25.8 132.0   1.0
Mn(I) half-set correlation CC(1/2) 1.000 1.000 0.771
Completeness99.8  97.6 100.0
Multiplicity16.8  14.1  17.7

Re: [ccp4bb] CYS modification and choice of PEG

[Keller, Jacob]

If you were collecting at a long-ish wavelength (for NaI phasing, perhaps?) you 
might be able to see a peak there in an anomalous difference Fourier map. This 
would be a pretty cool discovery!

JPK

From: Keller, Jacob
Sent: Wednesday, May 17, 2017 4:52 PM
To: Antonio Ariza <antonio.ar...@path.ox.ac.uk>; CCP4BB@JISCMAIL.AC.UK
Subject: RE: CYS modification and choice of PEG

Where would it get the sulphate to make sulphonate? Was there sulphate 
somewhere in the purification?

Maybe it's a phosphate gotten off the FMN? I guess the phospho-cys bond might 
be a bit longer?

JPK

Analyst.<https://www.ncbi.nlm.nih.gov/pubmed/25011562> 2014 Sep 
7;139(17):4118-23. doi: 10.1039/c4an00724g.
Puzzling over protein cysteine phosphorylation--assessment of proteomic tools 
for S-phosphorylation profiling.
Buchowiecka 
AK<https://www.ncbi.nlm.nih.gov/pubmed/?term=Buchowiecka%20AK%5BAuthor%5D=true_uid=25011562>1.
Author information<https://www.ncbi.nlm.nih.gov/pubmed/25011562>
Abstract
Cysteine phosphorylation has recently been discovered in both prokaryotic and 
eukaryotic systems, and is thought to play crucial roles in signaling and 
regulation of cellular responses. This article explores the topics of chemical 
stability of this type of structural modification and the resulting issues 
regarding affinity enrichment of S-phosphopeptides and their mass 
spectrometry-based detection in the course of general proteomics studies. 
Together, this work suggests that the current advances in phosphoproteomic 
methodologies provide adequate tools for investigating protein cysteine 
phosphorylation and appear to be immediately available for practical 
implementation. The article provides useful information necessary for designing 
experiments in the emerging cysteine phosphoproteomics. The examples of 
methodological proposals for S-linked phosphorylation detection are included 
herein in order to stimulate development of new approaches by the 
phosphoproteomic community.





From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Antonio 
Ariza
Sent: Wednesday, May 17, 2017 4:04 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] CYS modification and choice of PEG

I haven't asked anything for a loong time, so here are a couple of question 
"for the honourable members of the esteemed CCP4 bulletin board" ... or as I'd 
usually say: "for y'all".  ;)

1)  I have this modified CYS in one of the structures I'm working on and ... 
I'm quite unhappy with it (see attached pics). At first I thought it was 
cacodylate ... but alas, no cacodylate was used during purification or 
crystallisation. So I looked up possible modifications on CYS residues and I 
came up with cysteine-s-sulfonic acid (CSU). This looks good in principle but 
it doesn't quite fit the electron density as the bond between the two sulphur 
atoms is too short. The average length for an S-S bond is about 2.05 Ang and 
refmac refines this one to 1.97 Ang, but it looks like it should be at least 
2.5 Ang to sit correctly in the electron density. Also, there is some negative 
density in there, suggesting that maybe it's something with fewer electrons 
than a sulfonic acid ... or maybe it has less than 100% occupancy. Any 
suggestions?

The condition contains TRIS, bicine, NaCl, NaFl, NaI, DTT, FMN, PEG 500 MME and 
PEG 20,000.

2)  There are two partial PEG molecules in this structure. I've initially 
modeled two PEG 400 (PE4) molecules into the density (simply because I 
remembered the 3 -letter code for it) and removed the excess atoms from them. 
However, since there is a mixture of PEG 500 MME and PEG 20,000 in the 
condition, what would you recommend I use instead of PE4?

Cheers,

Tony

--

Dr. Antonio Ariza
University of Oxford
Sir William Dunn School of Pathology
South Parks Road
Oxford
OX1 3RE
e-mail: antonio.ar...@path.ox.ac.uk<mailto:antonio.ar...@path.ox.ac.uk>
Tel: 00 +44 1865 285655

Links to my public profiles:
ResearchGate<https://www.researchgate.net/profile/Antonio_Ariza>
LinkedIn<https://www.linkedin.com/in/antonioariza1>
GoogleScholar<https://scholar.google.co.uk/citations?user=9pAIKV0J=en>
Twitter<https://twitter.com/DrAntonioAriza?lang=en>

Check out my latest paper!!!
The toxin-antitoxin system DarTG catalyzes reversible ADP-ribosylation of 
DNA<http://www.cell.com/molecular-cell/abstract/S1097-2765(16)30722-5>

Re: [ccp4bb] CYS modification and choice of PEG

[Keller, Jacob]

Where would it get the sulphate to make sulphonate? Was there sulphate 
somewhere in the purification?

Maybe it's a phosphate gotten off the FMN? I guess the phospho-cys bond might 
be a bit longer?

JPK

Analyst. 2014 Sep 
7;139(17):4118-23. doi: 10.1039/c4an00724g.
Puzzling over protein cysteine phosphorylation--assessment of proteomic tools 
for S-phosphorylation profiling.
Buchowiecka 
AK1.
Author information
Abstract
Cysteine phosphorylation has recently been discovered in both prokaryotic and 
eukaryotic systems, and is thought to play crucial roles in signaling and 
regulation of cellular responses. This article explores the topics of chemical 
stability of this type of structural modification and the resulting issues 
regarding affinity enrichment of S-phosphopeptides and their mass 
spectrometry-based detection in the course of general proteomics studies. 
Together, this work suggests that the current advances in phosphoproteomic 
methodologies provide adequate tools for investigating protein cysteine 
phosphorylation and appear to be immediately available for practical 
implementation. The article provides useful information necessary for designing 
experiments in the emerging cysteine phosphoproteomics. The examples of 
methodological proposals for S-linked phosphorylation detection are included 
herein in order to stimulate development of new approaches by the 
phosphoproteomic community.





