Re: [ccp4bb] need help--Rfree is not decreasing

[Eleanor Dodson]

First suggestion - use WEIGHT AUTO in REFMAC - you may have restrained 
the geometry too tightly..


 And have you tried TLS ?
 And at what level do you see no density - remember sigma levels in 
maps need to vary with B factor - very easy to achieve this with COOT..


Eleanor

JOE CRYSTAL wrote:

Dear all,


I am refining a structure at 2.0 A.  The water molecules have been 
added using arp/warp resulting Rwork/Rfree=21/26% (about 370 HOH for 
360 residues).  After 10 cycles of refmac refinement (wt 0.3), 
Rwork/Rfree went up about 1.5% to 22.5/27.5%.  I did some minor 
adjustments and add/delete water in Coot followed by 10 cycles refmac 
refinement, but Rwork/Rfree are still around 22.5/27.5%.  I also 
noticed a few side chains without density.  Will setting those atoms 
to 0 occupancy or high B factor or mutating to Ala help decrease Rfree 
substantially?  If not, is there any better strategies to lower down R 
factors?  I will be very appreciative if you have any suggestions or 
comments to offer.  Thank you in advance. 



Best,


Joe

Re: [ccp4bb] refmac5 issues: C-terminal amidation and maltose library file

[Eleanor Dodson]

Q2

Copy the MAL entry into your own directory
cp $CLIBD/monomers/m/MALcif ./

Then correctt it in your directory

And assign LIBIN ./MAL.cif

The program will read your corrected version and ignore the distributed 
one.



Q1

If you run REFMAC the GUI under review restraints, it will detect and 
make a LINK entry for you
Then you will need to use the GUI task - merge monomer library to 
combine your corrected MAL with the new LINK


Run refmac again with XYZIN the output from "review restraints " task ( 
that will include a LINK record)

and it should/might! work..
Eleanor





Pioszak, Augie wrote:


Hello All,

I have two questions about refinement with Refmac5.

1. I have a protein structure that is C-terminally amidated. What is 
the best way to handle this?


I was trying to use the sketcher to create my own library definition 
for the modifed amino acid, but then I had issues with the modified 
amino acid not being covalently linked to the rest of the protein. I’m 
guessing there is a better way, such as creating a definition for just 
the additional NH2 group and telling refmac how to covalently link it 
to the last amino acid, but I’m not sure how to do this.


2. I have a structure with maltose in it. The refmac library 
definition for maltose has atoms “O1” and “O1,” mislabeled as Carbons. 
How can I correct this?


I know I can change it with the sketcher, but then it tells me the 
name MAL is already taken and I have to use another. I’d prefer to 
leave it as the standard MAL name.


Thanks for any help. I apologize if these issues have been covered before.

Augie

Augen Pioszak, Ph.D.

Postdoctoral Fellow

Laboratory of Structural Sciences

Van Andel Research Institute

333 Bostwick Ave. N.E.

Grand Rapids, MI 49503

phone: (616) 234-5399

email: [EMAIL PROTECTED] 


**
**This email message, including any attachments, is for the sole use 
of the intended recipient(s) and may contain confidential information. 
Any unauthorized review, use, disclosure or distribution is 
prohibited. If you are not the intended recipient(s) please contact 
the sender by reply email and destroy all copies of the original 
message. Thank you.**

Re: [ccp4bb] Stop the new PDB format!

[Clemens Vonrhein]

There is a collection of posts (unfortunately with a number of spam
messages) at

  http://wwpdb-remediation.rutgers.edu/mail-archive/

with various comments. Although I'm not familiar with the internal
workings of this remediation program, it seems indeed that the PDB
format is now largely auto-generated from the internally used
mmCIF. Unfortunately in my experience (having had a look at a few dozen
random entries of the new PDB files) this means that some of the new
PDB files of old entries will look very different from what you/we
deposited several years ago. The format seems better (internally
consistent) but the content has sometimes suffered.

But I guess there is always room for frictions when one side is mainly
interested in data format, storage and databases and the other mainly
interested in the crystallographic content. Finding a good compromise
between those two groups of experts is non-trivial.

At least the new databases will always have a link to the original
version of the PDB file - although it will still mean I can't now
search for an author name MUELLER (German U-umlaut transfered in the
proper ASCII format), since the PDB files now contain MULLER (because
PUBMED isn't able to properly translate non-ASCII names ...). Or an
analysis of programs used for structure solution will show a veri
different distribution - since the information has been significantly
changed.

Anyway, have a look at your favourite PDB file with the attached
script

  ./pdb23.sh 1abc

It is quite interesting sometimes. I haven't cehcked the mmCIF files -
maybe they are much better (as a 'hint' from the database people to
the crystallographers to stop using PDB format and switch to mmCIF,
maybe?).

