Re: [ccp4bb] Showing Stick Representation In Coot

[Edwin Pozharski]

I don't know the explicit answer to your question, but you can always copy
active site residues into a separate model and use different representation
for that.

On Tue, Aug 27, 2024 at 8:52 AM Firdous Tarique 
wrote:

> Hello everyone.
>
> Is there any way to show stick representation of the selected amino acid
> residues in Coot while everything else is represented in the form of
> C-alphas/Backbone. I want to show the amino acids of the catalytic centre
> in the stick form and not alter the C-alphas/Backbone conformation for the
> rest of the molecule. Just curious if it can be done in Coot or not.
>
> Best
>
> Firdous
>
> --
>
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Re: [ccp4bb] To Trim or Not to To Trim

[Edwin Pozharski]

The most pertinent question is, of course, what is the average frequency of
the disordered chain controversy flareup.  Once we figure that out, some
profound mysteries of the Universe will reveal themselves.  I am betting
it's a simple combination of the solar cycle, inflation adjusted price of a
bag of roasted chestnuts at Heinzels Wintermärchen and certainly the time
elapsed since the most recent cicada emergence in Montgomery
County, Maryland.  I am still working on the fudge factors, but maybe I
should just ask a neural network.

Aha, here is the (non)-answer to "What should I do about disordered
sidechains"

Disordered sidechains can be a common issue in protein structures,
> particularly in regions that are flexible or have low electron density. One
> approach is to manually adjust the position of the sidechain using software
> tools such as Coot or PyMOL. However, if the resolution of the protein
> structure is low or the disorder is extensive, it may be difficult to
> accurately position the sidechain. In such cases, it may be necessary to
> consider alternative approaches such as mutagenesis, site-directed labeling
> or multiple conformer modeling. The specific approach would depend on the
> particular protein structure being studied and the goals of the research
> project.


Joking aside, here is what I learned from the vigorous dead horse beatings
in the past.

- Some people like keeping adding invisible atoms to side chains and
letting B factors sort it out, Caedite eos. Novit enim Dominus qui sunt
eius.
- Some people like to remove invisible atoms.  These weirdos claim that all
atoms should be treated equally, and that adding a bunch of arginine atoms
is not different from completing the his-tag or adding hydrogens at medium
resolution.  They point out that adding disordered side chains (marginally)
increases statistical error on the rest of the well defined structure (
https://tinyurl.com/yvz73ak2) and refer to the practice as orwellian.
Snobs, basically.
- A small minority assigns zero occupancy to disordered atoms.  I admire
them as they have gone beyond agnosticism.  Let me give you coordinates for
the atom, they say.  Let me then tell you that atoms are never there and
thus have zero occupancy at that position.  Meaningless, you say?  Well, so
is your existence and Santa Claus is your parents.

Now that my telomeres are a few hundred units shorter than they were last
time I whipped myself up into a frenzy over disordered side chains, I would
summarize my views this way.

*People are free to choose how to interpret experimental data that they
themselves obtained. *
*Published structures are reported to the PDB, and if someone doesn't like
what I did to my arginines - well, interpret it your own way, I am not
stopping you. *

Also, this (with full appreciation of the formal ccp4bb focus on
computational crystallography - but come on, you are not here for the
maximum likelihood formalism)

*I am yet to see an example of when different modeling of a disordered side
chain 30 angstroms away from the area where actual molecular biology
happens has changed conclusions derived from protein structure in question.*

Special thanks to Adrian Goldman for changing my attitude on this specific
point years ago.

Cheers.

---
I don't know why the sacrifice didn't work. The science seemed so solid.
Julien XIII, Lord of the Lemurs

On Thu, Mar 9, 2023 at 8:26 PM Rhys Grinter <
22087c81e8c6-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hi All,
>
> I'm trying to crowdsource an opinion on how people deal with modelling
> side chains with poorly resolved electron or cryoEM density.
>
> My preference is to model the sidechain and allow the B-factors to go high
> in refinement to represent that the side chain is flexible. However, I'm
> aware that some people truncate sidechains if density is not present to
> justify modelling. I've also seen models where the sidechain is modelled
> but with zero occupancy if density isn't present.
>
> Is there a consensus and justifying arguments for why one approach is
> better?
>
> Cheers,
>
> Rhys
>
>
> --
>
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Re: [ccp4bb] High-order oligomers vs robust crystals - references?

[Edwin Pozharski]

One could use PISA to get a rough distribution of oligomerization states of
proteins in the PDB and compare bacterial vs mammalian... there always will
be a question though of whether any bias is inherent in proteins or driven
by crystallizability itself.

Personally, I always though that bacterial proteins crystallize better
because they express better and are more stable.  All of this is hand
waiving on my part, of course.

---
I don't know why the sacrifice didn't work. The science seemed so solid.
Julien XIII, Lord of the Lemurs

On Fri, Nov 12, 2021 at 9:52 AM Frank von Delft 
wrote:

> Hello all
>
> Two decades ago, I remember (!) much talk about a reason that bacterial
> proteins crystallize "more easily" is that they tend to come as
> oligomers (dimers and up), and that this internal symmetry made them
> happier to crystallize.
>
> Did anybody ever publish hard evidence?  Or even, is there a primary
> citation for the idea?
>
> Thanks
> Frank
>
> 
>
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Re: [ccp4bb] simulation of structural changes in the protein

[Edwin Pozharski]

Any molecular dynamics simulation would have a temperature parameter.
GROMACS is free and easy to use. And do find a computational biology expert
to consult with if this is more than an excercise.

On Wed, Nov 10, 2021, 11:20 PM Dr Muhammad Saleem 
wrote:

> Dear All,
>
> I was wondering if there is any webserver or software to simulate the
> structural changes in the protein at different temperatures?
>
>
> regards,
> Saleem
>
>
>
>
>
>
>
>
>
> --
>
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Re: [ccp4bb] Tools for measuring RMSD of entire molecule when a single domain is aligned

[Edwin Pozharski]

https://pymolwiki.org/index.php/Rms_cur

On Fri, Nov 12, 2021, 9:05 AM Kyle Gregory <
3632e92fcc15-dmarc-requ...@jiscmail.ac.uk> wrote:

> Dear CCP4 bulletin board,
>
> I was wondering if there are any tools to determine the RMSD of the entire
> molecule (two domains) when only aligning one domain, the point i'm trying
> to make is a variation in domain positioning relative to one another but
> I'd like to quantify it. LSQ alignment in coot reports RMSD for the aligned
> porition only, as does the alignment tools on ccp4 cloud (unless i've
> missed a feature)
>
> Any advice would be greatly appreciated.
>
> Kind regards,
> Kyle
>
> --
>
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Re: [ccp4bb] H-bond modelling

[Edwin Pozharski]

There are tools such as hbplus

https://www.ebi.ac.uk/thornton-srv/software/HBPLUS/

that will calculate predicted hydrogen bonds (including ligands, you just
need to define their chemistry) throughout a structure. These tools usually
go beyond simple distance considerations and analyze further geometry
details.

Proper donor acceptor pair at 2.6A is almost without doubt a hydrogen bond.

On Sun, Jun 6, 2021, 4:17 PM vivek sharma  wrote:

> Dear all,
>
> How does one validate if 2 atoms (non-hydrogen) which are close enough,
> actually participate in H-bonding?
> I have a recent data that i am currently working on, the NH2 of ARG is at
> 2.61A from ligand's oxygen atom (refer to attached image), since there is
> no electron density for hydrogens, how can i be sure if i am modelling
> ligand correctly?
>
> thanks
>
> --
>
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Re: [ccp4bb] Contagious, Self-Distributing "Vaccines?"

[Edwin Pozharski]

I guess for such vehicle to be "extremely contagious" (or contagious at all
for that matter) it should be capable of rapidly multiplying inside the
host, so that it outruns immune system mediated destruction for at least
some time in order to be present in high enough concentration to
effectively spread via aerosols.  Given the range of immunodeficiencies
present in any population, you are essentially guaranteed to kill at least
some people whose immune system will not be able to cope with rapidly
multiplying virus.  You can theoretically fine tune the lethality of such
virus to make sure that number of people you thus murder will be less than
those that die either in no vaccine or traditional vaccination scenario,
but that would be ethical equivalent of that modern crypto fascist
suggestion that we just have to take it easy until herd immunity is
established, even though few million grandparents will die in the process
while the rest of us enjoy indoor dining.



On Wed, Feb 17, 2021 at 12:33 PM Jacob Keller 
wrote:

> It would seem to me that it should be possible to generate versions of the
> Covid virus that would:
>
> A. be extremely contagious and yet
> B. be clinically benign, and
> C. confer immunity to the original covid virus.
>
> If, then, this virus could be released, with appropriate "kill switch"
> safeguards built in, would this not solve the world's pandemic problems? Is
> there any reason, practically, why this approach would not be feasible?
>
> Maybe we don't really know enough to manipulate A, B, C yet?
>
> Or maybe it's too scary for primetime...nightmare bio-warfare apocalypse?
>
> Has this sort of thing been done, or does it have a name?
>
> Jacob
> --
>
> +
>
> Jacob Pearson Keller
>
> Assistant Professor
>
> Department of Pharmacology and Molecular Therapeutics
>
> Uniformed Services University
>
> 4301 Jones Bridge Road
>
> Bethesda MD 20814
>
> jacob.kel...@usuhs.edu; jacobpkel...@gmail.com
>
> Cell: (301)592-7004
>
> +
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>



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[ccp4bb] postdoctoral position at University of Maryland - IBBR/Rockville, structural biology

[Edwin Pozharski]

Postdoctoral position is available at the University of Maryland School of
Medicine Center for Biomolecular Therapeutics.  Researcher will be located
at the IBBR in Rockville, Maryland (one hour train ride from downtown
Washington - yes, right now visiting its multiple attractions of various
kinds is tricky, but this shall pass).  Position will be closely associated
with recently established cryoEM capabilities and focus on several
structural biology projects.  Strong background in protein production in
context of structural biology, and previous experience in X-ray
crystallography, NMR spectroscopy and use of biophysical methods is
preferred, as is proficiency in computational aspects of structural
biology.  T32 eligibility (recent PhD and US citizenship/permanent resident
status) is considered but not strictly required.

Please submit a CV and (optionally) a short cover letter to Ed Pozharski (
epozhars...@som.umaryland.edu).  References will be requested as needed.

Edwin Pozharski, PhD
Assistant Professor
Department of Biochemistry and Molecular Biology
Center for Biomolecular Therapeutics
University of Maryland School of Medicine
Institute for Bioscience and Biotechnology Research
9600 Gudelsky Dr., 4127D Bldg 2
Rockville, MD 20850



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Re: [ccp4bb] number of frames to get a full dataset?

