Re: [ccp4bb] Combining MR and SIR phases

[Luke Smithers]

Thanks everyone for your suggestions, I have a lot to try!

Luke

On Tue, 23 Jul 2019 at 17:43, Eleanor Dodson 
wrote:

> Well - there are lots of ways to proceed. It doesnt really matter for the
> crystallography theory if the exptl phases are from SAD or SIR. Just harder
> to handle in some software
> I think I would start to refine the poor MR model with the xptl phases as
> restraints.
> REFMAC will do this. Then see if the maps look better - can you see
> features not in the model?..
> If they do you could use Buccaneer to try to rebuild the existing model
> using exptl phases..
> It might work!
> Eleanor
>
> On Tue, 23 Jul 2019 at 09:55, Luke Smithers <
> luke.smith...@research.uwa.edu.au> wrote:
>
>> Hi all,
>>
>> I have collected native data and data on a bromide derivative of one of
>> my crystals, but have struggled to get a second derivative. I went through
>> the SIR pipeline as the anomalous signal from the Br was weak and have
>> generated some not-so-great phases. I have also attempted MR with the
>> closest match in the PDB (which is only 22% sequence identity) and got some
>> more not-so-great phases. I have found plenty of programs that will combine
>> SAD/MR phases, is there anything that will do SIR/MR? I understand it's
>> likely I will need more and better data and ideally a second derivative but
>> thought it was worth putting it out there to see if there is something like
>> this.
>>
>> Luke
>>
>> --
>>
>> *Luke Smithers*
>>
>> *PhD Candidate*
>>
>> Protein Production and Structure Facility, School of Molecular Sciences
>>
>> *T *+61 8 6488 3163
>>
>> [image: The University of Western Australia]
>> 
>>
>> [image: Pursue Impossible]
>> [image:
>> Facebook]
>> [image:
>> Twitter]
>> [image:
>> Youtube]
>> 
>>
>> [image:
>> http://static.weboffice.uwa.edu.au/visualid/graphics/email/sig2015/campaign.gif]
>> 
>>
>>
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
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>

-- 

*Luke Smithers*

*PhD Candidate*

Protein Production and Structure Facility, School of Molecular Sciences

*T *+61 8 6488 3163

[image: The University of Western Australia]


[image: Pursue Impossible]
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Re: [ccp4bb] challenges in structural biology

[Newman, Janet (Manufacturing, Parkville)]

There are a bunch of people doing this – in the small molecule world. And a lot 
of work has been done on some very robust protein systems too. Can you guess 
which ones?

The real issue (at the moment) is that all the pre-work needed to predict if or 
how a protein might crystallise takes more work and more protein than setting 
up crystallisation experiments.
How many people do DSL on protein in a crystallisation screen, for example? Or 
do self-association chromatography to determine the B22 (which changes under 
different conditions, naturally). Or try mapping out a phase diagram (for each 
condition)?

Many people are not even aware that a simple PCT can help one work out a 
sensible starting concentration for crystallisation trials.

As for AI, at the moment unsupervised learning doesn’t seem to do much, which 
means we need vast, well annotated datasets to make progress. MARKO, which 
Sarah mentioned, required half a million scored images, which took years to get 
together.

Janet

From: CCP4 bulletin board  On Behalf Of Keller, Jacob
Sent: Wednesday, 24 July 2019 4:18 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] challenges in structural biology

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] challenges in structural biology

[Peat, Tom (Manufacturing, Parkville)]

Yes, but are we poets or scientists?
Wax lyrical in your poetry, but maybe have some standards in our science?
cheers, tom

Tom Peat
Proteins Group
Biomedical Program, CSIRO
343 Royal Parade
Parkville, VIC, 3052
+613 9662 7304
+614 57 539 419
tom.p...@csiro.au


From: CCP4 bulletin board  on behalf of Goldman, Adrian 

Sent: Wednesday, July 24, 2019 7:39 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] challenges in structural biology

..and responding in the same vein:

my OED says that its etymology also comes from the Latin sulfur, sulphura in 
the plural.  So there is an etymological basis for the ph, even if it doesn’t 
come from Greek.

Plus, since when has etymological logic has _anything_ to do with English 
spelling?

Finally, it may be how the RSC is spelling it, but I would take a fair bet that 
writers of English prose today (pace America), contemplating an stinky inferno, 
will write “sulphurous flames”, not the unattractive and less stinky “sulfurous 
ones”.

Adrian


On 23 Jul 2019, at 22:21, CCP4BB 
<193323b1e616-dmarc-requ...@jiscmail.ac.uk>
 wrote:

Hi

Going off at a tangent...

The accepted spelling by the Royal Society of Chemistry (i.e. the professional 
body representing chemists in the U.K.) since at least the early 1990s has been 
"sulfate" too. "Sulphur", etc, has been deprecated for quite some time. Why? 
Well, there's no good etymological reason for the "ph" in "sulphate". My 1984 
copy of Greenwood and Earnshaw's "Chemistry of the Elements", written in 
Yorkshire, uses "sulfur" etc throughout.

"Phosphorus" comes from the Greek, so retains the "ph"s on both sides of the 
pond.

Element 13 appears to have started life as "alumium", mutated to "aluminum", 
and finally (in the English speaking world outside North America) settled down 
as "aluminium".

Harry
--
Dr Harry Powell

On 23 Jul 2019, at 17:12, Engin Özkan 
mailto:eoz...@uchicago.edu>> wrote:

On 7/23/19 3:35 AM, 
melanie.voll...@diamond.ac.uk wrote:
No longer those 20 odd names for ammonium sulphate

You mean ammonium *sulfate*. As it is called across the pond. :)

On a related note on common nomenclature for recording crystallization
experiments that Janet brought up:

I find it odd that we still do not report cryo-protection methods and
conditions in PDB depositions. Given that a large fraction of the small
molecules observed in crystal structures are derived from the
cryo-protectants, one would think that reporting the contents of that
solution (and pH) would be paramount to a PDB deposition. Surely, the
crystallographic experiment has changed since 1990/use of synchrotron
sources, which PDB has adjusted well to in most other aspects (e.g.,
including reporting of synchrotron x-ray optics and all the new
detectors during submission).

Engin




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Re: [ccp4bb] challenges in structural biology

[Goldman, Adrian]

..and responding in the same vein:

my OED says that its etymology also comes from the Latin sulfur, sulphura in 
the plural.  So there is an etymological basis for the ph, even if it doesn’t 
come from Greek.

Plus, since when has etymological logic has _anything_ to do with English 
spelling?

Finally, it may be how the RSC is spelling it, but I would take a fair bet that 
writers of English prose today (pace America), contemplating an stinky inferno, 
will write “sulphurous flames”, not the unattractive and less stinky “sulfurous 
ones”.

Adrian


On 23 Jul 2019, at 22:21, CCP4BB 
<193323b1e616-dmarc-requ...@jiscmail.ac.uk>
 wrote:

Hi

Going off at a tangent...

The accepted spelling by the Royal Society of Chemistry (i.e. the professional 
body representing chemists in the U.K.) since at least the early 1990s has been 
"sulfate" too. "Sulphur", etc, has been deprecated for quite some time. Why? 
Well, there's no good etymological reason for the "ph" in "sulphate". My 1984 
copy of Greenwood and Earnshaw's "Chemistry of the Elements", written in 
Yorkshire, uses "sulfur" etc throughout.

"Phosphorus" comes from the Greek, so retains the "ph"s on both sides of the 
pond.

Element 13 appears to have started life as "alumium", mutated to "aluminum", 
and finally (in the English speaking world outside North America) settled down 
as "aluminium".

