Re: [ccp4bb] Summary - Valid to stop Refmac after TLS refinement?

[Dale Tronrud]

Bernhard Rupp wrote:

People also felt that the RMSD bond/angle of 0.016/1.6 was still a little

high.

This was subject of a discussion before on the board and I still don't 
understand it:


If I recall correctly, even in highly accurate and precise
small molecule structures, the rmsd of corresponding
bonds and angles are ~0.014A and 1.8deg. 


It always seems to me that getting these values much below is not a sign
of crystallographic prowess but over-restraining them?

Is it just that - given good resolution in the first place - the balance 
of restraints (matrix weight) vs low R (i.e., Xray data) gives the best 
Rfree or lowest gap at (artificially?) lower rmsd?


Is that then the best model?

I understand that even thermal vibration accounts for about 1.7 deg 
angle deviation -  are lower rmsd deviations then a manifestation

of low temp? But that does not seem to be much of an effect, if
one looks at the tables from the CSD small mol data (shown in 
nicely in comparison to the 91 Engh/Huber data in Tables F, pp385). 
 


   This is an on-going topic of discussion so let me put in my two cents.

   We calculate libraries of "ideal geometry" based on precise, small
molecule structures.  When these small molecule crystal structures are
compared to our derived libraries they are found to contain deviations.
These deviations are larger than the uncertainty in these models and
are presumed to reflect real features of the molecule; perturbations
due to the local environment in the crystal.

   These same perturbations are present in our crystals and we should
expect to find deviations from "ideal geometry" on the same scale as
that seen in the precise models.  This expectation lead to the practice
in the 1980's of setting r.m.s. targets of 0.02A and 3 degrees for
agreement to bond length and angle libraries.

   While this seems quite reasonable, we are left with the question:
Are the deviations from "ideal geometry" we see in a particular model
in any way related to the actual deviations of the molecule in the
crystal?  The uncertainties (su's) of the bond lengths in a model based
on 4A diffraction data are huge compared to the absolute value of the
true deviation.  For example, if the model had a deviation from "ideal
geometry" of 0.02A but the uncertainty of the distance is 0.2A can we
say that we have detected a signal that is significantly different than
zero, the null hypothesis?

   If we have a model with a collection of deviations from "ideal geometry"
but we have no expectation that those deviations are indicative of the
true deviations of the molecule in the crystal, are those deviations
serving any purpose?  If they do not reflect any property of the crystal
they are noise and should be filtered out.

   By this argument a model based on 4A resolution diffraction data should
have no deviation from "idea geometry" while one based on 0.9A diffraction
data should have no restraints on "ideal geometry" since the deviations
are probably all real and significant (except for specific regions of
the molecule that have problems).

   The problem we all face is the vast area between these extremes,
compounded by our inability to calculate proper uncertainties for the
parameters of our models.  The free R is our current tool-of-choice when
it comes to attempting to judge the statistical significance of aspects
of our model, without performing proper statistical tests which we don't
know how to do.  If we allow our model the freedom to deviate from our
library and the free R improves a "significant" (??) amount then the
resulting deviations must have some similarity to the true deviations
in the crystal, but if the free R does not improve then the deviations
must not be related to reality and should be suppressed.  This is the
type of assumption we make whenever we use the free R to make a choice.

   What we end of doing is not making a yes/no decision but instead we
variably suppress the amplitude of the deviations from "idea geometry"
and that is harder to justify.  I think a reasonable argument can be
made, but I have already written too many words in this letter.  It doesn't
really matter because we left the road of mathematical rigor when we took
the R free path.

   Unfortunately, many people have ignored what Brunger said in Methods
in Enzymology about choosing your X-ray/geometry weight based on the
free R and just starting saying "the rms bond length deviation must
be 0.007A".  The deviations from "idea geometry" of your model should be
no more or no less than what you can justifiably claim is a reflection
of the true state of the molecule in your crystal.

Dale Tronrud

Re: [ccp4bb] Strange behavior in R32

[Bart Hazes]

Hi Dan,

In hexagonal R32 setting there is translational crystallographic 
symmetry leading to systematic absences. If you think Xtal 5 is 
basically the same but with slightly different packing that break the 
symmetry then you will have pseudo-translational symmetry leading to one 
set of very strong intensities (those that obey the hexagonal R32 
lattice rules) and a set with very weak intensities (the once that are 
systematically absent in R32). That abundance of very strong and very 
weak intensities would give you a bimodal intensity distribution that 
would lead to the observed very large second moment in truncate. It is 
not immediately clear though what is going on with the other xtals. It 
may be of interest to look at the actual intensity distributions for 
different resolution slices to see if it is bimodal or not.


