Re: [ccp4bb] help with wwPDB validation warning

[Ezra Peisach]

Ok - I have tracked down where it is coming from.

The value being reported is sum(I)/sum(sigma_I).   This is not the same 
as Mean(I/sigmaI) - as I interpret the later as (Sum(I/sigma))/n.


Where I comes from is the intensity_meas, intensity_meas_au, or 
intensity column - whichever is present (priority left to right).


No averages here.

It is a simple diagnostic >80 or < 2 - report a warning.

This is from the old sf_convert program (written > 15 years ago) and the 
various warnings were carried over to a re-implementation in python.


At the time the program was implemented, the PDB had only started trying 
to make sense of the experimental data provided by the authors.  
Remember, experimental X-ray data were not required by the PDB until 
2008. Prior to that the data were optional, and some are a mess.  Use of 
experimental data in validation came afterwards.  MTZ files might have 
been accepted back then (I cannot remember) - so ensuring that the 
conversion did not result in incorrect translation was important.


The person doing the work at the time was a structural biologist, but 
may have come up with his own analyses to find conversion issues.  There 
will likely not be a reference.  Certainly the sources do not reference 
a methodology here.


So - what can we do moving forward?  Using community standards for 
identification of such errors should be incorporated. Changing the code 
is relatively easy.  Choosing the correct formulas would be the most 
meaningful.  And if sf_convert reports different data from AIMLESS - we 
should strive to understand why.



Ezra




On 6/10/24 2:15 PM, Gerard Bricogne wrote:

Dear Aline,

  This is an intriguing message: by what exact piece of software was it
produced?

  The notation I_avg/sigI_avg does not appear in the definition of the
closest item in the mmCIF dictionary, which would be

_reflns_shell.meanI_over_sigI_obs

that can be found at

https://mmcif.wwpdb.org/dictionaries/mmcif_pdbx_v50.dic/Items/_reflns_shell.meanI_over_sigI_obs.html

  As Kay explained, the quantity for which you are getting a warning is a
ratio of averages, which is not at all the same as the usual average of
(signal-to-noise) ratios, denoted Mean((I)/sd(I)) in AIMLESS.

  Much worse, in fact: that quantity (I_avg/sigI_avg) makes no sense
whatsoever in statistical terms. It must be a relic of a quantity that may
have seemed like a good idea to someone at some stage, and has since been
dutifully carried along forever after, and "gold-plated" so as to still be
present in the latest revision of the mmCIF dictionary.

  Perhaps you could request a reference to the publication in which this
quantity was proposed as a validation criterion and its acceptable limits
were derived :-) .

  This being said, if it is indeed the case that the average value of
your intensities is smaller than the average of their standard deviations,
there is definitely something wrong somewhere. Perhaps a confusion between
columns containing values pertaining to intensities vs. amplitudes?

  To sober me up from all this speculation, Clemens Vonrhein tells me
that it is very likely that it is not the I_avg/sigI_avg quantity that is
actually being calculated, and that it is simply a "normal" quantity (e.g.
Mean((I)/sd(I)) that is being mis-described in the warning message.


  With best wishes,

   Gerard.

--
On Fri, Jun 07, 2024 at 03:03:30PM +0100, Aline Dias da Purificação wrote:

Dear all,

I am currently validating a structure for deposition in the wwPDB and 
encountered the following warning in the validation system:

Warning: Value of (I_avg/sigI_avg = 0.83) is out of range (check Io or SigIo in 
SF file).

The Mean((I)/sd(I)) in the aimless log is 1.7 in the OuterShell, so I didn't 
understand the warning.

Has anyone experienced this before and could assist me?

Thank you.



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Re: [ccp4bb] Announcements: Impact of EDMAPS.rcsb.org shutdown and related June 13 Virtual Office Hour on Generating DSN6 and MTZ Files

[Ezra Peisach]

RCSB PDB is independent of PDBe with regards to this.  The  PDB archive 
does not distribute electron density maps.  Each site can provide 
"feature added" data content from their websites.


As you mentioned, PDBe runs a service that uses urls like 
https://www.ebi.ac.uk/pdbe/entry-files/1o08.ccp4 which is based on a 
re-implementation of the legacy EDS service at Uppsala. While I cannot 
speak for PDBe, I have no reason to believe that this service will be 
discontinued in the near future.


Both RCSB PDB and PDBe provide electron density volumes in BinaryCif 
format using a volume server.  These are used by Mol* on each of their 
sites. The goal for RCSB PDB is to serve up the electron density map 
contents based on the maps calculated by the validation process and not 
a separate process using the program DCC.  Validation report map 
coefficients are available for almost all X-ray structures in the PDB 
archive.  As software, such as Gemmi, makes it relatively 
straightforward for a user o convert the PDBx/mmCIF map coefficients to 
MTZ files or directly to a map, it no longer requires a large software 
installation.


I believe CCP4 maps are used for much of the graphical software. 
However, if you need to convert to a DSN6 file, you could use old MAPMAN 
binaries, use xdlmapman (part of CCP4),  sftools (part of CCP4) or 
compile mapman yourself using sources at 
https://github.com/martynwinn/Uppsala-Software-Factory. Up until know 
RCSB PDB has produced DSN6 formatted maps using an old MAPMAN binary.


I hope this clarifies the direction we are going in.