Cheers

Clemens

On Sat, Jul 21, 2007 at 12:05:35PM -0700, Ethan A Merritt wrote:
> On Saturday 21 July 2007 11:12, Joe Krahn wrote:
> > we all use in our daily research. They don't even want to keep the PDB
> > format at all. It's primary purpose now is for structural biologists.
> 
> That is inevitable.  The PDB format is simply not capable of representing
> the complexities of current crystallographic models, and will only become
> more obsolete as the state of the art progresses.  Because it is so wide-
> spread, it will remain a legacy format for import/export into programs 
> that are not up to the current crystallographic state of the art.  Yes,
> that means it will largely be used by non-crystallographers to import
> and view structures.
> 
> Thus I think the writing is on the wall that the PDB format as a primary
> working medium in crystallography is on its deathbed.  Of course it may
> linger there for a long while yet, and may be poked at from time to time
> in order to stave off its final expiration.
> 
> Having said that, I don't understand the motivation for changing this
> legacy format to something that the legacy programs will not recognize.
> That indeed seems self-defeating.
> 
>   Ethan Merritt
> 
> 
> 
> > The new PDB format (version 3) has a lot of very useful improvements,
> > and an update is long overdue. However, I am irate that RCSB chose NOT
> > to use the ACA meeting to discuss the changes. Instead, the format is
> > being put into production at the same time as the ACA meeting. It is
> > essentially stating that opinions expressed at the ACA do not count.
> > Their was a lot of conflict at their last attempt at an update. Instead
> > of working to better involve the structural biologist community, I feel
> > that they are intentionally discounting our interests because working
> > with the user community is too much effort.
> > 
> > Unfortunately, structural biologists generally do not want to spend time
> > arguing about file formats, while computer scientists can carry on for
> > weeks over minor details. This change is going to affect all of us. If
> > you have concerns about the new format that have not been addressed, it
> > is important to take action now. The PDB format is not just their
> > personal database format (that's what mmCIF is for), but the format that
> > we all use in our daily research. They don't even want to keep the PDB
> > format at all. It's primary purpose now is for structural biologists. It
> > is essential that we be part of the decision making process.
> > 
> > I just sent the following letter to the wwPDB, which is where
> > comments about the new format are supposed to go. If you will be at the
> > ACA meeting, I encourage you to complain loudly.
> > 
> > Joe Krahn
> > 
> > ---
> > To: [EMAIL PROTECTED]
> > Subject: The new PDB format is WRONG.
> > 
> > It seems obvious to me that the RCSB and wwPDB worked on the new format
> > to consider database users needs, but has intentionally ignored the rest
> > of the user community. RCSB manages mmCIF for database purposes, and has
> > declared a lack of interest in even keeping the PDB format. Obviously,
> > the primary purpose of the PDB format

Re: [ccp4bb] Stop the new PDB format!

[Clemens Vonrhein]

Hmmm - ccp4bb doesn't allow an attachement ... so script comes here:

-- cut here ---
#!/bin/sh

[ $# -eq 0 ] && echo " ERROR: give some PDB identifier as argument"

type tkdiff >/dev/null 2>&1
[ $? -eq 0 ] && diff=tkdiff || diff=diff

for id in $@
do
  id=`echo $id | tr '[A-Z]' '[a-z]'`
  [ ! -f pdb${id}.ent.Z ] && \
wget 
ftp://ftp.ebi.ac.uk/pub/databases/rcsb/pdb/data/structures/all/pdb/pdb${id}.ent.Z
  [ ! -f pdb${id}.ent.gz ] && \
wget 
ftp://ftp.ebi.ac.uk/pub/databases/rcsb/pdb-remediated/data/structures/all/pdb/pdb${id}.ent.gz
  zcat pdb${id}.ent.Z > ${id}.v2.pdb
  gunzip -c pdb${id}.ent.gz > ${id}.v3.pdb
  echo "$diff ${id}.v2.pdb ${id}.v3.pdb   Continue?"
  read m
  $diff ${id}.v2.pdb ${id}.v3.pdb
done
-- cut here ---


-- 

***
* Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
*
*  Global Phasing Ltd.
*  Sheraton House, Castle Park 
*  Cambridge CB3 0AX, UK
*--
* BUSTER Development Group  (http://www.globalphasing.com)
***

Re: [ccp4bb] CCP4 Wiki

[tadeusz . j . skarzynski]

Yes, this is how a wiki page for CCP4 might look like. However, to make a 
wiki successful we have to make sure it is secure and that there is a 
dedicated core team to maintain it. Kevin was asked by the CCP4 to lead 
the wiki project and while he is setting things up, it would be 
counterproductive to start setting up independent "CCP4 wiki" sites.

Tadeusz
 






"Artem Evdokimov" <[EMAIL PROTECTED]> 
Sent by: "CCP4 bulletin board" 
23-Jul-2007 00:29
Please respond to "Artem Evdokimov" <[EMAIL PROTECTED]>

 
To
CCP4BB@JISCMAIL.AC.UK
cc

Subject
[ccp4bb] CCP4 Wiki






Hi,

Would something like this work?

http://www.xtals.org/wiki/

I've only set it up today, but it seems to work just fine. Feel free to 
play
with it.

Artem

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of 
Lucas
Bleicher
Sent: Sunday, July 22, 2007 11:02 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Suggestion: Wiki -- was:Re: [ccp4bb] need 
help--Rfree
is not decreasing

That would be a great idea. In fact, I keep on my
mailbox dozens of great postings (most of them
summaries) in CCP4 which would be very useful to
everybody if there's an online resource, with
information organized in topics. I would gladly copy
them to this wiki.