Bart

[EMAIL PROTECTED] wrote:

Dear Friends,

I have some crystals of a small RNA in sg R32 that exhibit some bizzare
behaviors and fail to give phasing solutions. I am hoping someone out there
might be able to lend some insight. All crystals come out of the same condition
and look the same morphologically but, for reasons unknown and at this point
uncontrollable, have very different cell dimensions:

Xtal 1: R32 77 x 77 x 80 Ang
Xtal 2: R32 77 x 77 x 86 Ang
Xtal 3: R32 78 x 78 x 366 Ang
Xtal 4: R32 78 x 78 x 460 Ang
Xtal 5: P3(1)21 77 x 77 x 85 Ang

I've collect >95% complete datasets for each crystal form to resolutions between
2.2-3.0 Ang. Denzo/scalepack outputs look fine in each case. Overall R-sym's
are okay (range is 8-13%). The only apparent red flag is the 2nd moment of I
calculated in truncate. These values are much larger than those expected for
either twinned (1.5) or untwinned (2.0) data:

Xtal 1: 3.2
Xtal 2: 2.5
Xtal 3: 4.9
Xtal 4: 3.7
Xtal 5: 4.2

Also, use of the Yeates server to look for partial twinning tells me there are
no twin laws for R32, while the P3(1)21 data does not seem to be twinned. 


I'm guessing crystals 1 & 2 are very similar but still somewhat non-isomorphous,
and crystal 5 is also very similar with a breakdown in space group symmetry
that would put 3 mols/ASU instead of 1 mol/ASU as in R32. But I do not
understand what is going on with crystals 3 & 4. Has anyone out there
experienced similar non-integer multiples of a cell dimension for a given
crystal? I have covalently attached iodine in some of these crystal forms. But
no luck finding any sites by SAD, and I can't help but wonder if this funny
c-dimension behavior and/or the high /^2 values are indicative of some
greater crystal pathology.

Thanks in advance,
Dan





--

==

Bart Hazes (Assistant Professor)
Dept. of Medical Microbiology & Immunology
University of Alberta
1-15 Medical Sciences Building
Edmonton, Alberta
Canada, T6G 2H7
phone:  1-780-492-0042
fax:1-780-492-7521

==

Re: [ccp4bb] Summary - Valid to stop Refmac after TLS refinement?

[Bernhard Rupp]

>People also felt that the RMSD bond/angle of 0.016/1.6 was still a little
high.

This was subject of a discussion before on the board and I still don't 
understand it:

If I recall correctly, even in highly accurate and precise
small molecule structures, the rmsd of corresponding
bonds and angles are ~0.014A and 1.8deg. 

It always seems to me that getting these values much below is not a sign
of crystallographic prowess but over-restraining them?

Is it just that - given good resolution in the first place - the balance 
of restraints (matrix weight) vs low R (i.e., Xray data) gives the best 
Rfree or lowest gap at (artificially?) lower rmsd?

Is that then the best model?

I understand that even thermal vibration accounts for about 1.7 deg 
angle deviation -  are lower rmsd deviations then a manifestation
of low temp? But that does not seem to be much of an effect, if
one looks at the tables from the CSD small mol data (shown in 
nicely in comparison to the 91 Engh/Huber data in Tables F, pp385). 
 

Thx, br

 

[ccp4bb] Strange behavior in R32

[dklein]

Dear Friends,

I have some crystals of a small RNA in sg R32 that exhibit some bizzare
behaviors and fail to give phasing solutions. I am hoping someone out there
might be able to lend some insight. All crystals come out of the same condition
and look the same morphologically but, for reasons unknown and at this point
uncontrollable, have very different cell dimensions:

Xtal 1: R32 77 x 77 x 80 Ang
Xtal 2: R32 77 x 77 x 86 Ang
Xtal 3: R32 78 x 78 x 366 Ang
Xtal 4: R32 78 x 78 x 460 Ang
Xtal 5: P3(1)21 77 x 77 x 85 Ang

I've collect >95% complete datasets for each crystal form to resolutions between
2.2-3.0 Ang. Denzo/scalepack outputs look fine in each case. Overall R-sym's
are okay (range is 8-13%). The only apparent red flag is the 2nd moment of I
calculated in truncate. These values are much larger than those expected for
either twinned (1.5) or untwinned (2.0) data:

Xtal 1: 3.2
Xtal 2: 2.5
Xtal 3: 4.9
Xtal 4: 3.7
Xtal 5: 4.2

Also, use of the Yeates server to look for partial twinning tells me there are
no twin laws for R32, while the P3(1)21 data does not seem to be twinned. 

I'm guessing crystals 1 & 2 are very similar but still somewhat non-isomorphous,
and crystal 5 is also very similar with a breakdown in space group symmetry
that would put 3 mols/ASU instead of 1 mol/ASU as in R32. But I do not
understand what is going on with crystals 3 & 4. Has anyone out there
experienced similar non-integer multiples of a cell dimension for a given
crystal? I have covalently attached iodine in some of these crystal forms. But
no luck finding any sites by SAD, and I can't help but wonder if this funny
c-dimension behavior and/or the high /^2 values are indicative of some
greater crystal pathology.

Thanks in advance,
Dan

Re: [ccp4bb] cryo was:bigger size - > better diffraction?

