Re: [ccp4bb] 27.5% R/29% Rfree for 1.75A structure (details updated)

[Stuart Endo-Streeter]

How are you deciding your resolution limit?  I/sigma, Rmrgd-F, Rmeas, etc. 
etc.?  We recently had a person in a fellow lab that couldn't figure why 
their R/Rfree was mid- to high-twenties for their 1.8 angstrom structure.  
Turned out the data stopped at 2.1.

Stuart

On Tuesday 29 July 2008 17:34, JINJIN ZHANG wrote:
> Dear All,
>
>
> Here are updated details for the high R problem of my 1.75A structure. 
>
>
> 1. Protein 100 aa, peptide 6 aa, co-crystalized
> 2. Space group: P43212. Overall R-fac: 0.03. Redundancy: >5 for 98% of
> reflections. B-factor:55 3. CNS refinement
> 4. Phenix xtriage checked, no twinning is suspected.
> 5. Water molecules added
>
>
> Thanks a lot for all your comments and suggestions.
>
>
> Best,
> Jinjin Zhang

-- 








__
Stuart T. Endo-Streeter
Structural Biology and Biophysics
Dept. Biochemistry
LSRC C266
Duke University
919-681-1668
[EMAIL PROTECTED]

Re: [ccp4bb] multiple XNAME scala? and changing DNAMEs

[hari jayaram]

Hello Phil Evans,
Thanks a lot for your reply. Pointless worked great to edit XNAME and DNAME
for unmerged mtz files.

We are now taking multiple crystal data as unmerged mosflm output mtzs ,
squishing them together into one pseudo-crystal mtz with the same XNAME and
DNAME using Pointless. The mtz header does have correctly 1 dataset. The
only issue I guess is the CELL parameters are from one of the two datasets.

This does seem to be  quite strange but we are trying to mimic the "Scenario
4" as presented in the HKL-scalepack manual in the section combining
multiple native datasets together . In that scenario it asks you to take
multiple native data-sets together and combine their *.x files and fit one
a*, b* and c* and a batch mosaicity , rotx , roty .


Wondering what  is the correct way of getting the CELL parameters for this
multi-crystal dataset.


Hari




On Tue, Jul 29, 2008 at 5:06 PM, Phil Evans <[EMAIL PROTECTED]> wrote:

> The easiest way to combine multiple crystals and renaming datasets for
> Scala is to use Pointless, available from the CCP4 prerelease site. You
> can't use CAD on unmerged files: the older programs Rebatch & Sortmtz can
> also be used
>
> Note the if you are merging multiple crystals, you are essentially creating
> a composite "crystal" so all parts to be merged must have the same XNAME &
> DNAME
>
> Phil
>
>
>
>
>
> On 29 Jul 2008, at 20:56, hari jayaram wrote:
>
>  Sorry for that incomplete last post
>>
>> Thanks to everyone who wrote in with advice on scaling multi-crystal
>> spacegroup P1 datasets together to increase redundancy for MAD/SAD/SAS
>> phasing.
>>
>> I have three questions about scaling and merging  multi crystal datasets
>> in scala
>>
>> In our case we have the following
>> 1) Crystal 1 - XNAME - C1 - DNAME peak1
>>  DNAME  inf1
>> 2)Crystal 2 - XNAME- C2 DNAME peak1
>>
>> The two crystals have cell dimensions within 1-2 % and I have cadded the
>> output from mosflm for each of the crystals into one giant mtz file. No two
>> batch numbers are the same .When we run scala the Job fails and We get
>> errors like "Insufficient Data to determine parameters" erros - Too few
>> reflections
>>
>> Of course scala on each crystal separately works great and gives us
>> reasonable correlation coefficients ( 0.2 to 0.3 for Correlations within
>> half dataset for peak and inflection to 3.2 A. The overall redundancy is
>> around 6-8. Rpim around 0.05 )
>>
>> My question is can scala scale mutli-crystal datasets as I am attempting
>> to do.
>>
>>
>> 2) My second unrelated question is how can I DRENAME using cad on unmerged
>> data. If I have run mosflm and then want to change the DNAME of a dataset ,
>> CAD says it can DRENAME a dataset , but at the same time CAD has a problem
>> with unmerged mtz files. How can I edit the DNAME, XNAME, PNAME etc on an
>> mtzfile output from mosflm
>>
>> Your help is greatly appreciated.
>>
>> Hari Jayaram
>>
>>
>>

[ccp4bb] Registration deadline for Como Crystallography School extended

[Derek Logan]

Dear all,

The registration deadline for the Como Crystallography School has been  
extended to 28th August. For more details see the web site http://www.crystallographyschool.org 
. Please note that the deadline for booking accommodation has not been  
extended from the initial date of 25th July. You are welcome to  
continue using the hotel booking form provided by Centro Volta, but  
they are unable to guarantee that you will get the room of your choice  
from now on.


