Re: [ccp4bb] to determine missing atoms and residues in a PDB file

[Bernhard Rupp (Hofkristallrat a.D.)]

I do not seem to understand the meaning of ‘fixing’. Fixing something can
mean

 

a)  repairing it, implying that something was broken or amiss. Lack of
experimental information expressed as omission of atoms is not something
that needs fixing.  

b)  keeping it constant. Like in having one single energy minimized
conformation and accepting it once ‘fixed’ as in (a).

c)  injection of conscience-expanding drugs, often also hallucinogenic,
as in ‘fixing’ in (b)’.

d) removal of reproductive organs, as in possible punishment for
mutilating experimental structure models by fixing as in (a,b,c) 

 

A discrete ensemble of probable rotamers following a distribution with
constrained occupancy probabilities according to their empirically observed
frequency (optimally context sensitive) might be an approximate solution to
a to this date quite contentious issue. I am sure Paul will make it happen
;-)

 

Anyhow - beware of fixers.

 

BR   

 

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
debayan dey
Sent: Wednesday, May 30, 2012 10:59 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] to determine missing atoms and residues in a PDB file

 

The missing residues/atoms in the PDB file can be found out and  fixed in
Schrödinger program's Prime module refinement.It will automatically detect
the missing atoms in a residue and will fix it. After fixing it can energy
minimize it.For missing portions of residue it can build the missing
portions using homology model.The other option for fixing missing atoms is
to use the following server:
http://lorentz.immstr.pasteur.fr/pdb/frozen_submission.php


-Debayan Dey
 

On Wed, May 30, 2012 at 5:20 PM, sreetama das  wrote:

Dear All,

  I have a PDB file which does not have the REMARKS cards 465
(for missing residues) and 470 (for missing atoms). This is not a deposited
PDB file. Is there any program to figure out the missing residues and atoms
(some programs complain about missing atoms) ? Or do I have to check in any
particular file generated during the processing of the diffraction data? The
S2C program from Prof. Dunbrack's Lab does not show any option of uploading
pdb files (this solution was mentioned to a previous query on the BB).

Thanks in advance,

sreetama



-- 
Debayan Dey
Research Fellow
Dept. of Physics,
Biocrystallography and Computational Biology Laboratory
Indian Institute of Science
 India

Re: [ccp4bb] GFP membrane protein expression

[David Drew]

> Dear all
> 
> Is there any method to check membrane protein overexpression using GFP when 
> the C terminus is in periplasm? My reading so far all mention that for C 
> terminus fusion to work, it has to be cytoplasm.
> 
> Thank you.


Dear Theresa,

Superfolder GFP (sGFP) is reported to translocate and fold into the periplasm 
of E. coli using a secretary signal peptide (Aronson DE et al.Traffic. 2011), 
suggesting it would likewise be active when fused to the C-terminus of a 
membrane protein. Because sGFP is no longer sensitive to upstream membrane 
protein integration the formation of any aggregates (inclusion bodies) are 
likely to be fluorescent. Nonetheless, with FSEC you should be able to quickly 
estimate the amount of folded material. Alternatively if your membrane protein 
has an Nin topology you may consider the addition of an N-terminal GFP fusion. 
Again, you have the drawback that you can get uncoupled translation of the 
N-terminal fusion resulting in "free" GFP. However, as long as you know this 
then one can simply isolate membranes first before estimating overexpression 
levels. If you have an Nout topology the N-terminal GFP may affect 
folding/targeting and I would probably avoid this route. One last option it to 
consider the addition of an extra TM segment. Jeff Abramson's group has these 
vectors (Hseih JM et al. Prot. Sci. 2010).

Best of luck!

Cheers
David.

Dr. David Drew
Royal Society University Research Fellow
Division of Molecular Biosciences
Imperial College London
South Kensington Campus
London
SW7 2AZ
d.d...@imperial.ac.uk
Tel: 020-7594-9624  (ext. 49624)

Re: [ccp4bb] to determine missing atoms and residues in a PDB file

[debayan dey]

The missing residues/atoms in the PDB file can be found out and  fixed in* *
*Schrödinger program's Prime module refinement.It will automatically detect
the missing atoms in a residue and will fix it. After fixing it can energy
minimize it.For missing portions of residue it can build the missing
portions using homology model.The other option for fixing missing atoms is
to use the following server:**
**http://lorentz.immstr.pasteur.fr/pdb/frozen_submission.php*

*-Debayan Dey
*
On Wed, May 30, 2012 at 5:20 PM, sreetama das  wrote:

> Dear All,
>   I have a PDB file which does not have the REMARKS cards 465
> (for missing residues) and 470 (for missing atoms). This is not a deposited
> PDB file. Is there any program to figure out the missing residues and atoms
> (some programs complain about missing atoms) ? Or do I have to check in any
> particular file generated during the processing of the diffraction data?
> The S2C program from Prof. Dunbrack's Lab does not show any option of
> uploading pdb files (this solution was mentioned to a previous query on the
> BB).
> Thanks in advance,
> sreetama
>


-- 
Debayan Dey
Research Fellow
Dept. of Physics,
Biocrystallography and Computational Biology Laboratory
Indian Institute of Science
 India

[ccp4bb] GFP membrane protein expression

[Theresa Hsu]

Dear all

Is there any method to check membrane protein overexpression using GFP when the 
C terminus is in periplasm? My reading so far all mention that for C terminus 
fusion to work, it has to be cytoplasm.

