Re: [ccp4bb] Phantom Crystals

[Daniel Picot]

The object that approaches the most a ghost to my mind is a picture of
a bubble trapped into a lipidic cubic phase of the detergent 
beta-octyl-glucoside, it can be found in the article  from P. Sakya, 
J.M. Seddon & R. Templer (1994) J.Phys II France 4:1311. I got also 
crystals of membrane protein (i.e. not bubble) in the same detergent 
that exhibited the same faceted cubic habits, they often disappeared 
quickly (a few days) and were to soft to be manipulated.

Daniel

George DeTitta a écrit :
I’d appreciate it if people could tell me their experiences with what I 
would call “phantom crystals”, or “ghost crystals”.  These are objects 
that display the seeming morphology of crystals (clear facets, sharp 
edges) but do not diffract X-rays AT ALL.  I would not count objects 
that diffract to 30 A in this category.  I mean objects that don’t show 
a single Bragg spot.


 

**George T. DeTitta, Ph.D.** 


**Principal Research Scientist**

**Hauptman-Woodward Institute** 


**Professor and Chairman**

**Department of Structural Biology**

**SUNY at Buffalo**

**700 Ellicott Street Buffalo NY 14203-1102 USA**

**(716) 898-8600 (voice)**

**(716) 898-8660 (fax)**

**www.hwi.buffalo.edu** 

 

Re: [ccp4bb] [COOT] Novel maps...

[Dirk Kostrewa]

Dear Eleaonor and CCP4ers,

I always wondered, why there are phase columns PHWT, PHDELWT (Refmac)  
or PH2FOFCWT, PHFOFCWT (phenix.refine) in addition to the PHIC column  
of the atomic model. In absence of other phase information, the phase  
should come from the atomic model, only.


Best regards,

Dirk.

Am 18.06.2009 um 10:24 schrieb Eleanor Dodson:


It would be a cock-up  map. Unless there is exptl phasing

PHDELWT = PHIC  when mFo-DFc is positive, and mFo-DFc is negative it  
equals PHIC + 180


Eleanor


Simon Kolstoe wrote:

Dear cootbb,

When building into density I use a map generated from the FWT  
amplitudes column and PHWT phases columns which I think is my 2Fo- 
Fc, and a second map generated from DELFWT and PHDELWT which I  
think is my Fo-Fc. This keeps me happy and in known territory.  
However today someone in the lab asked what it means to generate a  
map using FWT and PHDELWT. Does anyone know what type of map this  
would be in the Fo Fc terminology?


Thanks,

Simon





***
Dirk Kostrewa
Gene Center, A 5.07
Ludwig-Maximilians-University
Feodor-Lynen-Str. 25
81377 Munich
Germany
Phone:  +49-89-2180-76845
Fax:+49-89-2180-76999
E-mail: kostr...@lmb.uni-muenchen.de
***

Re: [ccp4bb] [COOT] Novel maps...

[Eleanor Dodson]

Well, it makes for consistency between the output when there is not, and 
is, exptl phase information - in the 2nd case the PHWT is a weighted 
derivative of the two phase estimates.


You could get away with just one of PHWT and PHDELWT since they can 
only differ bu 180, but that would require writing mFO-DFC as a signed  
number , and there were concerns about what programs would do with 
negative "F" estimates at one point..

  Eleanor



 Dirk Kostrewa wrote:

Dear Eleaonor and CCP4ers,

I always wondered, why there are phase columns PHWT, PHDELWT (Refmac) 
or PH2FOFCWT, PHFOFCWT (phenix.refine) in addition to the PHIC column 
of the atomic model. In absence of other phase information, the phase 
should come from the atomic model, only.


Best regards,

Dirk.

