[ccp4bb] Some advices on model modification

[allen price]

Dear all:
I got a dataset at 2.8 angstron. I have tried several ways such as phaser, 
MRBUMP,BALBES,but still can't solve the 
data,which means I have to edit my model. Maybe I'd better cut it off or delete 
the water or loop. I really have no 
idea,as it is my first time to do such things, I alway used the whole model to 
mr. Could anyone give me some advice? 
what kind  of software do you guys use? really need you help!
Best regards,
Allen

Re: [ccp4bb] Rant: B vs TLS, anisou, and PDB headers

[price]

In the end, we're solving all these structures because we believe (or 
at least hope) that they'll be useful for understanding 
biology.  That means that biologists should be able to understand 
what we deposit.
When I've tried to teach undergraduates "what to make of" structural 
models, I find I can give (most of) them an intuitive feel for what B 
tells or doesn't tell them.  I don't have time to even bring up TLS parameters.
The file that gets downloaded from the databank and displayed by 
pymol or PDBviewer needs to be as simple as possible while still 
being true.  Exactly how those Bs were derived should be included in 
the file, but in a way that the non-specialist users can get by 
without reading it all (since they most certainly won't ;-) ).


Phoebe

At 03:56 PM 3/29/2008, you wrote:

I believe the simplest and most honest thing to deposit are the 
parameters of your model,

viz the TLS parameters and the residual B factors.
Derived quantities should be calculated as and when you need them.


---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123

RNA is really nifty
DNA is over fifty
We have put them
  both in one book
Please do take a
  really good look
http://www.rsc.org/shop/books/2008/9780854042722.asp

Re: [ccp4bb] Model ensemble for x-ray crystallography

[price]

Didn't that trick very successfully lower the 
R-factors of the completely wrong models that led 
to the Great Pentaretraction?  Unless you have 
stunningly high resolution, beware.

Phoebe

At 10:13 AM 3/28/2008, you wrote:

Some time ago I've heard about the idea of proposing
an ensemble of models (as in NMR), instead of a single
model for x-ray crystallography structures. If I
remember correctly, this idea has been published
somewhere. Can anyone tell me what article is that?

Lucas


  Abra sua conta no Yahoo! Mail, o único 
sem limite de espaço para armazenamento!

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---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123

RNA is really nifty
DNA is over fifty
We have put them
  both in one book
Please do take a
  really good look
http://www.rsc.org/shop/books/2008/9780854042722.asp

Re: [ccp4bb] finicky protein

[price]

Just beware that changing how you break the cells open can 
change the average size of chromosome chunks, which can 
change how DNA binding proteins behave in the lysate.


 Original message 
>Date: Mon, 3 Mar 2008 15:21:15 +
>From: Mads Gabrielsen <[EMAIL PROTECTED]>  
>Subject: [ccp4bb] finicky protein  
>To: CCP4BB@JISCMAIL.AC.UK
>
>I am not a big fan of sonication. Try changing your way of 
disrupting the
>cells.
>
>I have compared sonication vs mechanical stress on several 
unrelated proteins,
>and for me a good old french press wins every time. If you 
want to get all
>modern and fancy, a cell disruptor gives similar results.
>
>Cheers,
>
>Mads Gabrielsen
>
>
>[Hide Quoted Text]
>
>On Mar 2, 2008, at 11:47 PM, Tim Gruene wrote:
>
>Hi all
>
>sorry, for offtopic query...
>
>I am trying to purify my protein by Ni-NTA affinity 
chromatography.  After
>sonication as i centrifuge bacterial lysate, soon after 10 
min  whole
>lysates
>get precipitated during loading on the column and some time 
it remain
>soluble too. if i get purified through the column without  
precipitation,
>it
>gets precipitated during dialysis.
>I have tried lot, by chnaging buffers, increasing salt or  
deacreasing salt
>or no salt at are helpless.
>I do purifiaction in cold room.
>
>can any one suggest some solution?
>
>Thanks in advance.
>
>NSH
>
>
>-- 
>Dr. Mads Gabrielsen
>
>GBRC, B217
>Division of Biochemistry and Molecular Biology
>IBLS
>University of GlasgowPhone Office: 01413308119
>G12 8QQ  Phone Lab: 01413306449
>UK   E-mail: 
[EMAIL PROTECTED]
>
>

>This message was sent using IMP, the Internet Messaging 
Program.

Re: [ccp4bb] crashing-out protein eluted from Nickel column

[price]

An added benefit of EDTA is that it inhibits some proteases - for one 
of our wimpier proteins, spiking each fraction collector tube with a 
little EDTA before running the Ni column really helped reduce keep 
the sample in one piece.

Phoebe

At 01:18 PM 2/19/2008, Sophia Tsai wrote:

Hi,

Agreed on this. I used to have issues with aggregation due to the 
Nickel being stripped off the column (happens with elution in 
imidazole). Adding 10mM EDTA to the elution immediately AFTER it has 
come out of the column will chelate the Ni++ and prevents 
aggregation (at least in my case).  Afterwards, I use gel filtration 
to remove the Ni++, as well.


Hope that helps!
Sophia

On Mon, Feb 18, 2008 at 4:48 AM, Ngo Duc Tri 
<[EMAIL PROTECTED]> wrote:

Hi,
I used another way to deal with this problem. You can try to elute 
your protein with the buffer containing 50mM EDTA (You need at least 
10CV to elute completely). Then use gel filtration to remove the Ni.


I applied this method with two proteins and it showed good results.
Good luck!

TriNgo



---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123

RNA is really nifty
DNA is over fifty
We have put them
  both in one book
Please do take a
  really good look
http://www.rsc.org/shop/books/2008/9780854042722.asp

Re: [ccp4bb] an over refined structure

[price]

Rotational near-crystallographic ncs is easy to handle this way, but 
what about translational pseudo-symmetry (or should that be 
pseudo-translational symmetry)? In such cases one whole set of spots 
is systematically weaker than the other set.  Then what is the 
"theoretically correct" way to calculate Rfree?  Write one's own code 
to sort the spots into two piles?