Thank you.

Re: [ccp4bb] Einstein would be proud

[Bosch, Juergen]

Thanks for sharing Raji, this is indeed a great visualization.
Very talented student, I'm sure we will read more about his research in the 
future.

Jürgen

On May 30, 2012, at 12:19 PM, Raji Edayathumangalam wrote:

Hi Folks,

Sorry for a non-CCP4 post but I simply couldn't resist.

Here's a video that's simply out of this world! This video was made by an 
undergraduate student at Northeastern and it just won him a trip to outer 
space. Yes, outer space! Not only did the student write the script and make the 
video but he also did all the chalk art and composed and played the music in 
the video. The narration is slow but totally funky! Well worth the fifteen 
minutes, in my opinion...

http://www.northeastern.edu/insolution/other/2012/04/rocket-man/

Please DO NOT miss it if you like science or art or music or imagination or 
philosophy or some vague combination of the five...

Enjoy!
Raji

--
Raji Edayathumangalam
Instructor in Neurology, Harvard Medical School
Research Associate, Brigham and Women's Hospital
Visiting Research Scholar, Brandeis University



..
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://web.mac.com/bosch_lab/

Re: [ccp4bb] to determine missing atoms and residues in a PDB file

[Paul Emsley]

On 30/05/12 12:50, sreetama das wrote:

Dear All,
  I have a PDB file which does not have the REMARKS cards 
465 (for missing residues) and 470 (for missing atoms). This is not a 
deposited PDB file. Is there any program to figure out the missing 
residues and atoms (some programs complain about missing atoms) ? Or 
do I have to check in any particular file generated during the 
processing of the diffraction data? The S2C program from Prof. 
Dunbrack's Lab does not show any option of uploading pdb files (this 
solution was mentioned to a previous query on the BB).




The fill-partial-residues function or the Rotamer probability validation 
tool in Coot will find standard residues with missing atoms.

Re: [ccp4bb] to determine missing atoms and residues in a PDB file

[Edward A. Berry]

sreetama das wrote:

Dear All,
I have a PDB file which does not have the REMARKS cards 465 (for missing 
residues) and 470
(for missing atoms). This is not a deposited PDB file. Is there any program to 
figure out
the missing residues and atoms (some programs complain about missing atoms) ? 
Or do I have
to check in any particular file generated during the processing of the 
diffraction data?
The S2C program from Prof. Dunbrack's Lab does not show any option of uploading 
pdb files
(this solution was mentioned to a previous query on the BB).
Thanks in advance,
sreetama


I think if you generate SEQRES records for the sequence of your protein (or grab them from 
another pdb if another structure has been deposited), add them to your raw pdb,

and submit to the validation server at the PDB, the validation report will
include detailed listing of missing residues and missing atom in partial
residues.

[ccp4bb] Einstein would be proud

[Raji Edayathumangalam]

Hi Folks,

Sorry for a non-CCP4 post but I simply couldn't resist.

Here's a video that's simply out of this world! This video was made by an
undergraduate student at Northeastern and it just won him a trip to outer
space. Yes, outer space! Not only did the student write the script and make
the video but he also did all the chalk art and composed and played the
music in the video. The narration is slow but totally funky! Well worth the
fifteen minutes, in my opinion...

http://www.northeastern.edu/insolution/other/2012/04/rocket-man/

Please DO NOT miss it if you like science or art or music or imagination or
philosophy or some vague combination of the five...

Enjoy!
Raji

-- 
Raji Edayathumangalam
Instructor in Neurology, Harvard Medical School
Research Associate, Brigham and Women's Hospital
Visiting Research Scholar, Brandeis University

Re: [ccp4bb] MAD data process problem

[Jrh]

Dear Qixu Cai,
The following paper should be informative for your query:-
http://dx.doi.org/10.1107/S0909049595013288
Best wishes,
John
Prof John R Helliwell DSc 
 
 

On 29 May 2012, at 10:11, Qixu Cai  wrote:

> Dear all,
> 
> Sorry for the question from MAD beginner.
> 
> When we process the MAD datasets, including the peak-data, edge-data and 
> remote-data, which datasets need to be process with anomalous?
> 
> I know peak-data obviously need data processing with anomalous, but what 
> about edge-data and remote-data when we want to use MAD method?
> 
> Thank you very much!
> 
> Best wishes,
> 
> Qixu Cai

[ccp4bb] Three Senior Faculty Positions in X-ray Crystallography, Cryo-Electron Microscopy, and NMR

[Mark A. White]

University of Texas Medical Branch

   Senior Faculty in X-ray Crystallography
   Sealy Center for Structural Biology & Molecular
Biophysics

 UTMB Health seeks senior faculty applicants in structural biology
in the Sealy Center for Structural Biology and Molecular Biophysics
(SCSBMB). The Center supports a graduate program in molecular biophysics
and five well-run core laboratories in X-ray, Cryo-EM, NMR, Computation
and Solution Biophysics, each with an excellent PhD-level manager. For
details see: http://www.scsb.utmb.edu/  Core facility instrumentation
includes: newly purchased Rigaku Ultimate Homelab Plus, comprising the
FR-E+DW SuperBright x-ray source with the R-AXIS IV and the BioSAXS-1000
Kratky camera; plus a Bruker M06HF high-brilliance source with the
Bruker platform-CCD; new 2200FS, 2100, & 1400 JEOL Cryo-EM instruments
(the 2200FS is in BSL/3 containment); newly upgraded 800-, and 600- MHz
NMR spectrometers with Bruker Avance III consoles and solution
biophysics instrumentation including a Biacore T-100, a Thermofluor
high-throughput-screening instrument, plus spectroscopic, kinetic,
calorimetric, analytical UC, and mass spectrometry instrumentation.

 The successful candidate must be a highly motivated individual with
a PhD or MD degree, a strong publication record, and a record of
independent well-funded grant support. The ideal candidate will have
extensive experience in crystallography applied to the study of the
structure, dynamics, novel mechanisms, and functions of
bio-macromolecules, viruses, assemblies etc. Candidates for positions in
either the department of Biochemistry and Molecular Biology or
Pharmacology and Toxicology should also have or seek overlap with the
highly collaborative established biomedical research community in UTMB’s
basic science departments, and centers and programs of excellence such
as the Institute for Human Infection and Immunity, the Galveston
National Laboratory, the Center for Tropical Diseases, the Institute for
Translational Sciences, the Sealy Center for Cancer Cell Biology, the
Sealy Center for Environmental Health and Medicine, the Sealy Center on
Aging, the George P. and Cynthia Woods Mitchell Center for
Neurodegenerative Diseases, the Moody center for Brain and Spinal Cord
Injury Research, the Sealy Center for Molecular Medicine and the
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variety of core services, in recombinant DNA, genomics, proteomics,
high-throughput drug screening, mass spectrometry, membrane protein
crystallization, and protein expression and purification. Excellent
collaborative opportunities also exist through UTMB’s participation in
the Gulf Coast Consortia and the Keck Center for Interdisciplinary
Bioscience http://www.gulfcoastconsortia.org/home.aspx 

 Applicants are requested to submit electronically: a cover letter
expressing interest in being considered, a curriculum vitae, current
funding, a summary of research accomplishments, and future goals to
mailto: scsbmb.recruit...@utmb.edu

 Direct inquiries to Dr. B. Montgomery Pettitt, mpett...@utmb.edu,
409-772-0723.

UTMB has been aggressively recruiting in a large number of research
areas and is an equal opportunity, affirmative action institution that
proudly values diversity. Candidates of all backgrounds are encouraged
to apply.

Faculty Positions

  * Cryo-Electron Microscopy: Announcement
  * NMR: Announcement
  * X-ray Crystallography: Announcement



-- 
Yours sincerely,

Mark A. White, Ph.D.
Associate Professor of Biochemistry and Molecular Biology, 
Manager, Sealy Center for Structural Biology and Molecular Biophysics
X-ray Crystallography Laboratory,
Basic Science Building, Room 6.660 C
University of Texas Medical Branch
Galveston, TX 77555-0647
Tel. (409) 747-4747
Cell. (281) 734-3614
Fax. (409) 747-1404
mailto://mawh...@utmb.edu
http://xray.utmb.edu
http://xray.utmb.edu/~white

Re: [ccp4bb] TCEP resistant disulfide

[Sean Seaver]

Hi Jose,

>Has anyone worked with, or seen an example of, a solvent accessible disulfide 
>that cannot be reduced with TCEP?

Something to keep in mind is that TCEP is not very stable with phosphate 
buffers near neutral pH.

>Alternatively solvent accessible disulfides that resist reduction with DTT but 
>can be reduced with TCEP?

This could be due to the effective pH of TCEP HCl (~1.5-8.5) being wider than 
DTT ~7.