Harry
--
Dr Harry Powell

On 23 Jul 2019, at 17:12, Engin Özkan 
mailto:eoz...@uchicago.edu>> wrote:

On 7/23/19 3:35 AM, 
melanie.voll...@diamond.ac.uk wrote:
No longer those 20 odd names for ammonium sulphate

You mean ammonium *sulfate*. As it is called across the pond. :)

On a related note on common nomenclature for recording crystallization
experiments that Janet brought up:

I find it odd that we still do not report cryo-protection methods and
conditions in PDB depositions. Given that a large fraction of the small
molecules observed in crystal structures are derived from the
cryo-protectants, one would think that reporting the contents of that
solution (and pH) would be paramount to a PDB deposition. Surely, the
crystallographic experiment has changed since 1990/use of synchrotron
sources, which PDB has adjusted well to in most other aspects (e.g.,
including reporting of synchrotron x-ray optics and all the new
detectors during submission).

Engin




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Re: [ccp4bb] challenges in structural biology

[CCP4BB]

Hi

Going off at a tangent...

The accepted spelling by the Royal Society of Chemistry (i.e. the professional 
body representing chemists in the U.K.) since at least the early 1990s has been 
"sulfate" too. "Sulphur", etc, has been deprecated for quite some time. Why? 
Well, there's no good etymological reason for the "ph" in "sulphate". My 1984 
copy of Greenwood and Earnshaw's "Chemistry of the Elements", written in 
Yorkshire, uses "sulfur" etc throughout.

"Phosphorus" comes from the Greek, so retains the "ph"s on both sides of the 
pond.

Element 13 appears to have started life as "alumium", mutated to "aluminum", 
and finally (in the English speaking world outside North America) settled down 
as "aluminium".

Harry
--
Dr Harry Powell

> On 23 Jul 2019, at 17:12, Engin Özkan  wrote:
> 
>> On 7/23/19 3:35 AM, melanie.voll...@diamond.ac.uk wrote:
>> No longer those 20 odd names for ammonium sulphate
> 
> You mean ammonium *sulfate*. As it is called across the pond. :)
> 
> On a related note on common nomenclature for recording crystallization 
> experiments that Janet brought up:
> 
> I find it odd that we still do not report cryo-protection methods and 
> conditions in PDB depositions. Given that a large fraction of the small 
> molecules observed in crystal structures are derived from the 
> cryo-protectants, one would think that reporting the contents of that 
> solution (and pH) would be paramount to a PDB deposition. Surely, the 
> crystallographic experiment has changed since 1990/use of synchrotron 
> sources, which PDB has adjusted well to in most other aspects (e.g., 
> including reporting of synchrotron x-ray optics and all the new 
> detectors during submission).
> 
> Engin
> 
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1



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Re: [ccp4bb] challenges in structural biology

[Mark J van Raaij]

A recent paper in my favourite journal :-) suggests there is no correlation in 
crystallisation conditions even for similar proteins:
http://scripts.iucr.org/cgi-bin/paper?S2053230X19000141 

As you write, surface loops are likely to be different even for similar 
proteins and those are likely to be important for crystal contacts.



> On 23 Jul 2019, at 10:35, melanie.voll...@diamond.ac.uk 
>  wrote:
> 
> I don't think AI will do our job in future as it heavily relies on the 
> crystal structures for training. However, as a community we should embrace 
> this technology/method to help us solve our structures. And why not start 
> with crystallisation? And again the PDB is in a good position here to enforce 
> standards which will pave the way to make use of all the information in the 
> database to train AI. All chemicals must be IUPAC conform an then the PDB can 
> decide about trivial names based on a set of rules for humans (AI doesn't 
> care how you name it as long as it is consistent). No longer those 20 odd 
> names for ammonium sulphate as Janet pointed out years ago.
> 
> 
> And regarding a magic bullet (as in one size fits all), why should a kinase 
> crystallise in the same condition as a polymerase? They do different jobs in 
> a different micro-environment within the cell so their chemical properties 
> will be different. Perhaps there could be some common ground for evolutionary 
> related molecules but a conserved active site doesn't mean a similar surface 
> for crystal contacts which is the key bit in crystallisation, right?
> 
> 


> But as Kay pointed out, crystallisation is a whole field on its own and will 
> go beyond the GRC and the question asked by James.
> 
> 
> M
> 
> 
> From: CCP4 bulletin board  on behalf of Kay Diederichs 
> 
> Sent: 23 July 2019 08:59:10
> To: ccp4bb
> Subject: Re: [ccp4bb] challenges in structural biology
> 
> If you look at the nice figure at the top of the online article, do you 
> believe that this (or rather, the correct) arrangement of domains/ molecules 
> can be predicted from a couple of correlated mutations, and energy 
> minimization? I think AI is a long way from that.  Finding the correct fold 
> of a compact domain, yes I think it's getting there.
> 
> best,
> Kay
> 
> 
> On Tue, 23 Jul 2019 08:28:42 +0530, Nishant Varshney  wrote:
> 
>> What about AI doing our job in the future?
>> 
>> https://www.nature.com/articles/d41586-019-01357-6?utm_source=Nature+Briefing_campaign=4c1d57fdf3-briefing-dy-20190722_medium=email_term=0_c9dfd39373-4c1d57fdf3-44201949
>> 
>> Best Regards
>> Nishant
>> 
>> On Mon, 22 Jul 2019 at 11:30 PM, Sarah Bowman 
>> wrote:
>> 
>>> I'd like to point out that the MAchine Recognition of Crystallization
>>> Outcomes (MARCO) makes a start to 'deep learning applied to crystallization
>>> outcomes', at least in terms of being able to classify drop images
>>> efficiently.
>>> 
>>> 
>>> 
>>> There is obviously more work to be done to correlate these data with
>>> crystallization cocktail components (which Janet and Tom point out the
>>> difficulties with) and positive outcomes.  It seems the first step really
>>> needs to be consistent descriptions and vocabulary - I fully agree with
>>> Janet here!
>>> 
>>> 
>>> 
>>> Reference on MARCO for those interested: Bruno AE, Charbonneau P, Newman
>>> J, Snell EH, So DR, Vanhoucke V, et al. (2018) Classification of
>>> crystallization outcomes using deep convolutional neural networks. PLoS ONE
>>> 13(6): e0198883. https://doi.org/10.1371/journal.pone.0198883
>>> 
>>> 
>>> 
>>> Cheers,
>>> 
>>> Sarah
>>> 
>>> 
>>> 
>>> *Sarah EJ Bowman, PhD*
>>> 
>>> 
>>> 
>>> Associate Research Scientist, Hauptman-Woodward Medical Research Institute
>>> 
>>> Director, High-Throughput Crystallization Screening Center
>>> 
>>> Research Associate Professor, Department of Biochemistry, University at
>>> Buffalo
>>> 
>>> 
>>> 
>>> Research Webpage 
>>> 
>>> www.getacrystal.org
>>> 
>>> 
>>> 
>>> sbow...@hwi.buffalo.edu
>>> 716-898-8623
>>> 
>>> 
>>> 
>>> 
>>> 
>>> *From: *CCP4 bulletin board  on behalf of Bernhard
>>> Rupp 
>>> *Organization: *k.k. Hofkristallamt
>>> *Reply-To: *"b...@hofkristallamt.org" 
>>> *Date: *Monday, July 22, 2019 at 1:42 PM
>>> *To: *"CCP4BB@JISCMAIL.AC.UK" 
>>> *Subject: *Re: challenges in structural biology
>>> 
>>> 
>>> 
>>> What about 'deep learning' applied to crystallization outcomes? Can it
>>> guide individual trials better than intuition? Can it find previously
>>> unknown promising combinations on a larger scale?
>>> 
>>> 
>>> 
>>> I think several people were well aware of this need for some sort of sound
>>> machine learning already 15 years ago but we had no cloud based AI
>>> 
>>> services thenmaybe it is time to pick this up - particularly if face
>>> recognition can classify the fine detail in 

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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[ccp4bb] REFERENCE_DATA_SET in XDS-CORRECT

[wtempel]

Hi all,
I have a dataset that looks like from (a) primitive hexagonal crystal(s),
apparent (from merging stats) point group 32 or may be (twinned?) 3. During
CORRECT, I am imposing space group number 143 and specify a
REFERENCE_DATA_SET to determine the optimal reindexing operator between two
wedges of images that are separated by a wedge of images with horrible
patterns. Why does XDS prefer the first over the third reindexing choice,
given that .88 > .84?