[Tommi Kajander]

for cryoprotection just drag it through paratone-N (put next to your crystal
drop) and you dont have to  worry about ill effects on the crystal
(usually). this will cut down the long road with cryoprotectant search to
minimal. (..not sure if this had anything to do with the original topic, but
since it keeps coming up...) it doesnt penetrate the crystal and works and
you dont have to  waste time and crystals on finding a solution with some
soluble cryoprotection additive that doenst kill the crystal.. 

tommi

Quoting Leonard Thomas <[EMAIL PROTECTED]>:

> The first thing to try before going down the long road of fussing  
> with cryo is to take a shot at room temp. and see how your crystal  
> diffracts in general.  It is true it may be a cryo problem, but if  
> the non cryo protected crystals do not diffract then why would one  
> expect the cryo protected one to.
> 
> As for controlling crystal growth.  I would second what Shane wrote  
> and try seeding.   Also trying the usually additives and varying  
> protein concentration/precipitant concentration should help also so.
> 
> Len
> 
> 
> On Apr 4, 2007, at 8:56 AM, Shane Atwell wrote:
> 
> > Streak seeding all your trays should give you a better handle on
> > nucleation. You might be too high in protein or precipitant w/o the
> > seeding, hence the showering and rare nice crystals.
> >
> > Varying cryos, or cryo concentrations, or how the cryo is added can  
> > help
> > a lot. Try 5 or 6 different cryos at 3 concentrations each. When you
> > have the best nailed down then try sequential transfers of the  
> > crystals
> > from low to target concentrations (e.g. into 5% for a couple minutes,
> > then 10, 20, 25%). Also, you can try growing the crystals w/ a bit  
> > (5%)
> > or the final conc or cryo already present. Should help in getting them
> > habituated to the cryo.
> >
> > Shane Atwell
> >
> >> -Original Message-
> >> From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On
> >> Behalf Of Jenny
> >> Sent: Wednesday, April 04, 2007 5:50 AM
> >> To: CCP4BB@JISCMAIL.AC.UK
> >> Subject: [ccp4bb] bigger size - > better diffraction?
> >>
> >> Hi, All,
> >>
> >> I got a crystal that diffracts at 3.3A in house.The crystal
> >> size is about 0.2mm* 0.1mm * 0.2mm. At first I thought the
> >> size is fine,but it turns out the smaller ones diffract
> >> worse.I guess the reason is that
> >> the cell unit is really big (126.292   126.292   134.904  p4212,
> >> pretty big for a 10kD protein, isn't it?)
> >>
> >> So looks like I need to grow bigger crystals in order to get
> >> better diffractions.The problems is ,every time when I set up
> >> trays, the growing conditions is not exactly the same, so I
> >> have to set up a whole tray or maybe even 2 trays , then 2 or
> >> 3 conditions will jump out with good crystals ( 2 or 3
> >> nucleation site ) and some of the others will show lots lots
> >> of small crystals.I used NaCl as the salt, in a 4*6 tray, the
> >> [NaCl] is going from 2.0,2.05,2.1,2.15,something like
> >> that and 0.05M does make big difference.I used Urea as the
> >> additive in this case ( 25 m ~  100 mM) and tried
> >> 2+2,3+1,3+2, 3+1 ( 3 uL protein and 1 uL buffer ) is better
> >> than the other two cases.Right now it's growing in room temp
> >> in about a week.And crystals that not fresh got some bubbles
> >> around the edge and didn't diffract well.
> >>
> >> Does anyone have any suggestions that what I could do to
> >> improve the diffraction?
> >>
> >> Thanks a lot.
> >>
> >> Jenny
> >>
> 


-- 
Tommi Kajander, Ph.D.
Macromolecular X-ray Crystallography
Research Program in Structural Biology and Biophysics
Institute of Biotechnology
PO box 65 (Street address: Viikinkaari 1, 4th floor)
University of Helsinki
FIN-00014 Helsinki, Finland
Tel. +358-9-191 58903
Fax  +358-9-191 59940

Re: [ccp4bb] bigger size - > better diffraction?

[artem]

A small comment: crystals that do not diffract well at R.T. can still
diffract well when frozen. There are several reasons for this, including:

 * poor stability of crystal once the drop is open to the air

 * crystal has low tolerance for handling (capillary mount can be
challenging and cause more physical damage, especially if the
experimenter is rusty with capillary mounts)

 * the effect of freezing - it has been known to cause transitions between
space groups etc.

 * the effect of cryoprotectant (dehydration, and so forth)

So - even if your stuff doesn't diffract well in a capillary - it still
can be saved sometimes :) Or not.

Artem

> diffracts in general.  It is true it may be a cryo problem, but if
> the non cryo protected crystals do not diffract then why would one
> expect the cryo protected one to.

Re: [ccp4bb] bigger size - > better diffraction?

[Elspeth Garman]

For room temperature testing, it is much easuier to use a cryoloop 
enclosed in a capillary (with some mother liqour at the top) or the 
plastic tubes supplied by MiTeGen as per the method detailed in:


Skrzypczak-Jankun, E., Bianchet, M. A., Amzel, L. M. and Funk Jr., M. O. 
(1996). /Acta Cryst./,/ /*D52*, 959-965.


than to capillary mount.
Also, if you do sequential transfer into cryo, try it `in situ' by 
pipetting on increasing concetrations and removal of liquid rather than 
moving the crystal between wells of higher concentration - mosaicity 
should be less.

Good luck
Elspeth

Leonard Thomas wrote:

The first thing to try before going down the long road of fussing
with cryo is to take a shot at room temp. and see how your crystal
diffracts in general.  It is true it may be a cryo problem, but if
the non cryo protected crystals do not diffract then why would one
expect the cryo protected one to.