Phoebe

At 01:05 PM 2/8/2008, Axel Brunger wrote:

In such cases, we always define the test set first in the high-symmetry
space group choice.  Then, if it is warranted to lower the crystallographic
symmetry and replace with NCS symmetry, we expand the test set
to the lower symmetry space group.  In other words, the test set itself
will be invariant upon applying any of the crystallographic or NCS operators,
so will be maximally "free" in these cases.   It is then also possible to
directly compare the free R between the high and low crystallographic
space group choices.

Our recent Neuroligin structure is such an example (Arac et al., 
Neuron 56, 992-, 2007).



Axel




On Feb 8, 2008, at 10:48 AM, Ronald E Stenkamp wrote:


I've looked at about 10 cases where structures have been refined in lower
symmetry space groups.  When you make the NCS operators into 
crystallographic

operators, you don't change the refinement much, at least in terms of
structural changes.  That's the case whether NCS restraints have 
been applied
or not. In the cases I've re-done, changing the refinement program 
and dealing

with test set choices makes some difference in the R and Rfree values.  One
effect of changing the space group is whether you realize the copies of the
molecule in the lower symmetry asymmetric unit are "identical" or 
not.  (Where

"identical" means crystallographically identical, i.e., in the same packing
environments, subject to all the caveats about accuracy, precision, thermal
motion, etc).  Another effect of going to higher symmetry space groups of
course has to do with explaining the experimental data with simpler 
and smaller

mathematical models (Occam's razor or the Principle of Parsimony).

Ron


Axel T. Brunger
Investigator,  Howard Hughes Medical Institute
Professor of Molecular and Cellular Physiology
Stanford University

Web:http://atb.slac.stanford.edu
Email:  [EMAIL PROTECTED]
Phone:  +1 650-736-1031
Fax:+1 650-745-1463




---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html 

Re: [ccp4bb] an over refined structure

[price]

What is the resolution? Are you using ncs restraints (you probably 
should, on the coordinates but not on the Bs)?  How does your 
Ramachandran plot look?
You might tighten the geometry even more.  Aside from all theoretical 
arguments about how big the rmsd's should be, if they're rather 
loose, things that are built incorrectly may be shoved back into 
density, making mistakes harder to spot.
Have you made sure that all your side chains are not just in the 
density, but in nice, non-strained rotamers?
The backbone and side chains are rather couple in refinement, 
especially if you don't have very high resolution data - this means 
that if you originally had the backbone slightly wrong, you may have 
built the side chains in the wrong rotamers.  They'll look like they 
fit the density, but everything will be a bit strained until you fix 
them.  Particularly if your Ramachandran plot is a bit scruffy, try 
optimizing the backbone geometry (by something such as lego in O), 
then refitting the side chains.


Phoebe Rice


At 11:33 AM 2/4/2008, you wrote:

Hi Tim,

Thank you for your and information and suggestions. There are two 
indepdent molecules in the asymmetric unit and one molecule does not 
have very good density, especially in the N-terminus.


Do you think that I should remove the region in the refinement?

Best,

Sun

Tim Gruene <[EMAIL PROTECTED]> wrote:
I would agree that the difference is suspiciously high. I. Tickle and
others have published analytical expressions for how to estimate the ratio
between R and Rfree, just google for "tickle rfree" to find the
references.

You easily achieve a large difference by adding too many waters which just
model noise. There may be other reasons for which more knowledge about the
structure is required. Do you have large unmodelled regions, like loops
that do not show in the density map?

Tim


--
Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A


On Mon, 4 Feb 2008, Sun Tang wrote:

> Hello All,
>
> I refined a structure with Refmac in CCP4i and the R/Rfree is 
0.215/0.277. The difference between R and Rfree is too much even 
though I used 0.01 for weighting term in the refinement (the 
default value is 0.3). The RMSD for bond length and bond angle is 
0.016 A and 1.7 degree.

>
> What may be wrong with the over-refined structure? What is the 
reason for leading to an over-refined structure? How to avoid it?

>
> Best wishes,
>
> Sun Tang
>
>
> -
> Be a better friend, newshound, and know-it-all with Yahoo! 
Mobile. Try it now.




Be a better friend, newshound, and know-it-all with Yahoo! Mobile. 
Try 
it now.


---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html 

Re: [ccp4bb] Codon Optimized Expression

[price]

Rumor also has it that more than one "bad" codon in a row, particular 
near the beginning, can be extra-bad.  For one small, A/T rich 
protein, we simply "fixed" a pair of bad Arg codons near the 
N-terminus at the same time as recloning it, by using the N-terminal 
cloning primer to do the mutagenesis.  Combined with using codon-plus 
E coli, expression improved dramatically.

Good luck!
Phoebe Rice

At 09:29 AM 2/1/2008, Looney, Andrea Lynn (Andrea Hevrdeys) wrote:

Dear All,

I am getting very little (not zero) expression of my protein.  I am 
curious to know if it is worthwhile to codon optimize my gene, which 
is ~1200bp,  for E.coli.  Can you have "too much of a good 
thing"?  Can codon optimizing overwhelm the cell or in some way 
cause death or lowered expression?


Thank you all,

Andrea L. Hevrdeys



---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html 

Re: [ccp4bb] differences between Rsym and Rmerge

[price]

High R "merges" with no reasonable excuse can certainly be a useful 
red flag during data processing (along with the % of observations 
rejected, which I've never had a reviewer request).


Which brings up the point that one reasonable excuse is anisotropy - 
high Rs for merging random observations in the "imaginary" direction 
will be combined with lower Rs for merging decent data in the "real" 
direction.
Is there any extant software that will calculate directionally-binned 
Rmerges?  It would be useful both for re-assuring users that there 
isn't anything worse with their data, and for arguing with referees 
who don't read CCP4BB.