On 6/1/24 9:28 AM, Jon Cooper wrote:

It would be interesting to know if this will affect the the electron density 
maps which are downloadable from the EBI:

www.ebi.ac.uk/pdbe

They don't currently serve the older dsn6 format, only ccp4, I think.

Best wishes, Jon Cooper.
jon.b.coo...@protonmail.com

Sent from Proton Mail Android


 Original Message 
On 31/05/2024 16:36, Ezra Peisach 
<d150cd71a251-dmarc-requ...@jiscmail.ac.uk> wrote:


  In fall of 2024, electron density map coefficients will be available in
  the public PDB archive for all X-ray structures. These map coefficients
  will be the same as used in wwPDB Validation Reports.
  
  The new map coefficients files will replace the electron density maps

  and combined map coefficient files calculated and distributed by RCSB
  PDB and used by the NGLviewer at RCSB.org. These data (served by
  EDMAPS.rcsb.org) are calculated using publicly-available coordinate
  files and structure factor files and offered in DSN6 formatted map files
  and MTZ formatted map coefficient files. RCSB PDB plans to shutdown the
  NGL viewer by July 2024
  (https://www.rcsb.org/news/feature/65b42d3fc76ca3abcc925d15) and will no
  longer need the data served by EDMAPS.rcsb.org.
  
  RCSB PDB will be phasing out EDMAPS.rcsb.org:
  
    *  June 28, 2024: DSN6-formatted map files will no longer be

  provided. EDMAPS.rcsb.org will only serve MTZ files with map coefficients.
    *  Fall 2024: Electron density map coefficients will be available
  in the public PDB archive for all X-ray structures. At this point,
  EDMAPS.rcsb.org will be shut down, including access to MTZ files with
  map coefficients from this service.
  
  To help users with this transition, RCSB PDB will be holding a Virtual

  Office Hour on Thursday June 13, 2024 from 1:00pm-2:00pm Eastern,
  10:00am-11am Pacific. Please register online for this event at
  https://go.rutgers.edu/psx4pr63.
  
  Questions about this transition or the Virtual Office Hour can be sent

  to i...@rcsb.org.
  
  
  
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[ccp4bb] Announcements: Impact of EDMAPS.rcsb.org shutdown and related June 13 Virtual Office Hour on Generating DSN6 and MTZ Files

[Ezra Peisach]

In fall of 2024, electron density map coefficients will be available in 
the public PDB archive for all X-ray structures. These map coefficients 
will be the same as used in wwPDB Validation Reports.


The new map coefficients files will replace the electron density maps 
and combined map coefficient files calculated and distributed by RCSB 
PDB and used by the NGLviewer at RCSB.org. These data (served by 
EDMAPS.rcsb.org) are calculated using publicly-available coordinate 
files and structure factor files and offered in DSN6 formatted map files 
and MTZ formatted map coefficient files. RCSB PDB plans to shutdown the 
NGL viewer by July 2024 
(https://www.rcsb.org/news/feature/65b42d3fc76ca3abcc925d15) and will no 
longer need the data served by EDMAPS.rcsb.org.


RCSB PDB will be phasing out EDMAPS.rcsb.org:

 *  June 28, 2024: DSN6-formatted map files will no longer be 
provided. EDMAPS.rcsb.org will only serve MTZ files with map coefficients.
 *  Fall 2024: Electron density map coefficients will be available 
in the public PDB archive for all X-ray structures. At this point, 
EDMAPS.rcsb.org will be shut down, including access to MTZ files with 
map coefficients from this service.


To help users with this transition, RCSB PDB will be holding a Virtual 
Office Hour on Thursday June 13, 2024 from 1:00pm-2:00pm Eastern, 
10:00am-11am Pacific. Please register online for this event at 
https://go.rutgers.edu/psx4pr63.


Questions about this transition or the Virtual Office Hour can be sent 
to i...@rcsb.org.




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Re: [ccp4bb] Search for similar geometric arrangement in PDB database

[Ezra Peisach]

May I point you towards the shape similarity search available at the 
rcsb.org site?


https://www.rcsb.org/news/feature/63933da0e543b6038c4fc5dd

The news suggests that you need to upload via URL - but there is a file 
upload option in the source pull down.



On 5/22/24 5:03 PM, Das, Abhinaba wrote:


Dear community,

Is it possible to conveniently search the PDB for specific geometrical 
shapes, disregarding the input residues and specifically focusing on 
finding similar topologies?


Thanks in advance




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Re: [ccp4bb] a dinosaur asks ... PDB format query

[Ezra Peisach]

If you take a look at 
https://www.wwpdb.org/documentation/file-format-content/format33/sect9.html#ATOM


you will see the following:

77 - 78    LString(2)    element  Element symbol, right-justified.

Going by atom name will get you in trouble.  As you stated calcium vs 
Calpha.  The element symbol comes from the chemical component dictionary.



Ezra



On 5/15/24 6:28 AM, Harry Powell wrote:

Hi folks

I’m sure that this has been answered many times before (I’m sure that when I 
was young I even read it here…), and I *know* that we should all be using 
mmCIF, but I’m using PDB format files generated by a popular Python module and 
I wanted to check the output against a definitive format definition (if that’s 
not tautology).

I noticed this because I was encouraged to try Moorhen and found that a HEM 
(apparently written by this module) did not have the atoms connected with bonds 
in the display.