Lucas

--- Kay Diederichs <[EMAIL PROTECTED]>
escreveu:

> So - rather than repeat things that are obvious to
> some people, would it 
> not be good to have a crystallography-FAQ that one
> could point people 
> to? This should be part of a Wiki where "we
> crystallographers" could 
> collect our wisdom. This would be much more
> systematic, and less 
> volatile, than the postings of this mailing list
> (which to me _is_ a 
> very valuable ressource).
> 
> A Wiki is not difficult to set up. Maybe it could be
> part of the CCP4 
> pages? We set up a Wiki for our lab at the beginning
> of the year, and it 
> was a great success, in particular because it works
> the same way as 
> Wikipedia - anybody can contribute. There should be
> some means of 
> controlling "write access", but that could simply be
> granted to people 
> who are subscribed to the CCP4 mailing list.
> 
> I'd at least volunteer in helping to get a Wiki
> started. And one way to 
> get it filled with articles would be that those
> people who used to write 
> a "summary" of responses would simply compose a new
> Wiki article, and 
> report to the mailing list that this article exists,
> which could then be 
> expanded by others.
> 
> best,
> 
> Kay
> 
> 
> Anastassis Perrakis schrieb:
> > Sorry for the cliche, but *the goal of refinement
> is not to reduce R 
> > factors, but to produce a good model.*
> > 
> > ARP/wARP uses the 'WEIGHT AUTO' option of REFMAC5
> to get a good geometry.
> > You should set the weight to a value that produces
> 1-2 and 1-3 distances 
> > rms deviations similar to the ARP/wARP job, 
> > to be able to compare. The fact that weight is 0.3
> says nothing.
> > 
> > The correct weight can vary wildly from 0.02 to
> 0.5, in my experience. 
> > for 2.0 data 0.3 sound loose, 0.15-0.2 is what I
> am used to,
> > depending on dataset. But, The only way to tell
> what is right is 
> > inspecting the geometry and aim for a 'reasonable'
> rms 1-2 distances 
> > deviation.
> > 
> > What is 'reasonable',  can cause yet another long
> discussion, but my 
> > personal favorite for 1-2 distances rms deviation
> is between 0.015-0.020.
> > In Refmac these also give the lowest R factors, in
> my hands.
> > 
> > The invisible side chains is yet another long
> discussion that you can 
> > retrieve from the ccp4bb archives.
> > Again, my personal preference is to leave them in
> and let them get very 
> > high B factors, as long as they do not
> > get negative density in difference maps, that I
> presume you are inspecting.
> > I dont mind deleting them (but dont like it) and I
> think mutating to ALA 
> > is worse since its misleading to users.
> > 
> > Finally, given that you have 2.0 A data you should
> try and model not 
> > only waters, but also:
> > a. double conformations of side chains
> > b. solvent and cryoprotectant molecules; glycerol,
> SO4 etc should be 
> > different than waters and easy to model.
> > 
> > ... and I still cant help wondering how people do
> their phd's or 
> > post-docs in labs that no-one can explain
> > such trivialities. Or why people prefer not to ask
> their colleagues and 
> > supervisors, but to mail ccp4bb. 
> > Or why do I bother answering such emails on a
> Saturday morning, and then 
> > complaining, 
> > only to have the likes of Dr. Walsh commenting
> about my humor ;-)))
> > I find all these really scary.
> > 
> > Tassos
> > 
> > On 21 Jul 2007, at 0:29, JOE CRYSTAL wrote:
> > 
> >> Dear all,
> >>
> >>
> >> I am refining a structure at 2.0 A.  The water
> molecules have been 
> >> added using arp/warp resulting Rwork/Rfree=21/26%
> (about 370 HOH for 
> >> 360 residues).  After 10 cycles of refmac
> refinement (wt 0.3), 
> >> Rwork/Rfree went 

Re: [ccp4bb] FPLC vs Duo Flow

[Roger Rowlett]

Our AKTA FPLC has been trouble-free for 9 years, and we bought one of 
the early models. It has lived in a cold room, withstood periods of 
heavy use, periods of inactivity, and ham-handed undergraduates without 
incident. I think we have replaced only the injector channels and the 
solvent filters in those 9 years. These instruments are built like 
tanks, and at a small institution like ours, time is more important than 
money.


--

Roger S. Rowlett
Professor
Colgate University Presidential Scholar
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: [EMAIL PROTECTED]

Filip Van Petegem wrote:

Hi,
 
I believe this subject has been touched briefly before, but does 
anyone have any strong feelings before or against using an Akta 
FPLC/purifier versus a Biorad Duo Flow?  The Biorad Duo instruments 
are significantly cheaper; are they however also 'as good' as GE 
Healthcare? I'm especially interested in comments from people who have 
used both instruments before.
 
Cheers
 
Filip
 



--
Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3

phone: +1 604 827 4267
email: [EMAIL PROTECTED] 

Re: [ccp4bb] CCP4 Wiki

[artem]

Hey,

When was the announcement made? I can't claim to read all the CCP4 mail
very carefully, so I probably missed the it...

The wiki I've set up is essentially a sandlot - something to play with and
something to give people ideas (in other terms, not intended as a final
product), but I'll gladly remove it so as not to cause confusion.