Derek

[ccp4bb] 27.5% R/29% Rfree for 1.75A structure (details updated)

[JINJIN ZHANG]

Dear All,


Here are updated details for the high R problem of my 1.75A structure. 


1. Protein 100 aa, peptide 6 aa, co-crystalized
2. Space group: P43212. Overall R-fac: 0.03. Redundancy: >5 for 98% of 
reflections. B-factor:55
3. CNS refinement
4. Phenix xtriage checked, no twinning is suspected.
5. Water molecules added


Thanks a lot for all your comments and suggestions.


Best,
Jinjin Zhang

Re: [ccp4bb] 27.5% R/29% Rfree for 1.75A structure (details updated)

[Van Den Berg, Bert]

By R-fac you mean Rsym? If it is, a value of 0.3 seems way too high. How many 
rejections do you have with scaling?
 
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: [EMAIL PROTECTED]
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



From: CCP4 bulletin board on behalf of JINJIN ZHANG
Sent: Tue 7/29/2008 5:24 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] 27.5% R/29% Rfree for 1.75A structure (details updated)


Dear All,


Here are updated details for the high R problem of my 1.75A structure. 


1. Protein 100 aa, peptide 6 aa, co-crystalized
2. Space group: P43212. Overall R-fac: 0.3. Redundancy: >5 for 98% of 
reflections. B-factor:55
3. CNS refinement
4. Phenix xtriage checked, no twinning is suspected.
5. Water molecules added


Thanks a lot for all your comments and suggestions.


Best,
Jinjin Zhang 

[ccp4bb] 27.5% R/29% Rfree for 1.75A structure (details updated)

[JINJIN ZHANG]

Dear All,


Here are updated details for the high R problem of my 1.75A structure. 


1. Protein 100 aa, peptide 6 aa, co-crystalized
2. Space group: P43212. Overall R-fac: 0.3. Redundancy: >5 for 98% of 
reflections. B-factor:55
3. CNS refinement
4. Phenix xtriage checked, no twinning is suspected.
5. Water molecules added


Thanks a lot for all your comments and suggestions.


Best,
Jinjin Zhang

[ccp4bb] BioCARS (Sector 14, Advanced Photon Source) hosting Workshop on Time-resolved Macromolecular Crystallography, Nov 20-22, 2008

[vukica srajer]

Workshop on Time-resolved Macromolecular Crystallography
BioCARS, November 20-22, 2008

BioCARS is hosting a Workshop on Time-resolved Macromolecular Crystallography
from Thursday, November 20 to Saturday, November 22, 2008, at the Advanced
Photon Source, Argonne National Laboratory. The aim of the workshop is to
provide hands-on training in designing and conducting time-resolved experiments
and in Laue data processing and analysis. The workshop will also provide an
opportunity for participants to discuss application of the technique to their
own scientific projects with experts in the field. 


The BioCARS insertion device beamline 14-ID has recently undergone a complete
upgrade of both its X-ray and laser facilities. The beamline now includes two
new undulators that operate collinearly, a new Kirkpatrick-Baez mirror system, a
new picosecond laser system, and an upgraded ultra-fast X-ray chopper. As a
result, 100ps time-resolved experiments can now be conducted in both hybrid and
24-bunch APS mode, with crystals 10 times smaller and X-ray exposures 10 times
shorter than previously possible at BioCARS, while still obtaining excellent
data quality. As we resume time-resolved user operation on the upgraded 14-ID
beamline, we welcome new user groups interested in conducting time-resolved
experiments. At this workshop new and returning BioCARS users will be able to
learn about the exciting capabilities at beamline 14-ID.  


A workshop registration fee of $75 for students and $125 for others will be
charged. Workshop materials, meals, and lodging for three nights will be
provided by BioCARS, but travel costs to and from APS will be covered by
participants. Students who require it may apply to BioCARS for limited financial
assistance.

For additional information regarding the workshop and registration please visit:
http://cars9.uchicago.edu/biocars/pages/trworkshop08/index.shtml
or contact:
Vukica Srajer ([EMAIL PROTECTED]) or Jane Andrew ([EMAIL PROTECTED])

BioCARS and this workshop are supported by NCRR/NIH grant 5 P41 RR007707 to The
University of Chicago, Keith Moffat, PI.

We are looking forward to seeing you at the Workshop.

