Re: [ccp4bb] how to combine the experimental phase and molecular replacement phase

[Santosh Panjikar]

Dear Jiamu,

Try out MRSAD protocol of Auto-Rickshaw  
(http://www.embl-hamburg.de/Auto-Rickshaw). You can start this just by  
providing your selenomet
data, starting model, sequence information and space group on the  
Auto-Rickshaw server. It would do substructure determination based on  
your model phase, experimental phasing, phase combination, density  
modification, model building and refinement of the built model.  
Iteratively it improves phases and complete your model as much as  
possible.


Best
Santosh






Quoting "Jiamu Du" :


Dear All,
I am now working on a low resolution phase determination (around 3.3 A with
Se anomalous signal around 3.8 A).
I can find the Se site and get the phase, but the density map is not so
good.
Some part of the protein (about 1/3) has a homologue model which is also can
be found using Phaser. The homologue region has a good map while other
region only show a poor map.
I think the combination of experimental phase and MR phase might improve the
map. Is there anybody can help find which program can work on this?

Thanks.
--
Jiamu Du, Ph.D.
Postdoctoral Research Fellow
Laboratory of Structural Biology
Memorial Sloan-Kettering Cancer Center
RRL 269, 430 E 67th Street
New York, NY, 10021
E-mail: d...@mskcc.org
Tel: (217) - 417 - 9897





Santosh Panjikar, Ph.D.
Staff Scientist
EMBL-Hamburg Outstation
C/o DESY
Notkestrasse 85
22603 Hamburg (Germany)
panjikar at embl-hamburg.de
http://www.embl-hamburg.de/~panjikar

[ccp4bb] Postdoc position and R&D position at the MAX IV Laboratory

[Thomas Ursby]

TWO POSITIONS AVAILABLE AT THE MAX IV LABORATORY

POSTDOC POSITION (2-year position, application deadline September 1st 2011)
A postdoc position focusing on studies of how to best take radiation 
damage into account when collecting data, in particular at microfocus 
beamlines. This project is in collaboration with Dr. Thomas Schneider 
and Dr. Gleb Bourenkov at EMBL@PETRA III.
More info see: 
http://www.lunduniversity.lu.se/o.o.i.s?id=24914&Dnr=412211&Type=EU 

For questions concerning the research project please contact Dr. 
Marjolein Thunnissen (marjolein.thunnissen_at_biochemistry.lu.se)


R&D POSITION (2-year position, application deadline September 1st 2011)
A research and development position focusing on instrumentation and 
methods important for data collection quality, both at the present 
MAX-lab macromolecular crystallography beamlines 
(http://cassiopeia.maxlab.lu.se) and the future MX beamlines at the MAX 
IV facility (http://www.maxlab.lu.se/maxlab/max4/index.html):
More info see: 
http://www.lunduniversity.lu.se/o.o.i.s?id=24914&Dnr=412212&Type=EU 



Both positions include supporting operation of the present MAX-lab 
macromolecular crystallography beamlines.


FURTHER INFO AND APPLICATION
Follow the links above for some more information and please note that 
applications must be done on-line. Informal enquiries are welcome to 
Thomas Ursby (thomas.ursby_at_maxlab.lu.se).


SCIENTIFIC ENVIRONMENT
The MAX IV Laboratory is constructing a new facility and the same time 
operating the present MAX-lab facilities including two beamlines in user 
operation and a crystallization facility. The tunable I911-3 beamline 
has recently been upgraded with a completely new set-up including a MD2 
micro-diffractometer, a CATS sample changer and a HC1 humidity 
controller device. The new facility will include a 3 GeV storage ring, a 
1.5 GeV storage ring and a 3 GeV linac injector also serving a Short 
Pulse Facility. The construction started this year and the 3 GeV ring 
beamlines, including at least one MX beamline, are expected to be in 
user operation 2015-2016. The new MAX IV facility is being built next to 
the planned location of the European Spallation Source ESS 
(http://ess-scandinavia.eu). The MAX IV Laboratory is hosted by Lund 
University (http://www.lunduniversity.lu.se/about-lund-university) 
giving opportunities for collaborations with research at one of the 
largest, oldest and broadest universities in Scandinavia that is 
consistently ranked among the world's top 100 universities.


<>

Re: [ccp4bb] how to combine the experimental phase and molecular replacement phase

[Randy Read]

Hi,

Most of the answers are interpreting your question as follows, that you have a 
single Se-SAD data set that goes to 3.3A but only has anomalous signal to 3.8A. 
 Is that correct?  If so, then there are various ways to combine the MR and SAD 
phase information.  As you might imagine, we like the procedure in Phaser.  To 
expand a bit on what Nat said, Phaser takes the MR solution and treats it like 
a substructure (albeit one that has no anomalous scattering), then it uses 
log-likelihood-gradient (LLG) maps to find where anomalous scatterers should be 
added to improve the agreement with the data.  In our experience, including the 
MR model in this way can give you a more complete anomalous substructure 
(including minor sites from statistical disorder of the Se-Met side chains), 
which should improve the phases somewhat.  Because the anomalous scatterer 
sites and the MR model are treated as part of the same structure, and refined 
simultaneously, the phase information is combined naturally, without having to 
export part of the information through Hendrickson-Lattman coefficients.  
Refmac has many powerful features but, as far as I know, SAD LLG maps are not 
among them, so it's worth trying this even if you subsequently move to 
refinement in Refmac.

