Re: [ccp4bb] phasing with se-met at low resolution

[Engin Ozkan]

Here is a summary of responses to the thread I recently started on "phasing 
with se-met at low resolution":

There were many suggestions. They can be grouped into two categories:
1. Aim for very high redundancy at one wavelength (peak) and watch out for 
radiation damage. Sacrificing resolution is OK.
2. Collect two, three, or four wavelength MAD datasets. Helps break phase 
ambiguity (but is it better at finding sites compared to redundant peak SAD?).

Collecting in wedges with inverse beam is widely recommended, but it was also 
pointed out that its usefulness has not been systematically established. No 
matter what, one should watch out for radiation damage: Follow your scaling 
B-factors in scalepack.

Obviously, high solvent content helps with SAD/density modification. NCS 
averaging of low resolution, low quality maps can yield interpretable maps. 
Cross crystal averaging is also useful, if applicable. B factor averaging may 
help at lower resolution (apparently mostly when there is phasing data, as 
opposed to pure MR solutions, according to DelaBarre and Brunger, Acta Cryst 
2006). Many of you pointed out that building all-beta would be a big pain even 
at 4 Angstroems, while alpha helical structures can be built fine up to 5 
Angstroems.

However, it was pointed out by many of you that finding heavy metal sites 
appears to be the major problem, and where many get stuck. Many of you suggest 
very redundant SAD datasets could help with that.

Except for two (1rhz, 2oau), all cases sent to me used heavier metal soaks to 
find selenium sites  (or they were ~3.5 A datasets with I/sigma ~10 in the 
highest resolution bin, which might not be considered low resolution: in these 
cases finding heavy metal sites was expectedly straight-forward).

Summary of cases:

1rhz
Peak wavelength SAD with wedges combined with cross-crystal averaging.
Platinum-derivative might have confirmed heavy metal sites (not sure).
It was mentioned that low symmetry space groups do make life quite difficult.

2oau
Very redundant three wavelength data (high symmetry space group) to 5 A was 
amazingly enough to locate selenium sites (SOLVE located the sites). 7-fold NCS 
with native data to 3.5 A yielded "excellent" electron density maps. This one 
gives hope to all of us.

2jk4
"Together with phases from a Pt-derivative and after careful density 
modification, an initial low-resolution electron density map was obtained that 
clearly showed the protein envelope and the overall barrel dimensions."
Complementary NMR data (both NOE and dihedral restraints, secondary structure 
assignments and hydrogen bonds) had to be added to the refinement process to 
achieve interpretable density maps and reliable model.

1xdv
"A mercury derivative provided initial phases, which locate the positions of 
selenomethionines. Electron density maps calculated using experimentally 
determined phases to 4.3 Å, extended to 4.0 Å, were improved by B factor 
sharpening and by 2-fold noncrystallographic symmetry averaging."

And other cases where initial phases were obtained at very low resolution via 
heavy metal soaks (such as 3B8E).

Among to respondents were Kornelius Zeth, Partha Chakrabarti, Stephen Soisson, 
Leiman Petr, Artem Evdokimov, Dominika Borek, Clemens Vonrhein, Phil Jeffrey, 
Wladek Minor, Pete Meyer, James Holton, P. H. Zwart, Anastassis Perrakis, and 
others I forgot to mention. Many thanks.

[ccp4bb] CCP4 Northern Protein Structure workshop 'Carla 2009'

[Mads Gabrielsen]

Hello all and apologies for the cross posting and to non-UK residents for
the cluttering of their inboxes.


Just to let you know that we will be holding the popular Northern
universities CCP4-sponsored protein structure workshop again this September
(see advert below). This year it will again be in sunny Carlisle, which has
proved a successful alternative traditional Galashiels venue where no-one is
clear on what is going to happen.

I'd be grateful if you could also bring this advert to the attention of
anybody else who may be interested and is not on my current mailing list,
please (let me have their e-mail address as well if they don't mind).


An updated web page with registration details will appear soon at
http://www.chem.gla.ac.uk/protein/gala

Thanks

Mads Gabrielsen
(On behalf of the organizing committee)

#
ANNOUNCING THE 16TH CCP4 PROTEIN STRUCTURE WORKSHOP 2nd - 4th Sept 2009 at
St Martin's College in Carlisle

This is the first announcement of the annual meeting for crystallographers
and interested structural biologists from Northern UK laboratories to
present their latest research and hold a morning workshop on a special topic
of interest, all in a relatively informal atmosphere. This meeting has often
been known simply as "Galashiels" but will again return to last year's
successful venue in Carlisle.