Artem

> Yes, this is how a wiki page for CCP4 might look like. However, to make a
> wiki successful we have to make sure it is secure and that there is a
> dedicated core team to maintain it. Kevin was asked by the CCP4 to lead
> the wiki project and while he is setting things up, it would be
> counterproductive to start setting up independent "CCP4 wiki" sites.
>
> Tadeusz
>
>
>
>
>
>
>
> "Artem Evdokimov" <[EMAIL PROTECTED]>
> Sent by: "CCP4 bulletin board" 
> 23-Jul-2007 00:29
> Please respond to "Artem Evdokimov" <[EMAIL PROTECTED]>
>
>
> To
> CCP4BB@JISCMAIL.AC.UK
> cc
>
> Subject
> [ccp4bb] CCP4 Wiki
>
>
>
>
>
>
> Hi,
>
> Would something like this work?
>
> http://www.xtals.org/wiki/
>
> I've only set it up today, but it seems to work just fine. Feel free to
> play
> with it.
>
> Artem
>
> -Original Message-
> From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
> Lucas
> Bleicher
> Sent: Sunday, July 22, 2007 11:02 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Suggestion: Wiki -- was:Re: [ccp4bb] need
> help--Rfree
> is not decreasing
>
> That would be a great idea. In fact, I keep on my
> mailbox dozens of great postings (most of them
> summaries) in CCP4 which would be very useful to
> everybody if there's an online resource, with
> information organized in topics. I would gladly copy
> them to this wiki.
>
> Lucas
>
> --- Kay Diederichs <[EMAIL PROTECTED]>
> escreveu:
>
>> So - rather than repeat things that are obvious to
>> some people, would it
>> not be good to have a crystallography-FAQ that one
>> could point people
>> to? This should be part of a Wiki where "we
>> crystallographers" could
>> collect our wisdom. This would be much more
>> systematic, and less
>> volatile, than the postings of this mailing list
>> (which to me _is_ a
>> very valuable ressource).
>>
>> A Wiki is not difficult to set up. Maybe it could be
>> part of the CCP4
>> pages? We set up a Wiki for our lab at the beginning
>> of the year, and it
>> was a great success, in particular because it works
>> the same way as
>> Wikipedia - anybody can contribute. There should be
>> some means of
>> controlling "write access", but that could simply be
>> granted to people
>> who are subscribed to the CCP4 mailing list.
>>
>> I'd at least volunteer in helping to get a Wiki
>> started. And one way to
>> get it filled with articles would be that those
>> people who used to write
>> a "summary" of responses would simply compose a new
>> Wiki article, and
>> report to the mailing list that this article exists,
>> which could then be
>> expanded by others.
>>
>> best,
>>
>> Kay
>>
>>
>> Anastassis Perrakis schrieb:
>> > Sorry for the cliche, but *the goal of refinement
>> is not to reduce R
>> > factors, but to produce a good model.*
>> >
>> > ARP/wARP uses the 'WEIGHT AUTO' option of REFMAC5
>> to get a good geometry.
>> > You should set the weight to a value that produces
>> 1-2 and 1-3 distances
>> > rms deviations similar to the ARP/wARP job,
>> > to be able to compare. The fact that weight is 0.3
>> says nothing.
>> >
>> > The correct weight can vary wildly from 0.02 to
>> 0.5, in my experience.
>> > for 2.0 data 0.3 sound loose, 0.15-0.2 is what I
>> am used to,
>> > depending on dataset. But, The only way to tell
>> what is right is
>> > inspecting the geometry and aim for a 'reasonable'
>> rms 1-2 distances
>> > deviation.
>> >
>> > What is 'reasonable',  can cause yet another long
>> discussion, but my
>> > personal favorite for 1-2 distances rms deviation
>> is between 0.015-0.020.
>> > In Refmac these also give the lowest R factors, in
>> my hands.
>> >
>> > The invisible side chains is yet another long
>> discussion that you can
>> > retrieve from the ccp4bb archives.
>> > Again, my personal preference is to leave them in
>> and let them get very
>> > high B factors, as long as they do not
>> > get negative density in difference maps, that I
>> presume you are inspecting.
>> > I dont mind deleting them (but dont like it) and I
>> think mutating to ALA
>> > is worse since its misleading to users.
>> >
>> > Finally, given that you have 2.0 A data you should
>> try and model not
>> > only waters, but also:
>> > a. double conformations of side chains
>> > b. solvent and cryoprotectant molecules; glycerol,
>> SO4 etc should be
>> > different than waters and easy to model.
>> >
>> > ... and I still cant help wondering how people do
>> their phd's or
>> > post-docs in labs that no-one can explain
>> > such trivialities. Or why people prefer not to ask
>> their colleagues and
>> > supervisors, but to mail ccp4bb.
>> > Or why do I bother answering such emails on a
>> 

[ccp4bb] ARP/wARP 7.0 error

[Craig McElroy]

Hi all,
I am trying to use the new ARP/wARP to build a model starting from a
partially refined structure for the phases using the "Use pdb file as it is"
option. When I run the program it quits after/during the first REFMAC cycle
with the following message:


ERROR ('IndexError', ('list index out of range',), ['  File
"/Users/gerrit/CProg/ARP_svn/pyWARP/CArc.py", line 78, in
checkAndProcess\n', '  File
"/Users/gerrit/CProg/ARP_svn/pyWARP/CRefmacController.py", line 43, in
checkAndProcess\n', '  File
"/Users/gerrit/CProg/ARP_svn/pyWARP/CRefmacController.py", line 107, in
_parse\n']) 
--
ERROR => Ending JOB
--

I'm relatively sure I have everything installed properly, but it seems the
program is trying to reference scripts that do not exist (i.e. there is no
/Users/gerrit on my system and therefore no python scripts in
/Users/gerrit/CProg/ARP_svn/pyWARP/). Any help that you could provide would
be appreciated. Thanks in advance.
Craig

-- 
Craig McElroy, Ph.D.
Department of Molecular and Cellular Biochemistry
Ohio State University
483 Hamilton Hall
1645 Neil Ave.
Columbus, OH 43210
(614) 688-8630

Re: [ccp4bb] Stop the new PDB format!