The other way your question could be interpreted is to say that you have a 
native data set to 3.3A and a Se-SAD set to 3.8A.  If that's true, then the 
best approach would depend on how isomorphous the two data sets are.  If 
they're isomorphous, then you should treat it as SIRAS, but if they're not then 
you might want to do some cross-crystal averaging.  Again, in this scenario 
there are various ways of combining the experimental and model phase 
information.

However, completing the other 2/3 of the structure when you only have 
experimental phase information to 3.8A is a daunting challenge, if you don't 
have NCS or something else in your favour.  In your situation I'd still want to 
be working in the background on getting higher resolution native or SAD data, 
other crystal forms and/or other derivatives.

Regards,

Randy Read

On 13 Jul 2011, at 02:08, Jiamu Du wrote:

> Dear All,
> I am now working on a low resolution phase determination (around 3.3 A with 
> Se anomalous signal around 3.8 A). 
> I can find the Se site and get the phase, but the density map is not so good. 
> Some part of the protein (about 1/3) has a homologue model which is also can 
> be found using Phaser. The homologue region has a good map while other region 
> only show a poor map. 
> I think the combination of experimental phase and MR phase might improve the 
> map. Is there anybody can help find which program can work on this?
> 
> Thanks.
> -- 
> Jiamu Du, Ph.D.
> Postdoctoral Research Fellow
> Laboratory of Structural Biology
> Memorial Sloan-Kettering Cancer Center
> RRL 269, 430 E 67th Street
> New York, NY, 10021
> E-mail: d...@mskcc.org 
> Tel: (217) - 417 - 9897
> 

--
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research  Tel: + 44 1223 336500
Wellcome Trust/MRC Building   Fax: + 44 1223 336827
Hills RoadE-mail: rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.   www-structmed.cimr.cam.ac.uk

Re: [ccp4bb] how to combine the experimental phase and molecular replacement phase

[Navraj S. Pannu]

structure, and refined simultaneously, the phase information is combined 
naturally, without having to export part of
the information through Hendrickson-Lattman coefficients.  


I am very happy to see that phenix and phaser have adopted and are 
promoting using the SAD function.  Although, I must say that I am a bit 
surprised, as when we were first discussing the merits of combining all 
the information in this way, we found rather strong resistance:


http://www.iucr.org/news/newsletter/volume-14/number-1/congress-and-general-assembly

"R. Read and G. Bricogne (UK) suggested that the anomalous phasing 
information was well described by Hendrickson Lattman coefficients..."


Refmac does have the ability to output likelihood difference maps: we are 
all thankful for Gerard for introducing this powerful technique.  In 
future versions of CCP4 and Refmac this will be automated.

Re: [ccp4bb] Off topic - Streptactin Column

[Prof. Dr. Arne Skerra]

We have been using Strep-Tactin SuperFlow (high capacity) columns more than 100 
times, even when run under preparative conditions with protein extracts from E. 
coli fermentations.

Biotin binding to the engineered streptavidin is not irreversible but can be 
cured by extended washing. To check the column status I recommend our HABA 
binding procedure described in:

Schmidt, T. G. M. & Skerra, A. (2007) The Strep-tag system for one-step 
purification and high affinity detection or capturing of proteins. Nat. Protoc. 
2, 1528-1535.

Good luck,
Arne Skerra


Am 11.07.2011 um 22:56 schrieb D Bonsor:

> Does anyone know how many times roughly you can re-use a Streptactin column? 
> I know that contamination with biotin will destroy the column but the protein 
> that I am using has gone through both a GST and Nickel purification steps 
> before seeing the Streptactin column and I think lately that I have been 
> seeing decreased binding.
> 
> Thanks in advanced.


+  Prof. Dr. Arne Skerra  +
Lehrstuhl f. Biologische Chemie  +  Technische Universitaet Muenchen
Emil-Erlenmeyer-Forum 5  +  85350 Freising-Weihenstephan  +  Germany
Phone: +49 (0)8161 71-4351  +  Fax: -4352
http://www.wzw.tum.de/bc  +  eMail: ske...@tum.de

Re: [ccp4bb] how to combine the experimental phase and molecular replacement phase

[Soisson, Stephen M]

Seconding David's suggestion, I have had this issue on several occasions and I 
would highly recommend using SHARP.  In my experience, SHARP does a superior 
job handling this type of data.
 
Best of luck-

Steve
 
 


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of David 
Veesler
Sent: Tuesday, July 12, 2011 9:21 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] how to combine the experimental phase and molecular 
replacement phase


Hi Jiamu, 
to complete the Juergen answer, that's true that the MR-SAD approach is very 
powerful and can definitely help in your case. You can use Sharp and add the 
information from your partial molecular replacement solution encoded as HL 
coefficients and do a MR-SAD phasing.
Here is the procedure 
http://www.globalphasing.com/sharp/manual/chapter4.html#ExternalPhaseInformation
Cheers
David


Le 12 juil. 2011 à 18:08, Jiamu Du a écrit :


Dear All,
I am now working on a low resolution phase determination (around 3.3 A 
with Se anomalous signal around 3.8 A). 
I can find the Se site and get the phase, but the density map is not so 
good. 
Some part of the protein (about 1/3) has a homologue model which is 
also can be found using Phaser. The homologue region has a good map while other 
region only show a poor map. 
I think the combination of experimental phase and MR phase might 
improve the map. Is there anybody can help find which program can work on this?