[Edwin Pozharski]

Replicate is a good option with its own problems as it can be seen as
referring to exact copy which multiple measurements clearly aren't. It does
have an advantage of being the word used by non-crystallographers though.

As a lame attempt at joke, use of terms redundancy and multiplicity to
describe the same concept is by itself redundant.

On Mon, Jun 29, 2020, 7:21 PM Bernhard Rupp 
wrote:

> Ah…the rise of the replicants …
>
>
>
> https://www.youtube.com/watch?v=yWPyRSURYFQ
>
>
>
> …and don’t forget the and the Voight-Kampff Test results in Table 1.
>
>
>
> Best, BR
>
>
>
> *From:* Pierre Rizkallah 
> *Sent:* Monday, June 29, 2020 15:46
> *To:* b...@hofkristallamt.org; CCP4BB@JISCMAIL.AC.UK
> *Subject:* RE: [ccp4bb] number of frames to get a full dataset?
>
>
>
> You’re missing out on a grand opportunity for iconoclasm here. Try out
> ‘Degree of Replication’ or ‘Average Replication’ or ‘Replicate Frequency’.
> Any other offerings!
>
>
>
> P.
>
> ***
>
> Dr Pierre Rizkallah, Senior Lecturer Structural Biology
>
> Institute of Infection & Immunology, Sir Geraint Evans Building,
>
> School of Medicine, Heath Campus, Cardiff, CF14 4XN
>
> email: rizkall...@cardiff.ac.ukphone: +44 29 2074 2248
>
> http://www.cardiff.ac.uk/people/view/126690-rizkallah-pierre
>
>
>
> *From:* CCP4 bulletin board  *On Behalf Of *Bernhard
> Rupp
> *Sent:* 29 June 2020 23:36
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] number of frames to get a full dataset?
>
>
>
> I think it is time to escalate that discussion to crystallographic
> definition purists like Massimo or to a logical consistency proponent like
> Ian who abhors definitional vacuum 😊
>
>
>
> Cheers, BR
>
>
>
> *From:* CCP4 bulletin board  *On Behalf Of *Andreas
> Förster
> *Sent:* Monday, June 29, 2020 15:24
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] number of frames to get a full dataset?
>
>
>
> I like to think that the reflections I carefully measured at high
> multiplicity are not redundant, which the dictionary on my computer defines
> as "not or no longer needed or useful; superfluous" and the American
> Heritage Dictionary as "exceeding what is necessary or natural;
> superfluous" and "needlessly repetitive; verbose".
>
>
>
> Please don't use the term Needless repetitivity in your Table 1.  It sends
> the wrong message.  Multiplicity is good.
>
>
>
> All best.
>
>
>
>
>
> Andreas
>
>
>
>
>
>
>
> On Tue, Jun 30, 2020 at 12:03 AM James Holton  wrote:
>
> I have found that the use of "redundancy" vs "multiplicity" correlates
> very well with the speaker's favorite processing software.  The Denzo/HKL
> program scalepack outputs "redundancy", whereas scala/aimless and other
> more Europe-centric programs output "multiplicity".
>
> At least it is not as bad as "intensity", which is so ambiguous as to be
> almost useless as a word on its own.
>
> -James Holton
> MAD Scientist
>
> On 6/24/2020 10:27 AM, Bernhard Rupp wrote:
>
> > Oh, and some of us prefer the word 'multiplicity' ;-0
>
> Hmmm…maybe not. ‘Multiplicity’ in crystallography is context sensitive,
> and not uniquely defined. It can refer to
>
>1. the position multiplicity (number of equivalent sites per unit
>cell, aka Wyckoff-Multiplicity), the only (!) cif use of multiplicity
>2. the multiplicity of the reflection, which means the superposition
>of reflections with the same *d*  (mostly powder diffraction)
>3. the multiplicity of observations, aka redundancy.
>
> While (a) and (b) are clearly defined, (c) is an arbitrary experimental
> number.
>
> How from (a) real space symmetry follows (b) in reciprocal space
> (including the epsilon zones, another ‘multiplicity’) is explained here
>
> https://scripts.iucr.org/cgi-bin/paper?a14080
> 
>
> and also on page 306 in BMC.
>
> Too much multiplicity might create duplicity…
>
> Cheers, BR
>
>
>
> Jon Cooper
>
>
>
> On 23 Jun 2020 22:04, "Peat, Tom (Manufacturing, Parkville)" <
> tom.p...@csiro.au> wrote:
>
> I would just like to point out that for those of us who have worked too
> many times with P1 or P21 that even 360 degrees will not give you 'super'
> anomalous differences.
>
> I'm not a minimalist when it comes to data- redundancy is a good thing to
> have.
>
> cheers, tom
>
>
>
> Tom Peat
> Proteins Group
> Biomedical Program, CSIRO
> 343 Royal Parade
> Parkville, VIC, 3052
> +613 9662 7304
> +614 57 539 419
> tom.p...@csiro.au
>
>
> --
>
> *From:* CCP4 bulletin board  on behalf of
> 0c2488af9525-dmarc-requ...@jiscmail.ac.uk <
> 0c2488af9525-dmarc-requ...@jiscmail.ac.uk>
> *Sent:* Wednesday, June 24, 2020 1:10 A

Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] Better Beamline suggestion!

[Edwin Pozharski]

>
>
> In the absence of such you can resort to carefully bending the loop or
> bending the pin (Jim Holton made a nifty device for bending the pin) while
> keeping the xtal bathed in the cold stream.
>
>
 I would also mention these

https://hamptonresearch.com/product-Adjustable-Mounted-CryoLoop-385.html



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Re: [ccp4bb] Problem in real space - please sign & invite other scientists to sign this letter

[Edwin Pozharski]

Dear Daniel,

with all due respect, I do believe that you are making several mistakes in
your argument.

English is not my native tongue, but I suspect that there is a substantial
difference between "author" and "signatory".  What people are asked to do
here is essentially to sign a petition, not to become a co-author in
traditional sense.  There were 39 signatories to the US Constitution,
certainly not all of them are considered its authors.

Furthermore, most structural biologists are trained scientists and it is
rather routine part of our job to evaluate research we are not exactly
experts in.  I am not a climatologist, but I do take exception to your
assertion that I am therefore automatically too ignorant to understand
basic concepts that pertain to global warming and climate change.  A
climatologist wouldn't instantly know what B-factors are, but is certainly
capable of understanding the concept if you explain it.

Using physical science and its data to arrive at conclusions regarding
religion, politics and economic theory (!) is not at all embarrassing.
(And letter in question hardly does any "preaching", certainly not about
religion)

As for your demand that people stick to structural biology, may I suggest
that your reaction to exactly one entry in ccp4bb that elicited almost zero
follow up (until your comment) feels a bit overblown?  If you strongly
disagree with Dr Ripple and his colleagues, that is fine, but why shouldn't
people at ccp4bb occasionally share somewhat orthogonal information?  None
of us want to see inappropriate content, I am just not sure why you feel
that this specific post is something that needs to be purged.

Just to be clear - your post does create an impression that you might hold
the opinion that, as they say, "global warming is a hoax".  Please, let's
not have further discussion online on the specifics, but I think it might
be helpful if you could confirm or deny this.

Cheers,

Ed.

---
"I disapprove of what you say, but I will defend to the death your right to
say it"
Evelyn Beatrice Hall, "The Friends of Voltaire"

On Tue, Aug 20, 2019 at 9:23 PM Daniel M. Himmel, Ph. D. <
danielmhim...@gmail.com> wrote:

> Dear colleagues,
>
>
>
> Since when does being a structural biologist make us experts in
> climatology,
>
> and isn't it a breach of basic ethical practice and professionalism as
> scientists
>
> to sign on as authors to an article for which we have neither contributed
>
> research nor intellectual content of the manuscript?  Are we now going
> against
>
> the standard to which the editorial policies of leading reputable
> biological
>
> journals normally hold us as authors?  And doesn't it hurt the credibility
>
> of a serious scientific article, its authors, and the journal in which it
> appears
>
> if biologists with no expertise in earth science/astrophysics appear
>
> without humility as authors to such an article?
>
>
>
> Are you not embarrassed to put your name to an article that uses physical
>
> sciences data as a platform for preaching about religion, politics, and
> economic
>
> theory ("...social and economic justice for all...")?
>
>
>
> Does it not upset you when someone unfamiliar with structural biology draws
>
> firm conclusions that heavily depend on the part of a structural model
> that has high
>
> B-factors?  So why are you unconcerned that you may be guilty of an
> analogous
>
> error when, as structural biologists, you put your name to a controversial
> interpretation
>
> of selected earth science data?  See, for example,
>
> https://blogs.agu.org/geospace/2017/02/24/living-warm-peak-ice-ages/
> about the ways
>
> climate data can be misinterpreted by choosing too tight a time interval,
> and lets stick to
>
> structural biology and allied sciences in the CCP4 list, please.
>
>
>
> Respectfully,
>
> Daniel M. Himmel
>
>
>
>>
> --
>
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>



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Re: [ccp4bb] Mandatory mmCIF format for crystallographic depositions to the PDB

[Edwin Pozharski]

But my 80 symbols... :)



On Wed, Feb 20, 2019 at 3:08 AM John Berrisford  wrote:

> From July 1st 2019 onward, mmCIF format files will become mandatory for
> crystallographic depositions to the Protein Data Bank. PDB format files
> will no longer be accepted for deposition of structures solved by these
> techniques.
>
> PDBx/mmCIF will be the only format accepted for deposition of PDB
> structures resulting from macromolecular crystallography (MX), including
> X-ray, neutron, fiber, and electron diffraction methods *via *OneDep
> starting July 1st 2019. The deposition of PDBx/mmCIF format files will
> improve the efficiency of the deposition process and enhance validation
> through capture of the more extensive experimental metadata supported by
> PDBx/mmCIF, compared to the legacy PDB format.
>
> For information about these changes, please visit the wwPDB news page
> . If
> you have any queries or comments regarding these changes, please contact
> the wwPDB consortium via deposit-h...@mail.wwpdb.org.
>
> The wwPDB consortium
>
>
>
>
>
>
>
> --
>
> John Berrisford
>
> PDBe
>
> European Bioinformatics Institute (EMBL-EBI)
>
> European Molecular Biology Laboratory
>
> Wellcome Trust Genome Campus
>
> Hinxton
>
> Cambridge CB10 1SD UK
>
> Tel: +44 1223 492529
>
>
>
> http://www.pdbe.org
>
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>
> http://twitter.com/PDBeurope
>
>
>
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Re: [ccp4bb] SO4 or PO4

[Edwin Pozharski]

Consider removing sulfate from the mother liquor after crystals are
formed.  I recall having success replacing ammonium sulfate with sodium
malonate (it's a nice cryoprotectant as well).  This is assuming that
ammonium sulfate is your major precipitant.  If it's just an additive (say,
your conditions are some PEG + 0.2M ammonium sulfate), try transferring
crystal to whatever PEG/glycerol combo you need for cryoprotection minus
ammonium sulfate.