From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Antonio 
Ariza
Sent: Wednesday, May 17, 2017 4:04 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] CYS modification and choice of PEG

I haven't asked anything for a loong time, so here are a couple of question 
"for the honourable members of the esteemed CCP4 bulletin board" ... or as I'd 
usually say: "for y'all".  ;)

1)  I have this modified CYS in one of the structures I'm working on and ... 
I'm quite unhappy with it (see attached pics). At first I thought it was 
cacodylate ... but alas, no cacodylate was used during purification or 
crystallisation. So I looked up possible modifications on CYS residues and I 
came up with cysteine-s-sulfonic acid (CSU). This looks good in principle but 
it doesn't quite fit the electron density as the bond between the two sulphur 
atoms is too short. The average length for an S-S bond is about 2.05 Ang and 
refmac refines this one to 1.97 Ang, but it looks like it should be at least 
2.5 Ang to sit correctly in the electron density. Also, there is some negative 
density in there, suggesting that maybe it's something with fewer electrons 
than a sulfonic acid ... or maybe it has less than 100% occupancy. Any 
suggestions?

The condition contains TRIS, bicine, NaCl, NaFl, NaI, DTT, FMN, PEG 500 MME and 
PEG 20,000.

2)  There are two partial PEG molecules in this structure. I've initially 
modeled two PEG 400 (PE4) molecules into the density (simply because I 
remembered the 3 -letter code for it) and removed the excess atoms from them. 
However, since there is a mixture of PEG 500 MME and PEG 20,000 in the 
condition, what would you recommend I use instead of PE4?

Cheers,

Tony

--

Dr. Antonio Ariza
University of Oxford
Sir William Dunn School of Pathology
South Parks Road
Oxford
OX1 3RE
e-mail: antonio.ar...@path.ox.ac.uk
Tel: 00 +44 1865 285655

Links to my public profiles:
ResearchGate
LinkedIn
GoogleScholar
Twitter

Check out my latest paper!!!
The toxin-antitoxin system DarTG catalyzes reversible ADP-ribosylation of 
DNA

Re: [ccp4bb] peroxy-glutamate?

[Keller, Jacob]

I would think the first goal is to model the observed data correctly, and then 
afterwards an accurate "before" model could be inferred.

It seems that it would be extremely helpful to this end to add another column 
to the .pdb format: a "time constant" for radiation damage for each atom. When 
set to 0, there would be no decay (default, toggled off in refinement?), and 
negative and positive values could denote exponential decay (carboxyl) or 
appearance (CO2). But then, of course, one would also have to set up refinement 
to use unmerged data. But shouldn't this be done at some point anyway, now that 
we have the cyber-power to do it?

JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tristan 
Croll
Sent: Tuesday, May 09, 2017 11:45 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] peroxy-glutamate?

Hmm... this is a bit of a philosophical pickle in my mind. Do we want to model 
the structure as what it looks like after radiation damage has had its way with 
it, or what it must have looked like *before* the damage? I can see arguments 
both ways (and can sympathise with the former if you want to make radiation 
damage a subject of your manuscript), but this is going to lead to headaches 
for people who want to make use of the resulting coordinates to study the 
actual biology of your protein. 
Personally, I'd strongly prefer the latter approach.

Tristan

On 2017-05-09 16:06, Edward A. Berry wrote:
> On 05/09/2017 06:18 AM, Ian Tickle wrote:
>> We have seen almost identical density to Ed's for GLU side-chains, 
>> with what looks like a linear molecule (yes exactly the size of CO2!) 
>> where the carboxylate group would be and absolutely no density for 
>> the CG-CD bond.  So it's indeed very tempting to say that the CO2 is 
>> still there, and presumably making the same H bonds that the 
>> carboxylate was making to hold it there.  It would not be hydrated to 
>> carbonic acid, according to 
>> https://en.wikipedia.org/wiki/Carbonic_acid : "The hydration 
>>  equilibrium constant 
>>  at 25 °C is 
>> called K_h , which in the case of carbonic acid is [H_2 CO_3 ]/[CO_2 
>> ] ≈ 1.7×10^−3 in pure water^[5] 
>>  and ≈
>> 1.2×10^−3 in seawater .^[6]
>>  Hence, 
>> the majority of the carbon dioxide is not converted into carbo
> n
> ic
>> acid, remaining as CO_2 molecules.".
> 
> It looks like this ignores subsequent ionization of H2CO3 which would 
> be quite spontaneous at neutral pH.  However the Wikipedia article 
> also indicates the equilibrium is quite slow (which makes sense- 
> otherwise why would carbonic anhydrase exist?) and it would be a great 
> deal slower in vitreous ice at 100 K. Anyway, I had reached the same 
> conclusion and have modeled a number of the troublesome glutamates as 
> decarboxylated with CO2 hovering above. There is a problem that the 
> remaining CG tends to push the CO2 a little out of the density in some 
> cases, but not a severe clash and it may work itself out with further 
> refinement or manual assistance.
> eab

Re: [ccp4bb] peroxy-glutamate?

[Keller, Jacob]

Wouldn’t the not-bonded CO2 have a new steric clash with the CG, though? And 
what happened to the radical that was presumably generated?

Also, I would think solvent-exposed side chains would be more prone to 
diffusion than buried ones.

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ian Tickle
Sent: Tuesday, May 09, 2017 6:19 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] peroxy-glutamate?


Hi Andrew
We have seen almost identical density to Ed's for GLU side-chains, with what 
looks like a linear molecule (yes exactly the size of CO2!) where the 
carboxylate group would be and absolutely no density for the CG-CD bond.  So 
it's indeed very tempting to say that the CO2 is still there, and presumably 
making the same H bonds that the carboxylate was making to hold it there.  It 
would not be hydrated to carbonic acid, according to 
https://en.wikipedia.org/wiki/Carbonic_acid : "The 
hydration equilibrium 
constant at 25 °C is called 
Kh, which in the case of carbonic acid is [H2CO3]/[CO2] ≈ 1.7×10−3 in pure 
water[5] and ≈ 
1.2×10−3 in 
seawater.[6]
 Hence, the majority of the carbon dioxide is not converted into carbonic acid, 
remaining as CO2 molecules.".
Also, diffusion of hydrated HCl in crystalline (hexagonal) ice is apparently 
negligible at 110 K according to this paper: "Depth-Profiling and Diffusion 
Measurements in Ice Films Using Infrared Laser Resonant Desorption", F.E. 
Livingston, J.A. Smith & S.M. George, Anal. Chem., 2000, 72 (22), 5590–9,DOI: 
10.1021/ac000724t.  Quoting their observations: "at T = 110 K show that the HCl 
hydrate interlayer is initially well localized at t = 0.  The temperature of 
the H2O/ HCl/ H2O sandwich structure was then raised to T = 190 K for t = 120 s 
with a constant H2O backing pressure and subsequently cooled rapidly to ∼110 K 
to terminate further HCl diffusion.”.  Now of course measurements of hydrated 
HCl in crystalline ice may have absolutely no relevance to CO2 in a protein and 
vitreous ice.  It's known that ions diffuse more rapidly in vitreous than 
crystalline ice because the diffusion mechanism requires 'hopping' between H2O 
vacancies and there are far fewer of these in crystalline ice.
Cheers
-- Ian