Thanks.
-- 
Jiamu Du, Ph.D.
Postdoctoral Research Fellow
Laboratory of Structural Biology
Memorial Sloan-Kettering Cancer Center
RRL 269, 430 E 67th Street
New York, NY, 10021
E-mail: d...@mskcc.org 
Tel: (217) - 417 - 9897


-- 
This message has been scanned for viruses and 
dangerous content by MailScanner  , and 
is 
believed to be clean. 



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This message has been scanned for viruses and 
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Notice:  This e-mail message, together with any attachments, contains
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for affiliates is available at 
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[ccp4bb] how to define coordinate number of Heavy atom and identity of attached atoms

[Haytham Wahba]

 
Dear all

i have protein soaked in Dimethyltindibromide

in the active site 2 cysteine and 1 aspartic acid

by help of anomalous map i can see 2 Sn atoms in each subunit in AU

there is difference in both subunits, Sub-A shows acetate near Sn
and Sub-B show phosphate ion, even the position of one of the 
2 cystein is different from unit to other 
1-is it normal to see such difference between 2 subunit? 


also the distances between Sn and surrounding atoms does not suggest covalent 
binding

2-the important question is how to confirm if the 2 methyl groups or even one 
methyl group of dimethlytin are still there or cleaved

i mean one can not differentiate between density of water and methygroup.
How to define coordinate number of Heavy atom and identity of the attached atoms
as you will see in the link below (with water) and (with Methyl) the map are 
the same and as well r-factors

3-what can i do to confirm presence or cleavage of methyl: collect more data or 
using other technique like exafs, Mossbauer spectroscopy could help

(with water)

http://dl.dropbox.com/u/12001878/Subunit-A.png
 
http://dl.dropbox.com/u/12001878/subunit-A-anomalous.png
 
http://dl.dropbox.com/u/12001878/Subunit-B.png
 
http://dl.dropbox.com/u/12001878/subunit-B-anomalous.png
 
(with methyl)

 
http://dl.dropbox.com/u/12001878/DimethylTin-subunit-A-alt-conf.png
 
http://dl.dropbox.com/u/12001878/DimethylTin-subunit-B.png

N.B: resolution 1.7 A
r factors and molprop are perfect.


thank you for your time

haytham
biochimi
canada

[ccp4bb] Advanced Light Microscopy Facility at EMBL Heidelberg

[Wolfgang Huebner]

Light microscopy and fluorescence techniques permit the analysis not only of 
individual proteins but also functional macromolecular complexes, which have 
increasingly come into the focus of modern structural biology.

The Advanced Light Microscopy Facility (ALMF) at European Molecular Biology 
Laboratories (EMBL) in Heidelberg offers a collection of state-of-the-art light 
microscopy equipment. Access for up to 4 weeks to the facility is provided free 
of charge under the Advanced Infrastructure Initiative P-Cube, sponsored within 
the European FP7 program. 

A wide range of light microscopy techniques can be applied from the 
determination of protein localisation in cells or tissue, analysis of protein 
interactions and dynamics to the tracking of dynamic processes in live 
environment. 
We offer the possibility to perform free of charge sophisticated microscopy 
experiments using the adequate equipment with the support from experts.
For more information, please email wolfgang.hueb...@embl.de or visit the 
following websites : 
http://www.embl.de/research/units/scb/mueller_christoph/projects/index.html
http://www.p-cube.eu/

[ccp4bb] PhD and Postdoctoral Position / Helmholtz Centre for Infection Research

[Andrea Scrima]

Applications are invited for a PhD and a postdoctoral position in Protein
Crystallography and Biochemistry at the Helmholtz-Centre for Infection
Research, Braunschweig, Germany.


Topic:
Structural characterization of protein complexes involved in the regulation
of autophagy


Methods:
protein expression and purification,  X-ray crystallography, biophysical
methods (ITC, fluorescence, Biacore...), molecular biology


The Helmholtz Centre for Infection Research:
More than 600 people are employed at the Helmholtz Centre for Infection
Research (HZI) in Braunschweig, a research institution in the Helmholtz
Association of German Research Centres funded jointly by the Federal
Republic of Germany and the Federal State of Lower Saxony. The Department
of Molecular and Structural Biology offers excellent working conditions,
state-of-the-art technical platforms and equipment (including bacterial,
insect cell and mammalian protein expression systems, access to rotating
anode X-ray source and synchrotron), a lively scientific community and
excellent support for the development of personal soft-skills. The Centre’s
task is to undertake biomedical research in the field of infection biology
to expand our knowledge of the fundamental mechanisms behind medically
relevant infectious diseases with the aim of providing the key for the
development of new therapeutics and vaccines.