 Several solutions exist for the given space group that explain the data. The
 selected solution (marked "*") correlates best with the given
reference data set.
 The independent settings of the cell can be obtained by application of the
 REINDEXING TRANSFORMATION to the original indices H,K,L from file INTEGRATE.HKL

 CORRELATION = correlation factor with the reference data set
 NPAIR   = number of unique reflection pairs correlated
 Rmeas=Rrim  = redundancy independent R-factor (intensities)
   For definition see:
   Rmeas : Diederichs & Karplus,
   Nature Struct. Biol. 4, 269-275 (1997);
   Rrim  : Weiss & Hilgenfeld,
   J.Appl.Cryst. 30,203-205 (1997).
 COMPARED= number of reflections used for calculating Rmeas
 ESD = Agreement with given unit cell geometry

 CORRELATION  NPAIR  Rmeas  COMPARED  ESD   REINDEX TRANSFORMATION

  *   0.84  142   20.0 2690.00  1  0  0  0 -1 -1  0  0  0  0 -1  0
  0.25  141   20.0 2690.00  1  1  0  0  0 -1  0  0  0  0 -1  0
  0.88  142   20.0 2690.00  1  0  0  0  0  1  0  0  0  0  1  0
  0.23  141   20.0 2690.00  0 -1  0  0  1  1  0  0  0  0  1  0

Many thanks.
Wolfram Temp



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Re: [ccp4bb] Combining MR and SIR phases

[Phil]

Nomenclature: pure SIR or MIR without anomalous has almost never been used, 
since the beginning of macromolecular crystallography (why would you?). So 
those of us who are lazy have often used SIR/MIR when we meant SIRAS/MIRAS: we 
should be more precise. 
Phil 

Sent from my iPhone

> On 23 Jul 2019, at 15:23, Eleanor Dodson 
> <176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> Oops George - you are correct..
> I guess I never think of using either SIR or SAD phases without some 
> investigation into hand, and then only work with the better result.
> 
> The selection for SAD is rather straightforward - use SHELXE to do density 
> modification and if your sites are correct it is usually very clear which 
> gives the best result.
> 
> I dont think anyone now would just do SIR phasing without including the 
> anomalous contribution from the sub-structure as well, and with that extra 
> information you can choose the correct hand. 
> 
> An anomalous difference map with phases from the correct hand shows the 
> anomalous scatterers as strong peaks.
> Eleanor
> 
> 
> 
> 
> 
>> On Tue, 23 Jul 2019 at 15:03, George Sheldrick  
>> wrote:
>> I'm afraid that I have to disagree with Eleanor, a very rare event. Both 
>> pure SIR and pure SAD give you only half of the necessary phase information. 
>> The initial map will in both cases be a double image. One then tries to 
>> improve it by density modification. For pure SAD one image is positive and 
>> one is negative, so just setting negative density to zero is a good start. 
>> For pure SIR both images are positive so this does not help, the map remains 
>> centrosymmetric. This also explains why SAD works much better if the solvent 
>> content is high, there is less danger of the images canceling each other. 
>> This is much less of a problem for MAD or SiRAS, both of which could work 
>> for a bromine derivative. If you collected Friedel opposites you may still 
>> be able to try SIRAS.
>> 
>> George
>> 
>> 
>> 
>>> On 23.07.19 11:43, Eleanor Dodson wrote:
>>> Well - there are lots of ways to proceed. It doesnt really matter for the 
>>> crystallography theory if the exptl phases are from SAD or SIR. Just harder 
>>> to handle in some software 
>>> I think I would start to refine the poor MR model with the xptl phases as 
>>> restraints. 
>>> REFMAC will do this. Then see if the maps look better - can you see 
>>> features not in the model?..
>>> If they do you could use Buccaneer to try to rebuild the existing model 
>>> using exptl phases..
>>> It might work! 
>>> Eleanor
>>> 
 On Tue, 23 Jul 2019 at 09:55, Luke Smithers 
  wrote:
 Hi all,
 
 I have collected native data and data on a bromide derivative of one of my 
 crystals, but have struggled to get a second derivative. I went through 
 the SIR pipeline as the anomalous signal from the Br was weak and have 
 generated some not-so-great phases. I have also attempted MR with the 
 closest match in the PDB (which is only 22% sequence identity) and got 
 some more not-so-great phases. I have found plenty of programs that will 
 combine SAD/MR phases, is there anything that will do SIR/MR? I understand 
 it's likely I will need more and better data and ideally a second 
 derivative but thought it was worth putting it out there to see if there 
 is something like this.
 
 Luke
 
 -- 
 Luke Smithers
 
 PhD Candidate
 
 Protein Production and Structure Facility, School of Molecular Sciences 
 
 T +61 8 6488 3163  
 
 
 
 
 
 
  
 
 
 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
>>> 
>> -- 
>> Prof. George M. Sheldrick FRS
>> Dept. Structural Chemistry
>> University of Goettingen
>> Tammannstr.  4
>> D37077 Goettingen
>> Germany
>> Tel: +49 551 3933021 or +49 5594 227312
>> 
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>> 
> 
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Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] challenges in structural biology

[Reza Khayat]

Biophysical techniques used to screen samples (e.g. SEC, SEC-MALS, DLS, SAXS, 
CD...) before freezing are not as promising as many hope for. There are lots of 
examples where samples behave beautiful by multiple biophysics methods and then 
crash and burn on a cryo-EM grid. Consequently, screening freezing conditions 
become the bottle neck of cryo-EM.

?
Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

From: CCP4 bulletin board  on behalf of Patrick Shaw 
Stewart 
Sent: Tuesday, July 23, 2019 1:35 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] Re: [ccp4bb] challenges in structural biology



On a completely different tack, isn't the most pressing requirement in current 
structural biology a really good method of characterizing macromolecular 
samples before they are put onto cryoEM grids - ie analysing and screening them 
in solution.

For one thing I'm told those huge microscopes are quite prone to breaking down, 
which makes the queues (lines) to get onto them even longer.

That method might be (micro-scale) DLS - or something completely different.

Thx, Patrick


On Mon, Jul 15, 2019 at 8:44 PM Holton, James M 
<270165b9f4cf-dmarc-requ...@jiscmail.ac.uk>
 wrote:
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




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 Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
 Directors: Peter Baldock, Patrick Shaw Stewart

 
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Re: [ccp4bb] challenges in structural biology

[Patrick Shaw Stewart]

On a completely different tack, isn’t the most pressing requirement in
current structural biology a really good method of characterizing
macromolecular samples *before *they are put onto cryoEM grids – ie
analysing *and screening them *in solution.

For one thing I’m told those huge microscopes are quite prone to breaking
down, which makes the queues (lines) to get onto them even longer.

That method might be (micro-scale) DLS – or something completely different.

Thx, Patrick


On Mon, Jul 15, 2019 at 8:44 PM Holton, James M <
270165b9f4cf-dmarc-requ...@jiscmail.ac.uk> wrote:

> 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://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>


-- 
 patr...@douglas.co.ukDouglas Instruments Ltd.
 Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
 Directors: Peter Baldock, Patrick Shaw Stewart

 http://www.douglas.co.uk
 Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034
 Regd. England 2177994, VAT Reg. GB 480 7371 36



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Re: [ccp4bb] challenges in structural biology

[Patrick Shaw Stewart]

Hi James – thx for starting a riveting thread.

(Of course) I agree with Dom, Janet, Artem and the cosmic cats that
crystallization is key.

I also agree with Artem a relatively modest investment in the fundamentals
of crystallization could make a big difference – even a 10% improvement in
productivity would save the community $ millions.

A high proportion of Nobel prizes (and highly-cited papers) are essentially
about method development.  So why is it so hard to get grants for new
scientific methods?  It’s as though current funding is only about charging
up the scientific motorways (interstates/autobahns for US/German citizens)
while ignoring the side-roads.  You have to claim that you will cure
cancer, diabetes, Ebola *and *ageing in less than six months for your grant
application to be considered.