On 4 May 2017 at 11:25, Andrew Leslie 
> wrote:
Dear Ed,

  I find your electron density quite interesting, because generally 
(I think, I would be happy to be corrected on this) when de-carboxylation of 
Asp/Glu occurs due to radiation damage, there is no evidence of what happens to 
the resulting CO2 group. One interpretation of this is that it diffuses away 
from the side chain and is effectively totally disordered, so no electron 
density is seen, but I was surprised that this would always be the case, 
especially as I would have thought that diffusion would be quite limited at 
100K (maybe I’m wrong about that too, but that is supposed to be one reason why 
radiation damage is less at 100K).

If the residual density is due to partial de-carboxylation, then I would have 
expected density for the CG-CD bond, which is not present (at your chosen 
contour level).

Do many of your Glu side chains have the residual density?

Best wishes,

Andrew


> On 3 May 2017, at 22:19, Edward A. Berry 
> > wrote:
>
>
>
> On 05/03/2017 02:46 PM, Gerard Bricogne wrote:
>> Dear Ed,
>>
>>  Have you considered the possibility that it could be a water
>> stepping in to fill the void created by partial decarboxylation of the
>> glutamate? That could be easily modelled, refined, and tested for its
>> ability to flatten the difference map.
>>
>>  Gerard.
>>
> Actually some of them do appear decarboxylated. Is that something that can 
> happen? In the crystal, or as radiation damage?
> However when there is density for the carboxylate (figure), it appears 
> continuous and linear, doesn't break up into spheres at H-bonding distance - 
> almost like the CO2 is still sitting there- but I guess it would get hydrated 
> to bicarbonate. I could use azide. Or maybe waters with some disorder.
> Thanks,
> eab
>
> Figure- 2mFo-DFc at 1.3 sigma, mFo-DFc at 3 sigma, green CO2 is shown for 
> comparison, not part of the model.
>
> 

Re: [ccp4bb] BSA as additive

[Keller, Jacob]

Regarding that paper, I would point out that cytosols generally contain 50-100 
mM glutamate, so it makes sense that glutamate enhances solubility.

JPK


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Evans, 
Nicola
Sent: Monday, May 08, 2017 8:54 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] BSA as additive


I haven't used BSA, but I did recently use 50mM L-glutamic acid for this exact 
reason (and 5% glycerol in all buffers except the last one for crystallography) 
after reading this paper and it made a big difference to my last protein prep: 
https://www.ncbi.nlm.nih.gov/pubmed/15264823



For my final crystallography buffer I have tried with and without L-glutamic 
acid (as I am trying to optimise micro-crystals and worried the L-glu would 
make sample too soluble) but still waiting to see if I get any improvement. 
Both have drops with micro-crystals already (after 2 days), the L-glutamic acid 
sample has fewer, hoping some other drops will yield better crystals over time.



Hope that helps!



Nicola


From: CCP4 bulletin board > 
on behalf of Ha Sin >
Sent: 08 May 2017 12:32:44
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] BSA as additive

Dear CCP4BB members,

Sorry about my non crystallography question. Does anyone know of a reference 
where BSA has been used as an "additive" in the protein concentration step to 
prevent aggregation of the main protein?

I would appreciate your suggestions.

Thank you,

H.Sin

Re: [ccp4bb] peroxy-glutamate?

[Keller, Jacob]

I have seen anomalous "flecks" bespangling an protein's internal cavity which 
had a couple of cysteines in it, and I assumed that these were liberated 
sulphurs (they were not Fourier-truncation-like.) I would agree with Andrew 
that diffusion should not be large, but alighting on the nearest perch should 
be possible, no? Attractive and repulsive forces remain just as strong at 100K, 
so although motions are not driven by thermal sampling or random-walking, they 
would still be driven by local forces.

Further, I would think that the likeliest local perches would appear as new 
sites, for the CO2 as well as for sulphurs.

JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of DUMAS 
Philippe (VIE)
Sent: Thursday, May 04, 2017 6:57 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] peroxy-glutamate?

 
Le Jeudi 4 Mai 2017 12:25 CEST, Andrew Leslie  a 
écrit: 

Dear Andrew,
We looked in details to this problem of diffusion at ca. 100 K with bromine in 
"Ennifar et al., Acta D58(2002)1262" and we concluded

"It was attempted to derive a value for the diffusion coefficient of the free 
bromine species (most likely Br-) in amorphous ice at 100±110 K. This failed 
because the diffusion was much too rapid compared with both the radiolysis and 
datacollection timescales to permit such a determination."

Best regards
Philippe Dumas
 
> Dear Ed,
> 
>   I find your electron density quite interesting, because 
> generally (I think, I would be happy to be corrected on this) when 
> de-carboxylation of Asp/Glu occurs due to radiation damage, there is no 
> evidence of what happens to the resulting CO2 group. One interpretation of 
> this is that it diffuses away from the side chain and is effectively totally 
> disordered, so no electron density is seen, but I was surprised that this 
> would always be the case, especially as I would have thought that diffusion 
> would be quite limited at 100K (maybe I’m wrong about that too, but that is 
> supposed to be one reason why radiation damage is less at 100K).
> 
> If the residual density is due to partial de-carboxylation, then I would have 
> expected density for the CG-CD bond, which is not present (at your chosen 
> contour level).
> 
> Do many of your Glu side chains have the residual density?
> 
> Best wishes,
> 
> Andrew
> 
> 
> > On 3 May 2017, at 22:19, Edward A. Berry  wrote:
> > 
> > 
> > 
> > On 05/03/2017 02:46 PM, Gerard Bricogne wrote:
> >> Dear Ed,
> >> 
> >>  Have you considered the possibility that it could be a water

> >> stepping in to fill the void created by partial decarboxylation of 
> >> the glutamate? That could be easily modelled, refined, and tested 
> >> for its ability to flatten the difference map.
> >> 
> >>  Gerard.
> >> 
> > Actually some of them do appear decarboxylated. Is that something that can 
> > happen? In the crystal, or as radiation damage?
> > However when there is density for the carboxylate (figure), it appears 
> > continuous and linear, doesn't break up into spheres at H-bonding distance 
> > - almost like the CO2 is still sitting there- but I guess it would get 
> > hydrated to bicarbonate. I could use azide. Or maybe waters with some 
> > disorder.
> > Thanks,
> > eab
> > 
> > Figure- 2mFo-DFc at 1.3 sigma, mFo-DFc at 3 sigma, green CO2 is shown for 
> > comparison, not part of the model.
> > 
> > 
 
 
 
 

Re: [ccp4bb] peroxy-glutamate?