For further information and details of how to apply, please visit:

PhD:
http://www.helmholtz-hzi.de/de/job_und_karriere/diplom_und_doktorarbeiten/stellenangebote/masters_and_thesis_detail/job/details/292011-1/
Postdoc:
http://www.helmholtz-hzi.de/de/job_und_karriere/stellenangebote/jobs/ansicht/job/details/302011-1/


Application deadline:
23.07.2011

Re: [ccp4bb] how to combine the experimental phase and molecular replacement phase

[Jiamu Du]

Dear All,
Thank you so much.
Because the data quality is not so good (P1 space group, Rmerge 0.19,
redundancy 3.9). I would like to try all the methods one by one to see which
is better for my case.
Thanks again.


On Wed, Jul 13, 2011 at 8:01 AM, Soisson, Stephen M <
stephen_sois...@merck.com> wrote:

> **
> Seconding David's suggestion, I have had this issue on several occasions
> and I would highly recommend using SHARP.  In my experience, SHARP does a
> superior job handling this type of data.
>
> Best of luck-
>
> Steve
>
>
>  --
>  *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of
> *David Veesler
> *Sent:* Tuesday, July 12, 2011 9:21 PM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] how to combine the experimental phase and
> molecular replacement phase
>
> Hi Jiamu,
> to complete the Juergen answer, that's true that the MR-SAD approach is
> very powerful and can definitely help in your case. You can use Sharp and
> add the information from your partial molecular replacement solution encoded
> as HL coefficients and do a MR-SAD phasing.
> Here is the procedure
> http://www.globalphasing.com/sharp/manual/chapter4.html#ExternalPhaseInformation
> Cheers
> David
>
>
>  Le 12 juil. 2011 à 18:08, Jiamu Du a écrit :
>
> Dear All,
> I am now working on a low resolution phase determination (around 3.3 A with
> Se anomalous signal around 3.8 A).
> I can find the Se site and get the phase, but the density map is not so
> good.
> Some part of the protein (about 1/3) has a homologue model which is also
> can be found using Phaser. The homologue region has a good map while other
> region only show a poor map.
> I think the combination of experimental phase and MR phase might improve
> the map. Is there anybody can help find which program can work on this?
>
> Thanks.
> --
> Jiamu Du, Ph.D.
> Postdoctoral Research Fellow
> Laboratory of Structural Biology
> Memorial Sloan-Kettering Cancer Center
> RRL 269, 430 E 67th Street
> New York, NY, 10021
> E-mail: d...@mskcc.org
> Tel: (217) - 417 - 9897
>
>
> --
> This message has been scanned for viruses and
> dangerous content by *MailScanner* , and is
> believed to be clean.
>
>
>
> --
> This message has been scanned for viruses and
> dangerous content by *MailScanner* , and is
> believed to be clean.
>
> Notice:  This e-mail message, together with any attachments, contains
> information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station,
> New Jersey, USA 08889), and/or its affiliates Direct contact information
> for affiliates is available at http://www.merck.com/contact/contacts.html) 
> that may be confidential,
> proprietary copyrighted and/or legally privileged. It is intended solely
> for the use of the individual or entity named on this message. If you are
> not the intended recipient, and have received this message in error,
> please notify us immediately by reply e-mail and then delete it from
> your system.
>
>


-- 
Jiamu Du, Ph.D.
Postdoctoral Research Fellow
Laboratory of Structural Biology
Memorial Sloan-Kettering Cancer Center
RRL 269, 430 E 67th Street
New York, NY, 10021
E-mail: d...@mskcc.org
Tel: (217) - 417 - 9897

[ccp4bb] occupancy-refinement

[dhurjati putcha]

Dear CCP4ers,

While trying to refine a protein-ligand structure (reso=2.9A) I notice that
the density (2Fo-Fc)for the ligand is discontinuous. I also notice that the
density for the residues in the ligand binding pocket (LBP) is also very
feeble. And when I refine the ligand occupancy the density for the ligand
appears and covers almost all the molecule, and the occupancy refines to
<1.0.

However, I cannot refine the occupancy/B factors for the LBP residue side
chains because the occupancy stays at 1.0 (and if I change it manually, it
returns to 1) and the B factors (group & individual) remain close to the
average for the remainder of the molecule. If I omit the LBP residue side
chains, I get some Fo-Fc density suggesting that these side chains are real.


Is there previous evidence for such phenomena suggesting multiple
conformations/dynamics/loose binding? Is there a way to quantify the
dynamics associated with the side chains (as with the ligand where the
occupancy is <1.0) so I can argue it out with potential manuscript
reviewers? The R-factor/R free #s (22%/19%) suggest that the refinement is
nearing convergence.

Best regards,

Kumar.

[ccp4bb] large R-Rfree difference in "final" structure

[Careina Edgooms]

Dear ccp4 bulletin board

I just have a slight concern regarding my Rwork Rfree difference. I have a 
structure that I have solved. I am reasonably content that it is complete 
because it has refined well, it no longer has bad geometries and contacts and 
all the rotamers, ramachandra, bond lengths etc are good. It gives favourable 
scores on molprobity and procheck. My only concern is the R factor difference. 
The resolution of the structure is 2.3A. The R factor is 0.24 after refinement 
but the Rfree is 0.33 which seems to me to be rather high. Should I be 
concerned?