Take Care,

Sean Seaver

P212121
http://store.p212121.com/

Re: [ccp4bb] P321 space group reindex problem

[Thomas White]

On Wed, 30 May 2012 13:16:12 +0100
Ian Tickle  wrote:

> From the point of view of deciding which are the alternate settings I
> don't think it's helpful to consider polar directions anyway.  What
> matters is which symmetry axes of the lattice are not present in the
> point group. 

Possibly relevant here is a set of tables I put together for our
free-electron laser experiments, where tens or even hundreds of
thousands of patterns have to be indexed independently:

https://www.desy.de/~twhite/crystfel/twin-calculator.pdf

Underlying these tables is the same underlying information as
everything else mentioned on this thread, but these ones tell you
what the apparent symmetry would be if the intensities were mixed up
according to all the available indexing ambiguities.  So far, no-one
has been able to reliably resolve these ambiguities in our case: the
intensities are just obscured by too much noise, partiality, a spiky
X-ray spectrum and so on.  That's why we have to have so many
patterns.  For the time being, we just merge according to the higher
symmetry, accept that the data may be (perfectly) "twinned", and handle
it at the later stages.  Later on when we've hopefully solved this
problem, these tables will serve as a menu of options for doing the
whole thing backwards.

I agree that polarity isn't the right criterion.  Point group "2" is
polar but does not exhibit any indexing ambiguity.  Point group
4/m, which is most definitely not polar, does.  This paper, Le Page et
al., has some similar tables but lists the actual ambiguity operators:
http://scripts.iucr.org/cgi-bin/paper?S0108767384001392

Of course, all of this only covers "merohedral" ambiguities, not
"pseudo-merohedral" ones which might arise by accident in special
cases.

Comments on and corrections to the tables are welcome!

Tom

Re: [ccp4bb] P321 space group reindex problem

[Ian Tickle]

> It does, at least my edition (Vol. A: 5th ed., 2002, Table 10.2.1.2,
> p.806) does - ITC has everything you need to know about space groups
> (and a lot more besides)!

Actually, as the aforementioned table indicates, it's not correct to
talk about "polar and non-polar space groups", but only about "polar
and non-polar directions" in space groups.  Many space-groups have
both polar and non-polar directions, which would seem to imply that
these space groups are both polar and non-polar at the same time!!!
For example, P3 has a polar direction parallel to the 3-fold and no
non-polar directions, whereas P321, even though it's classed as a
non-polar space group, nevertheless has 3 polar directions [100],
[010], [-1-10] parallel to the 3 2-folds in the xy plane, plus 4
non-polar directions (including the 3-fold).  Note that any direction
perpendicular to an even-fold rotation axis is always non-polar: these
'trivial' cases are not shown explicitly in the above table.

>From the point of view of deciding which are the alternate settings I
don't think it's helpful to consider polar directions anyway.  What
matters is which symmetry axes of the lattice are not present in the
point group.  So in the case of P321 the hexagonal lattice has a
6-fold parallel to c which can be thought of as the product of a 3-
and a 2-fold both || c, and also 2-folds perp to the 6-fold.  The
3-fold and the perp 2-folds are all present in P321 but the 2-fold ||
c is not, so that is what gives rise to the 2 alternate settings
(h,k,l) and (-h,-k,l).  In P3 all 2-folds are missing so you get 4
alternate settings.  The same missing symmetry can also give rise to
merohedral twinning, so it's nice to "kill 2 birds with 1 stone"!

Cheers

-- Ian

[ccp4bb] to determine missing atoms and residues in a PDB file

[sreetama das]

Dear All,
  I have a PDB file which does not have the REMARKS cards 465 (for 
missing residues) and 470 (for missing atoms). This is not a deposited PDB 
file. Is there any program to figure out the missing residues and atoms (some 
programs complain about missing atoms) ? Or do I have to check in any 
particular file generated during the processing of the diffraction data? The 
S2C program from Prof. Dunbrack's Lab does not show any option of uploading pdb 
files (this solution was mentioned to a previous query on the BB).
    Thanks in advance,
sreetama

Re: [ccp4bb] P321 space group reindex problem

[Vellieux Frederic]

Hi Ian,

You're right the information is there... but not where I was expecting 
it (on the page corresponding to an individual space group). It had 
never occurred to me that it could be somewhere else.


So thanks, and regards to Jasmine.

Fred.

Ian Tickle wrote:

Hello Fred

On 30 May 2012 07:55, Vellieux Frederic  wrote:
  

Hi there,



  

But: trigonal (and hexagonal) space groups "are" (usually?) polar. The cell
axis "c"  can go "up" or can go "down", and in order to get a consistent
indexing you need to check both indexing systems when you scale additional
data to your native (the indexing chosen by your first crystals defines the
"standard" indexing - I must say that I haven't checked in the drawings of
the international tables if having c going up or going down leads to a
difference in that particular space group, P321, I'd need to draw both
possibilities and check but I'm sorry I do not have the time right now - in
fact it's too bad that the International Tables do not indicate "Polar" or
"Non-polar").