I’m particularly interested in metal atoms here, and want to be 100% sure that 
I’ve found a calcium, say, and not a C-alpha.

Q: Is it necessary to check columns 77-78 if I really want to be sure?

I’ve read the following, but can’t see anything obvious in “official” PDB 
documentation that what it says here is actually defined anywhere:


Atom names are composed of an atomic (element) symbol right-justified in 
columns 13-14, and trailing identifying characters left-justified in columns 
15-16. A single-character element symbol should not appear in column 13 unless 
the atom name has four characters (for example, see Hydrogen Atoms). Many 
programs simply left-justify all atom names starting in column 13. The 
difference can be seen clearly in a short segment of hemoglobin (entry 3hhb):

Correct:
HETATM 1071 FE   HEM A   1   8.128   7.371 -15.022 24.00 16.74  FE
HETATM 1072  CHA HEM A   1   8.617   7.879 -18.361  6.00 17.74   C
HETATM 1073  CHB HEM A   1  10.356  10.005 -14.319  6.00 18.92   C
HETATM 1074  CHC HEM A   1   8.307   6.456 -11.669  6.00 11.00   C
HETATM 1075  CHD HEM A   1   6.928   4.145 -15.725  6.00 13.25   C

Incorrect:
HETATM 1071 FE   HEM A   1   8.128   7.371 -15.022 24.00 16.74  FE
HETATM 1072 CHA  HEM A   1   8.617   7.879 -18.361  6.00 17.74   C
HETATM 1073 CHB  HEM A   1  10.356  10.005 -14.319  6.00 18.92   C
HETATM 1074 CHC  HEM A   1   8.307   6.456 -11.669  6.00 11.00   C
HETATM 1075 CHD  HEM A   1   6.928   4.145 -15.725  6.00 13.25   C

I’m sure that someone here will say “why don’t you look at *, it’s 
obvious”, in which case - many thanks!

help

Harry



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Re: [ccp4bb] Crystal rescue

[Ezra Peisach]

MiTeGen (www.mitegen.com) sells a RT device  - plastic capillary to put 
over loop... I do not know how well it would work for a long data 
collection - but people here have used it to evaluate their crystals


Ezra


On 1/27/2010 3:58 AM, Flip Hoedemaeker wrote:

Zhiyi,

You can use a thin cap over your cryo loop, just put a drop of mother 
liquor in the top, place over the loop and make it airtight at the 
base.  Not sure who sells these things though, I guess you can make it 
from a capillary too. Then remove the cryo stream or put it at a temp 
above freezing, say 253K.


Flip

Zhiyi Wei wrote:

Thanks for so many quick responses!

Actually, I have test several different cryo-protectants, including
glycerol, EG, and PEG400. I did not see much differences between these
cryo conditions. So, I choose glycerol.

I would like to test my crystals in RT. But I don't know how to do
this. Just mount crystal to the X-ray machine without cryo stream? Or
I should use capillaries?

Zhiyi

On Wed, Jan 27, 2010 at 5:45 AM, Kelly Daughtry  wrote:

Tascimate can be used as the cryo as well. I have had experience with
crystals in similar condition and moved the crystals to a 20%
increased Tascimate solution and they froze well.

I agree with Ezra, room temperature mount your crystal before
freezing. It is the only way to know the true problem.


Kelly
***
Kelly Daughtry
PhD Candidate
Department of Physiology and Biophysics
Boston University School of Medicine
590 Commonwealth Ave
R 390
Boston MA, 02215
(P) 617-358-5548
***



On Tue, Jan 26, 2010 at 1:25 PM, Marcus Winter
 wrote:

Dear Zhiyi,


Ezra is exactly right, of course.  The Oxford Diffraction PX Scanner
system can assess the diffraction qualities of (putative) protein
crystals in situ - in the crystallisation plate.  So, directly, you
would discover if your 'big and beautiful' crystals actually diffract
well... in their mother liquor under ambient conditions and before the
addition of any cryo-protect.  Do you have a friend or neighbour with
a PX Scanner ?  If not, please feel most welcome to contact
Oxford Diffraction: we would be pleased to assist if at all possible.


Good Luck and Best Wishes,

Marcus Winter.

www.oxford-diffraction.com




-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Ezra Peisach
Sent: 26 January 2010 16:01
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Crystal rescue

First you need to establish if it is your cryo conditions or the
crystals.  Depending where you are - they might have the equipment 
to do


a wet mount - without freezing.  Yes the crystal will not last - but
then you know if the problem is in the
crystal.  If it is - you need better crystals.  If it is the cryo - 
you

need to work on that.  Tacsimate is mixture of alot of different
compounds - but the smears are too close together to be a small salt
crystal on top...

Good luck,

Ezra

On 1/26/2010 10:42 AM, Zhiyi Wei wrote:

Dear all,

I got a problem with my crystals. I have two total different proteins
that both can be crystallized in the condition with PEG3350 and

Tacsimate
(although the concentrations are different) with different shapes. 
The

crystals look big and beautiful. However, when I test them in

synchrotron,

both of these two types of crystals showed poor diffractions. As

showed in

the attached diffraction image, the diffraction is up to ~4 A but

smear in

one direction while<8 A in the other direction. The interesting thing

is

that the diffraction pattern is similar for all crystals (from two

different

proteins) that I tested without exception although they belong to

different

space groups. So, I wonder whether these kind of pattern is caused by
Tacsimate (I don't know what it is) and how to rescue these crystals.