During refinement Rfree only drops from about 0.36 to 0.33 while the R factor 
drops from 0.31 to 0.24.. I have removed automatic weighting in refmac in order 
to constrain my bond lengths and angles during a couple of rounds of 
refinement. 
This did not have any effect on the R factors, however. I am fairly content 
that 
the space group I have chosen is correct so I am not sure what else could cause 
the big difference in R factors? There is no twinning. 

Can I be satisfied that my structure is correct despite the high R free or 
should I be doing other checks/ trying other things before I can submit this 
structure?

Thank you for any help
Careina

Re: [ccp4bb] large R-Rfree difference in "final" structure

[Pavel Afonine]

Hi Careina,

I just have a slight concern regarding my Rwork Rfree difference. I have a
> structure that I have solved. I am reasonably content that it is complete
> because it has refined well, it no longer has bad geometries and contacts
> and all the rotamers, ramachandra, bond lengths etc are good. It gives
> favourable scores on molprobity and procheck. My only concern is the R
> factor difference. The resolution of the structure is 2.3A. The R factor is
> 0.24 after refinement but the Rfree is 0.33 which seems to me to be rather
> high. Should I be concerned?
>

looking at all similar to yours structures in PDB (*):

phenix.r_factor_statistics 2.3


Histogram of Rwork for models in PDB at resolution 2.20-2.40 A:
 0.126 - 0.147  : 29
 0.147 - 0.168  : 253
 0.168 - 0.189  : 958
 0.189 - 0.210  : 1649
 0.210 - 0.232  : 1440
 0.232 - 0.253  : 549 <<< your structure
 0.253 - 0.274  : 125
 0.274 - 0.295  : 12
 0.295 - 0.316  : 1
 0.316 - 0.337  : 4
Histogram of Rfree for models in PDB at resolution 2.20-2.40 A:
 0.165 - 0.188  : 31
 0.188 - 0.211  : 223
 0.211 - 0.235  : 860
 0.235 - 0.258  : 1733
 0.258 - 0.281  : 1508
 0.281 - 0.304  : 542
 0.304 - 0.328  : 97
 0.328 - 0.351  : 19 <<< your structure
 0.351 - 0.374  : 6
 0.374 - 0.397  : 1
Histogram of Rfree-Rwork for all model in PDB at resolution 2.20-2.40 A:
 0.001 - 0.011  : 53
 0.011 - 0.021  : 172
 0.021 - 0.031  : 524
 0.031 - 0.041  : 960
 0.041 - 0.050  : 1264
 0.050 - 0.060  : 1050
 0.060 - 0.070  : 592
 0.070 - 0.080  : 224
 0.080 - 0.090  : 114
 0.090 - 0.100  : 67 <<< your structure
Number of structures considered: 5020

(*): *Crystallographic model quality at a glance*. Acta Cryst. D65, 297-300

It seems like you should be concerned about the high Rfree and Rfree-Rwork.

The second question about how to address it is program specific in some
sense and I hope Refmac experts will address it.

Pavel.

Re: [ccp4bb] large R-Rfree difference in "final" structure

[Ian Tickle]

What leads you to believe Rfree is too high? - without knowing your
data/parameter ratio it's impossible to tell.  If you look at our paper Acta
Cryst. (1998). D54, 547-557 (see Fig 1), then calculate the ratio y =
Rfree/Rwork: in this case it is 0.33/0.24 = 1.375.  Now look at the curve
labelled 'a=2' (for normal restrained isotropic refinement) for the
theoretically expected ratio and the green dots for the observed ratios
(structures in the PDB at resolutions between 2.0 and 2.5 A).  Then you need
to calculate the ratio x = (no of atoms in the a.u.) / (no of reflections in
the working set) and see where the point (x,y) falls relative to the curve
and the green dots.  If it's on or near the curve and in the middle of the
green dots it's fine, if not then we need to work out why.

-- Ian

On Wed, Jul 13, 2011 at 4:38 PM, Careina Edgooms
wrote:

> Dear ccp4 bulletin board
>
> I just have a slight concern regarding my Rwork Rfree difference. I have a
> structure that I have solved. I am reasonably content that it is complete
> because it has refined well, it no longer has bad geometries and contacts
> and all the rotamers, ramachandra, bond lengths etc are good. It gives
> favourable scores on molprobity and procheck. My only concern is the R
> factor difference. The resolution of the structure is 2.3A. The R factor is
> 0.24 after refinement but the Rfree is 0.33 which seems to me to be rather
> high. Should I be concerned?
>
> During refinement Rfree only drops from about 0.36 to 0.33 while the R
> factor drops from 0.31 to 0.24.. I have removed automatic weighting in
> refmac in order to constrain my bond lengths and angles during a couple of
> rounds of refinement. This did not have any effect on the R factors,
> however. I am fairly content that the space group I have chosen is correct
> so I am not sure what else could cause the big difference in R factors?
> There is no twinning.
>
> Can I be satisfied that my structure is correct despite the high R free or
> should I be doing other checks/ trying other things before I can submit this
> structure?
>
> Thank you for any help
> Careina
>