Phoebe

At 01:18 PM 1/18/2008, Mischa Machius wrote:

OK, that brings us back to a more substantial question: is any of
these R values actually suitable to judge the quality of a given
dataset? Instead of introducing novel R factors, one could also simply
ignore them altogether, make sure that the error models have been
properly chosen and look at I/sigma(I) as the main criterion.
[QUOTE ]If anyone then still wants to present low R factors, one can
always divide by 2, if necessary. [/QUOTE]

Best - MM


On Jan 18, 2008, at 1:02 PM, Salameh, Mohd A., Ph.D. wrote:


Thank you all, it was very, very helpful discussion. However, I
collected crystal data and the Rmerge overall was very high around
0.17
at 2.6A resolution and I'm wondering what is the acceptable value
(range) of R-merge that worth the time to continue processing! Very
anxious to hear your thoughts. Thanks, M

Mohammed A. Salameh, Ph.D.
Mayo Clinic Cancer Center
Griffin Cancer Research Building
4500 San Pablo Road
Jacksonville, FL 32224
Tel:(904) 953-0046
Fax:(904) 953-0277
[EMAIL PROTECTED]



-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Chris Putnam
Sent: Friday, January 18, 2008 1:21 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] differences between Rsym and Rmerge

On Friday 18 January 2008 09:30:06 am Ethan A Merritt wrote:


Rmerge is an average over replicate measurements of the intensity for
identical [hkl]. Rsym is an average over the measurements for all

symmetry

equivalent reflections.

In the presence of anomalous scattering, Rsym will be higher than

Rmerge

because the Bijvoet pairs, although symmetry related, do not have

identical

intensities.

One might logically report two values for Rsym,  one which averages
over the Bijvoet-paired reflections and one which does not.


This has been an eye-opening discussion for me.  I've been really
surprised
that there's been such a diversity of opinion about what these common
terms ought to refer to, and the fact that my understanding was wrong.
I always thought that Rsym was an average over all symmetry equivalent
reflections from the same crystal (including Bijvoet pairs) and Rmerge
was
properly restricted to cases of multi-crystal averaging.  (My versions
of
Table 1's from single crystals have used "Rsym" rather than "Rmerge".)

I wonder if the problem here is that the terms have become overloaded
(and
hence non-specific).  In that sense "Rmerge" is a particularly
unfortunate
name as every R that we're discussing is a really a merge of some sort
or
another.  (In the most naive sense, "Rmerge" might be thought to be
the
R
for whatever variation of reflection merging the experimenter
chooses to
do.)

One possible solution would be to push the community towards a new set
of
terms with clearly defined meanings (and whose names would be used
explicitly by new releases of MOSFLM, HKL2000, etc. and changes for
new entries in the PDB).

If new terms were to be adopted, they ought to specifically
distinguish
between single crystal and multi-crystal merging.  I see three such
R values that might be useful (I've arbitrarily chosen names to
distinguish
them from each other and the older terms):

Rhkl - R of identical hkl's

Rrot - R of symmetry-related hkls, but not Bijvoet pairs
("rot" coming from the concept that all symmetry-related
reflections can be found via rotations in reciprocal space and
the fact that "sym" has already been used)

RBijvoet - R of symmetry-related and Bijvoet-related hkls
(including reflections related by both rotations and an inversion
center in reciprocal space)

Rhkl,multi - multi-crystal version of Rhkl

Rrot,multi - muti-crystal version of Rrot

RBijvoet,multi - multi-crystal version of RBijvoet

The downside of adopting new names is that it makes the previous
literature
obsolete, but I wonder if the older terms were ambiguous enough that
that's
not such a problem.


--
Christopher Putnam, Ph.D.
Assistant Investigator
Ludwig Institute For Cancer Research




Mischa Machius, PhD
Associate Professor
UT Southwestern Medical Center at Dallas
5323 Harry Hines Blvd.; ND10.214A
Dallas, TX 75390-8816; U.S

[ccp4bb] Zn fingers and Ni columns

[price]

This has probably been discussed before, so apologies in advance.
We're eyeing a protein that has a probable C4 Zn finger in the 
middle.  The collaborators who are nicely going to PCR it up want to 
know if we'd like it with or without a His tag.
Is it a bad idea to co-mingle Zn-binders and Ni columns?  Or is it 
likely to bind the column quite nicely without the tag?

thanks,
Phoebe


---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html 

Re: [ccp4bb] SUMMARY: PEG MW vs. cryoprotectivity

[price]

Many thanks to all who replied.  The answers were remarkably varied - 
see below.
My own two bits worth - vitrification of mother liquor doesn't always 
lead to a nice, low-mosaicity, ice-free crystal freeze in our hands, 
although we're not willing to sacrifice a statistically significant 
number of crystals to figure out why.  Most of our crystals are 
annoyingly fragile, which makes it hard to disentangle the effects of 
bad cryoprotection from those of mechanical damage.

Phoebe

From: [EMAIL PROTECTED]
PEG 4000 has worked for us at high concentration (35-40%)
depending on what else is in there. The same goes for
PEG 3000. You can try increasing the concentration of
the PEG in the reservoir gradually (over the course of many
days) from the % they are grown at up to 25-40%. Hopefully this
will not crack your crystals.


From: Anastassis Perrakis <[EMAIL PROTECTED]>
in my experience peg 4k reduces the amount of glycerol that you need
but cant act as cryo on its own.

From: Juergen Bosch <[EMAIL PROTECTED]>
the larger the worse for cryo. But PEG4000 >40% freezes well. PEG8000 
needs some addition of smaller PEGs/Glycerol etc.



From: Kevin Jude <[EMAIL PROTECTED]>
I have used 23% PEG 3350/5% glycerol as a cryoprotectant (JACS 2006 p 
3011).  The PEG on its own didn't work at that concentration.


It would be enough to test this by making up the solutions and 
shooting empty loops, like Elspeth Garman did for glycerol.


From: Ezra Peisach <[EMAIL PROTECTED]>
I have seen discussions in the past... The easiest thing to do is 
test it yourself. Try freezing a small loop of high concentrations of PEG.

If it forms a clear glass it is worth pursuing...

From: "Jan Abendroth" <[EMAIL PROTECTED]>
20% PEG 2000 just worked fine, 35% PEG 3350 seems ok too.
also depends on the size of the loop.

From: Edwin Pozharski <[EMAIL PROTECTED]>
I have used pegmme2000 as cryoprotectant in the past (some 45% of 
it), and it worked fine.  Indeed, PEG4K s included in Hampton's kit.


From: Buz Barstow <[EMAIL PROTECTED]>
In our experience with freezing protein crystals under high pressure,
we've found that mid weight PEGs do help a little to enhance the 
cryo- protective effect of high pressure, although are not terrifically

effective, especially when at a low concentration of around 5%.