Vukica Srajer

--
~~~
Vukica Srajer Ph.D.
University of Chicago/CARS
9700 South Cass Ave, Bldg 434B
Argonne, IL 60439
tel: (630) 252-0455
fax: (630) 252-0443

Re: [ccp4bb] multiple XNAME scala? and changing DNAMEs

[Phil Evans]

The easiest way to combine multiple crystals and renaming datasets for  
Scala is to use Pointless, available from the CCP4 prerelease site.  
You can't use CAD on unmerged files: the older programs Rebatch &  
Sortmtz can also be used


Note the if you are merging multiple crystals, you are essentially  
creating a composite "crystal" so all parts to be merged must have the  
same XNAME & DNAME


Phil




On 29 Jul 2008, at 20:56, hari jayaram wrote:


Sorry for that incomplete last post

Thanks to everyone who wrote in with advice on scaling multi-crystal  
spacegroup P1 datasets together to increase redundancy for MAD/SAD/ 
SAS phasing.


I have three questions about scaling and merging  multi crystal  
datasets in scala


In our case we have the following
1) Crystal 1 - XNAME - C1 - DNAME peak1
  DNAME  inf1
2)Crystal 2 - XNAME- C2 DNAME peak1

The two crystals have cell dimensions within 1-2 % and I have cadded  
the output from mosflm for each of the crystals into one giant mtz  
file. No two batch numbers are the same .When we run scala the Job  
fails and We get errors like "Insufficient Data to determine  
parameters" erros - Too few reflections


Of course scala on each crystal separately works great and gives us  
reasonable correlation coefficients ( 0.2 to 0.3 for Correlations  
within half dataset for peak and inflection to 3.2 A. The overall  
redundancy is around 6-8. Rpim around 0.05 )


My question is can scala scale mutli-crystal datasets as I am  
attempting to do.



2) My second unrelated question is how can I DRENAME using cad on  
unmerged data. If I have run mosflm and then want to change the  
DNAME of a dataset , CAD says it can DRENAME a dataset , but at the  
same time CAD has a problem with unmerged mtz files. How can I edit  
the DNAME, XNAME, PNAME etc on an mtzfile output from mosflm


Your help is greatly appreciated.

Hari Jayaram

Re: [ccp4bb] Alternate Conformation in CNS

[Pavel Afonine]

Hi Krystle,

you can do it easily in PHENIX: http://phenix-online.org/

Please let me know if you interested and have questions.

Pavel.


On 7/29/2008 1:29 PM, Krystle . wrote:

Hi all,
I am trying to model an alternate conformation in my structure, but 
the alternate conformation is a completely different residue. For 
example, if two different ligands bound in the same position just at 
shared occupancies.  I saw an old posting about this same issue and 
they recommended giving different segids to different ligands and 
omitting their interactions, and referred to this paper: Liu et al, 
biochemistry 2005, 44(8):2982-92.


I did this bu I am unable to get CNS to view them as alternate 
conformations, and during refinement it views them as steric clashes. 
My question is how/where/in what file do you omit the interactions? Do 
you modify the .mtf file or some input file?


Thanks!
Krystle

---
Krystle Williams
Dept. Biochemistry and Biophysics
School Of Medicine and Dentistry
University of Rochester
601 Elmwood Ave. Box 712
Rochester, NY 14642
Phone:585-276-3681





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Re: [ccp4bb] 27.5% R/29% Rfree for 1.75A structure

[Pavel Afonine]

Hi Jinjin Zhang,

yes, it is high indeed. Here is the distribution of R-factors for 
structures in PDB at resolution between 1.7 and 1.8A:


Distribution of R-work, resolution 1.7-1.8A
 6.00 -   8.50: 1
 8.50 -  11.00: 0
11.00 -  13.50:16
13.50 -  16.00:   166
16.00 -  18.50:  1064
18.50 -  21.00:   983
21.00 -  23.50:   626
23.50 -  26.00:65
26.00 -  28.50:15
28.50 -  31.00: 2

Distribution of R-free, resolution 1.7-1.8A
 7.00 -   9.80: 1
 9.80 -  12.60: 0
12.60 -  15.40: 6
15.40 -  18.20:   140
18.20 -  21.00:   481
21.00 -  23.80:  1283
23.80 -  26.60:   826
26.60 -  29.40:   168
29.40 -  32.20:27
32.20 -  35.00: 6

Distribution of R-free-Rwork, resolution 1.7-1.8A
 1.00 -   2.10:   645
 2.10 -   3.20:   950
 3.20 -   4.30:   670
 4.30 -   5.40:   369
 5.40 -   6.50:   180
 6.50 -   7.60:72
 7.60 -   8.70:32
 8.70 -   9.80:16
 9.80 -  10.90: 1
10.90 -  12.00: 3

A few obvious things:

1) Check for twinning using, for example, phenix.xtriage:

phenix.xtriage your_data.mtz

2) Did you add water molecules to your model?

3) Check for crystal anisotropy.

4) Make sure there are no outliers, like Fobs=1.e+10 (phenix.refine 
detects and removes them automatically).


Pavel.


On 7/29/2008 1:16 PM, JINJIN ZHANG wrote:

Hi All,
 
We have pretty much finished fitting and refining a protein-peptide 
complex structure, however, the 27.5% R/29% Rfree seem quite for a 
1.75A resolution data. Does anybody have some idea about this kind of 
problem? Thank you.
 
Best,
Jinjin Zhang 

[ccp4bb] Alternate Conformation in CNS