Any

suggestions or comments?

Thanks a lot!

Best,
Zhiyi

[ccp4bb] On N/C terminus disorder...

[Ezra Peisach]

Howdy,


Within the last few years - I remember a paper discussing an examination 
of disorder in the termini of structures found in the PDB.  Does anyone 
know the citation?


Thanks in advance,

Ezra

Re: [ccp4bb] Crystal rescue

[Ezra Peisach]

First you need to establish if it is your cryo conditions or the 
crystals.  Depending where you are - they might have the equipment to do 
a wet mount - without freezing.  Yes the crystal will not last - but 
then you know if the problem is in the
crystal.  If it is - you need better crystals.  If it is the cryo - you 
need to work on that.  Tacsimate is mixture of alot of different 
compounds - but the smears are too close together to be a small salt 
crystal on top...


Good luck,

Ezra

On 1/26/2010 10:42 AM, Zhiyi Wei wrote:

Dear all,

I got a problem with my crystals. I have two total different proteins
that both can be crystallized in the condition with PEG3350 and Tacsimate
(although the concentrations are different) with different shapes. The
crystals look big and beautiful. However, when I test them in synchrotron,
both of these two types of crystals showed poor diffractions. As showed in
the attached diffraction image, the diffraction is up to ~4 A but smear in
one direction while<8 A in the other direction. The interesting thing is
that the diffraction pattern is similar for all crystals (from two different
proteins) that I tested without exception although they belong to different
space groups. So, I wonder whether these kind of pattern is caused by
Tacsimate (I don't know what it is) and how to rescue these crystals. Any
suggestions or comments?

Thanks a lot!

Best,
Zhiyi
   

Re: [ccp4bb] pdb-l: Retraction of 12 Structures....

[Ezra Peisach]

A number of years ago - we were asked to setup an ftp site a particular 
reviewer could see the coordinates... I could have looked at the logs to 
figure out where they were coming in from but chose not to.  Some 
journals also allow the author to upload additional info - that would be 
available to the reviewer - but not included in the publication (I  
think Biochemistry is one of them)...  Authors could then upload SF's 
and coordinates... If the reviewer wants to look - he/she then has the 
option - while still maintaining the anonymity of the review process...


Ezra


Dyda wrote:

On Sat, 12 Dec 2009 11:58:27 +0530 Dr. Anthony Addlagatta wrote:

  

Bernhard,

I would be worried about sending the structure factors and the coordinates 
along with
the manuscript.




I wonder why?

Cheers

   Fred
***
Fred Dyda, Ph.D.   Phone:301-402-4496
Laboratory of Molecular BiologyFax: 301-496-0201
DHHS/NIH/NIDDK e-mail:fred.d...@nih.gov  
Bldg. 5. Room 303 
Bethesda, MD 20892-0560  URGENT message e-mail: 2022476...@mms.att.net

Google maps coords: 39.000597, -77.102102
http://www2.niddk.nih.gov/NIDDKLabs/IntramuralFaculty/DydaFred
***
  

Re: [ccp4bb] pdb-l: Retraction of 12 Structures

[Ezra Peisach]

Matthew Franklin wrote:



Once again, I'd like to get the community's thoughts: should we ask the PDB to 
stop using 0 and 1 in its IDs?

I'll get off the soapbox now.

  
I would be all for it... Having tried to downloade 1o08 and gotten it 
screwed up Especially when journals use a sans-serif font - things 
get interesting


Ezra

Re: [ccp4bb] Enzyme inhibitor

[Ezra Peisach]

Sivaraman Padavattan wrote:

Dear all,
I am performing an assay to screen for the inhibitor molecules. One of 
our compound shows inhibition at 100 μM and activation at 500μM conc. 
What could be the possible reason for the compound work differently in 
two different conc.


Thanks
Sivaraman


 



Not saying this is right - but how about product inhibition 
(competition) and allosteric activation?


Ezra

Re: [ccp4bb] hairpin formation in gene

[Ezra Peisach]

Maybe... Depending on the energetics - it could stall the translation - 
or prevent initiation if it is at the start of the ORF. If it is in the 
middle - you might be ok... Usually the gene synthesis company can 
recommend alternate sequences that do not run into this problem or rare 
codon issues...


You may wish to see:


Protein Expr Purif. Purif.');> 2006 Feb;45(2):374-80. Epub 2005 Aug 8.
Rapid and easy thermodynamic optimization of the 5'-end of mRNA 
dramatically increases the level of wild type protein expression in 
Escherichia coli.
Cèbe R 
, 
Geiser M 
.



You mileage may vary

Ezra


Sangeetha Vedula wrote:

Dear bb-ers,

I am trying to have a gene synthesized and found out that it forms an 
11-bp hairpin. Does that complicate expression? Would it be better to 
try and disrupt it by altering codon usage to improve expression?


Thank you in advance,

Sangeetha.

Re: [ccp4bb] Inclusion bodies centrifugation

[Ezra Peisach]

Artem has already responded - but I believe you will pull down the 
cellular debris with the IB. 

In my prior experience, post centrifugation, you can wash the IB/debris 
with increasing amounts of urea in you solubilization - and you may find 
that the other cellular debris stays in solution before the IB goes in. 
(1M, 2M, 4M urea) - and after recentrifugation, I found that does a 
pretty good job of cleaning up the media.