As for controlling crystal growth.  I would second what Shane wrote
and try seeding.   Also trying the usually additives and varying
protein concentration/precipitant concentration should help also so.

Len


On Apr 4, 2007, at 8:56 AM, Shane Atwell wrote:


Streak seeding all your trays should give you a better handle on
nucleation. You might be too high in protein or precipitant w/o the
seeding, hence the showering and rare nice crystals.

Varying cryos, or cryo concentrations, or how the cryo is added can
help
a lot. Try 5 or 6 different cryos at 3 concentrations each. When you
have the best nailed down then try sequential transfers of the
crystals
from low to target concentrations (e.g. into 5% for a couple minutes,
then 10, 20, 25%). Also, you can try growing the crystals w/ a bit
(5%)
or the final conc or cryo already present. Should help in getting them
habituated to the cryo.

Shane Atwell


-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On
Behalf Of Jenny
Sent: Wednesday, April 04, 2007 5:50 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] bigger size - > better diffraction?

Hi, All,

I got a crystal that diffracts at 3.3A in house.The crystal
size is about 0.2mm* 0.1mm * 0.2mm. At first I thought the
size is fine,but it turns out the smaller ones diffract
worse.I guess the reason is that
the cell unit is really big (126.292   126.292   134.904  p4212,
pretty big for a 10kD protein, isn't it?)

So looks like I need to grow bigger crystals in order to get
better diffractions.The problems is ,every time when I set up
trays, the growing conditions is not exactly the same, so I
have to set up a whole tray or maybe even 2 trays , then 2 or
3 conditions will jump out with good crystals ( 2 or 3
nucleation site ) and some of the others will show lots lots
of small crystals.I used NaCl as the salt, in a 4*6 tray, the
[NaCl] is going from 2.0,2.05,2.1,2.15,something like
that and 0.05M does make big difference.I used Urea as the
additive in this case ( 25 m ~  100 mM) and tried
2+2,3+1,3+2, 3+1 ( 3 uL protein and 1 uL buffer ) is better
than the other two cases.Right now it's growing in room temp
in about a week.And crystals that not fresh got some bubbles
around the edge and didn't diffract well.

Does anyone have any suggestions that what I could do to
improve the diffraction?

Thanks a lot.

Jenny





--
-
Dr. Elspeth F. Garman,
Reader in Molecular Biophysics,
Department of Biochemistry,
Rex Richards Building,
University of Oxford,  Tel: (44)-1865-275398
South Parks Road,  FAX: (44)-1865-275182
OXFORD, OX1 3QU, U.K.  E-mail: [EMAIL PROTECTED]

-

Re: [ccp4bb] bigger size - > better diffraction?

[Leonard Thomas]

The first thing to try before going down the long road of fussing  
with cryo is to take a shot at room temp. and see how your crystal  
diffracts in general.  It is true it may be a cryo problem, but if  
the non cryo protected crystals do not diffract then why would one  
expect the cryo protected one to.


As for controlling crystal growth.  I would second what Shane wrote  
and try seeding.   Also trying the usually additives and varying  
protein concentration/precipitant concentration should help also so.


Len


On Apr 4, 2007, at 8:56 AM, Shane Atwell wrote:


Streak seeding all your trays should give you a better handle on
nucleation. You might be too high in protein or precipitant w/o the
seeding, hence the showering and rare nice crystals.

Varying cryos, or cryo concentrations, or how the cryo is added can  
help

a lot. Try 5 or 6 different cryos at 3 concentrations each. When you
have the best nailed down then try sequential transfers of the  
crystals

from low to target concentrations (e.g. into 5% for a couple minutes,
then 10, 20, 25%). Also, you can try growing the crystals w/ a bit  
(5%)

or the final conc or cryo already present. Should help in getting them
habituated to the cryo.

Shane Atwell


-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On
Behalf Of Jenny
Sent: Wednesday, April 04, 2007 5:50 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] bigger size - > better diffraction?

Hi, All,

I got a crystal that diffracts at 3.3A in house.The crystal
size is about 0.2mm* 0.1mm * 0.2mm. At first I thought the
size is fine,but it turns out the smaller ones diffract
worse.I guess the reason is that
the cell unit is really big (126.292   126.292   134.904  p4212,
pretty big for a 10kD protein, isn't it?)

So looks like I need to grow bigger crystals in order to get
better diffractions.The problems is ,every time when I set up
trays, the growing conditions is not exactly the same, so I
have to set up a whole tray or maybe even 2 trays , then 2 or
3 conditions will jump out with good crystals ( 2 or 3
nucleation site ) and some of the others will show lots lots
of small crystals.I used NaCl as the salt, in a 4*6 tray, the
[NaCl] is going from 2.0,2.05,2.1,2.15,something like
that and 0.05M does make big difference.I used Urea as the
additive in this case ( 25 m ~  100 mM) and tried
2+2,3+1,3+2, 3+1 ( 3 uL protein and 1 uL buffer ) is better
than the other two cases.Right now it's growing in room temp
in about a week.And crystals that not fresh got some bubbles
around the edge and didn't diffract well.

Does anyone have any suggestions that what I could do to
improve the diffraction?

Thanks a lot.

Jenny

Re: [ccp4bb] bigger size - > better diffraction?