[mesters]

I like to think structural biologists are more than just another user 
group, they FEED the PDB!

Their needs should first and foremost be taken care off, I would think.

Also, it would indeed be a great loss if legacy programs can not be used 
anymore.


- Jeroen -

Clemens Vonrhein wrote:

There is a collection of posts (unfortunately with a number of spam
messages) at

  http://wwpdb-remediation.rutgers.edu/mail-archive/

with various comments. Although I'm not familiar with the internal
workings of this remediation program, it seems indeed that the PDB
format is now largely auto-generated from the internally used
mmCIF. Unfortunately in my experience (having had a look at a few dozen
random entries of the new PDB files) this means that some of the new
PDB files of old entries will look very different from what you/we
deposited several years ago. The format seems better (internally
consistent) but the content has sometimes suffered.

But I guess there is always room for frictions when one side is mainly
interested in data format, storage and databases and the other mainly
interested in the crystallographic content. Finding a good compromise
between those two groups of experts is non-trivial.

At least the new databases will always have a link to the original
version of the PDB file - although it will still mean I can't now
search for an author name MUELLER (German U-umlaut transfered in the
proper ASCII format), since the PDB files now contain MULLER (because
PUBMED isn't able to properly translate non-ASCII names ...). Or an
analysis of programs used for structure solution will show a veri
different distribution - since the information has been significantly
changed.

Anyway, have a look at your favourite PDB file with the attached
script

  ./pdb23.sh 1abc

It is quite interesting sometimes. I haven't cehcked the mmCIF files -
maybe they are much better (as a 'hint' from the database people to
the crystallographers to stop using PDB format and switch to mmCIF,
maybe?).

Cheers

Clemens

On Sat, Jul 21, 2007 at 12:05:35PM -0700, Ethan A Merritt wrote:
  

On Saturday 21 July 2007 11:12, Joe Krahn wrote:


we all use in our daily research. They don't even want to keep the PDB
format at all. It's primary purpose now is for structural biologists.
  

That is inevitable.  The PDB format is simply not capable of representing
the complexities of current crystallographic models, and will only become
more obsolete as the state of the art progresses.  Because it is so wide-
spread, it will remain a legacy format for import/export into programs 
that are not up to the current crystallographic state of the art.  Yes,

that means it will largely be used by non-crystallographers to import
and view structures.

Thus I think the writing is on the wall that the PDB format as a primary
working medium in crystallography is on its deathbed.  Of course it may
linger there for a long while yet, and may be poked at from time to time
in order to stave off its final expiration.

Having said that, I don't understand the motivation for changing this
legacy format to something that the legacy programs will not recognize.
That indeed seems self-defeating.

Ethan Merritt





The new PDB format (version 3) has a lot of very useful improvements,
and an update is long overdue. However, I am irate that RCSB chose NOT
to use the ACA meeting to discuss the changes. Instead, the format is
being put into production at the same time as the ACA meeting. It is
essentially stating that opinions expressed at the ACA do not count.
Their was a lot of conflict at their last attempt at an update. Instead
of working to better involve the structural biologist community, I feel
that they are intentionally discounting our interests because working
with the user community is too much effort.

Unfortunately, structural biologists generally do not want to spend time
arguing about file formats, while computer scientists can carry on for
weeks over minor details. This change is going to affect all of us. If
you have concerns about the new format that have not been addressed, it
is important to take action now. The PDB format is not just their
personal database format (that's what mmCIF is for), but the format that
we all use in our daily research. They don't even want to keep the PDB
format at all. It's primary purpose now is for structural biologists. It
is essential that we be part of the decision making process.

I just sent the following letter to the wwPDB, which is where
comments about the new format are supposed to go. If you will be at the
ACA meeting, I encourage you to complain loudly.

Joe Krahn

---
To: [EMAIL PROTECTED]
Subject: The new PDB format is WRONG.

It seems obvious to me that the RCSB and wwPDB worked on the new format
to consider database users needs, but has intentionally ignored the rest
of the user community. RCSB manages mmCIF for databa

Re: [ccp4bb] CCP4 Wiki

[tadeusz . j . skarzynski]

Hi Artem,

There was a decision by the CCP4 Executive at the last CCP4 developers 
meeting in March to develop the wiki. Kevin was asked to coordinate this 
after his presentation of the wiki proposal and analysis of the issues 
involved, based on the experience gained from the York wiki. The 
developers meeting minutes are not available yet, but the ones for the 
Exec and STAB meetings have been posted at the CCP4 website. See 
http://www.ccp4.ac.uk/wg/datafiles/Exec_STAB_2007mar.doc.
Contributions from volunteers to establish and maintain the CCP4 wiki will 
be definitely appreciated.