Ezra


megha goyal wrote:



Dear All,
 
Our protein is expressed as inclusion bodies and I want to separate 
inclusion bodies from E.coli from the cellular debris after* *lysis of 
the cells by sonication.


Can I do this by normal centrifugation? and if yes, at what speed?

Our centrifuge has maximum speed of 14000 rpm. Can we do the 
separation using this centrifuge and if so how.


 


Thanking in anticipation.

Re: [ccp4bb] measure detergent concentration

[Ezra Peisach]

Regarding concentrating detergents w/ MWCO concentrators - may I suggest 
the following reference:


Refractive index-based determination of detergent concentration and its 
application to the study of membrane proteins

Pavel Strop and Axel T. Brunger
Protein Sci. 2005 August; 14(8): 2207–2211.


Michael Matho wrote:

Weikai,
 
We did it using NMR but you asked for a simple way so I guess I'm out 
of topic.
 
Anyway, since I believe it is the most accurate method, here it is: 
using a high detergent concentration stock solution you can assign 
resonance peaks to your detergent molecule bonds.
 
Then you can set up a standard curve using different known detergent 
concentrations (for example from 10% down to 0.1%) by calculating the 
surface of your peak(s) which is directly related to your detergent 
concentration.
 
Each time you need to know the concentration of a new sample, you just 
need to record the peaks, and use the three-click rule to deduct the 
unknown value.
 
As a colleague answered you earlier, we noticed that a 50kDa cutoff 
withheld a lot of detergent during concentration process and 
consequently your final concentration might increase significantly. 
For example we started with 0.25% DES and noticed increases of above 
1%. Of course this will depend on the concentration factor.
 
This did not happen when using a 100kDa cutoff, and DES concentration 
remain pretty much constant.
 
Now, it will depend on your system: what detergent you are 
using, since micel size and CMC are obviously the critical parameters 
here -- but also what maximal cutoff you can use w/o loosing your 
membrane protein in the flow through...
 
Good luck,

Michael

- Original Message -
*From:* Patrick Loll 
*To:* CCP4BB@jiscmail.ac.uk 
*Sent:* Friday, October 23, 2009 1:12 PM
*Subject:* Re: [ccp4bb] measure detergent concentration

I'll second this.  We've done this as an exercise in NSLS Membrane
Protein Crystallization workshop for a few years, and it works
like a charm. You can stain in a warm iodine chamber and visualize
by scanning the TLC plate on a garden variety scanner (we use an
inexpensive Canon LIDE that probably cost less than USD 60 five
years ago). We quantify the spot intensity with NIH Image or
equivalent, and get lovely linearity down to the CMC, spotting
only 1 uL of sample--so we haven't seen any need to concentrate.

On 23 Oct 2009, at 3:41 PM, Edward A. Berry wrote:


Only easy if you happen to have silica gel TLC plates and
a chromatography jar lying around, perhaps from some
phospholipid analysis:

A strategy for identification and quantification of
detergents frequently used in the purification of membrane proteins
Laura R. Eriks, June A. Mayor, and Ronald S. Kaplan
Analytical Biochemistry 323 (2003) 234–241

This paper recommends spotting on a TLC plate and running
beside standard amounts of the same detergent. From intensity/size
of the detergent spot after developing you can bracket the detergent
concentration. (And by the way they found that detergents are
concentrated by ultrafiltration). To increase sensitivity,
speedvac a volume too large to
spot on the plate, dissolve the residue in Me0H.

Ed
wei...@crystal.harvard.edu  wrote:

Hi Folks:
After concentrating a membrane protein, is there a (easy) way of
measuring
the detergent concentration in the sample?
Regards,
Weikai



---

Patrick J. Loll, Ph. D.  


Professor of Biochemistry & Molecular Biology

Director, Biochemistry Graduate Program

Drexel University College of Medicine

Room 10-102 New College Building

245 N. 15th St., Mailstop 497

Philadelphia, PA  19102-1192  USA


(215) 762-7706

pat.l...@drexelmed.edu 

Re: [ccp4bb] Reducing Agent Tips?

[Ezra Peisach]

Have you considered seeing if anyone has an anaerobic chamber you can 
work in?


Ezra


On 10/05/2009 11:39 AM, Roger Rowlett wrote:
Of these, typically TCEP>DTT>BME in terms of effectiveness in 
maintaining reduced Cys groups. Some proteins, when purified, require 
obscenely large reducing agent concentrations to keep them stable. One 
of our "horror show" proteins required 100 mM DTT + 10 uM EDTA to 
remain stable. You should consider including EDTA in the solution to 
sequester metal ions (if this is compatible with your protein). Metal 
ions, including the ubiquitious trace Zn2+, efficiently catalyze the 
oxidation of sulfhydryls. Include 1 uM or more if your protein will 
tolerate it.


Cheers.

Jeremiah Farelli wrote:

Hello all,

Anyone have any tips for reducing agents for use with the following
crystallization conditions:

100 mM hepes, pH 7.5, 24% PEG 1500

or

100 mM Tris, pH 8.0, 24% PEG 1500

I've tried BME and DTT (1 mM).  Currently trying out TCEP.  The protein
seems to be sensitive to oxidation, with a selenomet-derivative even more so.

Thanks!
   