Strictly speaking, this is not answering your original question, as it
suggests to recollect data instead of advice on how to distinguish sulfate
from phosphate in your density.

On Sat, Feb 16, 2019 at 4:07 AM 张士军 <21620150150...@stu.xmu.edu.cn> wrote:

> Dear all
>
> I have got a crystal grown at the condition both have ion of SO4 and PO4,
> and the diffraction resolution is very well, but the problem is coming: how
> to tell which is which just from electron density? I think they are exactly
> same. Thanks a lot !!!
>
> Beat Regards
>
> Shijun
>
> --
>
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Re: [ccp4bb] [EXTERNAL] [ccp4bb] refmac same residue different names

[Edwin Pozharski]

Herman,

thanks - however, it seems like I have poorly worded my question.  I do
know how to generate alternate conformers, what the PDB ATOM record format
is etc.  The point was that when I have alternate conformers that carry the
same residue ID but different residue types, Refmac exits with the error.
The question was whether there is a "native" solution to this that does not
include some pdb file acrobatics (i.e. separating the alternative type into
a separate residue and enforcing connectivity using elaborate LINK
records).   Based on what I see so far, there likely isn't any such native
option.  Whether these situations are common enough to warrant (possibly
elaborate) software changes is a separate question.

Cheers,

Ed.

---
I don't know why the sacrifice didn't work. The science seemed so solid.
Julien XIII, Lord of the Lemurs

On Wed, Feb 6, 2019 at 2:36 AM  wrote:

> Dear Edwin,
>
>
>
> I do not know whether your question has been answered already, but the
> answer is simple: you have to define alternative conformations. Easiest is
> to generate them in coot with the “add alternate conformation” option in
> the right panel. You may have to delete the original unlabeled alternative
> conformation first though.
>
> Alternatively, if you want to keep the original coordinates, or if the
> alternative residue is different: say a Leu versus a Phe you can open the
> pdb file with an editor and generate the alternative conformation yourself:
>
> One of the residues gets an “A” in front of the residue name, e.g. ALEU,
> and the alternative residue a “B”, say BLEU. You also have to reset the
> occupancies to 0.5 for both conformations (or different fractions which add
> up to one).
>
>
>
> Good luck!
>
> Herman
>
>
>
> *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von
> *Edwin Pozharski
> *Gesendet:* Montag, 4. Februar 2019 22:35
> *An:* CCP4BB@JISCMAIL.AC.UK
> *Betreff:* [EXTERNAL] [ccp4bb] refmac same residue different names
>
>
>
> Belated happy 2019, everyone.
>
>
>
> For whatever obscure reason, I need to refine a model that has two
> different residue types as alternate conformers with the same residue ID.
> Presented with a pdb file that has such feature, Refmac fails saying this
>
>
>
>  ERROR: in chain A residue: 443
> different residues have the same number
>
> There is an error in the input coordinate file
> At least one the chains has 2 residues with the same number
> Check above to see error
> ===> Error: Problem with coordinate file
>
>
>
> There are several ways of getting around this I can think of.  Perhaps
> duplicate chain with strict NCS for all but the residue in question could
> work.  Perhaps adding this residue as two separate chains and then adding
> enough LINK records to keep things in place could.  Either solution here is
> inelegant and requires reformating pdb file back to sanity prior to
> deposition.
>
>
>
> Is there some way to allow different geometries for alternate conformers
> that is native to Refmac?
>
>
>
> Cheers,
>
>
>
> Ed.
>
>
>
> PS.  I know that phenix.refine takes the mixed name pdb file straight up.
> I still want to be able to refine such structure with refmac (and buster,
> actually, but that's a question I already asked in the appropriate forum.
>
>
>
>
> Edwin Pozharski, PhD, Assistant Professor
> University of Maryland, Baltimore
> --
> When the Way is forgotten duty and justice appear;
> Then knowledge and wisdom are born along with hypocrisy.
> When harmonious relationships dissolve then respect and devotion arise;
> When a nation falls to chaos then loyalty and patriotism are born.
> -- / Lao Tse /
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] Turning off the bulk solvent modelling in Refmac5 to generate Polder maps?

[Edwin Pozharski]

It should be according to the manual

http://www.ccp4.ac.uk/html/refmac5/keywords/xray-principal.html#solv

If you using CCP4i, I believe this is done by unchecking the "Calculate the
contribution from the solvent region" box in Scaling section.

---
I don't know why the sacrifice didn't work. The science seemed so solid.
Julien XIII, Lord of the Lemurs

On Mon, Feb 4, 2019 at 6:50 AM Samuel Davis (PG Research) <
s.w.da...@dundee.ac.uk> wrote:

> Hi,
>
> I'm wondering if anyone knows if it is possible to turn off the bulk
> solvent modelling in Refmac5, for the purpose of generating Polder maps? I
> know that an option for Polder maps is directly implemented in Phenix, but
> we ideally want to use Refmac5, as we have used it for the rest of our
> refinement and want to keep it consistent if possible.
>
> Thanks,
>
> Samuel.
>
> Samuel Davis
> MRC 3.5 Year Programme PhD Student
> Life Sciences, Biological Chemistry and Drug Discovery, University of
> Dundee
> +44 (0)1382 388325 | swda...@dundee.ac.uk
>
> Scottish University of the Year
> The Times / Sunday Times Good University Guide 2016 and 2017
>
> Follow my blog: https://musingsofanearlycareerscientist.wordpress.com
>
> The University of Dundee is a registered Scottish Charity, No: SC015096
>
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[ccp4bb] refmac same residue different names

[Edwin Pozharski]

Belated happy 2019, everyone.

For whatever obscure reason, I need to refine a model that has two
different residue types as alternate conformers with the same residue ID.
Presented with a pdb file that has such feature, Refmac fails saying this

 ERROR: in chain A residue: 443
> different residues have the same number
>
> There is an error in the input coordinate file
> At least one the chains has 2 residues with the same number
> Check above to see error
> ===> Error: Problem with coordinate file


There are several ways of getting around this I can think of.  Perhaps
duplicate chain with strict NCS for all but the residue in question could
work.  Perhaps adding this residue as two separate chains and then adding
enough LINK records to keep things in place could.  Either solution here is
inelegant and requires reformating pdb file back to sanity prior to
deposition.

Is there some way to allow different geometries for alternate conformers
that is native to Refmac?

Cheers,

Ed.

PS.  I know that phenix.refine takes the mixed name pdb file straight up.
I still want to be able to refine such structure with refmac (and buster,
actually, but that's a question I already asked in the appropriate forum.


Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
-- / Lao Tse /



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[ccp4bb] include corners in mosflm

[Edwin Pozharski]

By default, iMosflm excludes corners from processing.  Is there a simple
way to make it the default to go all the way to the corner instead of
detector edge?  I could of course set the max resolution for processing to
some outrageous value that is guaranteed to be outside of the image, but
perhaps I am missing a more intelligent option in the gui.  (I vaguely
recall HKL2000 having a Edge/Corner/Other) radiobutton).

There is a whole separate question as to wisdom of including corners, of
course.  Yes, adding a resolution shell with robust data will improve model
quality even if such shell is woefully incomplete. On the other hand, it's
possible that fill-in option for missing reflections in map calculation may
make maps more biased. A reasonable solution to this would be to use 2
different resolution limits in refinement and map calculation - not hard to
script for that yet I don't know if any refinement software provides such
option natively.

Ed.

Re: [ccp4bb] refmac output

[Edwin Pozharski]

Just to clarify, how do you use the extra columns in this scenario?  My
suggestion was to have the output file that includes only the map
coefficient columns, so you still can look at the map.  IIRC, FP/SIGFP
columns from refmac output are actually modified from the input (scaled
with Boverall), so it was not recommended to use refmac output as input of
any kind.

Also, to provide context, my comment resulted from dealing with a large
chunk of data that included ~200 output mtz files - which is Gb-sized
tarball that had to be uploaded/downloaded.  Not the end of the world, but
cutting it in half seemed like a good idea at the time. :)  Not hard to
script that, of course.

I am not necessarily advocating for skinny files to be the default, but as
it stands, refmac/buster/phenix do not even provide the option of doing it
(It's entirely possible that I am wrong here on specifics and will get
corrected by Garib, Gerard and Pavel).

Cheers,

Ed.

Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
-- / Lao Tse /

[ccp4bb] refmac output

[Edwin Pozharski]

I know space is cheap these days, but is there a reason for Refmac to
generate all those extra columns in the output mtz file?  Refmac (as well
as phenix.refine and buster-tnt) output mtz file is almost always used for
only one purpose - look at the map in coot.  You only need 4 columns for
that, not 14.  Other columns are useful for testing, but why not make them
optional?

This would certainly be a low priority - one can easily delete extra
columns using, say, sftools.

Cheers,

Ed.

---
Hurry up, before we all come to our senses!
Julien, King of Lemurs

[ccp4bb] ccp4 package manager

[Edwin Pozharski]

CCP4 package manager doesn't work for me when I try a fresh ccp4
installation.  It goes through all the question dialogs, but then fails to
download the actual installation package, complaining of broken network
connection.  Multiple tries don't help.  Obviously, I can get around this
by downloading the full 750Mb suite directly, which works fine. This might
be something on my end, of course.  However, I've seen this twice now on
quite different network connections.  In fact, I haven't installed CCP4
using the package manager since what could have been couple of years.

Cheers,

Ed.
---
Hurry up, before we all come to our senses!
Julien, King of Lemurs

[ccp4bb] cif-file macro?

[Edwin Pozharski]

I expect this not to exist, but is there a way to define a variable in a
cif-file (e.g. a global esd target for, say, angles)?

I am certainly capable of putting together a bash script to emulate this,
so please don't bother with suggesting a workaround unless you really enjoy
that kind of thing :)

Cheers,

Ed

[ccp4bb] freerflag bug

[Edwin Pozharski]

FREERFLAG fails as part of the uniqueify script to complete a test set on
a cif-file downloaded from the PDB.  My observations:

1)
The bug is version-dependent - freerflag 6.2 succeeds, freerflag 1.1 fails
(comes with ccp4 6.3, apparently newer than "version 6.2" from
ccp4 6.2)
2) The input cif-file has 105382 working and 502 test
reflections (deposited pdb file confirms that as a relatively small 0.5%
test set size).
3) freerflag throws this error

Error in
LWREFL_NOEXIT: mindx 2 not open for write!
 FREERFLAG: 
LWREFL: failed to write reflection

I have not done any updates
since 6.3.0, but I do not see any mention of freerflag in updates
summary.

I can supply the input mtz file that produces the
error if needed.

Thanks,

Ed.