Re: [ccp4bb] large R-Rfree difference in "final" structure

[Gregory T Costakes]

Hi Careina, 

Your starting R/Rfree values seem just fine. The reason for the large disparity 
after refinement is because the weighting factor is way too large during the 
first rounds of refinement. For each round of refinement, have a few different 
runs which test weighting factors between 0.01-0.2. I have found that during 
the first round of refinement, running sequential rounds of 1 cycle refinements 
with gradually increasing weighting terms (0.01, 0.015, 0.02, 0.025, etc), 
works really well. This is a similar approach to how BALBES does its initial 
refinement. The smaller the weighting factor, the smaller the amount of change 
you will see between R/Rfree... at the expense of decreasing the amount that 
R/Rfree go down during each refinement. Another thing you could try if you are 
using Refmac is to switch from "Simple" to "Babinet" scaling. Hope this helps 

--- 
Greg Costakes 
PhD Candidate 
Department of Structural Biology 
Purdue University 
Hockmeyer Hall, Room 320 
240 S. Martin Jischke Drive, West Lafayette, IN 47907 


 


- Original Message -
From: "Careina Edgooms"  
To: CCP4BB@JISCMAIL.AC.UK 
Sent: Wednesday, July 13, 2011 11:38:38 AM 
Subject: [ccp4bb] large R-Rfree difference in "final" structure 



Dear ccp4 bulletin board 


I just have a slight concern regarding my Rwork Rfree difference. I have a 
structure that I have solved. I am reasonably content that it is complete 
because it has refined well, it no longer has bad geometries and contacts and 
all the rotamers, ramachandra, bond lengths etc are good. It gives favourable 
scores on molprobity and procheck. My only concern is the R factor difference. 
The resolution of the structure is 2.3A. The R factor is 0.24 after refinement 
but the Rfree is 0.33 which seems to me to be rather high. Should I be 
concerned? 


During refinement Rfree only drops from about 0.36 to 0.33 while the R factor 
drops from 0.31 to 0.24.. I have removed automatic weighting in refmac in order 
to constrain my bond lengths and angles during a couple of rounds of 
refinement. This did not have any effect on the R factors, however. I am fairly 
content that the space group I have chosen is correct so I am not sure what 
else could cause the big difference in R factors? There is no twinning. 


Can I be satisfied that my structure is correct despite the high R free or 
should I be doing other checks/ trying other things before I can submit this 
structure? 


Thank you for any help 
Careina 

Re: [ccp4bb] large R-Rfree difference in "final" structure

[aaleshin]

Careina,
I recommend to compare the quality of your data (Rmerge) to that of an average 
data set of same resolution. Do you have meaningful data to 2.3A resolution? 
Another possibility is the anisotropicity of your data. Try this server 
http://services.mbi.ucla.edu/anisoscale/, if the anisotropicity is significant.

Alex


On Jul 13, 2011, at 8:38 AM, Careina Edgooms wrote:

> Dear ccp4 bulletin board
> 
> I just have a slight concern regarding my Rwork Rfree difference. I have a 
> structure that I have solved. I am reasonably content that it is complete 
> because it has refined well, it no longer has bad geometries and contacts and 
> all the rotamers, ramachandra, bond lengths etc are good. It gives favourable 
> scores on molprobity and procheck. My only concern is the R factor 
> difference. The resolution of the structure is 2.3A. The R factor is 0.24 
> after refinement but the Rfree is 0.33 which seems to me to be rather high. 
> Should I be concerned?
> 
> During refinement Rfree only drops from about 0.36 to 0.33 while the R factor 
> drops from 0.31 to 0.24.. I have removed automatic weighting in refmac in 
> order to constrain my bond lengths and angles during a couple of rounds of 
> refinement. This did not have any effect on the R factors, however. I am 
> fairly content that the space group I have chosen is correct so I am not sure 
> what else could cause the big difference in R factors? There is no twinning. 
> 
> Can I be satisfied that my structure is correct despite the high R free or 
> should I be doing other checks/ trying other things before I can submit this 
> structure?
> 
> Thank you for any help
> Careina

Re: [ccp4bb] large R-Rfree difference in "final" structure

[Robbie Joosten]

Hi Careina,

 

Assuming you don't suffer from a very poor data parameter ratio that would lead 
to such a large R-free/R, you need to improve your refinement. If you have NCS 
you should use local NCS restraints. You could also try jelly-body restraints, 
although they may not work at your resolution.