Am 18.06.2009 um 10:24 schrieb Eleanor Dodson:


It would be a cock-up  map. Unless there is exptl phasing

PHDELWT = PHIC  when mFo-DFc is positive, and mFo-DFc is negative it 
equals PHIC + 180


Eleanor


Simon Kolstoe wrote:

Dear cootbb,

When building into density I use a map generated from the FWT 
amplitudes column and PHWT phases columns which I think is my 
2Fo-Fc, and a second map generated from DELFWT and PHDELWT which I 
think is my Fo-Fc. This keeps me happy and in known territory. 
However today someone in the lab asked what it means to generate a 
map using FWT and PHDELWT. Does anyone know what type of map this 
would be in the Fo Fc terminology?


Thanks,

Simon





***
Dirk Kostrewa
Gene Center, A 5.07
Ludwig-Maximilians-University
Feodor-Lynen-Str. 25
81377 Munich
Germany
Phone: +49-89-2180-76845
Fax: +49-89-2180-76999
E-mail:kostr...@lmb.uni-muenchen.de
***

Re: [ccp4bb] [COOT] Novel maps...

[Ian Tickle]

The SIGMAA program has since it was written in the 80's (mainly by Randy
of course, but also in part by Eleanor I believe?) used only 1 phase
column (= PHIC) for the case where the phases of FWT & DELFWT are equal
to PHIC: obviously if they are unequal (e.g. as a result of phase
combination) then additional phase columns are needed.  The FWT & DELFWT
columns may be negative, and (to my knowledge) this has never caused a
single problem.  In fact when I wrote the original CCP4 interface to
Lynn ten Eyck's FFT I specifically ensured that negative amplitudes did
not cause any problems.  Since programs such as Refmac were developed
*after* Sigmaa it's hard to understand the basis of this irrational fear
of negative numbers!

According to Wikipedia the ancient Greeks didn't accept the existence of
negative numbers either, in fact to quote from:

http://en.wikipedia.org/wiki/Negative_numbers#History

"For a long time, negative solutions to problems were considered
"false". In Hellenistic Egypt, Diophantus in the third century A.D.
referred to an equation that was equivalent to 4x + 20 = 0 (which has a
negative solution) in Arithmetica, saying that the equation was
absurd.".

Hopefully we've come a long way since then!

Cheers

-- Ian

> -Original Message-
> From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk]
On
> Behalf Of Eleanor Dodson
> Sent: 18 June 2009 09:50
> To: Dirk Kostrewa
> Cc: CCP4BB@jiscmail.ac.uk
> Subject: Re: [ccp4bb] [COOT] Novel maps...
> 
> Well, it makes for consistency between the output when there is not,
and
> is, exptl phase information - in the 2nd case the PHWT is a weighted
> derivative of the two phase estimates.
> 
>  You could get away with just one of PHWT and PHDELWT since they can
> only differ bu 180, but that would require writing mFO-DFC as a signed
> number , and there were concerns about what programs would do with
> negative "F" estimates at one point..
>Eleanor
> 
> 
> 
>   Dirk Kostrewa wrote:
> > Dear Eleaonor and CCP4ers,
> >
> > I always wondered, why there are phase columns PHWT, PHDELWT
(Refmac)
> > or PH2FOFCWT, PHFOFCWT (phenix.refine) in addition to the PHIC
column
> > of the atomic model. In absence of other phase information, the
phase
> > should come from the atomic model, only.
> >
> > Best regards,
> >
> > Dirk.
> >
> > Am 18.06.2009 um 10:24 schrieb Eleanor Dodson:
> >
> >> It would be a cock-up  map. Unless there is exptl phasing
> >>
> >> PHDELWT = PHIC  when mFo-DFc is positive, and mFo-DFc is negative
it
> >> equals PHIC + 180
> >>
> >> Eleanor
> >>
> >>
> >> Simon Kolstoe wrote:
> >>> Dear cootbb,
> >>>
> >>> When building into density I use a map generated from the FWT
> >>> amplitudes column and PHWT phases columns which I think is my
> >>> 2Fo-Fc, and a second map generated from DELFWT and PHDELWT which I
> >>> think is my Fo-Fc. This keeps me happy and in known territory.
> >>> However today someone in the lab asked what it means to generate a
> >>> map using FWT and PHDELWT. Does anyone know what type of map this
> >>> would be in the Fo Fc terminology?
> >>>
> >>> Thanks,
> >>>
> >>> Simon
> >>>
> >>>
> >
> >
> > ***
> > Dirk Kostrewa
> > Gene Center, A 5.07
> > Ludwig-Maximilians-University
> > Feodor-Lynen-Str. 25
> > 81377 Munich
> > Germany
> > Phone: +49-89-2180-76845
> > Fax: +49-89-2180-76999
> > E-mail:kostr...@lmb.uni-muenchen.de
> > ***
> >
> >