[Krystle .]

Hi  all,
I am trying to model an alternate conformation in my
structure, but the alternate conformation is a completely different
residue. For example, if two different ligands bound in the same position just 
at shared occupancies.  I saw an old posting about this same issue and they
recommended giving different segids to different ligands and omitting
their interactions, and referred to this paper: Liu et al, biochemistry
2005, 44(8):2982-92. 

I did this bu I am unable to get CNS to view them as alternate conformations, 
and during refinement it views them as steric clashes. My question is 
how/where/in what file do you omit the interactions? Do you modify the .mtf 
file or some input file? 

Thanks!
Krystle
 
---Krystle WilliamsDept. 
Biochemistry and BiophysicsSchool Of Medicine and DentistryUniversity of 
Rochester601 Elmwood Ave. Box 712Rochester, NY 14642
Phone:585-276-3681
 



_
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It's easy!
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[ccp4bb] 27.5% R/29% Rfree for 1.75A structure

[JINJIN ZHANG]

Hi All, We have pretty much finished fitting and refining a protein-peptide complex structure, however, the 27.5% R/29% Rfree seem quite for a 1.75A resolution data. Does anybody have some idea about this kind of problem? Thank you. Best,Jinjin Zhang

[ccp4bb] multiple XNAME scala? and changing DNAMEs

[hari jayaram]

Sorry for that incomplete last post

Thanks to everyone who wrote in with advice on scaling multi-crystal
spacegroup P1 datasets together to increase redundancy for MAD/SAD/SAS
phasing.

I have three questions about scaling and merging  multi crystal datasets in
scala

In our case we have the following
1) Crystal 1 - XNAME - C1 - DNAME peak1
  DNAME  inf1
2)Crystal 2 - XNAME- C2 DNAME peak1

The two crystals have cell dimensions within 1-2 % and I have cadded the
output from mosflm for each of the crystals into one giant mtz file. No two
batch numbers are the same .When we run scala the Job fails and We get
errors like "Insufficient Data to determine parameters" erros - Too few
reflections

Of course scala on each crystal separately works great and gives us
reasonable correlation coefficients ( 0.2 to 0.3 for Correlations within
half dataset for peak and inflection to 3.2 A. The overall redundancy is
around 6-8. Rpim around 0.05 )

My question is can scala scale mutli-crystal datasets as I am attempting to
do.


2) My second unrelated question is how can I DRENAME using cad on unmerged
data. If I have run mosflm and then want to change the DNAME of a dataset ,
CAD says it can DRENAME a dataset , but at the same time CAD has a problem
with unmerged mtz files. How can I edit the DNAME, XNAME, PNAME etc on an
mtzfile output from mosflm

Your help is greatly appreciated.

Hari Jayaram

[ccp4bb] Scaling multiple "native" datasets together

[hari jayaram]

Thanks to everyone who wrote in with advice on scaling multi-crystal P1
datasets together to increase redundancy for MAD/SAD/SAS phasing.

I have  a question about scaling and merging these multiple datasets in
scale/scaleit.

In our case we have the following
1) Crystal 1 - CNAME - C1

Re: [ccp4bb] Beamline Stability Issues

[James Holton]

Well, thanks!  But sorry to have doubled the number of things you have 
to do now.  I hate when that happens... :)


To answer your questions:

The layout of my beamline is documented in MacDowell. et. al. JSR 
(2004).  I can provide you with a reprint if you don't have access.  To 
summarize, we have:

source:
superconducting bending magnet.  source size 0.2 x 0.02 x ~2 mm.
Mirror 1:
parabolic collimating mirror: Rh over Pt coated water cooled invar steel 
flat mirror bent into a parabola.

motors: 1 stepper: for tilt (we move this about once or twice a year)
monochromator:
Khozu.  Two flat crystals.  First is water cooled, second is not 
cooled.  Motors are protected from Compton from crystals by a 
water-cooled copper wall.
motors: 5; 1 servo "Theta" (using Heidenhein encoder), 4 steppers to 
rotate (Theta2, Chi2) and translate (Y2, Z2) the second crystal.  Chi2 
is used for steering (many moves/day).  Theta2 moves during tune-ups 
(1-4 moves/day).  Theta and Y2 move with every energy change.  Z2 moves 
are reserved for large changes in energy.  Other than the Heidenhein 
encoder for Theta, there is no feedback.

Mirror 2:
toroidal focusing mirror: Rh over Pt coated uncooled silicon mirror. 2:1 
demagnification to eliminate spherical aberrations.
motors: 3 steppers; 2 to bend (moved once or twice a year) and 1 to tilt 
(moved all the time).

Feedback:
Beam is actively steered vertically (M2 Tilt) and horizontally (Chi2) 
using a video camera pointed at a phosphor on the back of the fast 
shutter as a beam position monitor.


Apertures:
the superbend has an exit aperture
the M1 mirror has an entrance aperture that defines the input acceptance 