It does, at least my edition (Vol. A: 5th ed., 2002, Table 10.2.1.2,
p.806) does - ITC has everything you need to know about space groups
(and a lot more besides)!

See also this table that I made where all polar & non-polar SGs are
listed individually:

http://www.ccp4.ac.uk/dist/html/alternate_origins.html

Counting only the non-enantiomorphic ones, PG3 (4 SGs) and PG6 (6 SGs)
are polar, whereas PG321 (3 SGs), PG312 (3 SGs), PG32 (1 SG) and PG622
(6 SGs) are non-polar.  So in all 10 are polar and 13 are non-polar.
A 2-fold axis perp to another axis always implies that there's no
preferred direction along the other axis, so it's non-polar.

Cheers

-- Ian


  

Re: [ccp4bb] P321 space group reindex problem

[Ian Tickle]

Hello Fred

On 30 May 2012 07:55, Vellieux Frederic  wrote:
> Hi there,

> But: trigonal (and hexagonal) space groups "are" (usually?) polar. The cell
> axis "c"  can go "up" or can go "down", and in order to get a consistent
> indexing you need to check both indexing systems when you scale additional
> data to your native (the indexing chosen by your first crystals defines the
> "standard" indexing - I must say that I haven't checked in the drawings of
> the international tables if having c going up or going down leads to a
> difference in that particular space group, P321, I'd need to draw both
> possibilities and check but I'm sorry I do not have the time right now - in
> fact it's too bad that the International Tables do not indicate "Polar" or
> "Non-polar").

It does, at least my edition (Vol. A: 5th ed., 2002, Table 10.2.1.2,
p.806) does - ITC has everything you need to know about space groups
(and a lot more besides)!

See also this table that I made where all polar & non-polar SGs are
listed individually:

http://www.ccp4.ac.uk/dist/html/alternate_origins.html

Counting only the non-enantiomorphic ones, PG3 (4 SGs) and PG6 (6 SGs)
are polar, whereas PG321 (3 SGs), PG312 (3 SGs), PG32 (1 SG) and PG622
(6 SGs) are non-polar.  So in all 10 are polar and 13 are non-polar.
A 2-fold axis perp to another axis always implies that there's no
preferred direction along the other axis, so it's non-polar.

Cheers

-- Ian

[ccp4bb] TCEP resistant disulfide

[Seijo, Jose A. Cuesta]

Has anyone worked with, or seen an example of, a solvent accessible
disulfide that cannot be reduced with TCEP?
Alternatively solvent accessible disulfides that resist reduction with
DTT but can be reduced with TCEP?

Thanks in advance,

Jose.


Jose Antonio Cuesta-Seijo, PhD
Carlsberg Laboratory
Gamle Carlsberg Vej 10
DK-1799 Copenhagen V
Denmark

Tlf +45 3327 5332
Email josea.cuesta.se...@carlsberglab.dk
 

Re: [ccp4bb] P321 space group reindex problem

[Clemens Vonrhein]

Hi Fred,

On Wed, May 30, 2012 at 08:55:35AM +0200, Vellieux Frederic wrote:
> For practical purposes, a "derivative" is considered non isomorphous
> when the differences in unit cell parameters exceed ca. 1% (this is
> because if you take 2 crystals from the same crystallisation drop
> and collect and process diffraction crystals from these 2 crystals,
> you will never get exactly the very same values for the unit cell
> parameters; non-isomorphism effects start at ca. 1% change and
> you'll never get 2 perfectly isomorphous crystals - even if you
> collect diffraction data twice from the same crystals you will not
> get "perfect isomorphism").
> 
> From the values mentioned, 1% of the cell parameters of the native
> for a and b is 1.81 Angstroem and for c 1.1 Angstroem (the angles do
> not matter for a trigonal space group).
> 
> Had you obtained a value for a, b larger than ca. 183 Angstroem, or
> below ca. 109.2 Angstroem (only in the direction indicated by the
> changes mentioned in your mail - I ignored changes in the opposite
> direction) then you would have been able to say that the crystals
> were non-isomorphous to each other. For me they are isomorphous to
> each other and I ignore these small differences in unit cell
> parameters.

I would be careful with the (popular) percentage-rule here: the
absolute value of cell differences is much more important. At least if
we assume that the change in cell parameters roughly corresponds with
a shift in actual atoms.  If you have a 1000A cell then a 1%
difference could mean a shift of 10A ... clearly, a helix moved 10A
away results in something completely different. But with a cell of 20A
you could have a 0.2A shift, which you might hardly notice.