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Re: [ccp4bb] Phantom Crystals

[Clemens Grimm]

Zitat von George DeTitta :


I'd appreciate it if people could tell me their experiences with what I
would call "phantom crystals", or "ghost crystals".  These are objects
that display the seeming morphology of crystals (clear facets, sharp
edges)


... There is also a second type of "ghost crystals", those which do  
not have edges at all, with a ball- or egg-like apearance and which  
diffract perfectly well!


but do not diffract X-rays AT ALL.  I would not count objects

that diffract to 30 A in this category.  I mean objects that don't show
a single Bragg spot.



George T. DeTitta, Ph.D.

Principal Research Scientist

Hauptman-Woodward Institute

Professor and Chairman

Department of Structural Biology

SUNY at Buffalo

700 Ellicott Street Buffalo NY 14203-1102 USA

(716) 898-8600 (voice)

(716) 898-8660 (fax)

www.hwi.buffalo.edu 

[ccp4bb] PhD Position in Structural Biology, Wuerzburg, Germany

[Clemens Grimm]

A PhD position in structural biology is available in the group of  
Prof. Utz Fischer at the Institute of Biochemistry, Biocentre of the  
Julius Maximilans-University, Wuerzburg/Germany.


The successful applicant will work on the crystallographic structure  
determination of the Survival Motor Neuron (SMN) complex, the key  
player during the pathogenesis of Spinal Muscular Atrophy (SMA).  
Protocols for bacterial expression and reconstitution of the SMN  
complex are established and first crystals are already available.


Please send your application including a letter of interest,  
curriculum vitae, academic certificates and the names and contact  
details of two referees to:




Dr. Clemens Grimm
e-mail: clemens.gr...@biozentrum.uni-wuerzburg.de
Institut für Biochemie
Biozentrum der Universität Würzburg
Am Hubland
D-97074 Würzburg
Germany



web: www.biozentrum.de

Re: [ccp4bb] Phantom Crystals

[Martin.Caffrey]

A few more examples of what Daniel refers to can be seen in Figure 11 - Nature 
Protocols 4:706-731, 2009.  We encounter them from time to time using the in 
meso (lipidic cubic phase) method.  With experience you get to recognize them 
for what they are.

Martin

-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Daniel 
Picot
Sent: 18 June 2009 08:21
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Phantom Crystals

The object that approaches the most a ghost to my mind is a picture of
a bubble trapped into a lipidic cubic phase of the detergent 
beta-octyl-glucoside, it can be found in the article  from P. Sakya, 
J.M. Seddon & R. Templer (1994) J.Phys II France 4:1311. I got also 
crystals of membrane protein (i.e. not bubble) in the same detergent 
that exhibited the same faceted cubic habits, they often disappeared 
quickly (a few days) and were to soft to be manipulated.
Daniel

George DeTitta a écrit :
> I'd appreciate it if people could tell me their experiences with what I 
> would call "phantom crystals", or "ghost crystals".  These are objects 
> that display the seeming morphology of crystals (clear facets, sharp 
> edges) but do not diffract X-rays AT ALL.  I would not count objects 
> that diffract to 30 A in this category.  I mean objects that don't show 
> a single Bragg spot.
> 
>  
> 
> **George T. DeTitta, Ph.D.** 
> 
> **Principal Research Scientist**
> 
> **Hauptman-Woodward Institute** 
> 
> **Professor and Chairman**
> 
> **Department of Structural Biology**
> 
> **SUNY at Buffalo**
> 
> **700 Ellicott Street Buffalo NY 14203-1102 USA**
> 
> **(716) 898-8600 (voice)**
> 
> **(716) 898-8660 (fax)**
> 
> **www.hwi.buffalo.edu** 
> 
>  
> 