-- 
Edwin Pozharski, PhD
University of Maryland, Baltimore

Re: [ccp4bb] Etiquette on publishing if there is a crystallization report from someone else.

[Edwin Pozharski]

Tim,

On 09/25/2012 09:51 AM, Tim Gruene wrote:

I would assume that someone who publishes crystallisation conditions has
given up solving the structure or some other reason to encourage others
to pick up the project


there could be several situations when this is not so.  Sometimes a 
crystallization report and preliminary structure may be needed for 
another publication.  Also, current NIH rules disallow proposal updates 
other than reference to an accepted manuscript, and so this may be a way 
to get information to reviewers.  It may also be a gambit to dissuade 
your competition, although that may backfire if such competition already 
has the structure done and was simply procrastinating.


With that said, I personally see no ethical issue here other than one 
absolutely must acknowledge the existence of published crystallization 
report.


Cheers,

Ed.

Re: [ccp4bb] Space group choice with respect to physical plausability

[Edwin Pozharski]

On 09/24/2012 07:09 AM, Harm Otten wrote:

Why is it so hard to break the symmetry for two (seemingly) different monomers?


Because it's present in your data?

In P1, do you still have the electron density that suggests the 
"overlap" (it's not entirely clear to me what you mean by that - a 
figure could help)?


Twinning?

An outside chance that you have a mix of the full-length and N-terminal 
truncation?

Re: [ccp4bb] Off Topic: help locating CNS data

[Edwin Pozharski]

If you are looking for reflection files, the tricky part is that in CNS 
format they did not include the unit cell parameters.  Just to locate 
them, it may be helpful to know that they contain a header that looks 
like this


NREFlection= 49238
ANOMalous=FALSe { equiv. to HERMitian=TRUE}
DECLare NAME=FOBS   DOMAin=RECIprocal   TYPE=REAL END
DECLare NAME=SIGMA  DOMAin=RECIprocal   TYPE=REAL END
DECLare NAME=TEST   DOMAin=RECIprocal   TYPE=INTE END

so if you are in *nix environment something like this

egrep -rl '^[ ]DECLare NAME.*DOMAin=RECIprocal' .

I am sure perl masters will laugh at my awkward attempts at using 
regular expressions.


Cheers,

Ed.

On 09/21/2012 06:10 AM, Rex Palmer wrote:
I have been presented with the problem of locating protein data for a 
structure which was refined here ten years ago with the CNS program.
Unfortunately I have never used this program so do not know what type 
of files I am looking for (or how many files).

Any suggestions please
Rex Palmer
http://www.bbk.ac.uk/biology/our-staff/emeritus-staff
http://rexpalmer2010.homestead.com

Re: [ccp4bb] off topic: protein peptide binding

[Edwin Pozharski]

This may be tricky if binding is too weak, but could you use some 
ultrafiltration with, say, 5kDa filters?  The idea is that peptide will 
not go through if it is associated with protein.  Another option is size 
exclusion, although that is not always straightforward as weakly bound 
ligands may come off upon dilution due to peak broadening.  You are 
using tris-tricine buffers, right?


On 09/14/2012 09:46 AM, anita p wrote:

Hi All,
I wanted some advice regarding mapping out Protein-peptide 
interaction. The peptide is a 12 mer and the protein is 15kDa.
Invivo studies suggest that the peptide is binds the protein and helps 
in transport.

Hence I feel it would perhaps transient binding.
I know that I should do ITC or BIAcore to show binding, but before 
going to those techniques, I feel, running a native gel would perhaps 
help. So the native gel can have lane1: protein, lane 2: peptide, lane 
3: protein+peptide.


If the protein binds to the peptide then I should not see a band 
corresponding to the peptide in lane 3.


But before I start this experiment, I wonder if any body has run 12 
mer  peptide on native gel, How long should I run...  How much 
quantity of peptide I should for the gel.


I wont be able to do western or pull-down, Equipment for native gel is 
available to me.

kindly advice,
regards
Anita

Re: [ccp4bb] Off-topic: Best Scripting Language

[Edwin Pozharski]

On 09/14/2012 12:30 AM, Eric Bennett wrote:

Actually it's a bit of a hindrance.  In Perl I can call the int function on 
anything and get a sensible answer.  In python if you call int on a string that 
contains a floating point number the default behavior is that it will crash:


The sensible answer you describe may be considered a buggy behavior.  If 
you use int() converter in your code, my guess is that you anticipate it 
will be processing a bunch of strings that are expected to represent 
integers.  If you have a string and you want it to be converted to 
integer even if it actually looks like a float, you can do this


number = int(float(example_string))

or this

import math
number = math.floor(float(example_string))

or perhaps this (more sensible)

number = round(float(example_string))

I wonder what situation you have in mind when forcing non-integer string 
data to become integers with a slightly shorter expression gives an 
advantage. On a broader point, I am sure that perl-bashers can come up 
with examples of said language behavior they may want to ridicule.  
Different computer languages will exhibit different behavior because 
they are, well, different.

That's brain dead.  IMHO of course.

Name-calling is not an argument.  It's not quite Godwin's rule, but still.

Cheers,

Ed.

Re: [ccp4bb] Ligand geometry obs. vs. ideal

[Edwin Pozharski]

You can do unrestrained refinement in refmac, at your resolution it may 
be OK.   If you want to keep protein restrained, you can either use 
harmonic restraints or come up with a special cif-file for your ligand 
with large esd targets.  There is no direct way to tell refmac to 
exclude specific residue from restraints, at least to my knowledge.


Cheers,

Ed.

On 09/12/2012 02:44 PM, Yuri Pompeu wrote:

What is the best way to refine the ligand unrestrained and then generate 
measurements?

Re: [ccp4bb] Off-topic: Best Scripting Language

[Edwin Pozharski]

Ethan,

I think majority of your complaints about python result from its very 
purpose - to be readable/portable for the sake of facilitating rapid 
implementation.  There are many other languages that provide tools to 
accomplish what Jacob wants to do (well, I would stay away from P''), 
but python definitely is a good option for casual calculations.


On 09/12/2012 02:28 PM, Ethan Merritt wrote:

I'm sure you can google for many "reasons I hate Python" lists.

Mine would start
1) sensitive to white space == fail
every language has a way to group lines of code.  Curly brackets are 
fine, but python is designed to force code readability, and preceding 
white space (btw, everywhere else it is ignored) does that.



2) dynamic typing makes it nearly impossible to verify program correctness,
and very hard to debug problems that arise from unexpected input or
a mismatch between caller and callee.


While indeed 1/3=0 (but so it will be in C), I think it's a bit of an 
overstatement that python code execution is "nearly impossible to verify".
Another goal of python is to accelerate implementation, and dynamic/duck 
typing supposedly helps that.  The argument is simply that weak typing 
favours strong testing, which should be a good thing.



3) the language developers don't care about backward compatibility;
it seems version 2.n+1 always breaks code written for version 2.n,
and let's not even talk about version 3


I don't think that's entirely true either, why would they then backport 
certain features from v3?  The decision to not provide backward 
compatibility was well explained.  While 2to3 converter may potentially 
fail on complex code, the very fact that it was implemented confirms 
that python developers do care about the issue to some extent. While I 
definitely agree that it is annoying when a module you rely on is 
deprecated, there is a strong argument that a clean break is sometimes 
better than continuous patching of a code that outlived its initial design.



4) slw unless you use it simply as a wrapper for C++,
in which case why not just use C++ or C to begin with?


Native python is not meant for number-crunching, but wrappers such as 
scipy allow one to combine python flexibility/readability with speed of 
compiled binaries.  One reason to use python over C/C++ is portability.



5) not thread-safe
I am definitely not an expert on this (or anything else), but afaiu this 
is not unique to python.


Cheers,

Ed.

Re: [ccp4bb] Off-topic: Best Scripting Language

[Edwin Pozharski]

All you need is scipy library to get those pesky statistic functions :)

On 09/12/2012 11:11 AM, Pete Meyer wrote:
Python's relatively easy to learn, and more flexible than octave/R; 
but it doesn't have the built-in statistic functions that octave and R 
do. 

Re: [ccp4bb] poorly diffracting and twinned trigonal crystal

[Edwin Pozharski]

Matt,

On 09/07/2012 09:56 AM, Matthew Franklin wrote:
I'm also a bit dubious about the 4.3 A limit; your useful data may be 
ending around 4.6 instead, despite the high I/sigma numbers. 


Why?  I would rather suggest Qing extends resolution to where 
I/sigma~1.  Other than Rmerge, I don't see what else you may be looking 
at, and that is "traditional but inferior" way to determine resolution 
cutoff.


Cheers,

Ed.

Re: [ccp4bb] poorly diffracting and twinned trigonal crystal

[Edwin Pozharski]

And also - I presume p6 does not work?

On 09/06/2012 07:48 PM, Qing Luan wrote:

which I can scale in P3, P31, P32, P321, P3121 and P3221 with similar 
statistics:

Re: [ccp4bb] poorly diffracting and twinned trigonal crystal

[Edwin Pozharski]

On 09/06/2012 07:48 PM, Qing Luan wrote:

I built a molecular replacement model

What is the model based on (i.e. how much sequence identity you have)?
Did you try something other than CNS (specifically for twinning detection)?
Did you check the patterson map and/or self-rotation for off-origin peaks?

36% solvent is highly unlikely given the resolution.

Did you try molecular replacement with individual domains?

etc, etc, etc

Re: [ccp4bb] scala & scaleit problems

[Edwin Pozharski]

Mary,

consider re-posting with log-files attached

Cheers,

Ed.

On 09/05/2012 08:44 AM, Mary Ortmayer wrote:

Hi,

My scala and scaleit programmes are failing with "bad input labels" for both my 
data and the ccp4 tutorial data. I would be grateful for any help.

Thanks,

Mary

Re: [ccp4bb] Determining Rpim value

[Edwin Pozharski]

Michelle,

On 09/04/2012 06:14 PM, Michelle Deaton wrote:
Is there a straightforward way to obtain this value from my data?  
From what I understand, most of my options involve going back and 
obtaining unmerged intensities.  I am hoping there may be a way for me 
to avoid having to backtrack that far, as this data is now very far 
along in the refinement process.
The simple answer is no, you cannot avoid going back to unmerged data, 
because the variation among "redundant" measurements is what Rpim etc 
reflects, and once that information is gone upon merging, it's gone. As 
others have pointed out, you can either


a) run scalepack with "NO MERGE ORIGINAL INDEX" option and then use 
scala.  There was also a separate program called RMERGE by Manfred 
Weiss, but the embl link does not work.  For this, you need at least the 
original denzo output, i.e. *.x files.
b) if you have original images, you can use mosflm/scala or xds/scala to 
re-process your data.  (Then choose the one that gave you the lowest 
R-value ;)


Perhaps one day scalepack will report Rpim.