--

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

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu

Re: [ccp4bb] Preparation of seed-stocks without seed-beads

[Ezra Peisach]

We used to use a needle to crush the crystals - and then make serial 
dilutions


ucbccka wrote:

sonication


  

Dear CCP4bbers,

Can anyone suggests how to make seed-stocks if one is not having
seed-beads... Is there any other methods to crush the crystals for the
same
purpose. What if it is simple vortexed. Off-course there wold be all sorts
of sizes, the intact crystals as well.
Please suggest.

Thanks a lot more for previous help.
James.





  

Re: [ccp4bb] r-factor does not reduce

[Ezra Peisach]

On 09/10/2009 12:17 PM, Sean Seaver wrote:

Hello Sylvia,

Two issues I would consider:
1)  recheck the space group
2)  make sure that I had selected a good model for my MR search

All the Best,

Sean
   

Hi Sylvia,

If you have just used rigid body refinement - the Rfactor might be high 
until you have allowed the individual atoms to move.


You did not indicate the resolution you are using - but every crystal is 
slightly different and parts of your model will likely need to move 
some.  The higher the resolution - the larger the effects of slightly 
incorrect positions will be.


The best criteria would be to look at the electron density maps and 
evaluate what you see.  Is there good backbone connectivity?  Do you see 
improvements to make in the model? Is there difference in sequence that 
is clearly shown - say a small sidechain changed to something large and 
bulky?  If so - you probably have the correct MR solution...


Ezra

Re: [ccp4bb] Distinguishing between P6(5)22 and P6(1)22

[Ezra Peisach]

William G. Scott wrote:


On Sep 8, 2009, at 4:29 PM, Ezra Peisach wrote:

If you want to gamble - go w/ P6122... Data from the PDB  indicates 
that there are 1.5x as many proteins w/ P6122 vs P6522...


So if you believe in the overall anisotropy of the universe 
(neglecting the weak interaction), it is time for some more P6(5)22 
examples.


Similarly, you should bring a bomb on your next airplane flight (but 
don't detonate it), because the probability that there will be two on 
any flight is astronomically low.



Hmm - if it was my data - I would probably go with P6522.  While the 
probability is higher for P6122, the maximum likelihood is if the data 
were mine - it would not go with the majority.


With regards to the bomb on a plane... This is an interesting service I 
can offer... By my taking a bomb on a plane - I can pretty much 
guarantee there not be another one there - so all the passengers 
benefit. But what is the probability that I will get the bomb on the 
plane - without getting caught?  If I fail to get it on the plane - have 
I really done anyone a service? (well besides giving the media something 
to report on, the police who get to wave their guns around, etc.).


Ezra

Re: [ccp4bb] Distinguishing between P6(5)22 and P6(1)22

[Ezra Peisach]

Rafael Couñago wrote:

Hi,

I am in doubt between the enamtiomorph space groups, p6522 and p6122. 
Is there a way to distinguish the correct one in the absence of a 
molecular replacement model?


Cheers.

Rafael.
You cannot distinguish by absences. How do you plan to phase? I would 
say choose  one space group - and if you get left  handed helicies - you 
guess wrong... (yes - the automated phasing software will try both and 
calculate which you should use based on other statistics)...


If you want to gamble - go w/ P6122... Data from the PDB  indicates that 
there are 1.5x as many proteins w/ P6122 vs P6522...


Ezra

Re: [ccp4bb] Weird expression behavior

[Ezra Peisach]

A little tangental  You mentioned lowering the temperature - how 
low... Stratagene markets a call line - Arctic Express - that adds 
chaperones that are more active at lower temps.  I know someone who 
overcame inclusion body problems by expression at 16 using these cells. 
(I know someone else who regularly induces o/n at 16C w/ regular 
cells).  The only draw back is that these cells produce a protein that 
binds to NiNTA resin...


Good luck...

Re: [ccp4bb] Weird expression behavior

[Ezra Peisach]

Been there, done that, got the T-shirt.

I do not believe there is a protease like 3C in E. coli.

That said - do you have any rare codons in your protein - that might 
cause the stall/termination in expression - leaving you a large amount 
of tag.  Have you followed through with purifictaion to see if you have 
low levels of full length expression?  If rare codons are a problem - 
multiple vendors have cell lines [such as BL21(DE3) RILP,  codonplus, etc.)


With regards to His tag causing inclusion bodies - there have been 
publications regarding solubility issues w/ His tags (I think Helena 
Berglund has a paper on this).


Have you considered (after taking rare codons into account), expression 
with no tag at all? pET3, pET11, etc.




Ezra

Israel Sanchez wrote:
Hello crystallographers in general and E.coli-protein producers in 
particular,


I would like to share with all of you a strange behavior of two of my 
expression constructs, looking for some advice or just know if anybody 
has experienced something similar:


The scenario is the following one, I am trying to produce a NTPase 
domain of around 20KDa of a human protein in E.coli. Initial cloning 
in a T7-based vector with a N-terminal-hexa histidine tags produced 
big quantities of an unfolded protein, in inclusion bodies. I tried 
all normal approaches to try to make the construct soluble: lowing 
expression  temperature, lowing the concentration of IPTG, different 
growing mediums, different E.coli strains, ... no success.
Then I decided to try some fusion-protein strategies, I cloned the 
same construct as a fusion protein with GST and MBP. Then, I could see 
a good soluble expression BUT only of the carrier protein (GST or 
MBP). Lowing expression temperature or lowering IPTG concentration 
does not produce any improvement. Both fusion-protein construct 
contain between the fusion partner and my protein a 3C site for 
cleavage with this protease.