See eg. 5.2 in Garman & Murray (2003):

  http://journals.iucr.org/d/issues/2003/11/00/ba5042/index.html

which shows

  5.2. Non-isomorphism

One of the biggest problems of heavy-atom derivatization is that
incorporation of a heavy atom into the lattice often induces a
change in the unit cell away from the native crystal values,
i.e. the derivatized crystal is non-isomorphous to the native
crystals. The heavy atom may perturb the arrangement of protein
molecules in the crystal or distort the protein molecule, causing
a change in unit-cell lengths. Note, however, that it is also
possible for the protein to move within the original unit cell
(resulting in a different sampling of the molecular
transform). The same unit cell is thus a necessary but not
sufficient condition for isomorphism.

Crick & Magdoff (1956[Crick, F. H. C. & Magdoff,
B. S. (1956). Acta Cryst. 9, 901-908.]) calculated that a 0.5 Å
change in all three unit-cell edges of a 100 Å cubed unit cell
would change the diffraction intensities by an average of 15% in a
3 Å resolution sphere. The predicted intensity changes induced by
non-isomorphism increase at higher resolution. When faced with a
non-isomorphous derivative, it is the absolute change in the cell
which should be considered compared with the working resolution,
rather than the relative change, i.e. a change of 1.0% in a 100 Å
unit cell edge has a similar effect to that of a 0.5% change in a
200 Å unit cell edge, if compared at similar resolutions. As a
general rule of thumb, a change in cell dimensions of dmin/4 is
tolerable, where dmin is the resolution limit (Drenth,
1999[Drenth, J. (1999). Principles of Protein X-ray
Crystallography, 2nd ed. Berlin: Springer-Verlag.]). For instance,
for 2.5 Å data, a 0.6 Å change in the unit cell might be
acceptable, whereas at 3.5 Å this could rise to 0.8 Å.

Cheers

Clemens

-- 

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

Re: [ccp4bb] Moleculardimensions kits

[Faisal Tarique]

hi

i dont know about the midas but proplex is good..but if u really wanna go
for some molecular dimension screen then opt for morpheus..it is damn
good..if any protein is ever going to crystallize, it will also give
crystal in this screen too.. u could find a hit in this screen also..

best of luck

On Tue, May 29, 2012 at 9:55 PM, Barbara Medagli <
barbara.meda...@elettra.trieste.it> wrote:

> Dear all,
> This is Barbara Medagli, working in the structural biology lab in Italy
> Surfing in the molecular dimensions web site
> I found this two screens: MIDAS and ProPlex.
> I was thinking in buy them as I have to order other
> items to this company
> Some of you already used those kits?
> Any experience with them?
> Thanks for the help
>
>
>
> Barbara Medagli, PhD
> Structural Biology Lab
> Sincrotrone Trieste SCpA
> S.S. 14, km 163.5, Loc. Basovizza
> 34012 Trieste/Italy
> phone +39040 3758807/8537
> fax +39040 3758029
>



-- 
Regards

Faisal
School of Life Sciences
JNU

Re: [ccp4bb] Akta vs HPLC

[Tommi Kajander]

if you have "peek" surface or titanium parts (if i recall right)  there are no 
problems with salt solutions.

tommi


On May 30, 2012, at 3:39 AM, aaleshin wrote:

> Back in Iowa State University we used Waters HPLC for protein purification 
> during many years without noticeable damage to the stainless steel tubings. 
> But Dan was right about the pumps, someone in the lab forgot to flush the 
> high salt pump with water after its use  and damaged the pump...
> 
> Alex
> 
> On May 29, 2012, at 5:14 PM, Daniel Anderson wrote:
> 
>> Hi, Ho,
>> 
>> Your question has a lot of variables.
>> 
>> "HPLC" columns should not be used on the Akta within my field of view 
>> because the Akta within my field of view does not have gradual pump 
>> acceleration and deceleration. "HPLC" columns can be damaged by sudden 
>> changes in pressure or composition.
>> 
>> The "HPLC" within my field of view has wetted stainless steel surfaces 
>> and the mobile phase should not contain chloride ion or reductant. 
>> Chloride ion would accelerate corrosion of the stainless steel (and 
>> result in metal ions in the protein). Reductant would strip off the 
>> "passivation" (during maintenance I soak the stainless parts in nitric 
>> acid to keep them stainless) later resulting in corrosion.
>> 
>> The Waters sales representative once told me that the pumps have to be 
>> salt-free and methanol-flushed at the end of every working day. Good 
>> luck implementing that policy.
>> 
>> -Dan
>> 
>> Ho Leung Ng wrote:
>>> Hello,
>>> 
>>>My Akta Purifier is being repaired, and I'm thinking about 
>>> borrowing a colleague's HPLC in the interim. What makes the Aktas 
>>> different from HPLCs? I've used HPLCs for purifying small molecules 
>>> and peptides but not proteins. Anything I should be careful about 
>>> regarding keeping the machines, columns, and proteins happy?
>>> 
>>> 
>>> Thank you,
>>> Ho
>>> 
>>> Ho Leung Ng
>>> University of Hawaii at Manoa
>>> Assistant Professor, Department of Chemistry
>>> h...@hawaii.edu 

Re: [ccp4bb] P321 space group reindex problem

[Adrian Goldman]

If the data sets are twinned, large differences between derivatives are to be 
expected unless the twin fraction is very, very low (>1-2%).  Given the above, 
I think nothing can be said until the data are all detwinned - and of course 
the correct axial interchange done.