of the beamline

the mono has an entrance aperture that serves as a guide for tilting M1
the M2 mirror has adjustable entrance slits for defining the beam 
convergence angle on the sample

there is a pinhole (round 100 um) just before the sample position

That is pretty much it.

WRT the second crystal:
Motion of the second crystal CAN cause small changes in energy.  This is 
because the Bragg reflection from the first crystal has some width to 
it, and also has long "tails".  Rotating the second crystal (what we 
call Theta2) will cause it's rocking curve (Si111) to select from these 
available energies.  However, the first thing you will notice from the 
second crystal rotating is a VERY significant drop in flux, combined 
with beam drift.  Just as though you had intentionally moved the crystal 
with a motor.  Since you didn't mention a drop in flux, I think you can 
eliminate your mono "falling off the rocking curve" as the source of 
your energy drift.  If you are still not sure, try intentionally tilting 
your second crystal to see if it changes your photon energy.


The only way to get an energy change without a loss of flux is for the 
two crystals to remain parallel.  In other words, the whole 
monochromator must tilt relative to the incoming beam.  Note that this 
will not change the angle of the exiting beam relative to the incident beam.


I am assuming you are using fluorescence scans to assay your photon 
energy, but you can also see the energy dispersion in the tails of your 
beam at the sample position.  Take out any pinholes or sample slits you 
might have and put a phosphor screen in the beam (at a relatively steep 
angle to spread out the vertical structure of the beam spot).  Then put 
in a selenium absorber foil and dial up the selenium edge.  As you pass 
through the Se edge, you will not see the intensity of the beam drop all 
at once, but rather a dark "curtain" will sweep through the beam 
profile.  This is because different parts of the beam actually have 
different photon energies!  Especially any long scatter "tails".  The 
reason why I mention this is because I think you could use a method of 
checking your photon energy that does not involve moving motors.


The best advice I can give anyone who is trying to solve any kind of 
drift problem is: DON'T TOUCH ANYTHING!  It is very tempting to keep 
trying to fiddle with one thing after another, but if your drift is on a 
timescale of 1 day, then you can only do one controlled experiment each 
day.  This is because you are effectively dealing with a different 
beamline each passing hour, and you are also changing things every time 
you fiddle.  There is no way to know if the results of your changes will 
take seconds, minutes, or days to have an effect.  Heat takes time to 
conduct, and once you have "exercised" a motor, it can take up to an 
hour or more for the heat you created to find its way into something 
that will move the beam when it expands, and even longer for the heat to 
dissipate completely.  For my "Z2" motor heat problem, it took ~8 hours 
for the beamline to "settle down" after that motor was "exercised".


For sake of example, I have the graph from my mono motor heating 
diagnostics here:

http://bl831.als.lbl.gov/~jamesh/old_pickup/Theta.bmp
http://bl831.als.lbl.gov/~jame

[ccp4bb] Preventing close contact between protein and ligand

[Sangeetha Vedula]

Dear bb users,

I am refining a protein-ligand complex (at 1.68 A resolution) in which the
ligand lies on a 2-fold crystallographic symmetry axis. The ligand occupancy
is, therefore, 0.5 in each asymmetric unit.

I am almost at the end of the refinement but one problem has me stumped.
Refmac keeps moving a carbon in the ligand too close to a serine OG and an
oxygen too close to an arginine CD. Given that the ligand is at the
interface, the density is not perfect. However, I rebuild the ligand to
eliminate close contacts and still be within density and refmac pulls it
right back close to the protein. The refined position does not even look
better than the rebuilt one! It almost always looks worse! Would refmac put
less weight on close contacts with the ligand because it is only partially
occupied?

I tried to use external restraints between the ligand and the residues so
that they are kept further away.

Upon searching the net, I found this command line:

*external distance first chain [ch] residue [res] insertion [ins] -
atom [n] [altcode [a]] second chain [ch] residue [res] insertion [ins]-
atom [n] [altcode [a] ] value [v] sigma [s] [symm y/n]

*I thought (hoped) that the distance herein is the minimum distance of
approach between the specified atoms, I added these lines from within
"Developer options" in refmac interface:

exte dist first chain A resi 59 atom CD seco chain X resi 2001 atom O1 valu
3.2 sigm 0.02 symm Y
exte dist first chain A resi 27 atom OG seco chain X resi 2001 atom C10 valu