So, the question is, does E.coli may posses protease similar to 3C 
that may explain the self-cleavage? why my ribosomes are not reaching 
until the end of the construct?


Thank you so much for your attention, any comment and/or suggestion 
would be highly apreciated 




--
PhD. Israel Sanchez Fernandez
EM-lab
Departamento de Ciencia de Proteinas
CIB-CSIC Madrid España

Re: [ccp4bb] refmac5 error message

[Ezra Peisach]

Vesna Serrano wrote:

Dear all,
I am using Refmac_5.5.0088 in CCP4 6.1.0, linux version. The program runs
well normally, but all of a sudden it failed with the following message:
Refmac_5.5.0088:   Open failed: File: /tmp/vesna/refmac5_temp1.06489.txt.
Here is the log file of the run:

#CCP4I VERSION CCP4Interface 2.0.4
#CCP4I SCRIPT LOG refmac5
#CCP4I DATE 05 Aug 2009  02:04:38
#CCP4I USER vesna
#CCP4I PROJECT wtDHPAB
#CCP4I JOB_ID 19
#CCP4I SCRATCH /tmp/vesna
#CCP4I HOSTNAME localhost.localdomain
#CCP4I PID 6486

 



 ###
 ###
 ###
 ### CCP4 6.1: Refmac_5.5.0088 version 5.5.0088 : 08/12/08##
 ###
 User: unknown  Run date:  5/ 8/2009 Run time: 02:04:38


 Please reference: Collaborative Computational Project, Number 4. 1994.
 "The CCP4 Suite: Programs for Protein Crystallography". Acta Cryst. D50,
760-763.
 as well as any specific reference in the program write-up.

 $TEXT:Reference1: $$ comment $$
   "Refinement of Macromolecular Structures by the  Maximum-Likelihood
Method:"
   G.N. Murshudov, A.A.Vagin and E.J.Dodson,(1997)
   Acta Crystallogr. D53, 240-255
   EU  Validation contract: BIO2CT-92-0524

 $$
 $SUMMARY :Reference1:  $$ Refmac: $$
 :TEXT:Reference1: $$

 Open failed: Unit:   7, File: /tmp/vesna/refmac5_temp1.06489.txt
(logical: /tmp/vesna/refmac5_temp1.06489.txt)
 Refmac_5.5.0088:   Open failed: File: /tmp/vesna/refmac5_temp1.06489.txt
Times: User:   0.0s System:0.0s Elapsed: 0:00


***
* Information from CCP4Interface script
***
The program run with command: /usr/local/xtal/ccp4-6.1.1/bin/refmac5 XYZIN
"/home/vesna/wtDHPAB/ABwt4ID50-adj1.pdb" XYZOUT
"/home/vesna/wtDHPAB/wtDHPAB_19_2_pdb_1.tmp" HKLIN
"/home/vesna/wtDHPAB/full_ABwtID4.mtz" HKLOUT
"/home/vesna/wtDHPAB/wtDHPAB_19_3_mtz_1.tmp" LIBOUT
"/home/vesna/wtDHPAB/wtDHPAB_19_lib.cif"
has failed with error message
Last system error message: Permission denied
 Refmac_5.5.0088:   Refmac_5.5.0088
***


#CCP4I TERMINATION STATUS 0 Last system error message: Permission denied 
Refmac_5.5.0088:   Open failed: File: /tmp/vesna/refmac5_temp1.06489.txt

#CCP4I TERMINATION TIME 05 Aug 2009  02:04:38
#CCP4I MESSAGE Task failed


I would very much appreciate help in solving this problem.

Regards,

Vesna Serrano

Dept. of Chemistry
NC State Univ.
Raleigh, NC 27695
  
Can you write to the directory /tmp/vesna? Does it exist?  The last 
error is permission denied...  This could happen if something wiped the 
files in /tmp - including the directory, or if someone else created the 
directory there and you cannot write to it  I would check "ls -ld 
/tmp/vesna" to see that the directory exists and the permissions are 
correct.


Another possibility is that someone changed the permissions on the 
directory /tmp - and made it not world writable... "ls -ld /tmp" would 
tell you if this was the problem.


Good luck...

Ezra

Re: [ccp4bb] Definition of Fourier coefficients

[Ezra Peisach]

They are SigmaA weighting coefficients.  See the sigmaa documentation - 
and references there in by Read. (I think the 1986 reference is the 
correct one).


Ezra


Mayer, Mark (NIH/NICHD) [E] wrote:

Hi,

What do m and D indicate in the Fourier coefficients for a 2mFo-DFc map?

I've dug a bit in web and CCP4 doc  but not found an explanation, though I'm 
sure its there.

Thanks,

Mark
  

Re: [ccp4bb] Parameter MAXSAVE exceeded

[Ezra Peisach]

I do not know off hand what a .cv file is - unless it is a cns/xplor 
reflection file.  Instead of using f2mtz - try sftools.


Ezra

Jayashankar wrote:

Dear scientists and friends,

When I try to convert a .cv file to .mtz by f2mtz I got the following 
error,what it means ans what should I do to get rid of it.