From: "Li Sheng" <[EMAIL PROTECTED]>
I used 40% w/v PEG 4000 as cryoprotectant.

From: "Moody, Dr P.C.E." <[EMAIL PROTECTED]>
my experience is that anything over PEG 600 is likely not to be 
a  reliable cryoprotectant, and 400 is the maximum safe 
size.can't comment on the commercial kits, my cynical nature 
would suggest that testing may not be an important part of the 
"product pipeline"Peter


From: Remy Loris <[EMAIL PROTECTED]>
PEG 4000 is a very good cryoprotectant in the range of 30-35%. If 
your crystallization condition includes PEG4000, it is a good idea 
for a first trial to find a good cryo condition raise the PEG4000 
concentration to 30-35%. Some of the Hampton ctrystal screen 
conditions (and other commercial kits as well) that contain PEG4000 
do even not need further addition of cryoprotectant (even if in the 
corresponding Hampton cryo screen they are diluted with glycerol, one 
of the most horible cryoprotectants in common use)
Lower MW PEGS can be useful as well, but the required concentrations 
will be higher. Please be aware that in quite a number of cases the 
ideal cryo solution can be very far away from the condition in which 
the protein was crystallized!


From: "gengxiang zhao" <[EMAIL PROTECTED]>
Before, I crystallized the complex of a protein with some small 
molecules. I use the PEG3350 as a precipatate. When I collect the 
dataset using X-ray detector. I only use 23% PEG3350 plus 5% PEG400 
as a cryoprotectants. Fortunately, it has been successful.


So, I think that the PEG3350 functions some cryo-protectants.

From: Florian Schmitzberger <[EMAIL PROTECTED]>
This might not be a direct answer to your question; I have found that 
PEG3350 at around 20-25 % (w/v) concentration (JSCG+ screen) - in 
combination with ~10 % (v/v) glycerol (which came from the protein 
sample buffer) was sufficient to cryoprotect crystals rather well; 
without further soaking or handling being necessary.


From: Jennifer Cash <[EMAIL PROTECTED]>
In our recent experience, larger PEGs work similarly to smaller PEGs 
as far as vitrification goes.  Additionally, transferring crystals to 
a solution of increased PEG concentration (as compared to mother 
liquor) can substantially reduce the amount of cryo needed for 
freezing and can be more gentle on crystals than just using a high 
concentration of cryo.  Here is a reference that tests cryoprotective 
ability of PEG 2000 & 2.

J. Appl. Cryst.  (2006)  39, 244-251




At 03:55 PM 12/4/2007, [EMAIL PROTECTED] wrote:
We've been having a discussion in the lab about whether or not 
middle-sized PEGs such as 4000 can be expected to serve as 
cryoprotectants (and if not, why

[ccp4bb] PEG MW vs. cryoprotectivity

[price]

We've been having a discussion in the lab about whether or not 
middle-sized PEGs such as 4000 can be expected to serve as 
cryoprotectants (and if not, why certain commercial kits are 
formulated the way they are).  Can anybody shed some light / 
references on the question of the size of PEGs vs. their ability to 
help in freezing?

thanks,
Phoebe


---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html 

Re: [ccp4bb] elusive DNA density

[price]

All statistics aside, I never believe a molecular 
replacement solution until it produces an Fo-Fc 
map with peaks for something that should have 
been there but wasn't in the model.


If you know roughly where the DNA should bind, 
you could try making a solvent mask that includes 
that region (plus the protein of course), and 
then see if you can pull up some vaguely 
believable density by density modification.


Do the crystals pack reasonably without the DNA there?

If you think the DNA is poorly ordered or at low 
occupancy, the most definitive test would 
probably be to collect a new set with iodine on 
the DNA.  If that doesn't show up, you're sunk!


Good luck,
Phoebe

At 02:08 AM 11/7/2007, Serge Cohen wrote:

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Hi;

You might also consider if it is likely that you have statistical
disorder, causing a blur in the DNA. This could be the case if your
binding is not specific, in which case it is possible that DNA is
shifted by a couple of bases (or if your DNA is "nearly palindromic"
you might have some AsU containing the DNA in one direction and other
in the reverse direction).

Obviously, for this to be the reason of the weak density you observe,
you should first rule out the model bias introduced by MR (as
mentioned by Debanu), an dalso consider what is the sequence of your
DNA, it's structure, and the likeliness that it is not bound to the
same way to the protein in each copy.

Serge.

PS : If you expect the DNA to be making some fibbers in the crystal,
you should definitely be able to see from the diffraction images that
you DNA is present by a strong SF along the fibber axis around the
base stacking distance. This would also be a good proof that you
indeed have diffracting DNA.

Le 7 nov. 07 à 04:36, Melody Lin a écrit :


Dear all,

I've been working on a series of DNA-protein complex structures. In
my recently acquired data sets, I got almost no density for DNA if
I do molecular replacement or rigid body fitting with the protein
structure, although I am 100% sure I have DNA in the structure by
indepenent means. If I use models with DNA, I could find some DNA
density with those data sets, but as I refine the structure, the
density became very poor. The resolutions for those data sets are
between 2.0-2.4 A.  Also, if I use the scaled data from synchrotron
rather than the re-scaled data at home, I got better DNA density,
although for re-scaling, I used site parameters that I copied done
from synchrotron. The only differences between those two sets of
scaled data are: (1) the original scaled data take into account all
reflections, including high resolution data 
with low completeness/ redundancy, which are 
cut in the re-scaling; (2) error models were

changed so chi squares for each bin are 0.8-1.2 for re-scaling.

My (very naive) questions are: (1) Does the DNA density I saw in
the cases where I use models with DNA for MR/rigid body fitting
only reflect model bias? (2) are simulated annealing or cycles of
coordinate/B factor refinement enough to get rid of model bias? (3)
Does weak DNA density have to do with data processing?

Thanks very much for any suggestion,
Melody Lin


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---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html 

Re: [ccp4bb] protein degradation?

[price]

Some proteases are metal-dependent, and inhibitors for those aren't 
Ni-column-compatible.  "We" (meaning my students) found that it helps 
to (1) work very quickly and (2) put EDTA into the fraction collector 
tubes before eluting from the Ni column.