Tadeusz


 




[EMAIL PROTECTED] 
Sent by: "CCP4 bulletin board" 
23-Jul-2007 15:24
Please respond to [EMAIL PROTECTED]

 
To
CCP4BB@JISCMAIL.AC.UK
cc

Subject
Re: [ccp4bb] CCP4 Wiki






Hey,

When was the announcement made? I can't claim to read all the CCP4 mail
very carefully, so I probably missed the it...

The wiki I've set up is essentially a sandlot - something to play with and
something to give people ideas (in other terms, not intended as a final
product), but I'll gladly remove it so as not to cause confusion.

Artem

> Yes, this is how a wiki page for CCP4 might look like. However, to make 
a
> wiki successful we have to make sure it is secure and that there is a
> dedicated core team to maintain it. Kevin was asked by the CCP4 to lead
> the wiki project and while he is setting things up, it would be
> counterproductive to start setting up independent "CCP4 wiki" sites.
>
> Tadeusz
>
>
>
>
>
>
>
> "Artem Evdokimov" <[EMAIL PROTECTED]>
> Sent by: "CCP4 bulletin board" 
> 23-Jul-2007 00:29
> Please respond to "Artem Evdokimov" <[EMAIL PROTECTED]>
>
>
> To
> CCP4BB@JISCMAIL.AC.UK
> cc
>
> Subject
> [ccp4bb] CCP4 Wiki
>
>
>
>
>
>
> Hi,
>
> Would something like this work?
>
> http://www.xtals.org/wiki/
>
> I've only set it up today, but it seems to work just fine. Feel free to
> play
> with it.
>
> Artem
>
> -Original Message-
> From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
> Lucas
> Bleicher
> Sent: Sunday, July 22, 2007 11:02 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Suggestion: Wiki -- was:Re: [ccp4bb] need
> help--Rfree
> is not decreasing
>
> That would be a great idea. In fact, I keep on my
> mailbox dozens of great postings (most of them
> summaries) in CCP4 which would be very useful to
> everybody if there's an online resource, with
> information organized in topics. I would gladly copy
> them to this wiki.
>
> Lucas
>
> --- Kay Diederichs <[EMAIL PROTECTED]>
> escreveu:
>
>> So - rather than repeat things that are obvious to
>> some people, would it
>> not be good to have a crystallography-FAQ that one
>> could point people
>> to? This should be part of a Wiki where "we
>> crystallographers" could
>> collect our wisdom. This would be much more
>> systematic, and less
>> volatile, than the postings of this mailing list
>> (which to me _is_ a
>> very valuable ressource).
>>
>> A Wiki is not difficult to set up. Maybe it could be
>> part of the CCP4
>> pages? We set up a Wiki for our lab at the beginning
>> of the year, and it
>> was a great success, in particular because it works
>> the same way as
>> Wikipedia - anybody can contribute. There should be
>> some means of
>> controlling "write access", but that could simply be
>> granted to people
>> who are subscribed to the CCP4 mailing list.
>>
>> I'd at least volunteer in helping to get a Wiki
>> started. And one way to
>> get it filled with articles would be that those
>> people who used to write
>> a "summary" of responses would simply compose a new
>> Wiki article, and
>> report to the mailing list that this article exists,
>> which could then be
>> expanded by others.
>>
>> best,
>>
>> Kay
>>
>>
>> Anastassis Perrakis schrieb:
>> > Sorry for the cliche, but *the goal of refinement
>> is not to reduce R
>> > factors, but to produce a good model.*
>> >
>> > ARP/wARP uses the 'WEIGHT AUTO' option of REFMAC5
>> to get a good geometry.
>> > You should set the weight to a value that produces
>> 1-2 and 1-3 distances
>> > rms deviations similar to the ARP/wARP job,
>> > to be able to compare. The fact that weight is 0.3
>> says nothing.
>> >
>> > The correct weight can vary wildly from 0.02 to
>> 0.5, in my experience.
>> > for 2.0 data 0.3 sound loose, 0.15-0.2 is what I
>> am used to,
>> > depending on dataset. But, The only way to tell
>> what is right is
>> > inspecting the geometry and aim for a 'reasonable'
>> rms 1-2 distances
>> > deviation.
>> >
>> > What is 'reasonable',  can cause yet another long
>> discussion, but my
>> > personal favorite for 1-2 distances rms deviation
>> is between 0.015-0.020.
>> > In Refmac these also give the lowest R factors, in
>> my hands.
>> >
>> > The invisible side chains is yet another long
>> discussion that you can
>> > retrieve from the ccp4bb archives.
>> > Again, my personal preference is to leave them in
>> and let them get very
>> > high B factors, as long as they do no

Re: [ccp4bb] CCP4 Wiki

[James Stroud]

Would a "CCP4 wiki" be different from a general crystallography wiki? Would it 
reflect, for instance, the breadth of topics on the CCP4BB?


On Monday 23 July 2007 09:57, [EMAIL PROTECTED] wrote:
> Contributions from volunteers to establish and maintain the CCP4 wiki will
> be definitely appreciated.