''Parameter MAXSAVE exceeded''

--
S.Jayashankar
Research Student
Institute for Biophysical Chemistry
Hannover Medical School
Germany 

Re: [ccp4bb] Off topic: McDonalds Happy Meal and Crystallography

[Ezra Peisach]

I saw that toy - Personally, I do not like the idea of encouraging 
children to aim a light (low powered led) directly at one's eye...


Ezra


P Hubbard wrote:
I took my 5 year old niece to a McDonald's (in England) for a happy 
meal today - Spiderwick Chronicles promotion. The toy's light effect 
reminded me of a visual aid I saw to help introduce X-ray diffraction 
and the crystal lattice to students. My niece was more interested in 
using the visual effect to see if her toy was a goodie or baddie.


Cheers!


Going green? See the top 12 foods to eat organic. 

Re: [ccp4bb] PERL system call to CCP4

[Ezra Peisach]

You may wish to look at the occp4-pm perl package by E. Courcelle and 
J.P. Samama  (formerly available from ftp://ftp.ipbs.fr/pub/occp4 - but 
this no longer works...) I no longer have a copy but someone might...


Ezra


[EMAIL PROTECTED] wrote:

Dear CCP4 users,

I am writing a PERL script to execute a number of CCP4 commands (ncsmask,  
pdbset, and dm)  in succession.  I have tried using system call or PIPE 
command, neither of which work.  The ccp4 scripts generated work independently 
on the command line.



Any suggestions?

Thank you in advance!
  

Re: [ccp4bb] scalepack2mtz

[Ezra Peisach]

It depends on your path environment variable... It used to be that "." 
was by default in your path - then the more security conscious removed 
the current working directory from the default list of paths to search 
for commands.


Try ./scalepack2mtz.exam 
The ./ gives a path


U Sam wrote:

I used to run "scalepack2mtz" succefully. now it is not working. Can anybody 
suggest the way to short it out.

%chmod +x scalepack2mtz.exam   (% is a unix/linux prompt).
%scalepack2mtz.exam

message is as follows.
bash: scalepack2mtz.exam: command not found

Although I sourced the path /opt/ccp4/ccp4-6.0/ccp4-6.0/bin , I still get the 
same message.

Thanks any suggestion.
Sam
_
Gear up for Halo® 3 with free downloads and an exclusive offer. It’s our way of 
saying thanks for using Windows Live™.
http://gethalo3gear.com?ocid=SeptemberWLHalo3_WLHMTxt_2

Re: [ccp4bb] xplot84driver problems

[Ezra Peisach]

The file format is definitely machine byte order dependent. (see 
$CDOC/plot84.doc) A few years ago I was toying with the idea of making 
library code deal with swapping if need be...

Never did it though

William Scott wrote:

Did you make your plt file on the intel mac?  I've noticed that ones I made on 
ppc give that error on my otherwise functional xplot84driver (in the fink 
package).

I tried byte-swapping with dd but to no avail.

I guess this is still the cutting edge of 1984 software?

Bill

On Thu, 6 Sep 2007 15:02:01 +0200
Derek Logan <[EMAIL PROTECTED]> wrote:

 Hi,
 
 I've installed CCP4 on an Intel Mac running Mac OS X 10.4.10 using  
 the binary installer from the automatic download page. Everything  
 works fine and dandy except xplot84driver. This currently means I  
 have to convert every plot file on the command line using pltdev then  
 use Preview to view the PS as PDF, rather than just using the CCP4i  
 pulldown menu.
 
 Initially xplot84driver complains that libifcore.dylib is missing.  
 This appears to be an Intel compiler-specific library which I just  
 happen to have in /opt/intel/fc/9.1.024/lib, as I once evaluated the  
 beta release of the compiler. If I copy this to $CCPLIB or link from  
 there to it, it complains that it doesn't have libifm.dylib, which in  
 turn complains that it doesn't have libirc.dylib. Finally when all  
 these libraries are linked to and loaded, xplot84driver complains  
 about "Bad plot84 file format" and does not open the plot file. Does  
 anyone have an idea what is going on?
 
 Thanks

 Derek
 --
 Derek Logan tel: +46 46 222 1443
 Molecular Biophysicsfax: +46 46 222 4692
 Lund University
 Box 124, Lund, Sweden
 
 
  

Re: [ccp4bb] High overall b-factor

[ezra peisach]

I have never worked at 3.2A - but I suspect that the overal temperature 
factor is being determined from the slope of a Wilson plot.  However, 
Wilson plots only really "work" at higher resolution (2.8 or better). Look 
at the output from truncate and see what it is predicting - and look at 
the plot - it should be pretty obvious why you have such a high B.


Ezra


On Fri, 9 Mar 2007, George Lountos wrote:



Hello all:

I just recently collected data on initial crystals I grew of an enzyme
with inhibitor. The crystals diffract to only 3.2 A but I was able to get
phases by molecular replacement to see if there is any inhibitor bound.
Although the data processed well in HKL2000 with good statistics and the
current structure refinement is at R-factor of 22% and R-free 30% at 3.2
A, the overall B-factor of the protein is very, high (100 A^2). I can see
difference density for the ligand in the active site and after refinement
it fits well in the density but the B-factor for the ligand is 110. I
have not come across a refinement with such high B-factors where the
protein density and ligand density can be distinguished at such high
B-factors. Does anyone have any suggestions if there is something going
wrong here?

Thanks,

George



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