At 06:22 AM 11/4/2007, Vijay Kumar wrote:

Hi,

I have been trying purify a N-ter his-tagged protein over-expressed 
in E.coli. After purification (Ni-NTA or Co-Talon), I find two bands 
in SDS PAGE which are very close each other (top band in the right 
MW and more intense than the lower band). Western blot (for his-tag) 
of the gel gave signal for both the bands. Mass spec results 
confirmed both protein bands are the same. So I think it could be 
C-ter degradation of my protein. Also the 2 bands exist after 
ion-exchange and sizing column.


I use commercially available complete protease inhibitor tablets 
(increasing concentration has no effect) and sonication for lysis. I 
am wondering if people have encountered the same problem and got any 
suggestions?



Thanks in advance.

Regards,

Vijay


---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html 

Re: [ccp4bb] PDB Crazy or Me?

[price]

I don't see a concise list of changes there?

At 07:34 AM 10/9/2007, Eleanor Dodson wrote:

Did you go to this web site?
http://remediation.wwpdb.org/downloads.html

It has the new dictionary stuff
Eleanor


James Stroud wrote:

Hello All,

I noticed some things different about the PDB today causing me to 
rub my eyes vigorously and to put my nose right on my monitor in disbelief:


* -> ' for nucleic acid sugars
  O#P -> OP# for the phosphate oxygens
  C5A -> C7 for thymidine exocyclic methyl (didn't it used to be C5A?)

And who knows what else. In fact, my real question *is* "who knows 
what else?" I looked at the RCSB site and googled PDB changes, etc, 
but really couldn't find the low-down. Another question is how long 
before it all changes again? I noticed some threads here with PDB 
in the title recently, but didn't read them because I thought they 
discussed esoteric and philosophical issues that do not affect us common folk.


James

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

http://www.jamesstroud.com



---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html 

Re: [ccp4bb] solving structure of which 70% is known

[price]

Hi,
- I think someone already pointed out that you should try P6522.
- check that it isn't really a twinned P61 or P65 with 2 per asymmetric unit
- buy DNA with BrdU and some more with IdU, and/or grow your protein 
in SeMet - at your resolution, the more "real" phase info the better!

Phoebe

At 06:30 PM 8/29/2007, you wrote:

Hello,
 I am trying to solve a multi-protein DNA complex structure 
from a 3.6 A native data set. The target structure is a dimer (95 
aa in each monomer) in complex with DNA( 15 base pairs) plus a 
second protein of 131 aa. The data has been scaled to P6(1)22 sp. 
gr. and one target structure is expected to be in the asymmetric 
unit that corresponds to 68% solvent content. 70 % of the target 
structure is known in two parts(two different pdb structures 
previously solved contributing 55% and 15% of the target 
structure). I tried with molrep and phaser considering the first 
part(55%) as the search model but it turned out to no good solution 
which all clashes with symmetry related copies. If I assume the sp. 
gr. is not the case what else I can try. Any suggestion is well appreciated.

Thanks...
Raja


---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/index.html
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html 

[ccp4bb] water water everywhere

[price]

While we're still on the subject of good model-building habits and 
reviewing pitfalls, I've been shocked to download a couple of 
structures recently that seem to have solvent channels chock full of 
allegedly ordered water (many "layers" deep, and not exactly at 0.5A 
resolution).  To any new students out there:   making Rfree go down a 
bit by putting a water in every unexplained blob is NOT the same as 
building a good model!
I'm afraid I reviewed one of these (sans coordinates) ... so sorry to 
the community ... it wasn't obvious from table 1.

Phoebe


---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/index.html
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html 

Re: [ccp4bb] nature cb3 response

[price]

I don't think all journals have that policy, and even so, making the 
reviewers specifically request the data implies to the reviewees that 
somebody out there doesn't trust them.


You shouldn't have to insult the authors in order to do a proper 
reviewing job - you should just be able to download the data and 
coordinates right along with the pdf.


Phoebe

At 09:29 AM 8/17/2007, you wrote:

Phoebe,

Any and every reviewer has right to request the coordinate file as well
as sf file.
The other question is: Why most of them are not exercising their rights?



Vaheh Oganesyan

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Behalf Of
[EMAIL PROTECTED]
Sent: Thursday, August 16, 2007 8:10 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] nature cb3 response

A comment from my collaborator's student suggests a partial
answer.  This afternoon he happened to say "but of course the
reviewers will look at the model, I just deposited it!".  He was
shocked to find that "hold for pub" means that even reviewers can't
access the data.  Can that be changed?  It would take a bit of
coordination between journals and the PDB, but I think the student is
right - it is rather shocking that the data is sitting there nicely
deposited but the reviewers can't review it.
 Phoebe Rice