[Keller, Jacob]

Damage-Selective (DamSel) map?

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bellini, 
Dom
Sent: Wednesday, May 03, 2017 6:01 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] peroxy-glutamate?


or RDM (raddam detection map), better known as raddamap? :)



BW,



D


From: CCP4 bulletin board > 
on behalf of Pavel Afonine >
Sent: 03 May 2017 22:51:48
To: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] peroxy-glutamate?

Dear Gerard,

I am sure others
are certain to propose a cooler name for that very same type of map
some day ;-) .

a free tip: how about DDM (Decarboxylation Detector Map)?

All the best,
Pavel

Re: [ccp4bb] large number in ASU

[Keller, Jacob]

Use Zanuda to see whether the space group is actually a higher one—looks like a 
and c axes are pretty similar, and beta might be 120, suggesting a threefold. 
Otherwise it’s a pretty large beta. I wonder what the largest beta ever seen in 
the pdb is?

JPK


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Jademilson Santos
Sent: Wednesday, April 26, 2017 3:35 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] large number in ASU

Greetings all,

I am having trouble with a data set and would like to know if somebody can 
help. I'm working with a protein of approximately 50 kDa, which I have 
successfully crystallized. The crystals diffracted at a resolution of 3,65 
angstroms and upon initial processing using XDS i obtained the following 
information:

space group: P21
ISA = 33.3
cell unit: a=285.2, b=135.9, c= 287.5, α=90, β=117.5, γ=90
Matthews coefficient indicates that there are 40 molecules in the asymmetric 
unit

I am currently running the program Phaser (Phenix) to determine the phase via 
molecular replacement with a model that has 49% homology and query coverage of 
94% and the program is taking extremly long to finish. In this case in which 
there is an extremly high number of molecules in the asymmetric unit, is this 
actually possible? Does someone know how to work with these values and is there 
a specific strategy which i must follow?

Regards

Jademilson Celestino dos Santos
Laboratory of Applied Structural Biology
Department of Microbiology
Institute of Biomedical Sciences
University of São Paulo- USP

Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

[Keller, Jacob]

It’s p3221. Re-process your data forcing this space group, rebuild, and refine 
twin domains. Just do it—you won’t regret it!

Jacob

From: Eleanor Dodson [mailto:eleanor.dod...@york.ac.uk]
Sent: Friday, April 14, 2017 3:19 PM
To: Keller, Jacob <kell...@janelia.hhmi.org>
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

maybe attach your data processing log file..
e

On 14 April 2017 at 20:10, Eleanor Dodson 
<eleanor.dod...@york.ac.uk<mailto:eleanor.dod...@york.ac.uk>> wrote:
That twin factor list  means the apparent crystal symmetry must be P6/mmm.

You say you only have 2 molecules in the asymmetric unit of P32,therefor there 
must only be one in SGs P32 21 P32 12

So I dont understand why you have PHASER results like this:

SOLU SET  RFZ=4.4 TFZ=7.7 PAK=0 LLG=55 TFZ==9.6 LLG=350 TFZ==20.5 PAK=0 LLG=350 
TFZ==20.5..



Why so many TFZ here - is that achieved after refinement or something?



Eleanor



And what does the twinning analysis suggest?





On 14 April 2017 at 17:42, Keller, Jacob 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> wrote:
As I mentioned off-list, it would be helpful to know how many types of search 
models you are searching with—how many different molecules are in the complex? 
It’s hard to interpret MR results otherwise.

Also, since the higher-symmetry SG works in MR, you should try to refine the 
model in that SG, with only two twin domains, refining twin fraction. I can 
guarantee that a good reviewer will have you do this (if not, then not a “good 
reviewer.”)

JPK

From: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of Alex 
Lee
Sent: Friday, April 14, 2017 11:50 AM

To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Thanks Eleanor, I tried MR for P32 21 and P32 12.
SG P3221:  SOLU SET RFZ=5.3 TFZ=8.8 PAK=0 LLG=121 TFZ==11.2 LLG=944 TFZ==29.2 
PAK=0 LLG=944 TFZ==29.2

   SOLU SPAC P 32 2 1



SG P3212:

Solution #1 annotation (history):



   SOLU SET  RFZ=4.4 TFZ=7.7 PAK=0 LLG=55 TFZ==9.6 LLG=350 TFZ==20.5 PAK=0 
LLG=350 TFZ==20.5



   SOLU SPAC P 32 1 2



SG P32

SOLU SET RFZ=7.4 TFZ=10.4 PAK=0 LLG=187 TFZ==10.7 RF++ TFZ=17.0 PAK=0 LLG=436 
TFZ==17.8 LLG=1715 TFZ==34.3 PAK=0

LLG=1715 TFZ==34.3

   SOLU SPAC P 32



Based on TFZ and LLG, the P32 seems to be best. But I'll also try to refine and 
build P32 2 1 latter

On Fri, Apr 14, 2017 at 4:32 AM, Eleanor Dodson 
<eleanor.dod...@york.ac.uk<mailto:eleanor.dod...@york.ac.uk>> wrote:
First - four way twinning is possible but pretty rare for macromolecules

Pointless gives a very useful table of the CC agreement for each possible 
symmetry operator individually.
In this case with only two molecules in the asymmetric unit you you could only 
have a higher symmetry SG as
P32 21 P32 12 or P64

These would require as symmetry operators -
P32 21 - a three fold and a two fold k h -l
P32 12 - a three fold and a two fold -k -h -l

P64 - a six fold

If the scores for one set are better than the others you probably have that SG

However high degrees of twinning can disguise the symmetry scores of course..