-- 
James Stroud
UCLA-DOE Institute for Genomics and Proteomics
Box 951570
Los Angeles, CA 90095

http://www.jamesstroud.com

[ccp4bb] native PAGE for duplex DNA purification

[bputcha]

Thanks to all those who replied to my query about purification of protein-DNA 
complex for 
crystallization. I figured out that extraction from native PAGE is the best 
way to purify duplex DNA. 
Does any one have a protocol that had worked for you.

I found the following protocol that I am planning to use. Any ideas and 
precautions? 
I am concerned about the chances of Thymine dimer formation upon UV exposure 
during UV 
backshadowing. Are there any safer ways of visualizing unstained DNA on gels? 
Are PD-10 columns 
(Pharmacia) good for my application?



NATIVE PAGE gel - 15% polyacrylamide, , 1x TBE

Bio-Rad Protean II gel system
15 x 20cm plates
15% Acrylamide/ /1x TBE
5 well Prep Comb
340 volts

1) Run 4-6 ODs of oligo per well
2) Run gel with Xylene cyanol/Bromophenol Blue marker 
3) Run gel until leading dye is about 1 inch above bottom of gel
4) Remove gel to TLC plate (available from VWR; Catalog EM 16484-1)
5) UV back-shadow the gel to visualize the DNA bands. 
Use transilluminator gel box
6) Cut the correct band out of the acrylamide gel
7) Place the gel piece containing the DNA into a 15ml conical tube
8) Crush gel with pipet (wrap pipet tip in parafilm to keep gel pieces from 
getting into the pipet.) or 
autoclaved glass rod
9) Add 3mls HPLC grade Water.
10) Heat at 94*C for 15 minutes
11) Freeze at -70*C for 15 minutes
12) Heat at 94*C for 2 minutes
13) Centrifuge gel pieces to bottom of tube
14) Remove supernatant to a separate 1.5 ml centrifuge tubes (approx 1ml per 
tube) and Speedvac 
(lyophilize) down until only about 100ul of liquid remains in the tubes
15) Combine the products into one tube and wash other two tubes with 500ul 
(total) of water
16) Bring volume total up to 1ml with water
17) Load onto Sephadex G-25 column) 
18) Allow liquid to drip through column (by gravity), discard liquid
19) Add 1.5ml water to column 
20) Allow liquid to drip through column (by gravity) COLLECT THIS…..it has 
your oligo in it!
21) Measure A260 and calculate amount of oligo in solution (1 OD = 33ugs)
22) Dry oligo in speedvac/lyophilizer
23) Re-suspend oligos to desired concentration.

Dept. of Biochemistry, Cellular and Molecular Biology,
Walters Life Science, # 406,
University of Tennessee, TN, Knoxville, USA

Re: [ccp4bb] native PAGE for duplex DNA purification

[Artem Evdokimov]

If you're worried about UV all you need to do is to run multiple lanes of
the same stuff, shield all but one with some sort of UV-opaque material
(many kinds of plastics work very well), and cut the shielded lanes based on
what you see on the illuminated lane. In a properly cast gel, all the lanes
will run the same (within reason). This has been a standard technique in
molecular biology for decades. If you're paranoid you can shield all but TWO
lanes - one on each end of the gel, or one on one end and one in the center
(to account for 'smiling'). After cutting you can confirm that all the DNA
is removed by UV-ing the entire leftover gel.

Be prepared to run a pretty thick/large gel if you want to purify milligram
quantities of DNA this way. 

You can use other intercalator dyes to visualize DNA with e.g. blue light,
but then you have to get rid of the dyes so in the end this does not save
you much time.

Artem

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
bputcha
Sent: Monday, July 23, 2007 6:00 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] native PAGE for duplex DNA purification

Thanks to all those who replied to my query about purification of
protein-DNA 
complex for 
crystallization. I figured out that extraction from native PAGE is the best 
way to purify duplex DNA. 
Does any one have a protocol that had worked for you.

I found the following protocol that I am planning to use. Any ideas and 
precautions? 
I am concerned about the chances of Thymine dimer formation upon UV exposure

during UV 
backshadowing. Are there any safer ways of visualizing unstained DNA on
gels? 
Are PD-10 columns 
(Pharmacia) good for my application?



NATIVE PAGE gel - 15% polyacrylamide, , 1x TBE

Bio-Rad Protean II gel system
15 x 20cm plates
15% Acrylamide/ /1x TBE
5 well Prep Comb
340 volts

1) Run 4-6 ODs of oligo per well
2) Run gel with Xylene cyanol/Bromophenol Blue marker 
3) Run gel until leading dye is about 1 inch above bottom of gel
4) Remove gel to TLC plate (available from VWR; Catalog EM 16484-1)
5) UV back-shadow the gel to visualize the DNA bands. 
Use transilluminator gel box
6) Cut the correct band out of the acrylamide gel
7) Place the gel piece containing the DNA into a 15ml conical tube
8) Crush gel with pipet (wrap pipet tip in parafilm to keep gel pieces from 
getting into the pipet.) or 
autoclaved glass rod
9) Add 3mls HPLC grade Water.
10) Heat at 94*C for 15 minutes
11) Freeze at -70*C for 15 minutes
12) Heat at 94*C for 2 minutes
13) Centrifuge gel pieces to bottom of tube
14) Remove supernatant to a separate 1.5 ml centrifuge tubes (approx 1ml per

tube) and Speedvac 
(lyophilize) down until only about 100ul of liquid remains in the tubes
15) Combine the products into one tube and wash other two tubes with 500ul 
(total) of water
16) Bring volume total up to 1ml with water
17) Load onto Sephadex G-25 column) 
18) Allow liquid to drip through column (by gravity), discard liquid
19) Add 1.5ml water to column 
20) Allow liquid to drip through column (by gravity) COLLECT THIS...it has 
your oligo in it!
21) Measure A260 and calculate amount of oligo in solution (1 OD = 33ugs)
22) Dry oligo in speedvac/lyophilizer
23) Re-suspend oligos to desired concentration.