But the side-roads have often yielded the most important scientific
breakthroughs.


The current approach to crystallization is (I’m told) a type of
"martingale" – that is, a betting scheme/stochastic process where your next
bet is based on analysis of previous rounds of betting.  The problem is
that if your starting assumptions are flawed it may take many decades to
arrive at a good solution.

What would fundamental research into crystallization look like?  I used to
think it was a matter of “boiling down” the dimensions in a typical
crystallization screen to say 10 “underlying variables” using multivariate
analysis.  Now I think this view is wrong.  There are a few variables that
are common to all crystallization experiments (temperature, pH and
“saturation” – whatever that is) but we also have to explore the space of
all the possible small molecules that can interact with - and probably bind
to - our target protein and help it to crystallize.  So crystallization
space is similar to chemical space – very big indeed.

I (now) think the targets of crystallization experiments are like this
picture:

*https://www.douglas.co.uk/f_ftp1/How%20desperate%20are%20you.jpg
*


There are some proteins that just need to be pushed out of solution to
crystallize – like lysozyme.

Then there are others that need to combine somehow with one particular
small molecule to make crystals.  For example, thaumatin crystallizes very
easily if there’s tartrate in the drop.

Others need two small molecule additives.  Presumably still others could
crystallize if only we could find the right combination of 3, 4 or 5 small
molecules.

(Tartrate in the example above is in a sense a “silver bullet”.  Hampton
Research called their screen that because they had the idea the additives
would self-select, so you could put lots of them in each condition.
Strangely, their Silver Bullet screen never worked as well as some of us
expected - I don’t know why.  But there isn’t one silver bullet – there
are, or could be, thousands.)

Random microseeding including cross-seeding works really well and is one of
Artem's silver bullets of crystallization methods – but we still have to
get our first crystals somehow.

Practical questions that could (and should!) be answered include:

1. Which are the best precipitants and how many do we need?  Maybe 4 or 5
would be enough.
2. How can we identify the best set of several hundred small molecules to
use as additives?
3. How many small molecules should we put in each crystallization trial?
4. What should we do about pH and temperature?


I think these questions could be answered by one lab, with good
experimental design and automation, using say 25 target proteins – not
including lysozyme, see above - that can be bought from Sigma etc (Artem,
making them yourself is too much like hard work!)

This can make both screening and optimization more efficient.  Yes it would
be a lot of work, but the current approach is a lot of work for hundreds
(thousands?) of labs all over the world - work that is largely wasted.

But it needs proper funding – and I don’t know how to get that.

Best wishes to all,

Patrick

Ps Of course I agree with Janet, Tom and others that good record-keeping is
essential.  But there is a limit to what we will learn if we insist on
solving structures at the same time.  I say just buy in the model proteins
by the gram and focus what is *really *going on in crystallization.


On Sun, Jul 21, 2019 at 4:29 PM Artem Evdokimov 
wrote:

> Excellent question :)
>
> First of all, thank you for putting this out to the community!
>
> Secondly, I agree with several of us who've written that a single
> conference is not enough to discuss all the possible topics.
>
> Thirdly, in my opinion all the other problems are secondary to the main
> (and only remaining!) problem in crystallography: getting
> diffraction-quality protein crystals reproducibly and quickly
>
> The amount of funding for serious crystallization research seems to be
> close to non-existent. In general methodology funding is hard to get, but
> crystallization seems to me like the 

Re: [ccp4bb] challenges in structural biology

[Engin Özkan]

On 7/23/19 3:35 AM, melanie.voll...@diamond.ac.uk wrote:
> No longer those 20 odd names for ammonium sulphate

You mean ammonium *sulfate*. As it is called across the pond. :)

On a related note on common nomenclature for recording crystallization 
experiments that Janet brought up:

I find it odd that we still do not report cryo-protection methods and 
conditions in PDB depositions. Given that a large fraction of the small 
molecules observed in crystal structures are derived from the 
cryo-protectants, one would think that reporting the contents of that 
solution (and pH) would be paramount to a PDB deposition. Surely, the 
crystallographic experiment has changed since 1990/use of synchrotron 
sources, which PDB has adjusted well to in most other aspects (e.g., 
including reporting of synchrotron x-ray optics and all the new 
detectors during submission).

Engin




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Re: [ccp4bb] higher older tNCS

[Randy Read]

Dear Jessica,

There's a dedicated Phenix bulletin board, where it would be better to post 
Phenix-specific questions.

Nonetheless, you can do this in both the CCP4 and Phenix interfaces to Phaser, 
so I'll explain how to do this in all the interfaces.

First, I'm guessing that the order of the tNCS wasn't unambiguous, that Phaser 
chose something other than NMOL 3 in the automated run, but now you want to 
test the alternative of NMOL 3.

ccp4i: Under the "User parameters" tab, change "Number of assemblies related by 
TNCS vector" to 3.  The toggle should automatically be checked when you do that.

ccp4i2: In the Expert Mode Phaser MR GUI, under the "Keywords" tab, change 
"Number of molecules or complexes related by translation vector" to 3.

Phenix: in the Phaser-MR (full-featured) GUI, "Input and general options" tab, 
click on "Other settings".  In the "Translational NCS" section of the popup, 
set "Number of molecules or complexes related by translation vector" to 3.

And for completeness, if you're writing a command-line script, the command is 
"TNCS NMOL 3".

Best wishes,

Randy Read

> On 23 Jul 2019, at 16:06, Jessica Besaw  wrote:
> 
> Hello everyone, 
> 
> Does anyone know of a good phenix tutorial on how to deal with higher order 
> translational non-crystallographic symmetry (tNCS) in a structure? 
> 
> Ultimately, I need to know how to set TNCS NMOL to 3 in phaser of Phenix. I 
> am using the phenix GUI, and I am uncertain how to alter this parameter.  
> 
> Any help would be greatly appreciated
> 
> Cheers!
> 
> Jessica 
> 
>  
> 
>  
> 
>   Virus-free. www.avg.com 
> 
>  
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
> 
--
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research Tel: + 44 1223 336500
The Keith Peters Building   Fax: + 44 1223 336827
Hills Road   E-mail: 
rj...@cam.ac.uk
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[ccp4bb] higher older tNCS

[Jessica Besaw]

Hello everyone,

Does anyone know of a good phenix tutorial on how to deal with higher order
translational non-crystallographic symmetry (tNCS) in a structure?

Ultimately, I need to know how to set TNCS NMOL to 3 in phaser of Phenix. I
am using the phenix GUI, and I am uncertain how to alter this parameter.

Any help would be greatly appreciated

Cheers!

Jessica




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[ccp4bb] Postdoc position available in an enzymology laboratory at Florida State University

[Adarsh Kumar]

Dear all

One postdoctoral position is available immediately in an enzymology laboratory 
to elucidate mechanisms of DNA/RNA synthesis, DNA lesion bypass, and base 
excision repair catalyzed by various human and viral DNA/RNA polymerases, and 
to mechanistically investigate gene editing enzymes including CRISPR/Cas9 and 
Cas13a. Background in structural biology (X-ray crystallography and/or CryoEM), 
biophysics, protein purification, and recombinant DNA techniques is preferred. 
Prior experience with DNA binding proteins would be an added advantage. An 
important criterion for selection will be the ability of the desired individual 
to pursue independent research in an active multi-disciplinary setting. 

Interested candidates should send a CV and a statement describing experience, 
goals, and reasons for the interest in this position, and arrange to have three 
letters of recommendation sent to: 
Dr. Zucai Suo
Eminent Professor and Dorian and John Blackmon Chair of Biomedical Science
Department of Biomedical Sciences
Florida State University - College of Medicine
Tallahassee, FL
Email: zucai@med.fsu.edu

Further information about the research projects in the Suo Lab can be obtained 
from https://med.fsu.edu/suolab/research
The list of publications can be found at https://med.fsu.edu/suolab/publications

Job Description:
Employer: Florida State University College of Medicine
Location: Tallahassee, Florida
Salary: NIH guidelines for post-doctoral salaries + benefits
Discipline: Life Sciences, Biochemistry, Biology, Biophysics, Structural 
Biology, and Biomedical Sciences
Position Type: Full Time
Organization Type: Academia
Job Type: Postdoc



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[ccp4bb] PhD Positions: structural biology in biomedical research

[Oberer, Monika (m.obe...@uni-graz.at)]

Dear Colleagues,


The Medical University of Graz and the University of Graz, offer PhD positions 
in the international PhD programs including the framework of  the Special 
Research Program (SFB) Lipid Hydrolysis.