Re: [ccp4bb] Difference Map images

[Doug Ohlendorf]

Tim,

Not really. Fo's can be viewed as having 3 parts. 1) F from our modeled
structure, 2) F from what we can't model. This can be bulk solvent, partial
or multiple occupancies in low resolution structures, thermal anisotropy ,
etc. 3) F from random errors in measuring data. The first F is what we deal
with. The third F we can minimize by collecting high quality data. The
second F is usually crystal/space group dependent. It arises from
crystallization conditions, crystal morphology and intermolecular contacts.
Data from isomorphic crystals should have similar second F's so map (from
first F's) is cleaner.

Doug

-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Tim
Gruene
Sent: Wednesday, June 17, 2009 4:39 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Difference Map images

> I know that sometimes Fo(complex)-Fo(apo) cannot be done because of
> nonisomorphism. We've had a lot of success with this with the dioxygenases
> because there is no large scale alteration in the active site. As for the
> technique itself, Brian Matthews drilled this into me when I was a postdoc
> in his lab.
Wouldn't - in the case of non-isomorphsim - a molecular replacement with 
the apo-form come closest to a Fo(complex)-Fo(apo) map?

Just a thought.
Tim

Re: [ccp4bb] Phantom Crystals

[James Holton]

"Phantom Crystals" can easily be achieved by failing to cryoprotect most 
any crystal, or by a few other common crystal handling methods, but I 
don't think this is what you were asking about.


I do have users bring in what I call "plastic crystals" from time to 
time, but these crystals that don't diffract a single spot are 
relatively rare. Once every month or two I see a new one. (not counting 
repeat visits from users who are cursed with them) I always advise doing 
a 180-degree oscillation in such cases because sometimes it is a salt 
crystal that just happened to not have any reciprocal lattice points on 
the Ewald sphere. If this reveals a symmetrical pattern of a few very 
bright spots, then it can only mean a salt crystal. But, once in a while 
there really is no diffraction at all, and the user is left to ponder if 
it really was a very bad protein crystal or just a chunk of plastic that 
fell into their drop. Repeating the setup is the only way to really know.


Also, I suspect that small beamstops reduce the number of samples that 
meet your criterion, as I have also seen plenty of crystals that don't 
diffract beyond 30 A, or even 50 A or 100 A, but still give off a few 
spots in a region that would be behind the beamstop on many 
diffractometers. Such samples are common enough that Ana Gonzalez has 
coined the term "BBC" (behind-beamstop crystallography) to refer to such 
experiments.


Another way to make a "phantom crystal" is with radiation damage. If you 
blast any crystal long enough, all the spots will go away, leaving a 
SAXS pattern around the beamstop:

http://bl831.als.lbl.gov/~jamesh/ribo_blast/diffraction.gif
How useful this SAXS pattern is for deducing structural information 
remains to be seen, but since even "naturally phantom" crystals must 
still contain atoms, and atoms scatter x-rays, then SAXS signal from 
such objects could be useful.


Nevertheless, I think it is still up in the air how much diffraction 
tends to be degraded by crystal handling vs crystals just being "born 
ugly", as the proper control (shooting crystals without handling them) 
has not been done on anything but a few test cases. In fact, I have 
heard enough stories about ugly crystals diffracting very well and 
beautiful crystals diffracting poorly to wonder if these two qualities 
really are anticorellated. That is, beauty really is just "skin deep" 
(and ugly goes to the core). I think it will be telling to see what sort 
of results we get from the now several available "in-situ" diffraction 
systems one of which myself and others developed with 
Fluidigm, who are now selling them.