[Shane Atwell]

Streak seeding all your trays should give you a better handle on
nucleation. You might be too high in protein or precipitant w/o the
seeding, hence the showering and rare nice crystals.

Varying cryos, or cryo concentrations, or how the cryo is added can help
a lot. Try 5 or 6 different cryos at 3 concentrations each. When you
have the best nailed down then try sequential transfers of the crystals
from low to target concentrations (e.g. into 5% for a couple minutes,
then 10, 20, 25%). Also, you can try growing the crystals w/ a bit (5%)
or the final conc or cryo already present. Should help in getting them
habituated to the cryo.

Shane Atwell

> -Original Message-
> From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On 
> Behalf Of Jenny
> Sent: Wednesday, April 04, 2007 5:50 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] bigger size - > better diffraction?
> 
> Hi, All,
> 
> I got a crystal that diffracts at 3.3A in house.The crystal 
> size is about 0.2mm* 0.1mm * 0.2mm. At first I thought the 
> size is fine,but it turns out the smaller ones diffract 
> worse.I guess the reason is that
> the cell unit is really big (126.292   126.292   134.904  p4212,
> pretty big for a 10kD protein, isn't it?)
> 
> So looks like I need to grow bigger crystals in order to get 
> better diffractions.The problems is ,every time when I set up 
> trays, the growing conditions is not exactly the same, so I 
> have to set up a whole tray or maybe even 2 trays , then 2 or 
> 3 conditions will jump out with good crystals ( 2 or 3 
> nucleation site ) and some of the others will show lots lots 
> of small crystals.I used NaCl as the salt, in a 4*6 tray, the 
> [NaCl] is going from 2.0,2.05,2.1,2.15,something like 
> that and 0.05M does make big difference.I used Urea as the 
> additive in this case ( 25 m ~  100 mM) and tried 
> 2+2,3+1,3+2, 3+1 ( 3 uL protein and 1 uL buffer ) is better 
> than the other two cases.Right now it's growing in room temp 
> in about a week.And crystals that not fresh got some bubbles 
> around the edge and didn't diffract well.
> 
> Does anyone have any suggestions that what I could do to 
> improve the diffraction?
> 
> Thanks a lot.
> 
> Jenny
> 

Re: [ccp4bb] bigger size - > better diffraction?

[artem]

Hello Jenny,

0.2 Rmerge may be an indication of incorrectly assigned SG or perhaps a
misindexing by one, etc. Check what happens if you reprocess in P4.

For a 10kDa protein, your unit cell is kind of large - depending of course
on the symmetry, the higher obviusly the better. I would bet that you have
pretty high solvent content - nothing scientific about this prediction,
merely personal experience with several small proteins that crystallized
in high-symmetry SG and had >80% solvent.

What you may want to do is to very carefully study your cryo. When we were
working on a structure of FliS-FliC
complex(http://www.xtals.org/pdfs/FliC_FliS.pdf), the only way to freeze
the crystals correctly was to increase the concentration of sulfate ion in
the cryoprotectant. It later turned out that we had a sulfate ion bridging
a symmetry threefold, and in low sulfate the ion would leave, resultuing
in disorder in the crystals.

Ultimately, if you've already tried all the 'standard' tricks, such as
additives, glycerol/eg in the condition, replacing NaCl with an exotic
salt such as KCl, CsCl, RbCl, and so forth - perhaps you may want to
consider adjusting the surface of the protein. I would be glad to help you
with that.

Artem

[ccp4bb] Detwinning

[Sundaramoorthy, Munirathinam]

I am working with crystals with apparent I422 space group but it may be 
actually I4. Twinning analysis shows twinning fraction of 0.46. I tried to 
detwin the anomalous data in CCP4. Just to check the files were read and 
written properly, I wrote the detwinned data in ascii. The numbers look strange 
and needless to say the anomalous Patterson doesn't look good (The Patterson 
with untwinned data shows single strong site in both I4 and I422, but I 
couldn't solve the structure in either space group).

There are lot of negative intesities in the output. 
   0   0  72   -565.57   1621.53643.51   1700.95
   0   0  74  -2454.32   1778.62   3489.49   1845.18
   0   0  76   1161.54   1598.13   -657.16   1668.87
   0   0  78   1036.44   1492.05   -934.67   1488.42

If I write out Fs, the reflections with negative intensities have 0 for F(+) 
and value of the previous reflection for F(-). 
   0   0  720.000.00  244.28  203.06
   0   0  740.000.00  586.96  153.30
   0   0  76  311.74  194.46  586.96  153.30
   0   0  78  218.16  192.88  586.96  153.30

Did anyone face a similar problem? I am using version 6.0 of CCP4. The older 

Sundar

[ccp4bb] New crystal screening analysis web server...

[P Hubbard]

Hi all,

I've recently set up a web server to help in the initial crystal screening 
process, and thought it might be useful for other people too. It allows one 
to point-and-click to catalog and analyze the results of initial screens to 
help see what your protein does and doesn’t like, as well as a simple search 
engine and quick look-up of conditions from available screens.


It’s rather basic at the moment and may still have a few bugs, but if enough 
people like it (and its novel enough), I’ll add further utilities. If you 
find it useful, please feel free to link the site. Comments also welcome.


http://www.bioscienceforum.com/xtalwizard/xtalwizard.php

Cheers.