Adrian


On 30 May 2012, at 09:55, Vellieux Frederic wrote:

> Hi there,
> 
> I am not certain that the thread is "P321 space group reindex problem" any 
> more.
> 
> But: trigonal (and hexagonal) space groups "are" (usually?) polar. The cell 
> axis "c"  can go "up" or can go "down", and in order to get a consistent 
> indexing you need to check both indexing systems when you scale additional 
> data to your native (the indexing chosen by your first crystals defines the 
> "standard" indexing - I must say that I haven't checked in the drawings of 
> the international tables if having c going up or going down leads to a 
> difference in that particular space group, P321, I'd need to draw both 
> possibilities and check but I'm sorry I do not have the time right now - in 
> fact it's too bad that the International Tables do not indicate "Polar" or 
> "Non-polar").
> 
> For practical purposes, a "derivative" is considered non isomorphous when the 
> differences in unit cell parameters exceed ca. 1% (this is because if you 
> take 2 crystals from the same crystallisation drop and collect and process 
> diffraction crystals from these 2 crystals, you will never get exactly the 
> very same values for the unit cell parameters; non-isomorphism effects start 
> at ca. 1% change and you'll never get 2 perfectly isomorphous crystals - even 
> if you collect diffraction data twice from the same crystals you will not get 
> "perfect isomorphism").
> 
> From the values mentioned, 1% of the cell parameters of the native for a and 
> b is 1.81 Angstroem and for c 1.1 Angstroem (the angles do not matter for a 
> trigonal space group).
> 
> Had you obtained a value for a, b larger than ca. 183 Angstroem, or below ca. 
> 109.2 Angstroem (only in the direction indicated by the changes mentioned in 
> your mail - I ignored changes in the opposite direction) then you would have 
> been able to say that the crystals were non-isomorphous to each other. For me 
> they are isomorphous to each other and I ignore these small differences in 
> unit cell parameters.
> 
> The difference of 3% in Rfactor (I suppose this is |FP - FPH| / |FP|, no 
> "Sigma" character on my keyboard to indicate the summations over h k l) is I 
> think due to 2 different chemicals (heavy-atom compounds) in derivative 1 and 
> derivative 2. Differences in R-factors at low resolutions are often 
> associated with "solvent effects", and I think you will have 2 different 
> mother liquors and hence 2 different solvents in derivative 1 and in 
> derivative 2. That is assuming that derivative 1 and derivative 2 were 
> prepared using 2 different chemicals. And typically low-resolution data 
> (below 15 Angstroem resolution or so) is kept out during phasing by the MIR 
> method.
> 
> To locate the heavy atom constellations in the 2 derivatives, you could 
> compute and interpret difference Patterson maps - including automated 
> interpretation, vector search and the likes -, you could use "direct methods" 
> (the heavy atom constellation is similar to a small molecule because there 
> are far fewer atoms there than in the full macromolecule, and direct methods 
> work extremely well for small molecules - you would need to use the 
> isomorphous differences in order to use direct methods; no mention is made of 
> any anomalous signal so I do not know if you could this as well).
> 
> HTH,
> 
> Fred.
> 
> Qixu Cai wrote:
>> Why the 29% Rfactor indicate the derivatives are not isomorphous to native 
>> dataset?
>> 
>> Native dataset cell constant: 181.39 181.39 110.217 90 90 120
>> derivative1 cell constant: 181.909 181.909 109.62 90 90 120  
>>   Rfactor to native: 26%
>> derivative2 cell constant: 181.527 181.527 109.32 90 90 120  
>>   Rfactor to native: 29%
>> 
>> The Rfactor at low resolution is larger than in high resolution.
>> 
>> Could you please to help me figure out where the heavy atoms had been soaked 
>> into the crystal?
>> 
>> Thank you very much.
>> 
>> Best wishe,
>> 
>> Qixu Cai
>> 
>> 
>> 
>> 
>> 
>> 2012/5/30 Laurent Maveyraud > >
>> 
>>Hi,
>> 
>>it is therefore likely that your spacegroup is really P321...
>>hopefully, your data set is not twinned, did you check that ?
>> 
>>You are left with 2 possible indexing schemes, as already
>>mentionned. Chek scaling derivative / native scaling for each
>>indexation of the derivative : the lowest Rfactor will likely
>>indicate the right one.
>>It appears that you end up with Rfactor of about 29 %, which
>>suggest that your derivatives are not isomorphous to your native
>>dataset. How do cell parameters compare for each data set ?
>

Re: [ccp4bb] P321 space group reindex problem

[Vellieux Frederic]

Hi there,

I am not certain that the thread is "P321 space group reindex problem" 
any more.