On 14 April 2017 at 04:46, Keller, Jacob 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> wrote:
Try MR with one copy in all space groups of PG 321/312 using Phaser. Going from 
PG 3 to PG 32 should halve the number of copies per ASU. You may have to 
re-process your data in the higher point group to do this.

Or you might actually have a tetartohedral twin, but just try with the 
higher-symmetry point group first, see what happens.

JPK

From: Alex Lee [mailto:alexlee198...@gmail.com<mailto:alexlee198...@gmail.com>]
Sent: Thursday, April 13, 2017 11:32 PM

To: Keller, Jacob <kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>>
Cc: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Hi Keller,

Thanks for the suggestions! I only have two copies in ASU at SG P32. Zanuda 
also suggests P32 is the best SG.

On Thu, Apr 13, 2017 at 8:12 PM, Keller, Jacob 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> wrote:
Yes, this was my case exactly—it looks like there are two pairs of coupled twin 
domains: a,c and b,d. Assuming you have multiple copies of your model in the 
same ASU, try doing MR in higher symmetry space groups of point group 312 or 
321, like P3212 etc. There is this handy page with all the space groups and 
their possible twin operators: http://www.ccp4.ac.uk/html/twinning.html.

The twin fractions indicate a high twin fraction—~46% if actually hemihedral!

Also take a look at the paper I referenced for more info. I can send you a .pdf 
if you need me to.

Please let me know how it works out—I am interested in thes

Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

[Keller, Jacob]

As I mentioned off-list, it would be helpful to know how many types of search 
models you are searching with—how many different molecules are in the complex? 
It’s hard to interpret MR results otherwise.

Also, since the higher-symmetry SG works in MR, you should try to refine the 
model in that SG, with only two twin domains, refining twin fraction. I can 
guarantee that a good reviewer will have you do this (if not, then not a “good 
reviewer.”)

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Alex Lee
Sent: Friday, April 14, 2017 11:50 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Thanks Eleanor, I tried MR for P32 21 and P32 12.
SG P3221:  SOLU SET RFZ=5.3 TFZ=8.8 PAK=0 LLG=121 TFZ==11.2 LLG=944 TFZ==29.2 
PAK=0 LLG=944 TFZ==29.2

   SOLU SPAC P 32 2 1



SG P3212:

Solution #1 annotation (history):



   SOLU SET  RFZ=4.4 TFZ=7.7 PAK=0 LLG=55 TFZ==9.6 LLG=350 TFZ==20.5 PAK=0 
LLG=350 TFZ==20.5



   SOLU SPAC P 32 1 2



SG P32

SOLU SET RFZ=7.4 TFZ=10.4 PAK=0 LLG=187 TFZ==10.7 RF++ TFZ=17.0 PAK=0 LLG=436 
TFZ==17.8 LLG=1715 TFZ==34.3 PAK=0

LLG=1715 TFZ==34.3

   SOLU SPAC P 32



Based on TFZ and LLG, the P32 seems to be best. But I'll also try to refine and 
build P32 2 1 latter

On Fri, Apr 14, 2017 at 4:32 AM, Eleanor Dodson 
<eleanor.dod...@york.ac.uk<mailto:eleanor.dod...@york.ac.uk>> wrote:
First - four way twinning is possible but pretty rare for macromolecules

Pointless gives a very useful table of the CC agreement for each possible 
symmetry operator individually.
In this case with only two molecules in the asymmetric unit you you could only 
have a higher symmetry SG as
P32 21 P32 12 or P64

These would require as symmetry operators -
P32 21 - a three fold and a two fold k h -l
P32 12 - a three fold and a two fold -k -h -l

P64 - a six fold

If the scores for one set are better than the others you probably have that SG

However high degrees of twinning can disguise the symmetry scores of course..



On 14 April 2017 at 04:46, Keller, Jacob 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> wrote:
Try MR with one copy in all space groups of PG 321/312 using Phaser. Going from 
PG 3 to PG 32 should halve the number of copies per ASU. You may have to 
re-process your data in the higher point group to do this.

Or you might actually have a tetartohedral twin, but just try with the 
higher-symmetry point group first, see what happens.

JPK

From: Alex Lee [mailto:alexlee198...@gmail.com<mailto:alexlee198...@gmail.com>]
Sent: Thursday, April 13, 2017 11:32 PM

To: Keller, Jacob <kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>>
Cc: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Hi Keller,

Thanks for the suggestions! I only have two copies in ASU at SG P32. Zanuda 
also suggests P32 is the best SG.

On Thu, Apr 13, 2017 at 8:12 PM, Keller, Jacob 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> wrote:
Yes, this was my case exactly—it looks like there are two pairs of coupled twin 
domains: a,c and b,d. Assuming you have multiple copies of your model in the 
same ASU, try doing MR in higher symmetry space groups of point group 312 or 
321, like P3212 etc. There is this handy page with all the space groups and 
their possible twin operators: http://www.ccp4.ac.uk/html/twinning.html.

The twin fractions indicate a high twin fraction—~46% if actually hemihedral!

Also take a look at the paper I referenced for more info. I can send you a .pdf 
if you need me to.

Please let me know how it works out—I am interested in these types of things!

JPK

From: Alex Lee [mailto:alexlee198...@gmail.com<mailto:alexlee198...@gmail.com>]
Sent: Thursday, April 13, 2017 9:08 PM
To: Keller, Jacob <kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>>
Cc: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>

Subject: Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Hi Keller,

I do not how to check twin fraction after Refmac (I guess it's somewhere in log 
file). From the log file it seems I have four twin domain:

   Twin operators with estimated twin fractions 



Twin operator:  H,  K,  L: Fraction = 0.275; Equivalent operators:  K, -H-K,  
L; -H-K,  H,  L

Twin operator: -K, -H, -L: Fraction = 0.228; Equivalent operators: -H,  H+K, 
-L;  H+K, -K, -L

Twin operator:  K,  H, -L: Fraction = 0.270; Equivalent operators:  H, -H-K, 
-L; -H-K,  K, -L

Twin operator: -H, -K,  L: Fraction = 0.228; Equivalent operators: -K,  H+K,  
L;  H+K, -H,  L

On Thu, Apr 13, 2017 at 4:36 PM, Keller, Jacob 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> wrote:
What was the refined twin fraction after Refmac? It’s much more accurate than 
initial tests. Also, how many twin domains do you have? I

Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

[Keller, Jacob]

Try MR with one copy in all space groups of PG 321/312 using Phaser. Going from 
PG 3 to PG 32 should halve the number of copies per ASU. You may have to 
re-process your data in the higher point group to do this.