-James Holton
MAD Scientist

George DeTitta wrote:


I’d appreciate it if people could tell me their experiences with what 
I would call “phantom crystals”, or “ghost crystals”. These are 
objects that display the seeming morphology of crystals (clear facets, 
sharp edges) but do not diffract X-rays AT ALL. I would not count 
objects that diffract to 30 A in this category. I mean objects that 
don’t show a single Bragg spot.


**George T. DeTitta, Ph.D.**

**Principal Research Scientist**

**Hauptman-Woodward Institute**

**Professor and Chairman**

**Department of Structural Biology**

**SUNY at Buffalo**

**700 Ellicott Street Buffalo NY 14203-1102 USA**

**(716) 898-8600 (voice)**

**(716) 898-8660 (fax)**

**www.hwi.buffalo.edu** 

Re: [ccp4bb] Difference Map images

[Tim Gruene]

Hello Doug,

I understand your arguing, although I do not understand why Fo contains a 
contribution from the model(led structure). My idea was only supposed to 
be a backup in case the apo-form had not been measuredcor could not be 
used. I agree the Fo(complex)-Fo(apo) gives a much more realistic result 
than the MR-solution I suggested.


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

GPG Key ID = A46BEE1A


On Thu, 18 Jun 2009, Doug Ohlendorf wrote:


Tim,

Not really. Fo's can be viewed as having 3 parts. 1) F from our modeled
structure, 2) F from what we can't model. This can be bulk solvent, partial
or multiple occupancies in low resolution structures, thermal anisotropy ,
etc. 3) F from random errors in measuring data. The first F is what we deal
with. The third F we can minimize by collecting high quality data. The
second F is usually crystal/space group dependent. It arises from
crystallization conditions, crystal morphology and intermolecular contacts.
Data from isomorphic crystals should have similar second F's so map (from
first F's) is cleaner.

Doug

-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Tim
Gruene
Sent: Wednesday, June 17, 2009 4:39 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Difference Map images


I know that sometimes Fo(complex)-Fo(apo) cannot be done because of
nonisomorphism. We've had a lot of success with this with the dioxygenases
because there is no large scale alteration in the active site. As for the
technique itself, Brian Matthews drilled this into me when I was a postdoc
in his lab.

Wouldn't - in the case of non-isomorphsim - a molecular replacement with
the apo-form come closest to a Fo(complex)-Fo(apo) map?

Just a thought.
Tim

Re: [ccp4bb] Phantom Crystals

[V. Nagarajan]

Is there some sort of consensus on what properties make a crystal diffract
well or poorly? For example, solvent content is assumed to be critical. What
else? Can crystals even grow if there is short-range (less than beam radius)
disorder?

Thanks,
V. Nagarajan
JAN Scientific, Inc.

http://janscientific.com

-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of James
Holton
Sent: Thursday, June 18, 2009 11:04 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Phantom Crystals

[deleted]

Nevertheless, I think it is still up in the air how much diffraction 
tends to be degraded by crystal handling vs crystals just being "born 
ugly", as the proper control (shooting crystals without handling them) 
has not been done on anything but a few test cases. In fact, I have 
heard enough stories about ugly crystals diffracting very well and 
beautiful crystals diffracting poorly to wonder if these two qualities 
really are anticorellated. That is, beauty really is just "skin deep" 
(and ugly goes to the core). I think it will be telling to see what sort 
of results we get from the now several available "in-situ" diffraction 
systems one of which myself and others developed with 
Fluidigm, who are now selling them.

-James Holton
MAD Scientist

[ccp4bb] off topic

[Kn Ly]

Hello everyone,

I am trying to purify a 13 KDa membrane protein using Ni NTA. The protein is
solubilised in Triton X 100, 20 mM phosphate buffer, 150 mM NaCl and binds
very well to the column. However, it also turns the column brownish.
The protein contains 4 cysteine residues so I suspect that this causes
cross-linking with other proteins and thus brownish precipitation on the
column. So I included 5 mM beta-ME in my buffer to prevent disulfide bond
formation but this doesn't help. I tried 1 mM DTT and this ruined the column.
Help!! Is there anyway to prevent this brownish problem?

Thanks a lot in advance
Kien