3.2 sigm 0.02 symm Y

It didn't recognize these restraints at all.

However, when I change these lines to:

exte dist first chain A resi 59 atom CA seco chain X resi 2001 atom O1 valu
3.2 sigm 0.02 symm Y
exte dist first chain A resi 27 atom OG seco chain X resi 2001 atom C10 valu
3.2 sigm 0.02 symm Y

Refmac recognizes the first line but not the second - lines from log file:

Bond distance deviations from the ideal >10.000Sigma will be monitored

A 59 ARG CA  . - X   2001 DIE O1  . mod.= 5.024 id.= 3.200 dev= -1.824
sig.= 0.020

This raises two concerns:

Concern 1: From the first line of output: the restraints here don't seem to
be minimizing close contact at all; it seems to think they are bonded
somehow (the distance between these atoms is not 5.024; it is 6.26 A; I
don't know what 5.024 A is!).

I am missing something here. It'd be great if someone can tell me what that
is!

Concern 2: This command only works when the first atom specified is a
C-alpha atom (or maybe a main chain atom; I didn't try using other main
chain atoms). Why is that?

AND ULTIMATELY,

is there some way I can tell refmac not to make the ligand and protein
clash?

I'd really appreciate any help!

Thanks,

Sangeetha.

[ccp4bb] Postdoc Position in Protein Crystallography

[Tsai, Francis T.F.]

POSTDOCTORAL POSITION IN PROTEIN CRYSTALLOGRAPHY AT BAYLOR COLLEGE OF MEDICINE

An NIH-funded POSTDOCTORAL position is available immediately to investigate the 
structure and function of molecular chaperones and components of the protein 
quality control system. Diffracting crystals are available for some projects.

The position requires a doctoral degree in one of the natural sciences. 
Preference is given to applicants with 0-2 years of experience. Applicants must 
have demonstrated, relevant experience with crystallization and X-ray crystal 
structure determination (i.e. MAD/MIR) of proteins and enzymes.
Experience with gene cloning and methods used for protein expression and 
purification are an advantage. Salary for this position is highly competitive 
and is in addition to a generous fringe benefit package.

The Tsai lab is fully equipped for all aspects of molecular biology, protein 
chemistry, and structural biology (http://www.bcm.edu/labs/tsai/). Baylor 
College of Medicine is highly committed to support structural biology and has 
pledged over US$ 1 million in capital equipment to set-up a new
state-of-the-art X-ray crystallography facility in the Department of 
Biochemistry and Molecular Biology. This facility will nicely complement our 
existing strength in cryo-EM and functional proteomics.

To apply, please send a current CV, a brief description of your research 
interests and key accomplishments, and contact information of three to four 
referees to: Dr. Francis Tsai, Associate Professor, Departments of Biochemistry 
and Molecular Biology, and Molecular and Cellular Biology, Baylor
College of Medicine, One Baylor Plaza MS:BCM125, Room 315B, Houston, Texas 
77030, USA. E-mail: [EMAIL PROTECTED]

**
Francis T.F. Tsai, D.Phil.   Tel: +1 713 798-8668 (Office)
Associate Professor  Tel: +1 713 798-8565/5639 (Lab)
Department of Biochemistry Fax: +1 713 796-9438
Baylor College of Medicine  Email: [EMAIL PROTECTED]
One Baylor Plaza, BCM125   http://www.bcm.edu/labs/tsai/
Houston, TX 77030-3411, USA

Re: [ccp4bb] SUMMARY: synchrotron remote data collection

[John Rose]

Hi,

SER-CAT has been allowing remote access for about 18 months.
Both SER-CAT beamlines use a modified ALS-style crystal automounter  
and ALS style pucks.
SER-CAT 22ID can host 230 samples in its dewar and 22BM can host 96  
samples.
Both beamlines have remote data collection/data processing  
capabilities and SER-CAT offers a mail-in service to its members.
Several SER-CAT members have Rigaku ACTOR systems configured to host  
the ALS-style puck (<$10K field upgrade).


John Rose Ph.D.
Associate Professor
B204B, The Fred C. Davison Life Sciences Complex
120 Green Street
Department of Biochemistry and Molecular Biology
University of Georgia
Athens, GA 30602-7229
=
Phone: 706-542-1750
Fax:706-542-3077

Re: [ccp4bb] Rotation axis

[Borhani, David]

And the little jiffy program you want Phil is calc-ax (Version 951120)
by Joachim Meyer, University of Freiburg, Germany, which takes an RT mtx
(in OMAT format), and gives back all kinds of useful info.

A google search didn't turn this program up on the web, or Dr. Meyer;
original & my slightly modified code attached, & binary for linux.

Dave

David Borhani, Ph.D.
D. E. Shaw Research, LLC
120 West Forty-Fifth Street, 39th Floor
New York, NY 10036
[EMAIL PROTECTED]
212-478-0698
http://www.deshawresearch.com



> -Original Message-
> From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On 
> Behalf Of Ian Tickle
> Sent: Tuesday, July 29, 2008 5:18 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Rotation axis
> 
> Hi Phil
> 