Or you might actually have a tetartohedral twin, but just try with the 
higher-symmetry point group first, see what happens.

JPK

From: Alex Lee [mailto:alexlee198...@gmail.com]
Sent: Thursday, April 13, 2017 11:32 PM
To: Keller, Jacob <kell...@janelia.hhmi.org>
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Hi Keller,

Thanks for the suggestions! I only have two copies in ASU at SG P32. Zanuda 
also suggests P32 is the best SG.

On Thu, Apr 13, 2017 at 8:12 PM, Keller, Jacob 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> wrote:
Yes, this was my case exactly—it looks like there are two pairs of coupled twin 
domains: a,c and b,d. Assuming you have multiple copies of your model in the 
same ASU, try doing MR in higher symmetry space groups of point group 312 or 
321, like P3212 etc. There is this handy page with all the space groups and 
their possible twin operators: http://www.ccp4.ac.uk/html/twinning.html.

The twin fractions indicate a high twin fraction—~46% if actually hemihedral!

Also take a look at the paper I referenced for more info. I can send you a .pdf 
if you need me to.

Please let me know how it works out—I am interested in these types of things!

JPK

From: Alex Lee [mailto:alexlee198...@gmail.com<mailto:alexlee198...@gmail.com>]
Sent: Thursday, April 13, 2017 9:08 PM
To: Keller, Jacob <kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>>
Cc: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>

Subject: Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Hi Keller,

I do not how to check twin fraction after Refmac (I guess it's somewhere in log 
file). From the log file it seems I have four twin domain:

   Twin operators with estimated twin fractions 



Twin operator:  H,  K,  L: Fraction = 0.275; Equivalent operators:  K, -H-K,  
L; -H-K,  H,  L

Twin operator: -K, -H, -L: Fraction = 0.228; Equivalent operators: -H,  H+K, 
-L;  H+K, -K, -L

Twin operator:  K,  H, -L: Fraction = 0.270; Equivalent operators:  H, -H-K, 
-L; -H-K,  K, -L

Twin operator: -H, -K,  L: Fraction = 0.228; Equivalent operators: -K,  H+K,  
L;  H+K, -H,  L

On Thu, Apr 13, 2017 at 4:36 PM, Keller, Jacob 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> wrote:
What was the refined twin fraction after Refmac? It’s much more accurate than 
initial tests. Also, how many twin domains do you have? If you have many, it 
might be a higher space group but with less twinning. I recently had a case in 
which apparent tetartohedral (four-domain) twinning in P32 was really 
hemihedral (two-domain) twinning in P3212:

Acta Cryst.<http://journals.iucr.org/d> (2017). 
D73<http://journals.iucr.org/d/contents/backissues.html>, 22-31
https://doi.org/10.1107/S2059798316019318

Jacob

From: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of 
Eleanor Dodson
Sent: Thursday, April 13, 2017 3:11 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Twin refinement cannot be compared directly to untwinned - the R factors are 
between different parameters - without twinning it is assumed you have an 
amplitude obtained more or less from sqrt(I   But for a twinned data set that I 
is actually [ I1 + twin_factor I2 ] so the amplitude is not really correct and 
twinned refinement will give a much better estimate.

However you need to be careful that you have assigned the same FreeR flag to 
reflection pair related by the twin law. The modern program in the CCP4 data 
reduction pipeline looks after this pretty automatically - all possible 
symmetry equivalents are assigned the same FreeR but older software did not do 
this..

You can check it by looking at some twin equivalents - in SG P32 these could be 
h k l and -h, -k, l or h k l and k h -l  or h k l and -k, -h, -l .

Ideally they all should have the same Free R flag..

Eleanor

PS - the acid test is:  Do the maps look better?

E


On 13 April 2017 at 19:52, Robbie Joosten 
<r.joos...@nki.nl<mailto:r.joos...@nki.nl>> wrote:
Hi Alex,

You are not giving the number after  refinement without the twin refinement. 
Nevertheless, R-free drops like this are not unheard of. You should check your 
Refmac log file, it would warn you of potential space group errors. Refmac will 
also give you a refined estimate of the twin fraction.

Cheers,
Robbie

Sent from my Windows 10 phone

Van: Alex Lee<mailto:alexlee198...@gmail.com>
Verzonden: donderdag 13 april 2017 19:19
Aan: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Onderwerp: [ccp4bb] Refmac5 twi

Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

[Keller, Jacob]

Yes, this was my case exactly—it looks like there are two pairs of coupled twin 
domains: a,c and b,d. Assuming you have multiple copies of your model in the 
same ASU, try doing MR in higher symmetry space groups of point group 312 or 
321, like P3212 etc. There is this handy page with all the space groups and 
their possible twin operators: http://www.ccp4.ac.uk/html/twinning.html.

The twin fractions indicate a high twin fraction—~46% if actually hemihedral!

Also take a look at the paper I referenced for more info. I can send you a .pdf 
if you need me to.

Please let me know how it works out—I am interested in these types of things!

JPK

From: Alex Lee [mailto:alexlee198...@gmail.com]
Sent: Thursday, April 13, 2017 9:08 PM
To: Keller, Jacob <kell...@janelia.hhmi.org>
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Hi Keller,

I do not how to check twin fraction after Refmac (I guess it's somewhere in log 
file). From the log file it seems I have four twin domain:

   Twin operators with estimated twin fractions 



Twin operator:  H,  K,  L: Fraction = 0.275; Equivalent operators:  K, -H-K,  
L; -H-K,  H,  L

Twin operator: -K, -H, -L: Fraction = 0.228; Equivalent operators: -H,  H+K, 
-L;  H+K, -K, -L

Twin operator:  K,  H, -L: Fraction = 0.270; Equivalent operators:  H, -H-K, 
-L; -H-K,  K, -L

Twin operator: -H, -K,  L: Fraction = 0.228; Equivalent operators: -K,  H+K,  
L;  H+K, -H,  L