Cheers,
Ed.

Re: [ccp4bb] off topic: ITC or Biacore

[Edwin Pozharski]

On 08/09/2012 03:55 AM, rashmi panigrahi wrote:
Does any one have the experience of doing ITC or Biacore(SPR) at 10 or 
15 degrees?


You can do ITC at any temperature your instrument allows, certainly no 
problems at 4oC.  To share a trick, at least on a MIcrocal instrument 
cooling down is very slow, and I found it useful to wash the cell with 
ice cold water to quickly get down to 4 degrees.

Re: [ccp4bb] Pisa application

[Edwin Pozharski]

On 08/08/2012 09:00 AM, Jose Duarte wrote:

To my knowledge PISA by itself is not able to do interface prediction


To my understanding PISA is not *intended* to do interface prediction

Re: [ccp4bb] column profile

[Edwin Pozharski]

Note that gel filtration columns need to be calibrated separately for 
different buffer.  S100 is a preparative column not quite intended for 
molecular weight determination, but if that is what you are doing, it's 
important to do your own calibration.


On 08/07/2012 03:20 AM, MT wrote:

Have a look at this pdf.

Regards.

https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314750913712/litdoc28407384AA_20110831032125.pdf


Le 6 août 2012 à 10:23, sajid akthar  a écrit :


Hi all

If anyone have, please post the calibration profile for GE Sephacryl S100 26/60 
Hiprep column. Sorry I could not locate in the GE Healthcare site.

Thankx in advance

Sajid

Re: [ccp4bb] space group and multiplicity

[Edwin Pozharski]

On 08/02/2012 04:37 AM, Careina Edgooms wrote:

Dear ccp4

I ask a very fundamental question because I have not had formal 
training in this and I would like to understand.
How can I obtain the multiplicity (z) from the space group? So for 
example if the space group is P222 how do I know that there are 4 
monomers in the unit cell? Or if it is P422 then there is 8? I am only 
concerning myself with a primitive lattice for now because I am sure 
the others are more complicated.

thanks
Careina
If you want to derive this number yourself (instead of looking it up in 
ITC), do this:


1. Write down all the symmetry operators for the spacegroup.  To save 
time , I'll use P2 for an example:


(x,y,z)
(-x,y,-z)

2. Keep applying them until you get a closed list of symmetry mates:

(x,y,z) - primitive

(-x,y,-z) - second copy

Now apply second operator to the second copy and you get

(-(-x),y,-(-z)) = (x,y,z)  - but that is the same as the primitive 
operator, so further application of symmetry will not lead to new copies.


3.  Count the unique symmetry copies you found - in this case 2 of them 
and you are done.



Other space groups are not really more complicated, the same steps 
apply, you just have more operators.  Notice that symmetry mates should 
always be shifted back into the origin unit cell, e.g. in P21 the second 
operator is


(-x, y+1/2, -z)

which after two applications results in

(-(-x), (y+1/2)+1/2, -(-z)) = (x, y+1, z)

but this is the same as (x,y,z) after you translate it back by (0,-1,0).

Where do you find symmetry operators?  You can derive them yourself from 
the space group symbol or look them up in ITC.  Once you master this, 
you will be able to explain to others why there is no P22 space group :)


Cheers,

Ed.

Re: [ccp4bb] Process multiple data sets

[Edwin Pozharski]

> The unit cells is slightly different from each other. For example,
one has
> a/b/c @ 79/126/83. The other has a/b/c @ 84/127/90.
Although they are
> collected from the same crystal.

This is very substantial difference and with unit cell expanding by ~20%
one would expect scaling problems.  Try using the same unit
cell/orientation on all datasets, it's easy to do if you abandon gui and
feed input file directly to denzo.

-- 
Edwin Pozharski,
PhD
University of Maryland, Baltimore

Re: [ccp4bb] CNS installation

[Edwin Pozharski]

You won't have ia32-libs on a 32-bit OS - they provide libraries needed to
run 32-bit applications on a 64-bit system.  I doubt that reverse is
even possible, so as Ben said, you'd have to compile the binaries
yourself.  

> 
> Hello Ed, I have is 32 bit pc,
do I still need ia32-libs. When I searched
> "synaptic
package manager" for ia32-libs I dont see  any thing to install.
> 
> Date: Sat, 28 Jul 2012 16:39:07 -0400
>
From: epozh...@umaryland.edu
> Subject: Re: [ccp4bb] CNS
installation
> To: CCP4BB@JISCMAIL.AC.UK
> 
>
Rajesh,
> 
> this might be 32-vs-64 bit issue.  Make sure
that
> you are using correct binaries.  Installing ia32-libs may
also
> help.
> 
> Ed.
> 
>>
>> Dear All,
>>
>> I am trying to install
CNS in ubuntu 10.04 LTS. Even after reading
> all the
>> advice on CNS installation on CCP4bb, I have failed to
> get it working.
>> Both in bash and tcsh, cns_web works
but
> cns_solve gives error "command
>> not
found".
>>
> 
>>
yogesha@yogesha-laptop:~/work$ echo $CNS_SOLVE
>>
>>
>> /home/yogesha/sware/CNS/cns_solve_1.3
>>
>>
>> yogesha@yogesha-laptop:~/work$
cns_solve
>>
>>
>> cns_solve: command not
found
>>
>> I
> don't know how to trouble
shoot.
>> Any help is appreciated.
>>
>>
Thanks
>> Yogi
>>
> 
> 
>
--
> Edwin Pozharski, PhD
> University of Maryland,
Baltimore


-- 
Edwin Pozharski, PhD
University
of Maryland, Baltimore

Re: [ccp4bb] CNS installation

[Edwin Pozharski]

Rajesh,

this might be 32-vs-64 bit issue.  Make sure that
you are using correct binaries.  Installing ia32-libs may also
help.

Ed.

> 
> Dear All,
> 
> I am trying to install CNS in ubuntu 10.04 LTS. Even after reading
all the
> advice on CNS installation on CCP4bb, I have failed to
get it working.
> Both in bash and tcsh, cns_web works but
cns_solve gives error "command
> not found".
>

> yogesha@yogesha-laptop:~/work$ echo $CNS_SOLVE
> 
> 
> /home/yogesha/sware/CNS/cns_solve_1.3
> 
> 
> yogesha@yogesha-laptop:~/work$ cns_solve
> 
> 
> cns_solve: command not found
> 
> I
don't know how to trouble shoot.
> Any help is appreciated.
> 
> Thanks
> Yogi
> 


-- 
Edwin Pozharski, PhD
University of Maryland, Baltimore

Re: [ccp4bb] Structure Refinement Program

[Edwin Pozharski]

Scott,

you are asking for opinions when you should be asking for hard data.  
CNS, refmac and phenix all can regularize a structural model.  If your 
primary consideration is speed, you should simply try all three and 
compare cpu time.


Cheers,

Ed.

On 07/23/2012 12:50 PM, Scott Foy wrote:

Hello Everyone,

We are computationally averaging several homologous protein structures into a 
single structure. This of course will lead to a single protein structure that 
possesses poor biophysical characteristics of bond lengths, bond angles, steric 
hindrance, etc. Therefore, we will need a refinement program that is very rapid 
and that will restore optimal protein parameters upon input of a single PDB 
coordinate file. We are considering several programs such as Phenix and CNS and 
would appreciate any comments or opinions as to recommendations, advantages, 
and disadvantage for these, or other, programs. We will need to refine 
thousands of PDB files so speed is a significant consideration.

Thank you everyone for your input.

Sincerely,
Scott Foy
s...@mail.umkc.edu

Re: [ccp4bb] Structure alignment

[Edwin Pozharski]

One of many possible ways is to use rms_cur command in pymol.

But fundamentally such number is meaningless, since root mean square 
deviation is only useful when the average shift between the two 
structures is (close to) zero.



On 07/23/2012 11:06 AM, Theresa Hsu wrote:

Dear all

I have two proteins in PDB each with two subunits. One of the subunits can 
align well in both. How do I calculate rmsd for the aligned subunits and the 
other non-align subunits *separately*?

Thank you.

Re: [ccp4bb] off topic Thermal shift assay

[Edwin Pozharski]

My understanding is that the advantage of the thermofluor assay is that
you can test many conditions rapidly unless of course you have some kind
of high throughput CD spectrometer in mind.

> If you have a
CD available (not the one with music on it) you don't need a
> dye
just sufficient protein and you can thermal denature your protein
> assuming it contains some secondary structure elements.
>

> Jürgen
> 
> 
> On Jul 19, 2012, at
4:26 AM, anita p wrote:
> 
> Hi All,
> I want to
use a thermofluor for the thermal shift assay. My proteins are
>
cytoplasmic truncations of membrane protein. I have read about ANS,
> sypro-orange and CPM. Which is the once that is popularly used by
the
> crystallographers for condition optimization for
crystallization ??
> 
> I have read that it sypro orange
is not good for hydrophobic proteins and
> CPM can't be used with
DTT or bME in the buffer.
> I am a bit confused .
> Please
help
> thanks in advance
> Anita
> 
>
..
> Jürgen Bosch
> Johns Hopkins
University
> Bloomberg School of Public Health
>
Department of Biochemistry & Molecular Biology
> Johns Hopkins
Malaria Research Institute
> 615 North Wolfe Street, W8708
> Baltimore, MD 21205
> Office: +1-410-614-4742
>
Lab:  +1-410-614-4894
> Fax:  +1-410-955-2926
>
http://lupo.jhsph.edu
> 
> 
> 
> 
> 


-- 
Edwin Pozharski, PhD
University of
Maryland, Baltimore

Re: [ccp4bb] resolution limit

[Edwin Pozharski]

http://www.ysbl.york.ac.uk/ccp4bb/2001/msg00383.html


>
Rsym...what's that?
> 
> JPK
> 
> On Wed,
Jul 18, 2012 at 9:12 AM, Edwin Pozharski
>
wrote:
> 
>> As has been
shown recently (and discussed on this board), Rsym is not
>>
the
>> best measure of data quality (if any measure at all):
>>
>>
http://www.sciencemag.org/content/336/6084/1030.abstract
>>
>>
>>
>> > narayan viswam wrote:
>> >> Hello CCP4ers,
>> >> In my data, the
highest reolution shell 2.8-3.0 A has I/sigmaI 2.5 &
>>
>> Rsym 224.3 %
>> >> for multiplicity 7.8 and
completeness 98.2 %. I solved the structure
>> by
>>
>> MAD & refined it
>> >> to Rfree 27.3 %. Ths
crystal belongs to P622 space group and it is
>> not
>> >> twinned. The water
>> >> content is
68%. I loweredthe multiplicity to 4.1 by excluding few
>>
>> images but still the
>> >> Rsym is > 200 %
and I/sigmaI > 2.0. My rudimentary crystallography
>>
>> knowledge makes me
>> >> believe it's quite Ok
to use data upto 2.8 A and report the
>> statistics.
>> >> Could I request
>> >> people's views.
Thanks very much.
>> >> Narayan
>> >
>> > After refinement, what is R-free in the last shell? If it
is
>> significantly
>> > better
>>
> than random, say around .4 or less, that could be taken as
evidence
>> that
>> > there
>> > is
data in the last shell.
>> > Also check the error model-
Rsym >2 sort of implies the error is
>> greater
>> > than
>> > the signal, so I/sigI 2 seems
surprising.
>> > eab
>> >
>>
>>
>> --
>> Edwin Pozharski, PhD
>> University of Maryland, Baltimore
> 
> 
> 
> 
> --
>
***
> Jacob Pearson
Keller
> Northwestern University
> Medical Scientist
Training Program
> email: j-kell...@northwestern.edu
>
***
> 