This year the workshop will focus on "What next?" - after you have your
shiny new structure, then what?

Registrants are requested to provide a talk title (perhaps but not
necessarily related to the special topic) as the meeting will continue to
provide an invaluable opportunity for younger group members (PhD students
and postdocs) to present their work to a friendly external audience and pick
up useful tips.
Registrant costs will be subsidised by CCP4 and other external sponsors*.

*Interested commercial sponsors please contact the organisers for registrant
and exhibition costs.

Organising committee:
Dr Tina Howard - tina.how...@astrazeneca.com Dr Mads Gabrielsen -
m.gabriel...@bio.gla.ac.uk Prof Jim Naismith - j...@st-and.ac.uk Dr Richard
Pauptit - richard.paup...@astrazeneca.com


Mads Gabrielsen, MSc, PhD
Post-doctorial Research Associate
Microbiology, Division of Infection and Immunity
FBLS, University of Glasgow

B216
GBRC
120 University Place
G12 8TA

Phone: 01413308119
E-mail: m.gabriel...@bio.gla.ac.uk, mg...@chem.gla.ac.uk

Re: [ccp4bb] Problem with Rfree and Rfac

[Anastassis Perrakis]

hi Sravanti -

Something that is unclear from your message, is that if you used Phaser
for a SAD phasing or molecular replacement.

I assume that latter.

Starting from molecular replacement models at 3.0 A is very tough.
Unless the identity/similarity of the search model and expected model
is very high, re-building the model to represent your structure,
is a very tough job, even for experienced crystallographers at 3.0

I suspect that the reason your Rfree is not going down (which must be  
your main worry

right now) is that you are being too conservative during rebuilding.
Try to delete and add regions according to the density map and see how  
it goes.


A couple of tricks that are useful, is to do some density modification  
(dm, resolve, etc)
and/or run arp/warp (it will most likely get rid of most of your model  
at 3.0, but it
will likely produce a lower Rfree and better map that can help you  
build manually).


Tassos

On May 21, 2009, at 15:40, Sravanti Vaidya wrote:


Hello CCP4i GURUs,

I am working on a 3A resolution structure and I recently got a  
solution from
Phaser. I have just started building and refining the structure. I  
started
with Rfree-43 , R-41.7 and the first round of building and refining  
gave me
Rfree- 40 and Rfac- 35.5. The Rfree and R do not decrease to the  
same extent
during refinement (Refmac5). Further rounds of refinement increases  
the

difference between R and Rfree (Rfree-38 Rfac- 30)

I am worried that since this is just the beginning further rounds of
building and refinement might drastically increase the gap between R  
and
Rfree. On the positive note the FOM is increasing with refinement  
and the
maps look better. I also see new density at several regions where I  
can

build into.

I tried to change the weighting factor and geometric parameters  
after I read
the earlier posts here. The references noted earlier were extremely  
helpful.

Here are some other details of my structure which might help you

Space group- P1 21 1
Resolution- 3A
Solvent content- 39%
unit cell parameters- 63.3,  90.72 , 86.11, angles-  90, 91.5 , 90

Please enlighten me with your suggestions. Is it fine to go ahead  
with this
refinement or is there a way I can reduce this difference between R  
and Rfree??


Your help will be greatly appreciated. As this is my first structure  
I am

excited about it!!!

Thank you

Sravanti


P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

[ccp4bb] SCALEPACK2MTZ failed

[Raja Dey]

Dear Friends,
 I got the following error while running  SCALEPACK2MTZ in 
the latest version of CCP4 Program Suite 6.1.1 CCP4Interface 2.0.4 running on a 
macbook with intel core 2 duo. I also tried to run earlier SCALEPACK2MTZ job 
which was successfully finished before, but now failed with the same error 
message. Is there any clue or remedy?

SCALEPACK2MTZ:  Normal termination
Times: User:   0.1s System:0.0s Elapsed: 0:00  



#CCP4I TERMINATION STATUS 0 Error from script 
/sw/share/xtal/ccp4-6.1.1/ccp4i/scripts/import_scaled.script: can't read 
"dataset_name": no such variable
#CCP4I TERMINATION TIME 22 May 2009  09:38:45
#CCP4I MESSAGE Task failed

Thanks...