On Thu, Apr 13, 2017 at 4:36 PM, Keller, Jacob 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> wrote:
What was the refined twin fraction after Refmac? It’s much more accurate than 
initial tests. Also, how many twin domains do you have? If you have many, it 
might be a higher space group but with less twinning. I recently had a case in 
which apparent tetartohedral (four-domain) twinning in P32 was really 
hemihedral (two-domain) twinning in P3212:

Acta Cryst.<http://journals.iucr.org/d> (2017). 
D73<http://journals.iucr.org/d/contents/backissues.html>, 22-31
https://doi.org/10.1107/S2059798316019318

Jacob

From: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of 
Eleanor Dodson
Sent: Thursday, April 13, 2017 3:11 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Twin refinement cannot be compared directly to untwinned - the R factors are 
between different parameters - without twinning it is assumed you have an 
amplitude obtained more or less from sqrt(I   But for a twinned data set that I 
is actually [ I1 + twin_factor I2 ] so the amplitude is not really correct and 
twinned refinement will give a much better estimate.

However you need to be careful that you have assigned the same FreeR flag to 
reflection pair related by the twin law. The modern program in the CCP4 data 
reduction pipeline looks after this pretty automatically - all possible 
symmetry equivalents are assigned the same FreeR but older software did not do 
this..

You can check it by looking at some twin equivalents - in SG P32 these could be 
h k l and -h, -k, l or h k l and k h -l  or h k l and -k, -h, -l .

Ideally they all should have the same Free R flag..

Eleanor

PS - the acid test is:  Do the maps look better?

E


On 13 April 2017 at 19:52, Robbie Joosten 
<r.joos...@nki.nl<mailto:r.joos...@nki.nl>> wrote:
Hi Alex,

You are not giving the number after  refinement without the twin refinement. 
Nevertheless, R-free drops like this are not unheard of. You should check your 
Refmac log file, it would warn you of potential space group errors. Refmac will 
also give you a refined estimate of the twin fraction.

Cheers,
Robbie

Sent from my Windows 10 phone

Van: Alex Lee<mailto:alexlee198...@gmail.com>
Verzonden: donderdag 13 april 2017 19:19
Aan: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Onderwerp: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Dear All,

I have a protein/dna complex crystal and data collected at 3A and another set 
at 2.8A, space group P32. L test shows twinning (fraction around 0.11). The 
structure solved by MR and model building of the complex finish (no solvent 
built yet, I do not think it's good to build solvent in such low resolution 
data).

I did Refmac5 to refine my structure (restraint refinement) with or without 
twinning, to my surprise, the Rfree drops a lot after twin refinement of two 
data sets.  Summary below:

2.8A dataset: before twin refine 34%, 29%; after twin refine:24%, 19%
3A dataset: before twin refine 30%;26%; after refine 25%, 18%

I know that a lot of threads in CCP4bb talking about Rfree after twin refine 
and Rfree without twin refine can not compare directly. By drop R free this 
much by twin refine, it gives me a feeling of too good to be true (at such low 
resolution with such good Rfre

Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

[Keller, Jacob]

What was the refined twin fraction after Refmac? It’s much more accurate than 
initial tests. Also, how many twin domains do you have? If you have many, it 
might be a higher space group but with less twinning. I recently had a case in 
which apparent tetartohedral (four-domain) twinning in P32 was really 
hemihedral (two-domain) twinning in P3212:

Acta Cryst. (2017). 
D73, 22-31
https://doi.org/10.1107/S2059798316019318

Jacob

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Eleanor 
Dodson
Sent: Thursday, April 13, 2017 3:11 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Twin refinement cannot be compared directly to untwinned - the R factors are 
between different parameters - without twinning it is assumed you have an 
amplitude obtained more or less from sqrt(I   But for a twinned data set that I 
is actually [ I1 + twin_factor I2 ] so the amplitude is not really correct and 
twinned refinement will give a much better estimate.

However you need to be careful that you have assigned the same FreeR flag to 
reflection pair related by the twin law. The modern program in the CCP4 data 
reduction pipeline looks after this pretty automatically - all possible 
symmetry equivalents are assigned the same FreeR but older software did not do 
this..

You can check it by looking at some twin equivalents - in SG P32 these could be 
h k l and -h, -k, l or h k l and k h -l  or h k l and -k, -h, -l .

Ideally they all should have the same Free R flag..

Eleanor

PS - the acid test is:  Do the maps look better?

E


On 13 April 2017 at 19:52, Robbie Joosten 
> wrote:
Hi Alex,

You are not giving the number after  refinement without the twin refinement. 
Nevertheless, R-free drops like this are not unheard of. You should check your 
Refmac log file, it would warn you of potential space group errors. Refmac will 
also give you a refined estimate of the twin fraction.

Cheers,
Robbie

Sent from my Windows 10 phone

Van: Alex Lee
Verzonden: donderdag 13 april 2017 19:19
Aan: CCP4BB@JISCMAIL.AC.UK
Onderwerp: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Dear All,

I have a protein/dna complex crystal and data collected at 3A and another set 
at 2.8A, space group P32. L test shows twinning (fraction around 0.11). The 
structure solved by MR and model building of the complex finish (no solvent 
built yet, I do not think it's good to build solvent in such low resolution 
data).

I did Refmac5 to refine my structure (restraint refinement) with or without 
twinning, to my surprise, the Rfree drops a lot after twin refinement of two 
data sets.  Summary below:

2.8A dataset: before twin refine 34%, 29%; after twin refine:24%, 19%
3A dataset: before twin refine 30%;26%; after refine 25%, 18%

I know that a lot of threads in CCP4bb talking about Rfree after twin refine 
and Rfree without twin refine can not compare directly. By drop R free this 
much by twin refine, it gives me a feeling of too good to be true (at such low 
resolution with such good Rfree, maybe overrefined a lot?), but from the 
density map after twin refine, it does seem better than no twin refine map.

I do not know if reviewers are going to challenge this part.

Any input is appreciated.

Re: [ccp4bb] waters with positive FoFc peaks?

[Keller, Jacob]

It would be useful to know what wavelengths you were talking about. Also, try 
an anomalous difference Fourier map to see whether the atoms are weakly 
anomalous.

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Andrew 
Marshall
Sent: Thursday, April 13, 2017 2:00 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] waters with positive FoFc peaks?