_
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[ccp4bb] Scientist and intern positions open in crystallization group

[Shane Atwell]

We have two positions open in our crystallization group, one for a scientist 
and one for an intern. Please use our website to apply:

http://www.sgxpharma.com/careers/opportunities.php



Shane Atwell
Associate Director, Crystallization
SGX Pharmaceuticals
10505 Roselle Street
San Diego, CA 92121
phone: (858) 228-1622
fax: (858) 457-5362
[EMAIL PROTECTED]
 
This email message is for the sole use of the intended recipient(s) and may 
contain confidential and attorney-client privileged information.  Any 
unauthorized review, use, disclosure or distribution is prohibited.  If you are 
not the intended recipient, please contact the sender by reply email and 
destroy all copies of the original message. 

Re: [ccp4bb] Summary - Valid to stop Refmac after TLS refinement?

[NM Burton, Biochemistry]

Hello,

Thanks very much to all who replied with thoughts and suggestions.

The consensus was that my interpretation was not correct, and it is not 
valid to stop Refmac after TLS refinement.  People also felt that the RMSD 
bond/angle of 0.016/1.6 was still a little high.  Phenix.refine was 
suggested as a complete solution, with SA and TLS in the same package.


All the best,

Nick

--On 28 March 2007 16:34 +0100 "NM Burton, Biochemistry" 
<[EMAIL PROTECTED]> wrote:



Hello,

I've refined a structure with CNS to Rwork/free=0.226/0.273.  I switched
to Refmac5.2 to take advantage of TLS refinement and set up a run with 10
cycles of TLS refinement (groups as suggested by the TLSMD server)
followed by 10 cycles of restrained co-ordinate refinement.  After the
TLS cycles the model was improved (Rwork/free=0.222/0.241), however
during co-ordinate refinement Rfree refined up (final
Rwork/free=0.197/0.264).  My understanding would be that the TLS
refinement is modelling the ADPs most accurately, but that Refmac's
co-ordinate refinement is over-fitting slightly.  Would this seem
correct?  And if so, is it valid to run Refmac with no cycles of
co-ordinate refinement and take the resulting model as the final
structure?

Thanks very much,

Nick

--
NM Burton, Biochemistry
[EMAIL PROTECTED]




--
NM Burton, Biochemistry
[EMAIL PROTECTED]

[ccp4bb] CNS query

[Swanand Gore]

Dear All,

I have been experimenting with resolution limits in CNS in order to simulate
a low resolution scenario. I increase the high resolution till there are
thrice the reflections as number of atoms in the structure. Most of the
times this works but sometimes CNS aborts with this message:


Problems interpolating or extrapolating data.
Input data had the following distribution:
Data point 1 could not be used
Data point 2 could not be used
Data point 3 could not be used
Data point 4 could be used
Data point 5 could not be used
Data point 6 could be used
Data point 7 could not be used
Data point 8 could not be used
%NAYBRS error encountered: Less than two data points for
interpolation/extrapolation



Surprisingly I did not get anything useful after googling for this. Does
anybody know a wokaround? Or is this to be expected if number of reflections
is low?

Thanks in advance,
Swanand

[ccp4bb] POSTDOCTORAL POSITION IN PROTEIN COMPLEX CRISTALLOGRAPHY

[Vanessa Llobet]

POSTDOCTORAL POSITION IN PROTEIN COMPLEX CRYSTALLOGRAPHY
 

Applications are invited for a postdoctoral position in Prof. Miquel Coll’s
group at the Institute of Research in Biomedicine / Institut de Biologia
Molecular de Barcelona (CSIC) to work on the structural characterization of
protein complexes. The position is supported by a recently awarded
Consolider Project. A strong background in protein expression is required. 

 

The IRB/IBMB laboratories are equipped with state-of-the-art protein
expression and purification apparatus, including high-throughput facilities.
The crystallographic equipment includes nano-drop crystallization robots,
storage and visualization robots, two rotating anode generators (one a
MicroMax-007 micro-focus generator) with image plate detectors, a free
mounting system, and cutting-edge computational facilities. The Institute is
located in the stimulating scientific and cultural environment of the
Barcelona Science Park.

 
Those interested should contact (preferably by email):
 
Vanessa Llobet
Secretary
Structural and Computational Biology Programme 
Institute for Research in Biomedicine
Parc Científic de Barcelona, Josep Samitier 1-5 
08028 Barcelona, Spain
 
Email   [EMAIL PROTECTED]; 
URL:  www.irbbarcelona.org/mcoll 
 
Applicants should send a full curriculum vitae and the names and addresses
(including e-mail address) of two referees no later than April 30th, 2007. 

 

 

Vanessa Llobet
Secretary 

Structural &Computational Biology Programme 
Institute for Research in Biomedicine
Parc Científic de Barcelona
c/ Josep Samitier 1-5
08028 Barcelona - Spain
Tel. +34 93 403 99 53

Fax. +34 93 403 99 76
[EMAIL PROTECTED] ; [EMAIL PROTECTED] 

http://www.irbbarcelona.org 

 

Re: [ccp4bb] bigger size - > better diffraction?