--

Edwin Pozharski, PhD
University of Maryland, Baltimore

Re: [ccp4bb] resolution limit

[Edwin Pozharski]

As has been shown recently (and discussed on this board), Rsym is not the
best measure of data quality (if any measure at all):

http://www.sciencemag.org/content/336/6084/1030.abstract


> narayan viswam wrote:
>> Hello CCP4ers,
>>  
In my data, the highest reolution shell 2.8-3.0 A has I/sigmaI 2.5
&
>> Rsym 224.3 %
>> for multiplicity 7.8 and
completeness 98.2 %. I solved the structure by
>> MAD &
refined it
>> to Rfree 27.3 %. Ths crystal belongs to P622
space group and it is not
>> twinned. The water
>>
content is 68%. I loweredthe  multiplicity to 4.1 by excluding few
>> images but still the
>> Rsym is > 200 % and
I/sigmaI > 2.0. My rudimentary crystallography
>> knowledge
makes me
>> believe it's quite Ok to use data upto 2.8 A and
report the statistics.
>> Could I request
>>
people's views. Thanks very much.
>> Narayan
> 
> After refinement, what is R-free in the last shell? If it is
significantly
> better
> than random, say around .4 or
less, that could be taken as evidence that
> there
> is
data in the last shell.
> Also check the error model- Rsym >2
sort of implies the error is greater
> than
> the signal,
so I/sigI 2 seems surprising.
> eab
> 


--

Edwin Pozharski, PhD
University of Maryland, Baltimore

Re: [ccp4bb] CNS compliant addition of hydrogens to structure?

[Edwin Pozharski]

Oh, I know this one.  It's called CNS :)

You probably have
to set 

hydrogen_flag=true
in the generate.inp script - it's false by default.  If some other
atoms are missing and you want to keep it that way, use 


atom_build=(hydrogen)

Cheers,

Ed.

> Anyone know of a util that'll
add hydrogens in a naming scheme consistent
> with CNS? (reduce
doesn't do this).
> 
> 
> Thanks!
> 
> F
> 
> 
> 
> 
>
-
> Francis E. Reyes
PhD
> 215 UCB
> University of Colorado at Boulder
> 


-- 
Edwin Pozharski, PhD
University of
Maryland, Baltimore

Re: [ccp4bb] CNS installation

[Edwin Pozharski]

I think the bash-compatible startup script has disappeared from CNS 
distribution at some point.  The one that was distributed with cns 1.1 
still works though, and I attach my copy of it.


On 07/12/2012 11:24 AM, Dirk Kostrewa wrote:

Dear Fulvio Saccoccia,

along the lines of Ian Tickle's reply: there should be a script 
"cns_solve_env_sh" using /bin/sh, which is usually a soft-link to 
/bin/bash. I use this script for setup of CNS under bash.


Best regards,

Dirk.

Am 12.07.12 16:49, schrieb fulvio saccoccia:

Dear ccp4 users,
I tried to install CNS under Debian 64bit. I followed the 
installation

giude as reported by CNS developers but  I received the following
message when souurcing cns_solve_env:

bash: setenv: command not found
bash: setenv: command not found
bash: cns_solve_env: line 32: syntax error near unexpected token
`setenv'
bash: cns_solve_env: line 32: `  if ( ! $?CNS_ARCH ) setenv CNS_ARCH `
$CNS_SOLVE/bin/getarch`'

I know that the script would set all variables; it is indicated for csh
(or tcsh) shell. I tried to run the script under a csh shell but I
received a different error:

Word too long

The above statement is also received if a bash-optimized script is run.

Does anyone have experience in CNS installation and environment setting?
Any advice?

Thanks in advance

Fulvio Saccoccia
Dept. of Biochemical Sciences
Sapienza University of Rome, Italy





#!/bin/sh
#
# This file sets up the appropriate environmental variables and paths
# for CNSsolve. In the case of the same machines with different versions
# of the OS, backward compatibility is assumed - ie. a later version will
# be setup for a previous version of the OS if nothing else is available.
#
#   written by: Paul Adams
#
#   copyright Yale University
#
# ==
#
# >> Important: define the location of the CNSsolve directory <<
#
# CHANGE THE NEXT LINE TO POINT TO THE LOCATION OF THE CNSsolve DIRECTORY

CNS_SOLVE='/sware/cns'

#
# ==
#
# full expansion of the CNS_SOLVE variable prior to use.
#
export CNS_SOLVE; CNS_SOLVE=$CNS_SOLVE
#
# ==
#
# set the number of threads for SGI multiprocessors
# if this causes a problem on other systems it can be commented out
#
export MP_SET_NUMTHREADS; MP_SET_NUMTHREADS=1
#
# ==
#
# get the machine architecture
#
if [ -d $CNS_SOLVE ]; then
  if [ ! "$CNS_ARCH" ]; then
export CNS_ARCH; CNS_ARCH=`$CNS_SOLVE/bin/getarch`
  fi
else
  export CNS_ARCH; CNS_ARCH='unknown'
fi
#
# ==
#
# general environmental variables
#
export CNS_LIB; CNS_LIB=$CNS_SOLVE/libraries
export CNS_MODULE; CNS_MODULE=$CNS_SOLVE/modules
export CNS_TOPPAR; CNS_TOPPAR=$CNS_LIB/toppar
export CNS_CONFDB; CNS_CONFDB=$CNS_LIB/confdb
export CNS_XTALLIB; CNS_XTALLIB=$CNS_LIB/xtal
export CNS_NMRLIB; CNS_NMRLIB=$CNS_LIB/nmr
export CNS_XRAYLIB; CNS_XRAYLIB=$CNS_LIB/xray
export CNS_XTALMODULE; CNS_XTALMODULE=$CNS_MODULE/xtal
export CNS_NMRMODULE; CNS_NMRMODULE=$CNS_MODULE/nmr
export CNS_HELPLIB; CNS_HELPLIB=$CNS_SOLVE/helplib
#
# general user aliases
#
cns_web () { $CNS_SOLVE/bin/cns_web; }
cns_header () { $CNS_SOLVE/bin/cns_header; }
cns_info () { cat $CNS_SOLVE/bin/cns_info; }
cns_transfer () { $CNS_SOLVE/bin/cns_transfer; }
if [ -x $CNS_SOLVE/bin/cns_edit_local ]; then
  cns_edit () { $CNS_SOLVE/bin/cns_edit_local; }
else
  cns_edit () { $CNS_SOLVE/bin/cns_edit; }
fi
run_tutorial () { "csh -f tutorial.csh"; }
#
# g77 compilation and use
#
g77on () { CNS_G77=ON; . $CNS_SOLVE/.cns_solve_env_sh; }
g77off () { unset CNS_G77; . $CNS_SOLVE/.cns_solve_env_sh; }
#
# developer aliases
#
run_tests () { $CNS_SOLVE/bin/run_tests; }
run_diffs () { $CNS_SOLVE/bin/run_diffs; }
maketar () { $CNS_SOLVE/bin/maketar; }
create_patch () { $CNS_SOLVE/bin/create_patch; }
#
#
# ==
#
# to do expansions - unset noglob just in case user has it otherwise
#
set +f
#
# try to set up appropriate path
#
# first strip off any trailing information (eg. _g77)
#
CNS_ARCH=`echo ${CNS_ARCH} | sed -e 's/_g77//g'`
#
cns_vendor=`echo $CNS_ARCH | awk 'BEGIN{FS="-"}{print $1}'`
cns_cpu=`echo $CNS_ARCH | awk 'BEGIN{FS="-"}{print $2}'`
cns_os=`echo $CNS_ARCH | awk 'BEGIN{FS="-"}{print $3}'`
cns_major=`echo $CNS_ARCH | awk 'BEGIN{FS="-"}{print $4}'`
cns_minor=`echo $cns_major | sed -e 's/\./ /g'`
#
# if we are looking for a specific type of setup then limit search
#
cns_dirs=""
if [ ! "$CNS_G77" ]; then
  if /bin/ls -d $CNS_SOLVE/$cns_vendor-* >/dev/null 2>&1 ; then
cns_dirs="`/bin/ls -d $CNS_SOLVE/$cns_vendor-* 2>&1 | awk 
'BEGIN{FS="/"}{print $NF}' | sort -t\- +3 -4 -n -r`"
  fi
else
  CNS_ARCH="${CNS_ARCH}_g77"
  if /bi

Re: [ccp4bb] sulphur bridge

[Edwin Pozharski]

If you need reducing agent, the best choice is probably TCEP.  If you 
are to choose between the BME and DTT, I'd recommend DTT, since BME 
tends to modify cysteines (sometimes in active site which may be quite 
annoying, although post-crystallization DTT soaks can remove some of 
these).  On the other hand, I know of at least one case whereby CYS-BME 
modification has helped crystallization by mediating a crystal contact.


In summary, every protein is different and if you have capacity to try 
BME, DTT and TCEP, try all three.  And no reducing agent as well.



On 07/11/2012 04:50 AM, amro selem wrote:

Hallo every one,
i am working on one enzyme who has sulphur bridge , is using 2 
mercaptoethnol or DTT  during handling this protein for 
crystallography is not desirable .

best regards
Amr

Re: [ccp4bb] estimate of effective concentration

[Edwin Pozharski]

Filip,

if you have a way to measure the fraction bound (say you see two 
conformers and your data is good enough to refine occupancies), and if 
the binding constant for the two peptides in solution is measurable then 
you can derive your "effective concentration".  What would that really 
tell you I am not sure.  For all practical purposes, you have a 
monomolecular reaction, because the interacting components are held in 
proximity.  What is the effective concentration of individual amino 
acids in context of protein unfolding?


Cheers,

Ed.