> The rotation axis is the locus of points which the 
> transformation leaves
> unmoved, i.e. the eigenvector of the transformation matrix which has a
> unit eigenvalue.  So writing the transformation in 
> homogeneous form for
> convenience: x' = Sx you need to solve x' = x, or Sx = x, either
> analytically or just plug the matrix S into a canned eigenvector
> routine.
> 
> Cheers
> 
> -- Ian
> 
> > -Original Message-
> > From: [EMAIL PROTECTED] 
> > [mailto:[EMAIL PROTECTED] On Behalf Of Phil Evans
> > Sent: 29 July 2008 09:11
> > To: CCP4BB@JISCMAIL.AC.UK
> > Subject: Rotation axis
> > 
> > If I've go a superposition transformation (x' = Rx + t), as 
> > it happens  
> > from a superposition in ccp4mg, how do I get the position & 
> > direction  
> > of the rotation axis (to draw in a picture)?
> > I know that any (orthonormal) transformation can be 
> represented as a  
> > rotation about an axis + a screw translation along that axis
> > 
> > I'm sure I've done this before ...
> > 
> > thanks
> > Phil
> > 
> > 
> 
> 
> Disclaimer
> This communication is confidential and may contain privileged 
> information intended solely for the named addressee(s). It 
> may not be used or disclosed except for the purpose for which 
> it has been sent. If you are not the intended recipient you 
> must not review, use, disclose, copy, distribute or take any 
> action in reliance upon it. If you have received this 
> communication in error, please notify Astex Therapeutics Ltd 
> by emailing [EMAIL PROTECTED] and destroy all 
> copies of the message and any attached documents. 
> Astex Therapeutics Ltd monitors, controls and protects all 
> its messaging traffic in compliance with its corporate email 
> policy. The Company accepts no liability or responsibility 
> for any onward transmission or use of emails and attachments 
> having left the Astex Therapeutics domain.  Unless expressly 
> stated, opinions in this message are those of the individual 
> sender and not of Astex Therapeutics Ltd. The recipient 
> should check this email and any attachments for the presence 
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> Therapeutics Ltd only send and receive e-mails on the basis 
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> Astex Therapeutics Ltd., Registered in England at 436 
> Cambridge Science Park, Cambridge CB4 0QA under number 3751674
> 


calc-ax.tgz
Description: calc-ax.tgz

[ccp4bb] Mtccp4bb: remove

[Heinz J Weyer]

... please remove me from  your mailing list.
Thanks
Heinz J Weyer
--
PD Dr. Heinz J. WeyerEmail:  [EMAIL PROTECTED]
Paul-Scherrer Institut   Phone:  +41 (56) 310 3494
CH-5232 Villigen PSI Fax :   +41 (56) 310 3151
Switzerland

Re: [ccp4bb] Rotation axis

[Herman . Schreuder]

Dear Phil,
Because question keep popping up in the bullitin board about conversion
from a rotation matrix into rotation angles, I decided to take the
relevant subroutines from an old program from Groningen and make a jiffy
to do these conversions. It is a small fortran program and does not need
any additional libraries or subroutines. The program will take a
rotation matrix and translation vector and print all kind of rotation
angles and also the component of the translation vector parallel to the
rotation axis, which is the number you want. All other components of the
translation vector can be made zero by choosing the right position of
the rotation axis.

Best regards,
Herman Schreuder

 

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Phil Evans
Sent: Tuesday, July 29, 2008 10:11 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Rotation axis

If I've go a superposition transformation (x' = Rx + t), as it happens
from a superposition in ccp4mg, how do I get the position & direction of
the rotation axis (to draw in a picture)?
I know that any (orthonormal) transformation can be represented as a
rotation about an axis + a screw translation along that axis

I'm sure I've done this before ...

thanks
Phil


rotax.f
Description: rotax.f

Re: [ccp4bb] Rotation axis

[Ian Tickle]

Hi Phil

The rotation axis is the locus of points which the transformation leaves
unmoved, i.e. the eigenvector of the transformation matrix which has a
unit eigenvalue.  So writing the transformation in homogeneous form for
convenience: x' = Sx you need to solve x' = x, or Sx = x, either
analytically or just plug the matrix S into a canned eigenvector
routine.

Cheers

-- Ian

> -Original Message-
> From: [EMAIL PROTECTED] 
> [mailto:[EMAIL PROTECTED] On Behalf Of Phil Evans
> Sent: 29 July 2008 09:11
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Rotation axis
> 
> If I've go a superposition transformation (x' = Rx + t), as 
> it happens  
> from a superposition in ccp4mg, how do I get the position & 
> direction  
> of the rotation axis (to draw in a picture)?
> I know that any (orthonormal) transformation can be represented as a  