At 05:33 PM 8/16/2007, Bernhard Rupp wrote:
>Ok, enough political (in)correctness. Irrespective of fabricated or
not,
>I think this points to a general problem of commercial journals and
>their review process, as it seems that selling (.com) hot stuff
>induces an extraordinary capability of denial.
>
>The comment, as someone noted, does not address the allegations
>at all. This is reminiscent of my dealings with Nature in two
>related cases: They ignore or stonewall until the dispute is ended
>with an irrelevant comment. In one case, Axel B later proved
>with the correct structure that what we had commented on earlier
>was entirely correct.
>In the second case, the comment (by some of the leading experts,
>not just by me nobody) was rejected with no recourse based on another
>non-fact-addressing author comment and not published at all.
>
>Compare this to a similar case, when the Jacs editor (.org <--)
contacted me
>
>on its own accord to check for a related problem, leading to retraction
>of the paper after the editor (a scientist himself) evaluated
>facts and response.
>
>It also seems to depend on the handling Nature editor. I have made maps
of
>several structures from data unhesitantly provided by the editor when I
>had reason to ask for them during review. Those were also responsive to
>a mini-table-1-comment I sent on cb3, but I did not hear from the
editor
>assigned to cb3.
>
>This time again, the review completely failed (table 1 and comment
issues),
>and
>the editorial process failed as well, because the response is not
adequate.
>If someone - as tentatively and tactfully it may have been phrased -
accused
>
>me of faking data they'd eat shit until hell freezes over
>
>It is as simple as that: Extraordinary claim (super structure, bizarre
stats
>and properties) requires extraordinary proof. This rule has not been
>followed, which reflects poorly on the scientific process in this case.
>
>I also note that in no case known to me, persons involved in
irregularities
>have ever appeared as frequent (or at all) communicators on the ccp4bb.
>
>As long as grant review and tenure committees rely on automated
>bibliometrics
>and impact factors (and who knows who) to decide academic careers and
>funding,
>the big journals will remain the winners. The system has become
>self-perpetuating.
>
>Back to grant writing now.
>Need to get that paper out to nature...
>
>Cheers, br
>
>PS: it is pointless flaming me. I am the messenger only.
>
>-Original Message-
>From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
>Bernhard Rupp
>Sent: Thursday, August 16, 2007 2:03 PM
>To: CCP4BB@JISCMAIL.AC.UK
>Subject: Re: [ccp4bb] nature cb3 comment pdf
>
>thxthxthx to all the day and night owls for the many copies
>The winners have been selected, no more entries needed.
>thx again br
>
>-Original Message-
>From: Miriam Hirshberg [mailto:[EMAIL PROTECTED] On Behalf Of
Miriam
>Hirshberg
>Sent: Thursday, August 16, 2007 1:58 PM
>To: Bernhard Rupp
>Subject: Re: [ccp4bb] nature cb3 comment pdf
>
>
>attached, Miri
>
>
>On Thu, 16 Aug 2007, Bernhard Rupp wrote:
>
> > my nature web connection just died for good (probably a preventive
> > measure..)
> > Could someone kindly email me the pdfs of the comment and response?
> > Thx br
> > -
> > Bernhard Rupp
> > 001 (925) 209-7429
> > +43 (676) 571-0536
> > [EMAIL PROTECTED]
> > [EMAIL PROTECTED]
> > http://www.ruppweb.org/
> > -
> > People can be divided in three classes:
> > The few who make things happen
> > The many who watch th

Re: [ccp4bb] nature cb3 response

[price]

A comment from my collaborator's student suggests a partial 
answer.  This afternoon he happened to say "but of course the 
reviewers will look at the model, I just deposited it!".  He was 
shocked to find that "hold for pub" means that even reviewers can't 
access the data.  Can that be changed?  It would take a bit of 
coordination between journals and the PDB, but I think the student is 
right - it is rather shocking that the data is sitting there nicely 
deposited but the reviewers can't review it.

Phoebe Rice

At 05:33 PM 8/16/2007, Bernhard Rupp wrote:

Ok, enough political (in)correctness. Irrespective of fabricated or not,
I think this points to a general problem of commercial journals and
their review process, as it seems that selling (.com) hot stuff
induces an extraordinary capability of denial.

The comment, as someone noted, does not address the allegations
at all. This is reminiscent of my dealings with Nature in two
related cases: They ignore or stonewall until the dispute is ended
with an irrelevant comment. In one case, Axel B later proved
with the correct structure that what we had commented on earlier
was entirely correct.
In the second case, the comment (by some of the leading experts,
not just by me nobody) was rejected with no recourse based on another
non-fact-addressing author comment and not published at all.

Compare this to a similar case, when the Jacs editor (.org <--) contacted me

on its own accord to check for a related problem, leading to retraction
of the paper after the editor (a scientist himself) evaluated
facts and response.

It also seems to depend on the handling Nature editor. I have made maps of
several structures from data unhesitantly provided by the editor when I
had reason to ask for them during review. Those were also responsive to
a mini-table-1-comment I sent on cb3, but I did not hear from the editor
assigned to cb3.

This time again, the review completely failed (table 1 and comment issues),
and
the editorial process failed as well, because the response is not adequate.
If someone - as tentatively and tactfully it may have been phrased - accused

me of faking data they'd eat shit until hell freezes over

It is as simple as that: Extraordinary claim (super structure, bizarre stats
and properties) requires extraordinary proof. This rule has not been
followed, which reflects poorly on the scientific process in this case.

I also note that in no case known to me, persons involved in irregularities
have ever appeared as frequent (or at all) communicators on the ccp4bb.

As long as grant review and tenure committees rely on automated
bibliometrics
and impact factors (and who knows who) to decide academic careers and
funding,
the big journals will remain the winners. The system has become
self-perpetuating.

Back to grant writing now.
Need to get that paper out to nature...

Cheers, br

PS: it is pointless flaming me. I am the messenger only.

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Bernhard Rupp
Sent: Thursday, August 16, 2007 2:03 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] nature cb3 comment pdf

thxthxthx to all the day and night owls for the many copies
The winners have been selected, no more entries needed.
thx again br

-Original Message-
From: Miriam Hirshberg [mailto:[EMAIL PROTECTED] On Behalf Of Miriam
Hirshberg
Sent: Thursday, August 16, 2007 1:58 PM
To: Bernhard Rupp
Subject: Re: [ccp4bb] nature cb3 comment pdf


attached, Miri


On Thu, 16 Aug 2007, Bernhard Rupp wrote:

> my nature web connection just died for good (probably a preventive
> measure..)
> Could someone kindly email me the pdfs of the comment and response?
> Thx br
> -
> Bernhard Rupp
> 001 (925) 209-7429
> +43 (676) 571-0536
> [EMAIL PROTECTED]
> [EMAIL PROTECTED]
> http://www.ruppweb.org/
> -
> People can be divided in three classes:
> The few who make things happen
> The many who watch things happen
> And the overwhelming majority
> who have no idea what is happening.
> -
>


---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/index.html
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html 

Re: [ccp4bb] domains missing

[price]

Hi,
  Did you do rigid body refinement or regular minimization?  If over 
half your structure is missing, anything besides rigid body 
refinement will probably just bias the phases toward "nothing" in the 
rest of the asymmetric unit.
  I've had correct molrep solutions with R-factors that high, so 
there is hope.  But depending on how you refined your partial model, 
you could have a completely wrong solution with that R-factor as well.
  How sure are you that your solution is really correct?  If you 
take the rigid-body-refined-only model (to minimize phase bias), and 
delete something you know should be well-ordered (a fat side chain if 
you've got good data, a helix if you've got lousy data), then make an 
Fo-Fc map, does it reappear?  If not, forget all the statistics and 
start over with your searches.  If yes, can you see any sign of the 
other domains in that map, that solvent modification might help bring 
up from the land of noise?