We are hiring motivated PhD candidates for positions  and offer an in-depth, 
multidisciplinary training in biomedical research in a stimulating 
international environment. The thesis projects focus on aspects of metabolic 
and cardiovascular diseases, cancer, lipids and metabolism as novel therapeutic 
targets and diagnostic methods, and integrate basic research and 
clinically-oriented sciences utilizing a wide spectrum of state-of-the-art 
techniques including BIOMOLECULAR STRUCTURAL BIOLOGY.


Applicants must hold (or be close to obtaining) an undergraduate degree 
equivalent to a Master in any discipline of natural or life sciences or 
medicine. The selection procedure, all training activities and communications 
will be in English. Thus, excellent written and spoken English skills are 
required.

Besides being a dynamic place for science, Graz is regularly ranked among the 
European cities with the best quality of living, and a lively student city, not 
far from mountains and Mediterranean. There are  15 open positions, including 
two (PIs Oberer, Madl) using integrated structural biology approaches.

For more details please follow the links:

https://www.medunigraz.at/DK_MCD/Call_Open_Projects.htm
and
http://www.medunigraz.at/DK_MCD/Call.htm

and
https://www.medunigraz.at/lipid-hydrolysis/

Best wishes,
Monika Oberer
(PI in SFB Lipid Hydrolysis)


Assoc. Prof. Dr. Monika Oberer
Institute of Molecular Biosciences - Structural Biology
University of Graz
Humboldtstrasse 50/3
8010 Graz
Austria

Email: m.obe...@uni-graz.at
Phone: ++43-316-380-5431
*





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Re: [ccp4bb] Combining MR and SIR phases

[Eleanor Dodson]

Oops George - you are correct..
I guess I never think of using either SIR or SAD phases without some
investigation into hand, and then only work with the better result.

The selection for SAD is rather straightforward - use SHELXE to do density
modification and if your sites are correct it is usually very clear which
gives the best result.

I dont think anyone now would just do SIR phasing without including the
anomalous contribution from the sub-structure as well, and with that extra
information you can choose the correct hand.

An anomalous difference map with phases from the correct hand shows the
anomalous scatterers as strong peaks.
Eleanor





On Tue, 23 Jul 2019 at 15:03, George Sheldrick 
wrote:

> I'm afraid that I have to disagree with Eleanor, a very rare event. Both
> pure SIR and pure SAD give you only half of the necessary phase
> information. The initial map will in both cases be a double image. One then
> tries to improve it by density modification. For pure SAD one image is
> positive and one is negative, so just setting negative density to zero is a
> good start. For pure SIR both images are positive so this does not help,
> the map remains centrosymmetric. This also explains why SAD works much
> better if the solvent content is high, there is less danger of the images
> canceling each other. This is much less of a problem for MAD or SiRAS, both
> of which could work for a bromine derivative. If you collected Friedel
> opposites you may still be able to try SIRAS.
>
> George
>
>
> On 23.07.19 11:43, Eleanor Dodson wrote:
>
> Well - there are lots of ways to proceed. It doesnt really matter for the
> crystallography theory if the exptl phases are from SAD or SIR. Just harder
> to handle in some software
> I think I would start to refine the poor MR model with the xptl phases as
> restraints.
> REFMAC will do this. Then see if the maps look better - can you see
> features not in the model?..
> If they do you could use Buccaneer to try to rebuild the existing model
> using exptl phases..
> It might work!
> Eleanor
>
> On Tue, 23 Jul 2019 at 09:55, Luke Smithers <
> luke.smith...@research.uwa.edu.au> wrote:
>
>> Hi all,
>>
>> I have collected native data and data on a bromide derivative of one of
>> my crystals, but have struggled to get a second derivative. I went through
>> the SIR pipeline as the anomalous signal from the Br was weak and have
>> generated some not-so-great phases. I have also attempted MR with the
>> closest match in the PDB (which is only 22% sequence identity) and got some
>> more not-so-great phases. I have found plenty of programs that will combine
>> SAD/MR phases, is there anything that will do SIR/MR? I understand it's
>> likely I will need more and better data and ideally a second derivative but
>> thought it was worth putting it out there to see if there is something like
>> this.
>>
>> Luke
>>
>> --
>>
>> *Luke Smithers*
>>
>> *PhD Candidate*
>>
>> Protein Production and Structure Facility, School of Molecular Sciences
>>
>> *T *+61 8 6488 3163
>>
>> [image: The University of Western Australia]
>> 
>>
>> [image: Pursue Impossible]
>> [image:
>> Facebook]
>> [image:
>> Twitter]
>> [image:
>> Youtube]
>> 
>>
>> [image:
>> http://static.weboffice.uwa.edu.au/visualid/graphics/email/sig2015/campaign.gif]
>> 
>>
>>
>>
>> --
>>
>> 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
>
> --
> Prof. George M. Sheldrick FRS
> Dept. Structural Chemistry
> University of Goettingen
> Tammannstr.  4
> D37077 Goettingen
> Germany
> Tel: +49 551 3933021 or +49 5594 227312
>
>
> --
>
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[ccp4bb] Rigaku system up for grabs

[Thomas Edwards]

Dear CCP4bb

We are going through a large lab refurbishment programme and that, along with 
space costings for facilities, mean that sadly we will not be able to keep our 
local generator & detector.

If anybody can come to Leeds and take it all away (free!) then please do so!

The system:
Rigaku MicroMax 007 High-Flux generator with Raxis IV++ detector
About 6 years old.

Please do get in touch.

Ed

T.A.Edwards Ph.D.
Associate Professor of Biochemistry
Deputy Head of School
_
Astbury Centre for Structural Molecular Biology
School of Molecular and Cellular Biology
Faculty of Biological Sciences
===
Garstang 8.53d
University of Leeds, Leeds, LS2 9JT 
Telephone: 0113 343 3031
Faculty Staff 
Profile
Astbury Centre Web 
Page
[signature_1395366918]
Perturbation of Protein-Protein Interactions

Invention, my dear friends, is 93% perspiration, 6% electricity, 4% 
evaporation, and 2% butterscotch ripple. ~Willy Wonka

[/Users/bmbtae/bmbtae/0_Docs/Astbury/0_AstburyConversation/2020/01784_Astbury 
Conversation 2020 email signature 
(WR).png]



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Re: [ccp4bb] Combining MR and SIR phases

[George Sheldrick]

I'm afraid that I have to disagree with Eleanor, a very rare event. Both 
pure SIR and pure SAD give you only half of the necessary phase 
information. The initial map will in both cases be a double image. One 
then tries to improve it by density modification. For pure SAD one image 
is positive and one is negative, so just setting negative density to 
zero is a good start. For pure SIR both images are positive so this does 
not help, the map remains centrosymmetric. This also explains why SAD 
works much better if the solvent content is high, there is less danger 
of the images canceling each other. This is much less of a problem for 
MAD or SiRAS, both of which could work for a bromine derivative. If you 
collected Friedel opposites you may still be able to try SIRAS.


George


On 23.07.19 11:43, Eleanor Dodson wrote:
Well - there are lots of ways to proceed. It doesnt really matter for 
the crystallography theory if the exptl phases are from SAD or SIR. 
Just harder to handle in some software
I think I would start to refine the poor MR model with the xptl phases 
as restraints.
REFMAC will do this. Then see if the maps look better - can you see 
features not in the model?..
If they do you could use Buccaneer to try to rebuild the existing 
model using exptl phases..