> rotation about an axis + a screw translation along that axis
> 
> I'm sure I've done this before ...
> 
> thanks
> Phil
> 
> 


Disclaimer
This communication is confidential and may contain privileged information 
intended solely for the named addressee(s). It may not be used or disclosed 
except for the purpose for which it has been sent. If you are not the intended 
recipient you must not review, use, disclose, copy, distribute or take any 
action in reliance upon it. If you have received this communication in error, 
please notify Astex Therapeutics Ltd by emailing [EMAIL PROTECTED] and destroy 
all copies of the message and any attached documents. 
Astex Therapeutics Ltd monitors, controls and protects all its messaging 
traffic in compliance with its corporate email policy. The Company accepts no 
liability or responsibility for any onward transmission or use of emails and 
attachments having left the Astex Therapeutics domain.  Unless expressly 
stated, opinions in this message are those of the individual sender and not of 
Astex Therapeutics Ltd. The recipient should check this email and any 
attachments for the presence of computer viruses. Astex Therapeutics Ltd 
accepts no liability for damage caused by any virus transmitted by this email. 
E-mail is susceptible to data corruption, interception, unauthorized amendment, 
and tampering, Astex Therapeutics Ltd only send and receive e-mails on the 
basis that the Company is not liable for any such alteration or any 
consequences thereof.
Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, 
Cambridge CB4 0QA under number 3751674

[ccp4bb] Rotation axis

[Phil Evans]

If I've go a superposition transformation (x' = Rx + t), as it happens  
from a superposition in ccp4mg, how do I get the position & direction  
of the rotation axis (to draw in a picture)?
I know that any (orthonormal) transformation can be represented as a  
rotation about an axis + a screw translation along that axis


I'm sure I've done this before ...

thanks
Phil

Re: [ccp4bb] about microbatch

[Tom Caradoc-Davies]

Hi Jiamu,

I use 96-well V-well ELISA plates for microbatch and they work very well. I add 
paraffin until about 1mm from the top-edge of the well. You need 2-3mm of oil 
to reduce evaporation to very low rate. The drops will shrink over a few months 
as you loose water through the plastic of the plate as well.
When going from vapour diffusion to microbatch it is useful to screen a range 
of drop ratios. I use 1+2, 1.5+1.5 and 2+1 to go from a vapour diffusion "hit" 
to microbatch". This should give you an idea of a good starting ratio for 
further screens. You can use any drop size you like that you can accurately 
pipette.
Also, as pipetting error is now your main variable it is worth setting drops up 
in triplicate as you don't want to miss "good" conditions due to a bad drop.
If you have crystals that grow overnight (or within a day or so) and have PEG 
as your precipitant then it is worth trying microbatch. Under these conditions 
I have often gotten larger, better diffracting crystals than trying to optimise 
the very rapid growth in vapour diffusiion (your mileage may vary though :) ).
A problem with the ELISA plates is that it can be hard to fish crystals out of 
the drop using a pin. I have made some tools with 10mm steel Hampton tubes 
(that go into 18mm bases) and unmounted cryoloops (the ones in a paper slip) 
and with these you can put the tube down the side of the well to fish out the 
crystal (otherwise the pin base can block your view down the microscope). Some 
people pipette their crystals out of the microbatch experiment but I have lost 
a few crystals this way (the best ones of course) and so don't do that anymore.
Cheers,
Tom


Dr Tom Caradoc-Davies

Beamline Scientist

Protein Crystallography

Australian Synchrotron

800 Blackburn Road

Clayton, Victoria 3168

Australia.

Direct +61 3 8540 4187

Fax +61 3 8540 4200

Mobile +61 4 3430 7453

**

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intended recipient, you must not disclose, distribute, copy or use the 
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From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jiamu Du
Sent: Saturday, 26 July 2008 9:39 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] about microbatch

Dear all,
I want to screen crystal using microbatch method. I do not have standard 
microbatch plate, so I have to using a 96 well cell culture plate instead.
My question is below:
What is a typical drop size for microbatch? 1ul protein + 1ul mother solution 
or larger?
How much paraffin oil is sufficient for cover the drop?

Thanks.

--
Jiamu Du, Ph.D.
State Key Laboratory of Molecular Biology
Institute of Biochemistry and Cell Biology
Shanghai Institutes for Biological Sciences
Chinese Academy of Sciences
320 Yue-Yang Road
Shanghai 200031
P. R. China
Tel: +86-21-5492-1117
E-mail: [EMAIL PROTECTED]