   Phoebe Rice

At 02:38 PM 8/13/2007, you wrote:

Hi, ccp4 community,
I am solving my protein (300 aa) structure using molecular 
replacement. The space group is P622. There is only one molecule in 
the ASU. The protein is supported to have three domains. We have 
solved the domain 1 (120aa) structure; therefore we tried to use it 
as a model to solve the new structure. MolRep and Phaser can find 
the domain 1. We refined the model and  the R and Rfree factor was 
around 50%. When we used Coot to see the model and map, we found 
that the other two domains are missing. In the map, there is a big 
'hole' and little extra density (with 6-fold) inside. It is a big 
surprise to us. Is there any possibility that R is around 50 if the 
other two domains are missing?


Any suggests to figure out what happen to my structure?
Thanks a lot!

Mousheng Wu


---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/index.html
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html 

Re: [ccp4bb] resolution vs ramachandran

[price]

Every modern structure should be "above average" by procheck criteria.
For modest resolution structures, the availability of ncs restraints 
should have a big effect on one's Ramachandran expectations:  I've 
found ncs restraints at 3-3.5A do wonders for the Ramachandran plot 
even when they don't do much for Rfree.

Phoebe

At 11:44 AM 8/3/2007, Edward Berry wrote:

Procheck puts out such a correlation (% most favorable
vs resolution) in the _04.ps file. For example look at
page 7, first panel of the sample procheck output at:
http://sb20.lbl.gov/SQR/procheck-2H88.pdf
It appears that 83.5% would be well above average
for a 3 A structure according to procheck statistics.
(However I think most structures nowadays are
"above average" by procheck statistics)

Ed

Xiaofei Jia wrote:


Dear all,

I am now preparing my structure for deposition.  The
crystal diffracts to 3.0 A. R: 0.20; R free: 0.25.
What I am concerned with is the Ramachandran plot;
83.5% in core region, 15.8% in allowed, 0.5 % in
general allowed,0.2% in disallowed region. The model
fits in density pretty well and all the attempts to
improve Ramachandran have not been successful after
quite a few trials. I am wondering if 83.5 % in core
region is acceptable with 3.0 A resolution data? Moreover, is there 
some numeric correlation between

diffraction resolution and model Ramachandran?  Thank
you for your help.
Xiaofei



Moody friends. Drama queens. Your life? Nope! - their life, your 
story. Play Sims Stories at Yahoo! Games.

http://sims.yahoo.com/


---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/index.html
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html 

Re: [ccp4bb] Protein-DNA complex for crystallization

[price]

Dear Kumar,
One often has to try duplexes with many different 
ends before getting decent crystals (e.g. 18 for 
one project in my lab, even more for others).
Depending on your Kd, you might find that your 
complex falls apart during gel filtration.
How long are your oligos?  Gel purification 
sometimes helps us and sometimes doesn't, but 
longer ones (> 20-30nt) are more likely to benefit from it.
Did you check the concentrations yourself before 
annealing? (my students get better results when they do).
Can your oligos hairpin?  If you have a dimer 
that binds a symmetric DNA site, having 
individual monomers bound to hairpinned oligos 
would certainly make a mess of your xtals.
If you think you have an excess of one single 
strand, you could try annealing small amounts at 
several different ratios, and check how much is 
really duplex on a native agarose or acrylamide gel.
Have you checked your prep for nuclease 
contamination?  Just incubate some with a 
supercoiled plasmid and ~10mM Mg++ for a couple 
hours and see if the plasmid stays 
supercoiled.  This is a beautifully sensitive 
assay because cleaving only 1 bond out of 
thousands will change the plasmid's mobility - 
but bear in mind it will only reveal endonucleases, not exonucleases.
Finally, its always a good idea to pull up a pile 
of old papers and skim their methods sections for inspiration.

Good luck!
Phoebe Rice

At 11:01 AM 7/16/2007, you wrote:

Hi,
I am trying to crystallize a protein-DNA 
complex. I purify the protein finally

using gel filtration. I purchase
single stranded complementary oligos (desalting from idtdna.com), mix them up
and make DNA duplex by
heating to 95 degree C and cooling to room 
temperature. I mix protein and DNA,

concentrate and use it
for crystallization.
I am geting small crystals consistently under a specific condition. These
crystals take up IZIT dye but are
not well shaped. I am not able to improve the size and shape of the crystals
substantially even after
screening with additives (Hampton research).
I suspect that purity of the duplex DNA (presence of unpaired oligos) is
limiting the chances of obtaining
better crystals.

How can I purify the duplex DNA further?

Are there better ways of making protein-DNA complex for crystallization?

If I make the protein ­DNA complex and then do the gelfiltration, will the
complex purified so be a better
choice for crystallization?

Thank you
Kumar

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


---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/index.html
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html 

[ccp4bb] Post Doctoral Position: Microfluidic Protein Crystallization, University of Chicago

[Jessica Price]

Crystallization and structural biology of soluble and membrane
proteins using microfluidics. This NIH-funded project at the
University of Chicago aims to develop technologies for
crystallization and structural analysis of proteins in
nanoliter volumes (see PNAS 2006 103: 19243-19248). See
http://ismagilovlab.uchicago.edu/positions.html for details.

Experience in expression, purification, crystallization, and
structural analysis of macromolecules is essential. Experience
with membrane proteins, strong leadership and communication
skills, interest in technology development are highly
desirable. Experience in microfluidics is not required.

We welcome outstanding postdoctoral applications from
talented, motivated individuals. Respond with a CV and a brief
statement describing your expertise and interests to
[EMAIL PROTECTED]

Re: [ccp4bb] Highest shell standards

[price]

Isn't automatically included fabricated data for missing reflections 
a really bad idea for anisotropic data where most reflections are 
"missing" at high resolution?  Shouldn't there be a big flashing red 
flag alerting the user to what's been done?