 

Cheers,

Robbie


> Date: Wed, 13 Jul 2011 08:38:38 -0700 
> From: careinaedgo...@yahoo.com 
> Subject: [ccp4bb] large R-Rfree difference in "final" structure 
> To: CCP4BB@JISCMAIL.AC.UK 
> 
> Dear ccp4 bulletin board 
> 
> I just have a slight concern regarding my Rwork Rfree difference. I 
> have a structure that I have solved. I am reasonably content that it is 
> complete because it has refined well, it no longer has bad geometries 
> and contacts and all the rotamers, ramachandra, bond lengths etc are 
> good. It gives favourable scores on molprobity and procheck. My only 
> concern is the R factor difference. The resolution of the structure is 
> 2.3A. The R factor is 0.24 after refinement but the Rfree is 0.33 which 
> seems to me to be rather high. Should I be concerned? 
> 
> During refinement Rfree only drops from about 0.36 to 0.33 while the R 
> factor drops from 0.31 to 0.24.. I have removed automatic weighting in 
> refmac in order to constrain my bond lengths and angles during a couple 
> of rounds of refinement. This did not have any effect on the R factors, 
> however. I am fairly content that the space group I have chosen is 
> correct so I am not sure what else could cause the big difference in R 
> factors? There is no twinning. 
> 
> Can I be satisfied that my structure is correct despite the high R free 
> or should I be doing other checks/ trying other things before I can 
> submit this structure? 
> 
> Thank you for any help 
> Careina 

Re: [ccp4bb] large R-Rfree difference in "final" structure

[Adrian Goldman]

In my experience, the most common cause of this is the "locally correct, 
globally wrong" issue.  You could be in the wrong space group (i.e P21212 when 
you should be in P212121).  Another possibility is missing some element of 
local symmetry.  Is the packing in the unit cell appropriate or are you 
underpacked?  Do you have pseudosymmetry, which can mask the effects of 
merohedral twinning on certain measures? 

Adrian.


On 14 Jul 2011, at 04:54, Robbie Joosten wrote:

> Hi Careina,
> 
> 
> 
> Assuming you don't suffer from a very poor data parameter ratio that would 
> lead to such a large R-free/R, you need to improve your refinement. If you 
> have NCS you should use local NCS restraints. You could also try jelly-body 
> restraints, although they may not work at your resolution.
> 
> 
> 
> Cheers,
> 
> Robbie
> 
> 
>> Date: Wed, 13 Jul 2011 08:38:38 -0700 
>> From: careinaedgo...@yahoo.com 
>> Subject: [ccp4bb] large R-Rfree difference in "final" structure 
>> To: CCP4BB@JISCMAIL.AC.UK 
>> 
>> Dear ccp4 bulletin board 
>> 
>> I just have a slight concern regarding my Rwork Rfree difference. I 
>> have a structure that I have solved. I am reasonably content that it is 
>> complete because it has refined well, it no longer has bad geometries 
>> and contacts and all the rotamers, ramachandra, bond lengths etc are 
>> good. It gives favourable scores on molprobity and procheck. My only 
>> concern is the R factor difference. The resolution of the structure is 
>> 2.3A. The R factor is 0.24 after refinement but the Rfree is 0.33 which 
>> seems to me to be rather high. Should I be concerned? 
>> 
>> During refinement Rfree only drops from about 0.36 to 0.33 while the R 
>> factor drops from 0.31 to 0.24.. I have removed automatic weighting in 
>> refmac in order to constrain my bond lengths and angles during a couple 
>> of rounds of refinement. This did not have any effect on the R factors, 
>> however. I am fairly content that the space group I have chosen is 
>> correct so I am not sure what else could cause the big difference in R 
>> factors? There is no twinning. 
>> 
>> Can I be satisfied that my structure is correct despite the high R free 
>> or should I be doing other checks/ trying other things before I can 
>> submit this structure? 
>> 
>> Thank you for any help 
>> Careina

Re: [ccp4bb] large R-Rfree difference in "final" structure

[Matthias Zebisch]

Hi Careina,

in our lab we once had the problem, that the asymmetric unit contained 8 
molecules,
whereas 7 had only been modeled. Somehow the 8th monomer had evaded 
detection.

So be careful not to miss density.

Matthias


On 7/13/2011 7:54 PM, Robbie Joosten wrote:

Hi Careina,



Assuming you don't suffer from a very poor data parameter ratio that would lead 
to such a large R-free/R, you need to improve your refinement. If you have NCS 
you should use local NCS restraints. You could also try jelly-body restraints, 
although they may not work at your resolution.



Cheers,

Robbie



Date: Wed, 13 Jul 2011 08:38:38 -0700
From: careinaedgo...@yahoo.com
Subject: [ccp4bb] large R-Rfree difference in "final" structure
To: CCP4BB@JISCMAIL.AC.UK

Dear ccp4 bulletin board

I just have a slight concern regarding my Rwork Rfree difference. I
have a structure that I have solved. I am reasonably content that it is
complete because it has refined well, it no longer has bad geometries
and contacts and all the rotamers, ramachandra, bond lengths etc are
good. It gives favourable scores on molprobity and procheck. My only
concern is the R factor difference. The resolution of the structure is
2.3A. The R factor is 0.24 after refinement but the Rfree is 0.33 which
seems to me to be rather high. Should I be concerned?

During refinement Rfree only drops from about 0.36 to 0.33 while the R
factor drops from 0.31 to 0.24.. I have removed automatic weighting in
refmac in order to constrain my bond lengths and angles during a couple
of rounds of refinement. This did not have any effect on the R factors,
however. I am fairly content that the space group I have chosen is
correct so I am not sure what else could cause the big difference in R
factors? There is no twinning.

Can I be satisfied that my structure is correct despite the high R free
or should I be doing other checks/ trying other things before I can
submit this structure?