Dept. of Biochemistry, Cellular and Molecular Biology,
Walters Life Science, # 406,
University of Tennessee, TN, Knoxville, USA

[ccp4bb] resolution and solvent accessible surface area

[Hyunchul Kim]

Hi all, 

Resolution doesn't matter when solvent accessible surface area(SASA) is
calculated?

If resolution is poor, I think that the SASA is not likely to be
reliable. Then, is there any resolution cut-off for SASA calculation?

Also, when you calculate SASA, do you include NMR data, too?

Any comments are welcome :)

Thank you in advance.

Best,
Hyunchul Kim

Re: [ccp4bb] resolution and solvent accessible surface area

[Hyunchul Kim]

I am interested in the SASA per residue.

On Tue, 2007-07-24 at 14:43 +0900, Hyunchul Kim wrote:
> Hi all, 
> 
> Resolution doesn't matter when solvent accessible surface area(SASA) is
> calculated?
> 
> If resolution is poor, I think that the SASA is not likely to be
> reliable. Then, is there any resolution cut-off for SASA calculation?
> 
> Also, when you calculate SASA, do you include NMR data, too?
> 
> Any comments are welcome :)
> 
> Thank you in advance.
> 
> Best,
> Hyunchul Kim
> 

Re: [ccp4bb] resolution and solvent accessible surface area

[Miguel Ortiz-Lombardía]

Hello Hyunchul Kim,

I found this paper very interesting:

Novotny M, Seibert M, Kleywegt GJ.
On the precision of calculated solvent-accessible surface areas.
Acta Crystallogr D Biol Crystallogr. 2007 Feb;63(Pt 2):270-4. Epub 2007 Jan
16.
PMID: 17242521 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=ShowDetailView&TermToSearch=17242521&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

It discusses most of the topics you mentioned.
There are many programs around to do that type of calculations. I tend to
use naccess.

Best,



Miguel


2007/7/24, Hyunchul Kim <[EMAIL PROTECTED]>:


I am interested in the SASA per residue.

On Tue, 2007-07-24 at 14:43 +0900, Hyunchul Kim wrote:
> Hi all,
>
> Resolution doesn't matter when solvent accessible surface area(SASA) is
> calculated?
>
> If resolution is poor, I think that the SASA is not likely to be
> reliable. Then, is there any resolution cut-off for SASA calculation?
>
> Also, when you calculate SASA, do you include NMR data, too?
>
> Any comments are welcome :)
>
> Thank you in advance.
>
> Best,
> Hyunchul Kim
>





--
correo-e: [EMAIL PROTECTED]
~~~
Je suis de la mauvaise herbe,
Braves gens, braves gens,
Je pousse en liberté
Dans les jardins mal fréquentés!

Georges Brassens

Re: [ccp4bb] resolution and solvent accessible surface area

[Hyunchul Kim]

Hello Miguel,

Thank you for your reply.
It's related to the total SASA of a  protein structure but not that of
per residue. However, it still give me a hint. :)

Best,

Hyunchul Kim

On Tue, 2007-07-24 at 08:12 +0200, Miguel Ortiz-Lombardía wrote:
> Hello Hyunchul Kim,
> 
> I found this paper very interesting:
> 
> Novotny M, Seibert M, Kleywegt GJ.
> On the precision of calculated solvent-accessible surface areas.
> Acta Crystallogr D Biol Crystallogr. 2007 Feb;63(Pt 2):270-4. Epub
> 2007 Jan 16. 
> PMID: 17242521 [PubMed - indexed for MEDLINE]
> 
> http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=ShowDetailView&TermToSearch=17242521&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum
> 
> It discusses most of the topics you mentioned. 
> There are many programs around to do that type of calculations. I tend
> to use naccess.
> 
> Best,
> 
> 
> 
> Miguel
> 
> 
> 2007/7/24, Hyunchul Kim <[EMAIL PROTECTED]>:
> I am interested in the SASA per residue.
> 
> On Tue, 2007-07-24 at 14:43 +0900, Hyunchul Kim wrote: 
> > Hi all,
> >
> > Resolution doesn't matter when solvent accessible surface
> area(SASA) is
> > calculated?
> >
> > If resolution is poor, I think that the SASA is not likely
> to be
> > reliable. Then, is there any resolution cut-off for SASA
> calculation? 
> >
> > Also, when you calculate SASA, do you include NMR data, too?
> >
> > Any comments are welcome :)
> >
> > Thank you in advance.
> >
> > Best,
> > Hyunchul Kim
> >
> 
> 
> 
> -- 
> correo-e: [EMAIL PROTECTED]
> ~~~
> Je suis de la mauvaise herbe,
> Braves gens, braves gens, 
> Je pousse en liberté
> Dans les jardins mal fréquentés!
> 
> Georges Brassens