Phoebe

At 01:22 PM 3/26/2007, Edward A. Berry wrote:

Actually I was thinking of a somewhat earlier paper:

Rayment,I. Molecular relacement method at low resolution:
optimum strategy and intrinsic limitations as determined
by calculations on icosahedral virus models.
Acta Crystallogr. A 39, 102  116 (1983).

But thanks for bringing the Caliandro et al. paper to my attention.
Thanks also to Fred. Vellieux for his comments, and to Pete Dunton
for explaining to me that while fft doesn't do fillin by default,
the 2MFo-DFc map coefficients from refmac5 do have fillin values
for the missing reflection, making model bias a problem when
many missing residues are included.

Now I understand Petrus's question.

Ed

Michel Fodje wrote:

You are probably referring to the following works:
Caliandro et al, Acta Cryst. D61 (2005) 556-565
and Caliandro et al, Acta Cryst. D61 (2005) 1080-1087
in which they used density modification to calculate phases for
unmeasured reflections, and used the phases to extend the resolution by
calculating rough estimates unmeasured amplitudes. Using this technique
they actually could improve the electron density.
If I'm not mistaken, George Sheldrick has implemented this "Free Lunch"
algorithm in SHELXE.
/Michel
On Fri, 2007-03-23 at 08:05 -0800, Edward Berry wrote:


If instead you allow the missing F's
to "float", calculating them on each cycle from the previous map
using the fillin option, someone has shown (don't have the
reference handy at the moment) that the F's tend toward the true F's
(in the case that they weren't really missing but omitted as part
of the test).

Ed


---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/index.html
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html 

Re: [ccp4bb] protease cleavage sites

[price]

TeV is great but it isn't always a magic bullet for producing native 
N-termini:  we've experimentally determined the obvious, that if you 
bury part of its recognition site in secondary structure, it cleaves 
very very slowly.  We tried this on a protein where M1 is cleaved in 
vivo and aa#2 is the beginning of a nice helix, but the sequence 
itself is compatible with TeV sites.  Adding a couple small flexible 
residues between the cleavage site and the beginning of the folded 
structure did wonders for the cleavage rate.

Phoebe

At 04:01 AM 3/2/2007, Rene Frank wrote:

Hi,

A non-ccp4 Q. Sorry.

I would like to use a cleavable purification tag at the 
N-terminus/extracellular end of my membrane protein for 
purification. Before I start, I wonder if someone could recommend a 
particular protease site that I can engineer between the tag and my 
protein?  How about a proprietary cleavage system such as the 
PreScission protease (GE Healthcare)? I  would be grateful to hear 
success and horror stories in this area.


Best wishes,

Rene


Dr R.A.W. Frank, PhD
Royal Commission for the Exhibition of 1851 Research Fellow

Prof Seth Grant Lab / Genes to Cognition
Wellcome Trust Sanger Institute
Hinxton
Cambridge CB10 1SA

Work Tel: 0044 (0)1223 834244 ext. 7318
Cell No.: 0044 (0)7870 208280
===




---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/index.html
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html 

Re: [ccp4bb] Rfactor does not drop

[price]

I would take a well-ordered helix out of your 
allegedly-correctly-placed model (BEFORE doing any refinement other 
than rigid body), phase a 2Fo-Fc and an Fo-Fc map with it, and see if 
the helix shows up.  If it does, there is some reality in your 
solution.  If it doesn't, start over from scratch.
Actually, at your resolution a few fat side chains might do as a 
reality check rather than an entire secondary structure element.  In 
fact, you might start by looking at lower-resolution maps, since you 
almost certainly haven't phased the high-res terms properly.

Phoebe

At 05:26 PM 2/18/2007, Tim Gruene wrote:

Hi,

I dare say with an R-factor of 0.64, your MR solution is simply wrong.
Just into the blue, you could
1) try various MR-programs  (phaser, molrep, amore...)
2) try a different search model
3) try your best model but start with subdomains or chop off possibly
   flexible parts
4) have a look at the CaspR server
   http://www.igs.cnrs-mrs.fr/Caspr2/index.cgi
5) with crystals that give such good data try to get other pahse
   information (soaks, co-crystallisation, SAD (SAD at home), etc.)
Good luck, Tim

--
Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A


On Fri, 16 Feb 2007, Emmanuel Prata wrote:


Dear all,

I have a data set of a protein soaked with sacarose, and the statistic
data is good until the molecular replacement (Rmerge 0,07 (24%),
I/Sigma 7 (2,7), completness (99,8 (98%)), and resolution 1,98A. After
this step, I can not get the Rfactor (64%) to drop (I believe the
spacegroup is correct).
Could anyone give me suggestions?
Thank you in advance,
Emmanuel Prata


---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/index.html
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html 

Re: [ccp4bb] : misbound ligand examples?

[price]

Thanks!
Phoebe

At 05:56 PM 1/22/2007, you wrote:

Hi Pheobe,
I remember an interesting paper that described how a structure revealed a 
surprising role the buffer was playing in inhibition:


The 1.20 A resolution crystal structure of the aminopeptidase from 
Aeromonas proteolytica complexed with tris: a tale of buffer inhibition.


* Desmarais WT,
* Bienvenue DL,
* Bzymek KP,
* Holz RC,
* Petsko GA,
* Ringe D.


http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=12176384

-Original Message-
From: CCP4 bulletin board on behalf of [EMAIL PROTECTED]
Sent: Mon 1/22/2007 3:29 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: : misbound ligand examples?

A biochemist friend asked for examples of cases were a protein was
co-crystallized with or soaked in a ligand that bound in the wrong place -
say, because the ligand used wasn't quite the right one or because other
important ligands were absent.
I'm sure such examples are out there, especially when soaks were done at
high concentrations, but I'm having trouble thinking of concrete examples.
Help?
thanks,
Phoebe Rice


---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/index.html
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html


---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/index.html
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html 

: misbound ligand examples?

[price]

A biochemist friend asked for examples of cases were a protein was 
co-crystallized with or soaked in a ligand that bound in the wrong place - 
say, because the ligand used wasn't quite the right one or because other 
important ligands were absent.
I'm sure such examples are out there, especially when soaks were done at 
high concentrations, but I'm having trouble thinking of concrete examples.

Help?
thanks,
Phoebe Rice


---
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/index.html
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html