[Jenny]

oh, One thing forgot to mention is that I actually collected data in
house ( about 70 frames ), the completness is high ( 98%) but the
Rmerge is high ( 0.2 ), what does this suggest?

On 4/4/07, Jenny <[EMAIL PROTECTED]> wrote:

Hi, All,

I got a crystal that diffracts at 3.3A in house.The crystal size is
about 0.2mm* 0.1mm * 0.2mm. At first I thought the size is fine,but it
turns out the smaller ones diffract worse.I guess the reason is that
the cell unit is really big (126.292   126.292   134.904  p4212,
pretty big for a 10kD protein, isn't it?)

So looks like I need to grow bigger crystals in order to get better
diffractions.The problems is ,every time when I set up trays, the
growing conditions is not exactly the same, so I have to set up a
whole tray or maybe even 2 trays , then 2 or 3 conditions will jump
out with good crystals ( 2 or 3 nucleation site ) and some of the
others will show lots lots of small crystals.I used NaCl as the salt,
in a 4*6 tray, the [NaCl] is going from
2.0,2.05,2.1,2.15,something like that and 0.05M does make big
difference.I used Urea as the additive in this case ( 25 m ~  100 mM)
and tried 2+2,3+1,3+2, 3+1 ( 3 uL protein and 1 uL buffer ) is better
than the other two cases.Right now it's growing in room temp in about
a week.And crystals that not fresh got some bubbles around the edge
and didn't diffract well.

Does anyone have any suggestions that what I could do to improve the
diffraction?

Thanks a lot.

Jenny

[ccp4bb] bigger size - > better diffraction?

[Jenny]

Hi, All,

I got a crystal that diffracts at 3.3A in house.The crystal size is
about 0.2mm* 0.1mm * 0.2mm. At first I thought the size is fine,but it
turns out the smaller ones diffract worse.I guess the reason is that
the cell unit is really big (126.292   126.292   134.904  p4212,
pretty big for a 10kD protein, isn't it?)

So looks like I need to grow bigger crystals in order to get better
diffractions.The problems is ,every time when I set up trays, the
growing conditions is not exactly the same, so I have to set up a
whole tray or maybe even 2 trays , then 2 or 3 conditions will jump
out with good crystals ( 2 or 3 nucleation site ) and some of the
others will show lots lots of small crystals.I used NaCl as the salt,
in a 4*6 tray, the [NaCl] is going from
2.0,2.05,2.1,2.15,something like that and 0.05M does make big
difference.I used Urea as the additive in this case ( 25 m ~  100 mM)
and tried 2+2,3+1,3+2, 3+1 ( 3 uL protein and 1 uL buffer ) is better
than the other two cases.Right now it's growing in room temp in about
a week.And crystals that not fresh got some bubbles around the edge
and didn't diffract well.

Does anyone have any suggestions that what I could do to improve the
diffraction?

Thanks a lot.

Jenny

[ccp4bb] EBSA/BBS Eurobiophysics Congress, London, July 2007

[Elspeth Garman]

The 6th EUROBIOPHYSICS CONGRESS will take place in London, from 14 - 18

July 2007.


http://www.eurobiophysics.org


There are just 10 days left for abstract registration (deadline 14th 
April), and ~60 short oral presentations are still to be selected from 
the abstracts, which cover 26 themes. Late registration opens 30th April.



Registration includes lunches and refreshments, and some good value 
accommodation is still available.





  REGISTRATION  NOW OPEN FOR 


EUROBIOPHYSICS, 14 - 18 JULY 2007

http://www.eurobiophysics.org



-
Dr. Elspeth F. Garman,
Reader in Molecular Biophysics,
Department of Biochemistry,
Rex Richards Building,
University of Oxford,  Tel: (44)-1865-275398
South Parks Road,  FAX: (44)-1865-275182
OXFORD, OX1 3QU, U.K.  E-mail: [EMAIL PROTECTED]

-

Re: [ccp4bb] truncate error on x86_64

[Ian Tickle]

Hi Daniele

I recently made a change in truncate (added cumulative distribution for twinned 
intensities), however I'm not sure whether 6.0.2 incorporates that change.  
I've not seen either of the problems you report before, all I can suggest is 
you re-compile truncate with the debug (-g switch), with no optimisation (no 
-O), and also bounds-checking turned on might be informative (-fbounds-check 
assuming you're using g77).  Then run again with the debugger and post the 
traceback.  It shouldn't need the ulimit command to work correctly since there 
are no huge arrays in truncate, and the "segmentation violation" error usually 
indicates array bounds overflow (which programmers in general are very lax at 
checking!).