It might work!
Eleanor

On Tue, 23 Jul 2019 at 09:55, Luke Smithers 
> wrote:


Hi all,

I have collected native data and data on a bromide derivative of
one of my crystals, but have struggled to get a second derivative.
I went through the SIR pipeline as the anomalous signal from the
Br was weak and have generated some not-so-great phases. I have
also attempted MR with the closest match in the PDB (which is only
22% sequence identity) and got some more not-so-great phases. I
have found plenty of programs that will combine SAD/MR phases, is
there anything that will do SIR/MR? I understand it's likely I
will need more and better data and ideally a second derivative but
thought it was worth putting it out there to see if there is
something like this.

Luke

-- 


*Luke Smithers*

*PhD Candidate*

Protein Production and Structure Facility, School of Molecular
Sciences

*T *+61 8 6488 3163

The University of Western Australia





Pursue Impossible

Facebook

Twitter

Youtube



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Dept. Structural Chemistry
University of Goettingen
Tammannstr.  4
D37077 Goettingen
Germany
Tel: +49 551 3933021 or +49 5594 227312




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Re: [ccp4bb] Combining MR and SIR phases

[Eleanor Dodson]

Well - there are lots of ways to proceed. It doesnt really matter for the
crystallography theory if the exptl phases are from SAD or SIR. Just harder
to handle in some software
I think I would start to refine the poor MR model with the xptl phases as
restraints.
REFMAC will do this. Then see if the maps look better - can you see
features not in the model?..
If they do you could use Buccaneer to try to rebuild the existing model
using exptl phases..
It might work!
Eleanor

On Tue, 23 Jul 2019 at 09:55, Luke Smithers <
luke.smith...@research.uwa.edu.au> wrote:

> Hi all,
>
> I have collected native data and data on a bromide derivative of one of my
> crystals, but have struggled to get a second derivative. I went through the
> SIR pipeline as the anomalous signal from the Br was weak and have
> generated some not-so-great phases. I have also attempted MR with the
> closest match in the PDB (which is only 22% sequence identity) and got some
> more not-so-great phases. I have found plenty of programs that will combine
> SAD/MR phases, is there anything that will do SIR/MR? I understand it's
> likely I will need more and better data and ideally a second derivative but
> thought it was worth putting it out there to see if there is something like
> this.
>
> Luke
>
> --
>
> *Luke Smithers*
>
> *PhD Candidate*
>
> Protein Production and Structure Facility, School of Molecular Sciences
>
> *T *+61 8 6488 3163
>
> [image: The University of Western Australia]
> 
>
> [image: Pursue Impossible]
> [image:
> Facebook]
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> Twitter]
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> 
>
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> http://static.weboffice.uwa.edu.au/visualid/graphics/email/sig2015/campaign.gif]
> 
>
>
>
> --
>
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Re: [ccp4bb] Questionable Ligand Density: 6MO0, 6MO1, 6MO2

[Pearce, N.M. (Nick)]

Hi,

I agree with Melanie and I think we should also go further...

We could require reviewers to tick a box on a PDB reviewer page to confirm they 
have “checked” the structure. Whether this box has been ticked could then be 
displayed on the PDB page (of course the reviewer would remain anonymous) or 
sent to the journal that published the paper (which could them prompt the 
reviewer to do so). It would be transparent for both depositors, editors, and 
users whether the structure had been reviewed. In papers with multiple 
reviewers, of course not all reviewers would be expected to look (i.e. 
interdisciplinary papers). As John says, many journals already require 
validation reports — this is only one small step further and wouldn’t unduly 
increase the work load for reviewers, since we already expect them to check the 
structure. 

I’m not saying it would completely solve the problem as people could just log 
on and click the button without reading, but it would at least be the start of 
accountability or at least record keeping. If you forced (assigned) reviewers 
to visit the PDB webpage, seeing a large array of red sliders might at least 
trigger a reviewer to look more carefully. Even better, a potential reviewer 
page could contain automatically generated electron density pictures for 
ligands (as are already available on the pdb websites, so generating them isn’t 
extra work) — this would avoid the problem where images in the paper are 
inaccurately or incorrectly generated. Again, it might trigger reviewer 
investigation if they’re forced to click a button on the websites for “I am ok 
with this structure and it is of appropriate quality for the conclusions of the 
paper”. 

This would of course require journals to require links to the pdb depositions 
on manuscript submission rather than asking for them if a reviewer requests it 
— I don’t think that’s a big ask.

Thanks,
Nick

> On 23 Jul 2019, at 09:52, "melanie.voll...@diamond.ac.uk" 
>  
> Dear John,
> 
> 
> Yes, I think the PDB should be stricter. The PDB is in the position to 
> enforce compliance with rules and if they are not followed then one doesn't 
> get a validation report and in turn it would be difficult to publish (most 
> journals require a validation report). For years it has been tried to make 
> adherence to standards voluntary but there are still examples, like the one 
> that started the discussion, where people just don't bother.
> 
> 
> For these particular structures I actually suspect that even if there were 
> all reports given to the referees they may not have looked carefully enough. 
> The journal where they were published in is heavy on the chemistry side so 
> perhaps the referees focused on the synthesis of the compounds rather than 
> the structure and the interaction within the protein. Aside from that, at a 
> resolution of 2.7A to 3A discussing detailed chemical interactions and 
> placing a compound with confidence is questionable anyway.
> 
> 
> Cheers
> 
> 
> M
> 
> 
> From: CCP4 bulletin board  on behalf of John 
> Berrisford 
> Sent: 22 July 2019 22:23:47
> To: ccp4bb
> Subject: Re: [ccp4bb] Questionable Ligand Density: 6MO0, 6MO1, 6MO2
> 
> Dear Harry
> 
> We will be shortly making it mandatory for depositors to provide a value for 
> at least one of the merging statistics (Rmerge, Rpim, CC1/2 etc..). Most 
> depositors do, but we want to ensure that all depositors do provide at least 
> one value for a merging metric.
> 
> We would welcome feedback if we should be stricter and require a (or more 
> than one) specific metric (e.g. CC1/2) – please be aware that any required 
> metric must be available from all merging/scaling software.
> 
> Thanks
> 
> John
> 
> From: CCP4BB 
> Sent: 22 July 2019 11:32
> To: John Berrisford 
> Cc: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Questionable Ligand Density: 6MO0, 6MO1, 6MO2
> 
> Hi John
> 
> These are great, but the things that make me suspicious are the values of 
> overall R(merge); these are tucked away in the full reports, rather than 
> highlighted with all the other structural metrics in the validation sliders. 
> It would be wonderful to be able to see at a glance where overall R(merge) 
> values like these fit in with those of other deposited structures (even 
> better if it could be drawn to the attention of authors before final 
> deposition).
> 
> Kay and Gérard have already pointed out that the data processing here may 
> have some issues.
> 
> Of course, those of us involved in teaching data processing have been 
> emphasizing the importance of CC(1/2) rather than relying on R(merge) for 
> yonks, but if CC(1/2) isn't given in the report it's all we have to go
> 
> Harry
> --
> Dr Harry Powell
> 
> On 22 Jul 2019, at 10:05, John Berrisford 
> mailto:j...@ebi.ac.uk>> wrote:
> Dear Daniel
> 
> The issues you mentioned are highlighted in the wwPDB validation report
> 

[ccp4bb] Combining MR and SIR phases

[Luke Smithers]

Hi all,

I have collected native data and data on a bromide derivative of one of my
crystals, but have struggled to get a second derivative. I went through the
SIR pipeline as the anomalous signal from the Br was weak and have
generated some not-so-great phases. I have also attempted MR with the
closest match in the PDB (which is only 22% sequence identity) and got some
more not-so-great phases. I have found plenty of programs that will combine
SAD/MR phases, is there anything that will do SIR/MR? I understand it's
likely I will need more and better data and ideally a second derivative but
thought it was worth putting it out there to see if there is something like
this.