Hello all,

I have a 1.8A structure (Rfree/Rwork = 20.5/17.4) with 1420 water molecules 
modelled. There are approximately a dozen waters, all well structured with 
hydrogen bonds to protein atoms, with positive difference density (>4sigma, 
sometimes >5) at their centre. The only ions present in my buffer were Cs, Cl 
and a small amount of Na. I thought Cl ions might be a possibility, but many of 
them are in close proximity to acidic residues and/or one-another. It's 
probably worth noting that the same structure solved using data to 2.25A from 
the same crystal at a different wavelength doesn't contain these peaks (the 
offending waters look normal).

Has any come across this before? Thoughts?

Thanks,

Andrew Marshall
PhD Candidate
Laboratory of Protein Crystallography
Dept. of Molecular and Cellular Biology
School of Biological Sciences
The University of Adelaide

[ccp4bb] Voltage-Gated Structures

[Keller, Jacob]

Dear Crystallographers,

Is anyone aware of examples of membrane proteins for which voltage-activated 
and -inactivated structures are both known? Homologs would work too, but I 
found it difficult to sift through the PDB for this, even searching for 
"voltage-gated." Any help would be appreciated-I would like to see an example 
of what the voltage gating conformational change is like.

All the best,

Jacob Keller

***
Jacob Pearson Keller, PhD
Research Scientist
HHMI Janelia Research Campus / Looger lab
Phone: (571)209-4000 x3159
Email: kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>
***

Re: [ccp4bb] UVEX UV Fluorescence

[Keller, Jacob]

But I think he was asking about imaging of intrinsic fluorescence of protein 
crystals…

I like your idea about gel imaging, though.

JPK

From: Hughes, Jon [mailto:jon.hug...@bot3.bio.uni-giessen.de]
Sent: Wednesday, April 05, 2017 8:23 AM
To: Keller, Jacob <kell...@janelia.hhmi.org>; CCP4BB@JISCMAIL.AC.UK
Subject: AW: [ccp4bb] UVEX UV Fluorescence

for ethidium bromide you need a 590nm (50-100nm FWHH) bandpass interference 
filter. ccd chips are sensitive to infrared and transilluminators produce a lot 
of it!
best
j

Von: Keller, Jacob [mailto:kell...@janelia.hhmi.org]
Gesendet: Mittwoch, 5. April 2017 14:16
An: Hughes, Jon 
<jon.hug...@bot3.bio.uni-giessen.de<mailto:jon.hug...@bot3.bio.uni-giessen.de>>;
 CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Betreff: RE: [ccp4bb] UVEX UV Fluorescence

Why 590 nm? BP, SP, LP? I would have thought 390 nm LP or similar--was it a 
typo?

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hughes, 
Jon
Sent: Wednesday, April 05, 2017 6:43 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] AW: [ccp4bb] UVEX UV Fluorescence

you can save a fortune by just fitting a 590nm interference filter to the front 
of a ccd camera attached to a PC + a UV/B transilluminator. i set this up in my 
lab for about €1000 total 15 years ago and we've used it everyday since.
cheers
jon

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Cyprian 
Cukier
Gesendet: Mittwoch, 5. April 2017 12:26
An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Betreff: [ccp4bb] UVEX UV Fluorescence

Dear All,

We are about to buy a new imaging system for our laboratories and we consider 
UVEX UV Fluorescence Imaging from MD. Can anyone provide some comments about 
this equipment (eg. is it reliable, robust, no technical issues, 
low-maintenance etc.)? All comments will be appreciated.

Thanks,
Cyprian

Re: [ccp4bb] UVEX UV Fluorescence

[Keller, Jacob]

Why 590 nm? BP, SP, LP? I would have thought 390 nm LP or similar--was it a 
typo?

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hughes, 
Jon
Sent: Wednesday, April 05, 2017 6:43 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] AW: [ccp4bb] UVEX UV Fluorescence

you can save a fortune by just fitting a 590nm interference filter to the front 
of a ccd camera attached to a PC + a UV/B transilluminator. i set this up in my 
lab for about €1000 total 15 years ago and we've used it everyday since.
cheers
jon

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Cyprian 
Cukier
Gesendet: Mittwoch, 5. April 2017 12:26
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] UVEX UV Fluorescence

Dear All,

We are about to buy a new imaging system for our laboratories and we consider 
UVEX UV Fluorescence Imaging from MD. Can anyone provide some comments about 
this equipment (eg. is it reliable, robust, no technical issues, 
low-maintenance etc.)? All comments will be appreciated.

Thanks,
Cyprian

[ccp4bb] Off-topic: Attach His-tagged Protein to Coverslip

[Keller, Jacob]

Does anyone have a simple way to attach purified his-tagged protein solidly to 
a coverslip?

Thanks,

Jacob Keller

***
Jacob Pearson Keller, PhD
Research Scientist
HHMI Janelia Research Campus / Looger lab
Phone: (571)209-4000 x3159
Email: kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>
***

Re: [ccp4bb] Ambiguous crystal diffraction pattern

[Keller, Jacob]

It’s multiple crystals of salt.

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Prashant 
Deshmukh
Sent: Tuesday, April 04, 2017 8:40 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Ambiguous crystal diffraction pattern

Dear Crystallographers,
Please help us in figuring out whether the attached crystal diffraction 
screenshot belongs to a protein crystal or that of a salt crystal. The 
cryoprotection condition was not optimized for the crystal. The crystallization 
conditions for the crystal is :
Ammonium sulphate , lithium sulphate, Sodium Citrate buffer pH = 5.6.

Thanks.
Prashant Deshmukh
Dept. of Biophysics,
NIMHANS,
Bangalore 560 029,
E-mail:prashantbiophys...@gmail.com
Mob.No.: +919620986525

[ccp4bb] Helium-Temp Cryo-Cooling

[Keller, Jacob]

Dear Crystallographers,

It is my recollection from a while ago that cryocooling to Helium temperatures 
has modest if any effects on protein structure or diffraction data quality, and 
I've found a couple of papers just now to that effect. Does anyone know 
differently? Do the b-factors even change?

Further, is anyone aware of a beamline which has really low temperature 
capability, ideally in conjunction with spectroscopic analysis?

All the best,

Jacob Keller



***
Jacob Pearson Keller, PhD
Research Scientist
HHMI Janelia Research Campus / Looger lab
Phone: (571)209-4000 x3159
Email: kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>
***