 Raja


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Travel http://in.travel.yahoo.com/

Re: [ccp4bb] phaser

[Nathaniel Echols]

On Thu, May 21, 2009 at 11:04 PM, Tim Gruene wrote:

> this usually means that you have a second version of phaser installed, e.g.
> from the phenix package. To find out, go to a terminal from which you would
> start ccp4i and type 'which phaser'. My guess is that the answer to that
> command does not show phaser in the ccp4-directory.
>  To circumvent this problem you have to make sure that ccp4i 'sees' the
> phaser from ccp4 first. There are several ways to do this.
> 1) if you never use phaser from the phenix distribution, simply rename it
> to e.g. phaser-phenix or anything else.


I'm pretty sure this shouldn't be an issue - Phenix (at least version 1.4-3
and later) doesn't have a 'phaser' command, just 'phenix.phaser'.  However,
a standalone installation of Phaser could conflict with ccp4.

Re: [ccp4bb] SCALEPACK2MTZ failed

[Ronan Keegan]

Dear Raja,

This is a known bug in version 6.1.1. There is a fix for it available 
from the CCP4 problems pages. See item no. 7 in the following page:


http://www.ccp4.ac.uk/problems.php#6.1.1-ccp4i

You'll need to download "import_scaled.script" and replace the version 
that you have in:


/sw/share/xtal/ccp4-6.1.1/ccp4i/scripts/

Hope this helps.

Kind regards,

Ronan


Raja Dey wrote:


Dear Friends,
 I got the following error while running 
 SCALEPACK2MTZ in the latest version of CCP4 Program Suite 6.1.1 
CCP4Interface 2.0.4 running on a macbook with intel core 2 duo. I also 
tried to run earlier SCALEPACK2MTZ job which was successfully finished 
before, but now failed with the same error message. Is there any clue 
or remedy?


SCALEPACK2MTZ:  Normal termination
Times: User:   0.1s System:0.0s Elapsed: 0:00  




#CCP4I TERMINATION STATUS 0 Error from script 
/sw/share/xtal/ccp4-6.1.1/ccp4i/scripts/import_scaled.script: can't 
read "dataset_name": no such variable

#CCP4I TERMINATION TIME 22 May 2009  09:38:45
#CCP4I MESSAGE Task failed

Thanks...

 
Raja




Explore and discover exciting holidays and getaways with Yahoo! India 
Travel Click here! 

Re: [ccp4bb] SCALEPACK2MTZ failed

[Paul Leonard]

Dear Raja,

Where you have the input option

Use "dataset name" as identifier to append column labels.

Change this to "user defined identifier" and put a name in the box below.

I think Scalepack should then run ok after that.

Paul


> Dear Friends,
>  I got the following error while running
> SCALEPACK2MTZ in the latest version of CCP4 Program
> Suite 6.1.1 CCP4Interface 2.0.4 running on a macbook
> with intel core 2 duo. I also tried to run earlier
> SCALEPACK2MTZ job which was successfully finished
> before, but now failed with the same error message.
> Is there any clue or remedy?
>
> SCALEPACK2MTZ:  Normal termination
> Times: User:   0.1s System:0.0s Elapsed: 0:00
> 
> 
>
> #CCP4I TERMINATION STATUS 0 Error from script
> /sw/share/xtal/ccp4-6.1.1/ccp4i/scripts/import_scaled.script: can't read
"dataset_name": no such variable
> #CCP4I TERMINATION TIME 22 May 2009  09:38:45
> #CCP4I MESSAGE Task failed
>
> Thanks...
>
>  Raja
>
>
>   Explore and discover exciting holidays and getaways with Yahoo!
> India Travel http://in.travel.yahoo.com/


Department of Biochemistry
UMDNJ-Robert Wood Johnson Medical School
Center for Advanced Biotechnology and Medicine
679 Hoes Lane
Piscataway, New Jersey 08854-5627

Phone: (732) 235-4206
Email: leon...@cabm.rutgers.edu

Re: [ccp4bb] SCALEPACK2MTZ failed

[Raja Dey]

Dear Paul,Thanks. Now is running. Before in earlier version it was not required.