Luke

-- 

*Luke Smithers*

*PhD Candidate*

Protein Production and Structure Facility, School of Molecular Sciences

*T *+61 8 6488 3163

[image: The University of Western Australia]


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Re: [ccp4bb] challenges in structural biology

[melanie.voll...@diamond.ac.uk]

I don't think AI will do our job in future as it heavily relies on the crystal 
structures for training. However, as a community we should embrace this 
technology/method to help us solve our structures. And why not start with 
crystallisation? And again the PDB is in a good position here to enforce 
standards which will pave the way to make use of all the information in the 
database to train AI. All chemicals must be IUPAC conform an then the PDB can 
decide about trivial names based on a set of rules for humans (AI doesn't care 
how you name it as long as it is consistent). No longer those 20 odd names for 
ammonium sulphate as Janet pointed out years ago.


And regarding a magic bullet (as in one size fits all), why should a kinase 
crystallise in the same condition as a polymerase? They do different jobs in a 
different micro-environment within the cell so their chemical properties will 
be different. Perhaps there could be some common ground for evolutionary 
related molecules but a conserved active site doesn't mean a similar surface 
for crystal contacts which is the key bit in crystallisation, right?


But as Kay pointed out, crystallisation is a whole field on its own and will go 
beyond the GRC and the question asked by James.


M


From: CCP4 bulletin board  on behalf of Kay Diederichs 

Sent: 23 July 2019 08:59:10
To: ccp4bb
Subject: Re: [ccp4bb] challenges in structural biology

If you look at the nice figure at the top of the online article, do you believe 
that this (or rather, the correct) arrangement of domains/ molecules can be 
predicted from a couple of correlated mutations, and energy minimization? I 
think AI is a long way from that.  Finding the correct fold of a compact 
domain, yes I think it's getting there.

best,
Kay


On Tue, 23 Jul 2019 08:28:42 +0530, Nishant Varshney  wrote:

>What about AI doing our job in the future?
>
>https://www.nature.com/articles/d41586-019-01357-6?utm_source=Nature+Briefing_campaign=4c1d57fdf3-briefing-dy-20190722_medium=email_term=0_c9dfd39373-4c1d57fdf3-44201949
>
>Best Regards
>Nishant
>
>On Mon, 22 Jul 2019 at 11:30 PM, Sarah Bowman 
>wrote:
>
>> I'd like to point out that the MAchine Recognition of Crystallization
>> Outcomes (MARCO) makes a start to 'deep learning applied to crystallization
>> outcomes', at least in terms of being able to classify drop images
>> efficiently.
>>
>>
>>
>> There is obviously more work to be done to correlate these data with
>> crystallization cocktail components (which Janet and Tom point out the
>> difficulties with) and positive outcomes.  It seems the first step really
>> needs to be consistent descriptions and vocabulary - I fully agree with
>> Janet here!
>>
>>
>>
>> Reference on MARCO for those interested: Bruno AE, Charbonneau P, Newman
>> J, Snell EH, So DR, Vanhoucke V, et al. (2018) Classification of
>> crystallization outcomes using deep convolutional neural networks. PLoS ONE
>> 13(6): e0198883. https://doi.org/10.1371/journal.pone.0198883
>>
>>
>>
>> Cheers,
>>
>> Sarah
>>
>>
>>
>> *Sarah EJ Bowman, PhD*
>>
>>
>>
>> Associate Research Scientist, Hauptman-Woodward Medical Research Institute
>>
>> Director, High-Throughput Crystallization Screening Center
>>
>> Research Associate Professor, Department of Biochemistry, University at
>> Buffalo
>>
>>
>>
>> Research Webpage 
>>
>> www.getacrystal.org
>>
>>
>>
>> sbow...@hwi.buffalo.edu
>> 716-898-8623
>>
>>
>>
>>
>>
>> *From: *CCP4 bulletin board  on behalf of Bernhard
>> Rupp 
>> *Organization: *k.k. Hofkristallamt
>> *Reply-To: *"b...@hofkristallamt.org" 
>> *Date: *Monday, July 22, 2019 at 1:42 PM
>> *To: *"CCP4BB@JISCMAIL.AC.UK" 
>> *Subject: *Re: challenges in structural biology
>>
>>
>>
>> What about 'deep learning' applied to crystallization outcomes? Can it
>> guide individual trials better than intuition? Can it find previously
>> unknown promising combinations on a larger scale?
>>
>>
>>
>> I think several people were well aware of this need for some sort of sound
>> machine learning already 15 years ago but we had no cloud based AI
>>
>> services thenmaybe it is time to pick this up - particularly if face
>> recognition can classify the fine detail in faces maybe we finally could do
>> this with drop images as well...
>>
>>
>>
>> A summary of the state of affairs then is here:
>>
>>
>> http://www.ruppweb.org/cvs/br/rupp_2004_methods_predictive_models_crystallization.pdf
>>
>>
>>
>> LG BR
>>
>>
>>
>>
>>
>> Am 21.07.19 um 23:04 schrieb Artem Evdokimov:
>>
>> Dear Kay
>>
>>
>>
>> I disagree that 'magic bullet' is impossible. I think the definition is
>> wrong here - magic bullet to me is a rational set of methods that (when
>> executed with precision and care) enable crystallization to the maximum
>> possible benefit. This includes everything - constructs, crystallization
>> design, etc. Part of the magic bullet is also a precise 

Re: [ccp4bb] Density questionable?

[melanie.voll...@diamond.ac.uk]

Dear Peer, Engi,


Have you looked at the symmetry mates in Coot? If you look at the packing of 
your molecules and it makes sense to place an additional molecule in the gap in 
order to create crystal contacts then you should do so.


I would place the whole molecule there and start with an occupancy of say 20% 
for the whole chain and refine and check the B factors if they are more like 
the other molecules or higher/lower. If they are lower then you can increase 
the occupancy, if they are higher than you have to lower it. You will then 
probably see more undefined, bloby density which means you have additional 
conformations for loops or secondary structural elements or maybe the whole 
molecule is a bit twisted. Engi, at 1.8A you should be able to build 
alternative conformations, for the 2.3A of Peer this will be more difficult.


My guess is that your molecule is there but a bit "squashed" and hence has 
slightly different conformations in each unit cell and you will struggle to 
place just one model. You will also probably struggle to build side chains. Did 
you check for anisotropy? I would suspect that the resolution is lower in one 
or even two directions in connection with a large un-ordered/missing molecule.


Cheers


M


From: CCP4 bulletin board  on behalf of Kay Diederichs 

Sent: 23 July 2019 08:41:51
To: ccp4bb
Subject: Re: [ccp4bb] Density questionable?

Hi Engi,

I understand that you say that the 2nd molecule in your case is not real; it's 
just model bias?

I would not come to this conclusion. B values of NCS-related chains being quite 
different is not that rare. In my experience, at 1.8A and with a reasonably 
refined model (Rfree less than 30%), continuous electron density (even if it is 
visualized at low sigma) matching the model does not arise from model bias,. 
From your description, I infer that the second molecule exists in your crystal; 
it's "just" poorly ordered (or at least worse than the first) - otherwise 
ARP/wARP wouldn't build part of it. That ARP/wARP does not build the rest does 
not mean that a human cannot or should not build it, nor that it should not be 
built; the automatic programs typically only build the good parts.
The statistics of the MR solution should tell whether the 2nd molecule was 
found due to a significant signal (LLG increase in phaser; good contrast in 
molrep), or because the user was "asking" the program to position two molecules.
What is R/Rfree with/without the 2nd molecule? (Peer says that in his case the 
difference is 0.3% which I'd say is an insignificant difference)
Can the crystal lattice be continued in all 3 dimensions without the 2nd 
molecule?

best,
Kay

On Tue, 23 Jul 2019 06:22:31 +0200, Engi Hassaan  wrote:

>We faced a similar situation recently where our molecular replacement
>suggested 2 molecules in the asymmetric unit but the B factors of the
>second chain were twice as large as the first protien chain. At a
>resolution of 1.8 Å2 you could see the electron density for both chains. We
>performed ARP/wARP and realized only part of this second chain is resolved
>in our structure. It was model bias.
>
>Best,
>Engi
>
>
>Engi Hassaan
>PhD Student
>Research Group of Prof. Dr. G. Klebe
>Marburg, Germany
>
>
>On Mon, Jul 22, 2019, 19:34 Bernhard Rupp  wrote:
>
>> > Could there be two versions of each model: a "robustly-observed" and a
>> "most-likely" version?
>>
>> We tried/suggested something in this spirit once. Not sure how it was
>> received
>> http://journals.iucr.org/d/issues/2016/12/00/rr5136/index.html
>>
>> Best, BR
>> +
>> 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.
>>
>> -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 

Re: [ccp4bb] challenges in structural biology

[Kay Diederichs]

If you look at the nice figure at the top of the online article, do you believe 
that this (or rather, the correct) arrangement of domains/ molecules can be 
predicted from a couple of correlated mutations, and energy minimization? I 
think AI is a long way from that.  Finding the correct fold of a compact 
domain, yes I think it's getting there.

best,
Kay 


On Tue, 23 Jul 2019 08:28:42 +0530, Nishant Varshney  wrote:

>What about AI doing our job in the future?
>
>https://www.nature.com/articles/d41586-019-01357-6?utm_source=Nature+Briefing_campaign=4c1d57fdf3-briefing-dy-20190722_medium=email_term=0_c9dfd39373-4c1d57fdf3-44201949
>
>Best Regards
>Nishant
>
>On Mon, 22 Jul 2019 at 11:30 PM, Sarah Bowman 
>wrote:
>
>> I'd like to point out that the MAchine Recognition of Crystallization
>> Outcomes (MARCO) makes a start to 'deep learning applied to crystallization
>> outcomes', at least in terms of being able to classify drop images
>> efficiently.
>>
>>
>>
>> There is obviously more work to be done to correlate these data with
>> crystallization cocktail components (which Janet and Tom point out the
>> difficulties with) and positive outcomes.  It seems the first step really
>> needs to be consistent descriptions and vocabulary - I fully agree with
>> Janet here!
>>
>>
>>
>> Reference on MARCO for those interested: Bruno AE, Charbonneau P, Newman
>> J, Snell EH, So DR, Vanhoucke V, et al. (2018) Classification of
>> crystallization outcomes using deep convolutional neural networks. PLoS ONE
>> 13(6): e0198883. https://doi.org/10.1371/journal.pone.0198883
>>
>>
>>
>> Cheers,
>>
>> Sarah
>>
>>
>>
>> *Sarah EJ Bowman, PhD*
>>
>>
>>
>> Associate Research Scientist, Hauptman-Woodward Medical Research Institute
>>
>> Director, High-Throughput Crystallization Screening Center
>>
>> Research Associate Professor, Department of Biochemistry, University at
>> Buffalo
>>
>>
>>
>> Research Webpage 
>>
>> www.getacrystal.org
>>
>>
>>
>> sbow...@hwi.buffalo.edu
>> 716-898-8623
>>
>>
>>
>>
>>
>> *From: *CCP4 bulletin board  on behalf of Bernhard
>> Rupp 
>> *Organization: *k.k. Hofkristallamt
>> *Reply-To: *"b...@hofkristallamt.org" 
>> *Date: *Monday, July 22, 2019 at 1:42 PM
>> *To: *"CCP4BB@JISCMAIL.AC.UK" 
>> *Subject: *Re: challenges in structural biology
>>
>>
>>
>> What about 'deep learning' applied to crystallization outcomes? Can it
>> guide individual trials better than intuition? Can it find previously
>> unknown promising combinations on a larger scale?
>>
>>
>>
>> I think several people were well aware of this need for some sort of sound
>> machine learning already 15 years ago but we had no cloud based AI
>>
>> services thenmaybe it is time to pick this up - particularly if face
>> recognition can classify the fine detail in faces maybe we finally could do
>> this with drop images as well...
>>
>>
>>
>> A summary of the state of affairs then is here:
>>
>>
>> http://www.ruppweb.org/cvs/br/rupp_2004_methods_predictive_models_crystallization.pdf
>>
>>
>>
>> LG BR
>>
>>
>>
>>
>>
>> Am 21.07.19 um 23:04 schrieb Artem Evdokimov:
>>
>> Dear Kay
>>
>>
>>
>> I disagree that 'magic bullet' is impossible. I think the definition is
>> wrong here - magic bullet to me is a rational set of methods that (when
>> executed with precision and care) enable crystallization to the maximum
>> possible benefit. This includes everything - constructs, crystallization
>> design, etc. Part of the magic bullet is also a precise knowledge when
>> crystallization is unlikely (i.e. an actual proven predictor that
>> consistently discriminates between "you're going to succeed if you work
>> hard" and "it's doomed to fail, don't bother" scenarios in crystallization.
>>
>> The above is not sexy. It does not present itself as a lovely subject on
>> which to have international cocktail parties with politicians delivering
>> fancy speeches. But that is what is needed, and no one is funding that to
>> the best of my knowledge.
>>
>> What needs to be done is a significant amount of testing, standardization,
>> and methods development from the perspective of holistic outcome (i.e.
>> crystals that work) - and none of the previously advertised 'magic bullets'
>> work the way I just described.
>>
>> Having written this, I think you're right - this is a bit of a distraction
>> from James' original point. However it's a valid opportunity for a lively
>> discussion on its own :)
>>
>> Artem
>>
>> - Cosmic Cats approve of this message
>>
>> On Sun, Jul 21, 2019 at 4:52 PM Kay Diederichs <
>> kay.diederi...@uni-konstanz.de 
>> > wrote:
>>
>>  Dear Artem,
>>
>>  black or white is not my way of thinking, which is why I don't
>> believe in Hannibal's approach when it comes to crystallization.
>>
>>  None of the magic bullets that were advertised over the past decades
>> have proven generally applicable.  I believe more in incremental
>> 

Re: [ccp4bb] Density questionable?

[Kay Diederichs]

Hi Engi,

I understand that you say that the 2nd molecule in your case is not real; it's 
just model bias?

I would not come to this conclusion. B values of NCS-related chains being quite 
different is not that rare. In my experience, at 1.8A and with a reasonably 
refined model (Rfree less than 30%), continuous electron density (even if it is 
visualized at low sigma) matching the model does not arise from model bias,. 
From your description, I infer that the second molecule exists in your crystal; 
it's "just" poorly ordered (or at least worse than the first) - otherwise 
ARP/wARP wouldn't build part of it. That ARP/wARP does not build the rest does 
not mean that a human cannot or should not build it, nor that it should not be 
built; the automatic programs typically only build the good parts.
The statistics of the MR solution should tell whether the 2nd molecule was 
found due to a significant signal (LLG increase in phaser; good contrast in 
molrep), or because the user was "asking" the program to position two molecules.
What is R/Rfree with/without the 2nd molecule? (Peer says that in his case the 
difference is 0.3% which I'd say is an insignificant difference)
Can the crystal lattice be continued in all 3 dimensions without the 2nd 
molecule? 

best,
Kay

On Tue, 23 Jul 2019 06:22:31 +0200, Engi Hassaan  wrote:

>We faced a similar situation recently where our molecular replacement
>suggested 2 molecules in the asymmetric unit but the B factors of the
>second chain were twice as large as the first protien chain. At a
>resolution of 1.8 Å2 you could see the electron density for both chains. We
>performed ARP/wARP and realized only part of this second chain is resolved
>in our structure. It was model bias.
>
>Best,
>Engi
>
>
>Engi Hassaan
>PhD Student
>Research Group of Prof. Dr. G. Klebe
>Marburg, Germany
>
>
>On Mon, Jul 22, 2019, 19:34 Bernhard Rupp  wrote:
>
>> > Could there be two versions of each model: a "robustly-observed" and a
>> "most-likely" version?
>>
>> We tried/suggested something in this spirit once. Not sure how it was
>> received
>> http://journals.iucr.org/d/issues/2016/12/00/rr5136/index.html
>>
>> Best, BR
>> +
>> 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.
>>
>> -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
>>
>> 
>>
>> To unsubscribe from the CCP4BB list, click the following link:
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