On 06/20/2012 07:08 PM, Filip Van Petegem wrote:

Dear crystallographers,

I have a question concerning effective concentration. Say you have a 
crystal structure whereby two loops, each part of a different domain 
but within the same molecule happen to be juxtaposed and can form an 
interaction.  The loops have some degree of flexibility, but are 
ordered when interacting. The domains on which they are attached have 
a rigid configuration due to the remainder of the structure. The 
interaction is potentially very weak and mainly driven by the fact 
that the effective concentration is extremely high.


The question: how can one obtain a rough estimate of the effective 
concentration of these two juxtaposed loops?   The simple 
straightforward answer would be to just divide number (1 each) by 
volume (some box drawn around the loops), and convert this to molar. 
That's easy. However, this is over-simplified and really an 
underestimate of 'effective' concentration, because these loops cannot 
rotate freely when attached to the domains.  Hence, there are 
constraints that allow them to interact more readily compared to the 
isolated loops within the same box. So I'm looking for a model that 
also takes limited conformational freedom into account.


If anybody has any pointers to some reference text or paper that has 
performed such an analysis, I would be very interested.


Regards,

Filip

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

phone: +1 604 827 4267
email: filip.vanpete...@gmail.com 
http://crg.ubc.ca/VanPetegem/

[ccp4bb] ssrl

[Edwin Pozharski]

Anyone knows what happened at SSRL?  Remote access of all sorts appears 
to be down (including ssh, nx, website, webice).

Re: [ccp4bb] Rfrees

[Edwin Pozharski]

On Mon, 2010-11-29 at 14:30 +, Jyotica Batra wrote:
> is there
a way I can switch to phenix.refine by retaining the same
>
R-frees, I have from refmac

If you actually mean the test set,
then you don't need to do anything
other than use the same
mtz-file.  Keep in mind though that in phenix by
default (unless
it changed recently) the intensities will be converted
to amplitudes
internally using simple square root (i.e. no
French&Wilson) and
negative intensities discarded.  Hence the safe way
to minimize
changes would be to make sure that your mtz-file only
contains Fo's
and not intensities (there might be also some phenix
keyword to
rectify this, which I am sure you will be promptly informed
about).

But if you really mean getting the same R-free, I am
afraid it's almost
impossible and rather pointless.  There are
many things the two programs
do differently (measurement outlier
rejection, bulk solvent parameters
refinement, maximum likelihood
target construction, default restraint
targets, and even the target
minimization procedure itself, just to name
a few).

No
intent to start another round of the quarterly "who cares about
R-values" discussion.  Carthago delenda est.

Cheers,

Ed.

-- 
"I'd jump in myself, if
I weren't so good at whistling."
  
Julian, King of Lemurs

Re: [ccp4bb] Method to calculate the axis of an alpha helix

[Edwin Pozharski]

Interhlx may be what you are looking for

http://nmr.uhnres.utoronto.ca/ikura/resources/data+sw/interhlx/


> Dear all,
>I want to compare the conformational
change of two similar structures,
> using one alpha helix as the
reference. Then, how can I get a vector that
> can represent both
the position and direction of the helix? Is there any
> well-known
software can do this？
>Or, should I build a
cylinder model, with parameters [radius,bottom
>
center(x1,y1,z1),top center(x1,y2,z2)], using the coordinates of
>
C,C(alpha)
> and N to fit these parameters?
> Thanks for
any suggestions
> 
> Regards,
> Yuan SHANG
> 


-- 
Edwin Pozharski, PhD
University of
Maryland, Baltimore

Re: [ccp4bb] PEG in the pdb? zero occ

[Edwin Pozharski]

Perhaps I was confused by the "refinement exclude" keyword which
 explicitly says that atoms excluded from refinement will contribute to 
the mask calculation.  Thanks for the correction.
 
 I
would still object to the zero-occupancy atoms on semantic grounds. 
Partial occupancy means that an atom spends this fraction of time an this
position.  Zero occupancy thus says that the atom is never near this
particular point in space, which for disordered side chains is likely
untrue.

Cheers,

Ed.
 
> I agree that
zero occupancy is a bit ugly, but useful when not sure
> whether
you will ever see that LYS..
> 
> But I dont think it wlll
displace bulk solvent - at least not in REFMAC
> where an atom
with occ=0.0 will not contribute to the atom map. And I
> expect
this is true for all other structure factor calculations using
>
various programs..
> 
> Eleanor
> 
> Ed
Pozharski wrote:
>> On Thu, 2010-08-12 at 08:57 +, MARTYN
SYMMONS wrote:
>>> Zero occupancy is generally a deprecated
way of dealing with missing
>>> density as it is confusing
for less experienced user of the
>>> coordinates. I think
zero occupancy can be useful during refinement as
>>> the
atoms help fill space (or for  example satisfy NCS restraint
>>> format requirement) but then these atoms can be stripped
out before
>>> deposition. They should in any case never be
included in B-factor
>>> refinement as they will skew the
statistics and possibly the B-factor
>>> restraint model.
>>
>> Zero occupancy may be a bad idea for yet another
reason - the atoms will
>> displace bulk solvent and produce
what is essentially a hole in the
>> structure.  It may be
justified if you are trying to fill the empty
>> internal
cavities, but for atoms missing from density it seems like a
>>
wrong approach.
>>
> 


-- 
Edwin
Pozharski, PhD
University of Maryland, Baltimore

Re: [ccp4bb] Vista

[Edwin Pozharski]

I am sure Vista is probably just as good (or bad, depending on your 
point of view) as XP.  Most of this stuff should run just fine (I 
understand that Vista is probably loaded with features that will bring 
older computer to complete stop, but we are talking about brand new 
laptop here).  But let me emphasize an important point - you always run 
outdated versions on windows.  CCP4 is usually up-to-date, but if you 
want latest refmac - no windows binaries.  WinCoot is currently 0.3.3.1, 
not 0.4.1.  O - well, if you learn to work with it you are ready for 
transition to linux anyway.  X-plor (CNS) is advertised as compiled for 
WinNT/Win98, should be fine in Vista, but then the whole concept of CNS 
interface is unix-assuming (I couldn't imagine a diehard windows fan 
enjoying CNS for windows).  You can, of course, always compile (or at 
least try to compile) from source, if authors are kind enough to provide 
it.  But if you figure out how to compile coot in windows, I think you 
will not have any problem working in linux in general. 

Doing crystallography in windows is like playing hockey with a football 
- it sure does work but it's a wrong combination of tools.  The only 
reason I can think of is if laptop is always running windows because you 
use it for msword but want to open coot to take a quick look at the 
structure occasionally (without rebooting or running virtual machine).  
In which case there should be no crystallography-specific difference 
between XP and Vista.


deena wrote:

Hi All,
I am about to buy a laptop and find that the XP is twice the price of 
Vista. Does anyone have positive experience using Vista for 
crystallographic packages: CCP4, Coot, O, X-plor?  or must I still 
avoid it?


Thanks,

Deena

Deena Abells Oren, PhD
Manager, Structural Biology Resource Center
Rockefeller University
1230 York Avenue
New York, NY 10065-6399
phone: 212- 327-7429
fax: 212-327-7389



--
Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
--   / Lao Tse /

Re: [ccp4bb] Question about freeR tag

[Edwin Pozharski]

FREEFLAG (the program which is used to generate the test set) 
description says when describing the keyword SEED:


By default, for a given job on a given machine, the random number 
generator produces the same list of "random" free-R flags each time the 
job is run. Since you would generally only produce one list of free-R 
flags for each project, this is not usually a problem. However, if you 
specify the keyword SEED, then the random number generator is seeded 
with the current time, and will produce a different list of free-R flags 
each time the job is run.


So what you see is normal.

Zheng Zhou wrote:
Sorry that I didn't explain the situation clearly. I used only one 
output.sca file from HLK2000. I ran the scalepack2mtz (in CCP4i, data 
reduction, import merged data) several times, on both linux Fedora and 
window XP, the same computer though.


For the next step refinement, I mean add H2O, ion, ligands, double 
check certain sidechains In those refmac runs, I always use the 
same mtz file, which I generated from the beginning. I am as surprised 
as you are the Rfree flags are identical from those different "first" 
runs.


Thanks

On Jan 23, 2008 9:50 AM, Zheng Zhou <[EMAIL PROTECTED] 
<mailto:[EMAIL PROTECTED]>> wrote:


Hi, All

Could any tell me how CCP4 handle free R flag? I know It is
important to select the same FreeR reflections if I move to next
step of refinement. But everytime I start from fresh, the freeR
Flag remains unchanged. The Rwork and  Rfree of my models are fine
( 20.7% and 22.9%). I thought Rfree was randomly generated. I
asked more experienced people in the lab and neighbor labs, and
didn't get a straight answer. Did I do something wrong?

Thanks and sorry to bother others.

Zheng (Joe)

I cant understand this.
Do you mean you have multiple data sets and they all generate the same
FreeRs? If you have EXACTLY the same reflection list, and generate FreeR
flags on the same machine I guess they would be the same.. but it would
be surprising

Or if you are using the same file which already has FreeR flags then
they wont change of course - the default is always to use the
reflections flagged with FreeR = 0.  (Thats as it should be..)

By the by - your agreement between R and FreeR seems unusually low
unless you have very high resolution data or a great deal of
non-crystallographic symmetry.
Eleanor

Zheng  - I am not sure what you are doing, but as long as you work
with one dataset only, you generate the free reflections at the very
beginning when you import your data into CCP4. Then use the resulting
mtz file for all following refinement steps. There are situations
where one will have to deviate from this scheme, but those are rare.
If you feel you have such a case, then tell us more about it. Hope
that helps. Best - MM

Joe,


can you explain a bit clearer your problem?  what do you mean the 
'next step of refinement'?  are you just doing another round of 
refinement?  what program are you using, REFMAC5?  also, R/Rfree of 
20.7/22.9 is pretty good, depending on resolution.


yes, you are right, Rfree flags are generated randomly and can be 
anywhere from 5-10% of your reflections (your choice).  then, once the 
Rfree flagged reflections are selected, they will not change (nor be 
refined) throughout the rest of your structure determination.  they 
should have their own column in your *.mtz file called RfreeFlag or 
something like this (in CCP4 that is).  other programs handle this 
relatively the same, but may use various naming conventions.  one 
caveat is that CCP4 flags reflections using 0 by default (meaning 5% 
of your reflections are flagged with 0 and are the Rfree set).  Other 
programs such as CNS and PHENIX use 1 by default, so be careful when 
switching back and forth.  You can tell either program which to use 
for Rfree set, but needs to be set, since it is not default.


again, not exactly sure what the problem is, but i hope some of this 
helps.  feel free to email with further questions if needed.  best of 
luck!




cheers,
nick



--
Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
--   / Lao Tse /

Re: [ccp4bb] differences between Rsym and Rmerge

[Edwin Pozharski]

Bernie,

but my chi-squares are always near 1.0, so why would I report it?  How 
close they should be to 1 is open to discussion, of course.   The point 
is, it is assumed (at least in scalepack) that you adjust your error 
model until chi-square~1.  I have never seen a statistics table in a 
paper which would report chi-suqares. 