 Raja 




From: Paul Leonard 
To: Raja Dey 
Cc: ccp4bb@jiscmail.ac.uk
Sent: Friday, 22 May, 2009 10:31:51 AM
Subject: Re: [ccp4bb] SCALEPACK2MTZ failed

Dear Raja,

Where you have the input option

Use "dataset name" as identifier to append column labels.

Change this to "user defined identifier" and put a name in the box below.

I think Scalepack should then run ok after that.

Paul


> Dear Friends,
>  I got the following error while running
> SCALEPACK2MTZ in the latest version of CCP4 Program
> Suite 6.1.1 CCP4Interface 2.0.4 running on a macbook
> with intel core 2 duo. I also tried to run earlier
> SCALEPACK2MTZ job which was successfully finished
> before, but now failed with the same error message.
> Is there any clue or remedy?
>
> SCALEPACK2MTZ:  Normal termination
> Times: User:   0.1s System:0.0s Elapsed: 0:00
> 
> 
>
> #CCP4I TERMINATION STATUS 0 Error from script
> /sw/share/xtal/ccp4-6.1.1/ccp4i/scripts/import_scaled.script: can't read
"dataset_name": no such variable
> #CCP4I TERMINATION TIME 22 May 2009  09:38:45
> #CCP4I MESSAGE Task failed
>
> Thanks...
>
>  Raja
>
>
>   Explore and discover exciting holidays and getaways with Yahoo!
> India Travel http://in.travel.yahoo.com/


Department of Biochemistry
UMDNJ-Robert Wood Johnson Medical School
Center for Advanced Biotechnology and Medicine
679 Hoes Lane
Piscataway, New Jersey 08854-5627

Phone: (732) 235-4206
Email: leon...@cabm.rutgers.edu


  Explore your hobbies and interests. Go to 
http://in.promos.yahoo.com/groups/

[ccp4bb] issues with TLS refinement resolved

[Seema Mittal]

Hi All,

Thanks for all the help in fixing the refmac TLS refinement issue.   
The new refmac (5.5.0093) is working well so far.


Best,
Seema Mittal


On May 22, 2009, at 8:56 AM, Ronan Keegan wrote:



Hi Seema and Ben,

Garib has produced a fix for the TLS problem and put the update on  
his refmac page:


http://www.ysbl.york.ac.uk/~garib/refmac/latest_refmac.html

Get the current version (5.5.0093) for the system that you are  
using and replace your current executable or Ben might be able to  
create a new executable for you.


Ben, you might want to upgrade the version in SBGrid.

Thanks a lot for bringing this problem to our attention.

Best wishes,

Ronan

[ccp4bb] [offtopic?] putting b-factors from two pdbs on the same scale and interpretation

[Francis E Reyes]

Hi all,

Maybe this is more of a statistics/normalization question, but say you  
have the same molecule crystallized in two different states. How would  
you put their refined b-factors (directly from the pdb) on the same  
scale and say compare the b-factors of residues in the binding pocket?  
I figure some normalization scheme is involved, but are there any  
specific examples ? (quantile normalization? z-score?)


Are there any papers that discuss interpretation of b-factors (being a  
crystallographic property) to the relative dynamics/flexibility of the  
residue in the crystal (and subsequently in solution if the residue is  
not a crystal contact?). I'm particularly interested in the dangers of  
such an interpretation.


Thanks!

FR

-
Francis Reyes M.Sc.
215 UCB
University of Colorado at Boulder

gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D

8AE2 F2F4 90F7 9640 28BC  686F 78FD 6669 67BA 8D5D

Re: [ccp4bb] [offtopic?] putting b-factors from two pdbs on the same scale and interpretation

[Ethan Merritt]

On Friday 22 May 2009 11:10:16 Francis E Reyes wrote:
> Hi all,
> 
> Maybe this is more of a statistics/normalization question, but say you  
> have the same molecule crystallized in two different states. How would  
> you put their refined b-factors (directly from the pdb) on the same  
> scale and say compare the b-factors of residues in the binding pocket?  
> I figure some normalization scheme is involved, but are there any  
> specific examples ? (quantile normalization? z-score?)
> 
> Are there any papers that discuss interpretation of b-factors (being a  
> crystallographic property) to the relative dynamics/flexibility of the  
> residue in the crystal (and subsequently in solution if the residue is  
> not a crystal contact?). I'm particularly interested in the dangers of  
> such an interpretation.

[insert standard plug for TLSMD analysis]

I think that comparing individual B factors is the wrong way to go.