But: trigonal (and hexagonal) space groups "are" (usually?) polar. The 
cell axis "c"  can go "up" or can go "down", and in order to get a 
consistent indexing you need to check both indexing systems when you 
scale additional data to your native (the indexing chosen by your first 
crystals defines the "standard" indexing - I must say that I haven't 
checked in the drawings of the international tables if having c going up 
or going down leads to a difference in that particular space group, 
P321, I'd need to draw both possibilities and check but I'm sorry I do 
not have the time right now - in fact it's too bad that the 
International Tables do not indicate "Polar" or "Non-polar").


For practical purposes, a "derivative" is considered non isomorphous 
when the differences in unit cell parameters exceed ca. 1% (this is 
because if you take 2 crystals from the same crystallisation drop and 
collect and process diffraction crystals from these 2 crystals, you will 
never get exactly the very same values for the unit cell parameters; 
non-isomorphism effects start at ca. 1% change and you'll never get 2 
perfectly isomorphous crystals - even if you collect diffraction data 
twice from the same crystals you will not get "perfect isomorphism").


From the values mentioned, 1% of the cell parameters of the native for 
a and b is 1.81 Angstroem and for c 1.1 Angstroem (the angles do not 
matter for a trigonal space group).


Had you obtained a value for a, b larger than ca. 183 Angstroem, or 
below ca. 109.2 Angstroem (only in the direction indicated by the 
changes mentioned in your mail - I ignored changes in the opposite 
direction) then you would have been able to say that the crystals were 
non-isomorphous to each other. For me they are isomorphous to each other 
and I ignore these small differences in unit cell parameters.


The difference of 3% in Rfactor (I suppose this is |FP - FPH| / |FP|, no 
"Sigma" character on my keyboard to indicate the summations over h k l) 
is I think due to 2 different chemicals (heavy-atom compounds) in 
derivative 1 and derivative 2. Differences in R-factors at low 
resolutions are often associated with "solvent effects", and I think you 
will have 2 different mother liquors and hence 2 different solvents in 
derivative 1 and in derivative 2. That is assuming that derivative 1 and 
derivative 2 were prepared using 2 different chemicals. And typically 
low-resolution data (below 15 Angstroem resolution or so) is kept out 
during phasing by the MIR method.


To locate the heavy atom constellations in the 2 derivatives, you could 
compute and interpret difference Patterson maps - including automated 
interpretation, vector search and the likes -, you could use "direct 
methods" (the heavy atom constellation is similar to a small molecule 
because there are far fewer atoms there than in the full macromolecule, 
and direct methods work extremely well for small molecules - you would 
need to use the isomorphous differences in order to use direct methods; 
no mention is made of any anomalous signal so I do not know if you could 
this as well).


HTH,

Fred.

Qixu Cai wrote:
Why the 29% Rfactor indicate the derivatives are not isomorphous to 
native dataset?


Native dataset cell constant: 181.39 181.39 110.217 90 90 120
derivative1 cell constant: 181.909 181.909 109.62 90 90 
120Rfactor to native: 26%
derivative2 cell constant: 181.527 181.527 109.32 90 90 
120Rfactor to native: 29%


The Rfactor at low resolution is larger than in high resolution.

Could you please to help me figure out where the heavy atoms had been 
soaked into the crystal?


Thank you very much.

Best wishe,

Qixu Cai





2012/5/30 Laurent Maveyraud >


Hi,

it is therefore likely that your spacegroup is really P321...
hopefully, your data set is not twinned, did you check that ?

You are left with 2 possible indexing schemes, as already
mentionned. Chek scaling derivative / native scaling for each
indexation of the derivative : the lowest Rfactor will likely
indicate the right one.
It appears that you end up with Rfactor of about 29 %, which
suggest that your derivatives are not isomorphous to your native
dataset. How do cell parameters compare for each data set ?
Check also how the Rfactor varies with resolution, you might still
have usefull phasing info at low resolution.

hope this helps

laurent



Le 30/05/2012 07:50, Qixu Cai a écrit :

At first, I processed the data at P3 space group. But after
phenix.xtriage analysis, the Xtriage told me the space group
must be
P321, so I used P321 to process my data, and got an acceptable
Rmerge.

Qixu Cai



2012/5/29 Phil Evans mailto:p...@mrc-lmb.cam.ac.uk>