I am afraid I may be misinterpreting what you were trying to say - I 
apologize if that is the case.


Cheers,

Ed.

Santarsiero, Bernard D. wrote:

You know there is that other funny column with chi^2's. I like to quote
both. Half of the reviewers will know which column to look at, but you
will satisfy the other half.

Bernie

On Fri, January 18, 2008 1:39 pm, Edwin Pozharski wrote:
  

There are two opposing views on this.

First:  Rmerge doesn't matter.  Don't even look into that column in
scalepack output, you will be upset over nothing.  If you collect twice
as much data (360 sweep instead of 180) from the same crystal, your
Rmerge will go up due to higher redundancy, but the dataset will
actually get better because you measuring every reflection twice more
and your I/sigma will increase by ~40%.

Second:  Rmerge is very important, because if it is, say, 100% (oh,
those zeros in the scalepack output) it means that symmetry-related
reflections vary by about 100%, so your data is a pile of garbage (at
least in that resolution shell).  Cut your data at the resolution where
Rmerge is 30% and you will be rewarded by really low Rfactors for your
final model.  Plus, if you keep all the data to where I/sigma~1, your
Rmerge is guaranteed to be 0.00 in the output, and what are you going to
tell reviewers of your paper?

Of course, truth is somewhere in the middle.  If I collect on two
crystals of the same type (assuming everything else is the same, such as
redundancy), and one has much higher Rmerge, then I should probably
choose the other one.  If you cut resolution at I/sigma~1, and your
overall Rmerge is about 10%, I think it's normal.  But if it's 30%, you
may have some unusually high level of noise in your data (satellite
crystal?  twinning?  evil xray fairy messing with you?).  So Rmerge does
tell you something, but only in context with all the other information.
After all, the only thing that matters is if your electron density map
is interpretable.  I dare to say that the quality of the map you get
does correlate with Rmerge, but would I discard a dataset just because
Rmerge is high without trying to solve the structure and take a look at
the density?  Never.

Cheers,

Ed.

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 av

Re: [ccp4bb] differences between Rsym and Rmerge

[Edwin Pozharski]

Chris Putnam wrote:
I won't belabor this point (or defend this view) any further, 
though I will repeat my surprise at the lack of a clear

consensus for what Rsym and Rmerge actually mean,
as opposed to things like I/sigma, for example.
  
I/sigma is also open to interpretation.  Is it / or  
(averaged over all the reflection in a given resolution shell)?


--
Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
--   / Lao Tse /

Re: [ccp4bb] differences between Rsym and Rmerge

[Edwin Pozharski]

o 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.A.
Tel: +1 214 645 6381
Fax: +1 214 645 6353


--
Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
--   / Lao Tse /

Re: [ccp4bb] differences between Rsym and Rmerge

[Edwin Pozharski]

My understanding is(was) that Rsym refers to the merging of 
symmetry-related reflections during scaling whereas Rmerge refers, 
broadly, to any data merging process, but originally means merging of 
reflection with the same (hkl). Rcryst then should refer to the merging 
of data from different crystals. The final number reported by, say, 
scalepack will thus be both Rsym and Rmerge, but is referred to as 
Rmerge because Rsym is also an instance of Rmerge, at least 
semantically. I understand the usefulness of Rsym is limited to the 
process of space group identification, whereas overall Rmerge reflects 
(somewhat and in clearly relative fashion) upon the quality of your data.


At risk of injecting more controversy to this fairly benign discussion, 
let me quote Daniel Gewirth:


"In practice, there are two ways of assessing the high resolution limit 
of diffraction. The first is the ratio of the intensity to the error of 
the intensity, i.e. I/σ. The second way, which is traditional but 
inferior, is the agreement between symmetry related reflections, i.e. 
Rmerge."


So Rmerge is Rsym? What makes this question somewhat irrelevant, 
however, is this:


"One of the drawbacks of using Rmerge as a measure of the quality of a 
data set is that it can be

intentionally and unintentionally manipulated. "

Cheers,

Ed.

--
Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
--   / Lao Tse /

Re: [ccp4bb] The importance of USING our validation tools

[Edwin Pozharski]

Mischa,

I don't think that the field of nanotechnology crumbled when allegations 
against  Jan Hendrik Schon  (21 papers withdrawn, 15 in Science/Nature) 
turned out to be true.  I don't think that nobody trusts biologists 
anymore because of Eric Poehlman (17 falsified grants, 10 papers with 
fabricated data, 12 month in prison).  We are still excited to hear 
about stem cell research despite of what Hwang Woo-suk did or didn't 
do.  What recent events demonstrate is that in macromolecular 
crystallography (and in science in general) mistakes, deliberate or not, 
will be discovered. 
Ed.


Mischa Machius wrote:
Due to these recent, highly publicized irregularities and ample 
(snide) remarks I hear about them from non-crystallographers, I am 
wondering if the trust in macromolecular crystallography is beginning 
to erode. It is often very difficult even for experts to distinguish 
fake or wishful thinking from reality. Non-crystallographers will have 
no chance at all and will consequently not rely on our results as much 
as we are convinced they could and should. If that is indeed the case, 
something needs to be done, and rather sooner than later.  Best - MM


 


Mischa Machius, PhD
Associate Professor
UT Southwestern Medical Center at Dallas
5323 Harry Hines Blvd.; ND10.214A
Dallas, TX 75390-8816; U.S.A.
Tel: +1 214 645 6381
Fax: +1 214 645 6353


--
Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
--   / Lao Tse /

Re: [ccp4bb] RMSD

[Edwin Pozharski]

Does anyone know if Refmac includes riding hydrogens in rmsd 
calculation? Because if it does, then the rmsd will indeed be lower for 
Refmac, since these bonds are kept at ideal length.


Ed.


john kryst wrote:

Hi ccp4bb !!!

Does the rmsd estimation (for eg. rmsd_bonds ) depends on the program 
we use ??


Example : shifting from Refmac to CNS. There appears to be an increase 
in rmsd of bonds even without refining the structure in CNS. Is the 
estimation methods are different or am i doing something wrong !!


Thanks for your valuable inputs.

regards
john 


--
Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
--   / Lao Tse /

Re: [ccp4bb] How to get rid of Membrane formed on hanging droplets?

[Edwin Pozharski]

I had some luck once cutting the crystal out of skin with a razorblade.
When the skin is extremely sticky, you tend to get your crystal wrapped
up in it (which may be OK - you just get more background scatter) and
carefully cutting skin on all four sides may be a better option.

Wang, Yeming (NIH/NIEHS) [F] wrote:
> Dear everyone,
>
> I am working on crystallization of a protein/RNA complex recently. The 
> crystals were initially grown from BICINE(9.0) 0.1M, 1,4-Dioxane 2%(v/v) , 
> PEG 20,000 10%(w/v), at 10mg/ml. I noticed that there was a membrane formed 
> on the surface of the hanging droplets. This membrane seems very sticky. 
> Consequently, almost all of the crystals stick to this membrane and can't be 
> seperated for data collection. Sitting drops were also tried but crystals 
> stick to the bottom of the sitting well. Different buffer(Tris, CHES), 
> different PEG(PEG 8000, PEG3350, from 10%-1%) and different protein 
> concentration (10-3mg/ml) were also tried, but the sticky membrane was still 
> there. Does anyone have some experience solving this problem? Any suggestions 
> would be highly appreciated! 
>
> Yeming
> - 
> Yeming Wang, Ph.D. 
> Laboratory of Structural Biology: Macromolecular Structure Group 
> National Institute of Environmental Health Sciences 
> National Institute of Health 
> Mailing Address:   Street Address: 
> NIEHS, MD F3-05  NIEHS, Building 101, Room F363 
> P.O. BOX 12233 111 T.W. Alexander Drive 
> RTP, NC 27709   RTP, NC 27709 
> Tel (o): 919-316-4634 
> E-mail: [EMAIL PROTECTED]
>   

-- 
Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
--   / Lao Tse /

Re: [ccp4bb] Very weird

[Edwin Pozharski]

I believe Phaser places CRYST1 record at the top of an output 
pdb-file...  Yanming, did you try lower symmetry spacegroups?


Eleanor Dodson wrote:
It seems very odd..in fact almost impossible. You still have 3 correct 
molecules

I wonder if there is some error in the coordinate file?
Errors I make are:

 leaving and end record, between 2 parts.   - many programs stop 
reading coordinates at the first END


leaving CRYST1 and SCALE cards in the middle of the file..
Getting the CRYST record  wrong somewhere - REFMAC does not seem to 
check that the cell dimensions are consistent


Eleanor



Yanming Zhang wrote:

Dear all,

I am sorry to trouble you again because I am facing a very weird 
situation:


Three copies from Phaser are the right solutions based upon:
1, Rfree 42% R 39%
2, No packing clash
3, The packing within the 3 makes good sense
4, Density evenly distributed among the 3 copies even without NCS

Then fix these 3, asking Phaser to find one more (here I adopt the 
strategy one copy at a time). Phaser did find one more, So now I have 
4 copies in total.


Using the phase calculated from the previous 3 copies(because Rfree 
42%), the 4th copy can exactly match one junk of the density, even 
the side chain matchs, demonstrating the 4th copy is correct.


Checking the crystallographic packing, The density which the 4th 
occupies, is an extra density which did not account for by the 
previous 3 copies or their symmetry mates.


No Clash exists

Then I use the 4 copies to do the refinement, forseeing that there 
will be a huge Rfree drop from 42% because I account for the extra 
density using the 4th copy. BUT TO MY SURPRISE, THE RFREE INCREASE TO 
65%.


Can you teach me what is going wrong? Thank you
Yanming




--
Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
--   / Lao Tse /

Re: [ccp4bb] luzzati numbers in ccp4/refmac

[Edwin Pozharski]

I guess John was referring to PDB requirements.  CNS reports "sigmaa 
coordinate error", "cross-validated luzzati coordinate error" and 
"cross-validated sigmaa coordinate error" - I always thought this is 
what PDB means.  If I refined structure with refmac, I just leave these 
blank.


Ethan Merritt wrote:

On Thursday 01 March 2007 10:32, John Bruning wrote:
  

Where can I generate the following numbers in CCP4 coming with pdb and mtz from 
Refmac?

Luzzati SigmaA (obs)
Luzzati ESD (R-free set)
Luzzati SigmaA (R-free set).



The answer is that you should not be using Luzatti plots to estimate error.
(Actually, I'm not sure what "Luzatti SigmaA Rfree" means in the first 
place).


You should instead use the ESU values from either Maximum Likelihood
or from the Cruickshank DPI empirical indices.  These are given
in the PDB file output by refmac.



  


--
Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
--   / Lao Tse /