Cheers

-- Ian

> -Original Message-
> From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On 
> Behalf Of Daniele de Sanctis
> Sent: 04 April 2007 10:39
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] truncate error on x86_64
> 
> Dear all,
> 
> i installed  ccp4 6.0.2 on an amd64 machine running Ubuntu 6.10, 64bit
> version. Everything is running fine except a problem i have with
> truncate. At the beginning it failed giving a "segmentation violation"
> error which i overcame using "ulimit -s unlimited". Now I got another
> error (quite general indeed)
> 
> "The program run with command: truncate HKLIN
> "/home/daddah/kpn8/b4x4/kpn8_3_001_scala1.mtz" HKLOUT
> "/tmp/daddah/b4x4_15_1_mtz.tmp" PLOT /tmp/daddah/b4x4_15_falloff.plt
> has failed with error message
> Floating Exception"
> 
> any idea?
> 
> d
> 
> 
> -- 
> Daniele de Sanctis, PhD
> 
>  Homo sum humani nil alienum a me puto
> __
> phone ++ 351 21 4469 662fax ++ 351 21 4433 664
> 
> e-mail [EMAIL PROTECTED], [EMAIL PROTECTED]
> 
> Mailing address:
> ITQB, Av. República, Apartato 127
> 2781-901 Oeiras
> Portugal
> 
> 

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action in reliance upon it. If you have received this communication in error, 
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Cambridge CB4 0QA under number 3751674

[ccp4bb] question about structure analysis

[Marcela NUNEZ]

  Hi everybody
 I have a question about soluble structures analysis.
 I've been working on a heme protein who presents two isomers. These isomers 
can only be detected by NMR and its ratio varies among organisms. My goal is to 
explain how the isomer ratio is modulated and why one isomer is more stable 
than the other.  
 Someone think that the isomer ratio is modulated by steric interaction between 
the side chain of two non-conserved residues and heme subtituents. We suppose 
that the ratio is an effect of the heme cavity in the protein core. In order to 
prove this, we have analyzed the NMR structure of four organism (they are the 
only structures determined). Their rmsd is <= 1.8. We calculated the heme 
cavity using CASTp, but we didn't find any correlation between volume cavity 
and isomer ratio of the soluble structures. We also calculated distances 
between residue side-chain atoms and heme substituents atoms. We found a great 
correlation between some distances and isomer ratios; nevertheless, residues 
side-chain position in soluble structures varies even among the family of the 
best structures. So, I am not sure that the results that I obtained could be 
meaningful. Does anybody have an idea to prove that they could be???
 For the moment I don't have any other idea to explain the isomer ratios. Do 
you think that the analysis of soluble structure could let me to go forward in 
this explanation or should I stop here and use my results as a simple 
description??.
 Thanks in advance
 
 
 

Marcela Núñez
Tél: + 33 (0)6.12.67.38.80



-
 Découvrez une nouvelle façon d'obtenir des réponses à toutes vos questions ! 
Profitez des connaissances, des opinions et des expériences des internautes sur 
Yahoo! Questions/Réponses.

[ccp4bb] truncate error on x86_64

[Daniele de Sanctis]

Dear all,

i installed  ccp4 6.0.2 on an amd64 machine running Ubuntu 6.10, 64bit
version. Everything is running fine except a problem i have with
truncate. At the beginning it failed giving a "segmentation violation"
error which i overcame using "ulimit -s unlimited". Now I got another
error (quite general indeed)

"The program run with command: truncate HKLIN
"/home/daddah/kpn8/b4x4/kpn8_3_001_scala1.mtz" HKLOUT
"/tmp/daddah/b4x4_15_1_mtz.tmp" PLOT /tmp/daddah/b4x4_15_falloff.plt
has failed with error message
Floating Exception"

any idea?

d


--
Daniele de Sanctis, PhD

Homo sum humani nil alienum a me puto
__
phone ++ 351 21 4469 662fax ++ 351 21 4433 664

e-mail [EMAIL PROTECTED], [EMAIL PROTECTED]

Mailing address:
ITQB, Av. República, Apartato 127
2781-901 Oeiras
Portugal

[ccp4bb] Crystallization position, 25793BR, Novartis Pharma, Basel (Switzerland)

[Joerg Kallen]

Please apply via the web. Do not email replies to me. 


A global healthcare leader, Novartis has one of the most exciting product 
pipelines in the industry today. A pipeline of innovative medicines 
brought to life by diverse, talented, performance driven people. All of 
which makes us the most rewarding employer in our field. 

We are looking for a Scientific Associate in NIBR Research 

You will be working in the Protein Structure Unit in an interdisciplinary 
team of molecular biologists, biochemists and crystallographers. Your 
tasks will be the preparation and optimization of protein crystals for 
X-ray analysis, mainly using robotic systems. Characterization of protein 
crystals with X-rays, measurement of X-ray data and further 
crystallographic work. General lab management. 
Potential further developments in the job include: Application of 
biophysical techniques (isothermal titration calorimetry, differential 
scanning calorimetry, etc.) to measure/validate ligand binding. 
Purification and characterization of proteins and protein/ligand 
complexes. 

You have completed your apprenticeship in chemistry or biology and have a 
strong interest in further developing your skills in a challenging 
environment. Alternatively, you have completed your studies (Master, 
Diploma or equivalent, no Ph.D.) in biochemistry, biotechnology or a 
related field. You should be a good team player with an independent 
working style and be able to handle multiple projects in parallel. English 
is required. 

Please go to www.careers.novartis.com to apply directly online and quote 
Job ID 25793BR.



Dr. Joerg Kallen
Novartis Pharma AG
DT/PSU: LAB. KALLEN
CHBS, WSJ-088.9.08B
Novartis Pharma AG
Lichtstrasse 35
CH-4056 Basel
Switzerland
Phone: +41 61 3245579
Fax: +41 61 3242686
Email : [EMAIL PROTECTED]

_

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