There is much more statistical power in looking at the joint distribution
of B values in 3-space.  That is the basis for the analysis performed by
the TLSMD server

http://skuld.bmsc.washington.edu/~tlsmd

We have demonstrated that this kind of analysis can pick out similar
features (hinges, domain boundaries, flexible groups, vibrational modes)
not only in multiple crystal forms of the same protein but also across
structures of homologous proteins.

The TLSMD web site contains more discussion and links to publications.

Ethan



-- 
Ethan A Merritt
Biomolecular Structure Center
University of Washington, Seattle 98195-7742

Re: [ccp4bb] [offtopic?] putting b-factors from two pdbs on the same scale and interpretation

[Donnie Berkholz]

On 12:10 Fri 22 May , Francis E Reyes wrote:
> Maybe this is more of a statistics/normalization question, but say you 
> have the same molecule crystallized in two different states. How would 
> you put their refined b-factors (directly from the pdb) on the same 
> scale and say compare the b-factors of residues in the binding pocket? 
> I figure some normalization scheme is involved, but are there any 
> specific examples ? (quantile normalization? z-score?)
>
> Are there any papers that discuss interpretation of b-factors (being a 
> crystallographic property) to the relative dynamics/flexibility of the 
> residue in the crystal (and subsequently in solution if the residue is 
> not a crystal contact?). I'm particularly interested in the dangers of 
> such an interpretation.

No doubt this isn't the best approach, but it's given me useful results 
consistent with limited proteolysis, and I can actually understand and 
easily do the math. For each residue in each structure, I calculate the 
ratio of that residue's B to the average B for that structure. Then I 
just plot the two structures per-residue on a graph vs the ratios. I can 
look for gaps between lines to indicate differences, or just look at the 
patterns of the lines to look for more relatively disordered or static 
regions.

The goal is using the structure's average B to normalize for the 
difference between two structures, and use the ratio instead of a 
subtraction to try accounting for the difference in absolute B ranges 
between structures. For example, a structure with =30 might have 
individual B's ranging from 10 to 45, and a structure with =100 might 
have B's ranging from 30-150. For these cases, this calculation would 
give you 10/30 ~ 30/100, and 45/30 ~ 150/100, whereas a simple 
subtraction would give you 10-30 != 30-100, and 45-30 != 150-100.

-- 
Thanks,
Donnie

Donnie Berkholz
P. Andrew Karplus lab
Oregon State University


pgpFuOGMByPUC.pgp
Description: PGP signature

Re: [ccp4bb] MAD phasing

[Ho-Leung Ng]

You need to try alternative space groups, such as P6522, which has the
same extinctions and merging statistics as P6122.

What do the maps look like coming out of SOLVE/RESOLVE?


Ho
UC Berkeley

[ccp4bb] postdoc position available

[Wen Jiang]

A postdoc position is immediately available in the Jiang laboratory
(http://jiang.bio.purdue.edu) at the Markey Center of Structural
Biology and Department of Biological Sciences, Purdue University, West
Lafayette, Indiana, USA. Our research focuses on structural studies of
large macromolecular complexes and viruses using single particle
cryo-electron microscopy and 3-D reconstruction. Purdue is currently
equipped with two FEI FEG cryo-microscopes (200kV and 300kV) and will
install a FEI Titan Krios 300kV microscope later this year in our new
structural biology building. We are seeking a highly motivated
individual with extensive experience in structural biology and protein
biochemistry. The applicant should be a team player that enjoys
working in a collaborative environment. Please send cover letter, CV,
statement of research accomplishments and interests, and three
references to Dr. Wen Jiang (jian...@purdue.edu). Applications will be
reviewed until the position is filled.

[ccp4bb] Post-doctoral position available June 1st at UMDNJ

[Narayanan Ramasubbu]

A postdoc position is immediately available in my laboratory
at the Oral Biology Department, UMDNJ. The post-doc will work on the elucidation of 
structure of enzymes involved in modifying A. actinomycetemconitans biofilms. 
Crystals of two such enzymes are available.
Experience in expression and purification using his tag is essential. 
Additional experience in yeast expression is desirable. A highly motivated

individual with at least one year experience in structural biology and protein
biochemistry who is a team player is sought. This position is for one year with 
the potential for additional years depending upon funding.

Please send your CV and names of three
references to Dr. N. Ramasubbu (ramas...@umdnj.edu). 


Subbu