Thank you for any help
Careina 

[ccp4bb] Off topic - transformation problems

[Wonjin Bae]

Hi, all

Recently, some bactrial source enzyme(sialidase) was subcloned pET20b and  
pGEX4T3 vector.

Then, these two construct were transformed to BL21(DE3) expression host.
DNA sequencing results were accurate.

In case of pGEX, many colony was formed and shown the high level of  
expression.

But, colony was not shown in pET20b case. (no one colony)
In case of mock-pET20a vector transformed very well.

What's the problems? I never experieced transfomation problems.
Does anyone know how to solve the this problems?

Thanks a lot !!
Genie,

Re: [ccp4bb] How to evaluate Fourier transform ripples

[James Holton]

I think it is important to remember here that although 2Fo-Fc maps can
have "Fourier ripples" (AKA series termination errors) difference maps
are "resistant" to them.  This is because "leaving out" data in a map
calculation is equivalent to setting the coefficient to zero, and for
a difference map this is not such a bad guess, especially for
high-angle data.

However, difference maps are not "immune" to other kinds of errors,
like Fc not quite capturing the "real" electron density.  This can be
particularly apparent around heavy atoms, since they are "taller" in
electrons/A^3 than protein atoms, and so a ~10% percent error on a Zn
atom amounts to 3 electrons, or half a carbon atom!  One example of
where such a "bump" could come from is if the distribution of Zn
positions is not a perfect Gaussian.  There is no way to exactly
capture non-Gaussian shapes with a Gaussian atom model (B factor).
So, the refinement programs does the best it can to line up the
shapes, but then you see "ripples".  However, these ripples have
nothing to do with series-termination artifacts.

Note that some call non-Gaussian atomic displacement distributions
"anharmonic", but that word assumes the displacements are thermal in
origin, and I don't think that's appropriate for cryo-cooled protein
crystals.  "non-Gaussian" is more general.

-James Holton
MAD Scientist

On Wed, Jul 6, 2011 at 10:58 AM, Fischmann, Thierry
 wrote:
> Dear Zbyszek
>
> Wouldn't ripples be the results of calculating maps with truncated
> Fourier summations (unavoidably), and, consequently, be more obvious
> around a sharp feature such as an heavy atom metal center?
>
> The mathematic basis can be found here:
> http://en.wikipedia.org/wiki/Gibbs_phenomenon
>
> With best regards,
>
> Thierry
>
> Note: sent a 2nd time as it seems that it did not reach the BB the first
> time. Apologies if the message reaches you twice.
>
> -Original Message-
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
> Zbyszek Otwinowski
> Sent: Wednesday, July 06, 2011 1:19 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] How to evaluate Fourier transform ripples
>
> The question about Fourier transformation ripples has a straightforward
> answer in a fairly typical situation:
> A) data are collected to the resolution limit of diffraction,
> B) phases are uniform in quality across the resolution range, which is
> equivalent to R-free being uniform with respect to resolution within a
> factor of 2  or so,
> C) maps are not sharpened.
>
> The ripples originate from not including unobserved structure factors.
> The
> intensity of diffraction decreases rapidly past the measurability limit,
> so, in the above situation, the unobserved diffraction contributes very
> little. Consequently, the answer is that typically one should not see
> ripples.
> Ripples should not be confused with the effect of electron density maps
> being smoothed by vibrations and other forms of disorder.
>
> Zbyszek Otwinowski
>
>>
>> Dear All,         Hi. I was asked in a manuscript revision to discuss
>> about the possible effects of Fourier transformation ripples on the
>> crystallographic results. Specifically, the reviewers question whether
>> ripples may affect on the electron density around heavy metal center
> which
>> has a Mo-S-As connection. From which angle or in which way this
> problem
>> should be addressed most convincingly ?         Thank you for any
>> suggestion.Best,Conan
>
>
> Zbyszek Otwinowski
> UT Southwestern Medical Center at Dallas
> 5323 Harry Hines Blvd.
> Dallas, TX 75390-8816
> Tel. 214-645-6385
> Fax. 214-645-6353
> Notice:  This e-mail message, together with any attachments, contains
> information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station,
> New Jersey, USA 08889), and/or its affiliates Direct contact information
> for affiliates is available at
> http://www.merck.com/contact/contacts.html) that may be confidential,
> proprietary copyrighted and/or legally privileged. It is intended solely
> for the use of the individual or entity named on this message. If you are
> not the intended recipient, and have received this message in error,
> please notify us immediately by reply e-mail and then delete it from
> your system.
>

Re: [ccp4bb] Map Using Both Bijvoet and Dispersive Differences with Model Phases

[James Holton]

I believe the OP was asking how to best make an "element density map"
where the map value is proportional to the occupancy of not just any
anomalous scatterer, but a specific element of interest.  For example,
suppose you have Zn and Ni in your protein, but you are not sure which
atom is which.  If you have solved the structure and you have data
collected near the Zn and/or Ni edges, then you can make a "Zn density
map" and an "Ni density map" to resolve the "mysterious metal
problem".  I don't think there is a "canned" procedure for doing this
because you have to bear in mind what the f" and f' are for each
dataset, but below I describe something that might work.

Making an "element density map" from conventional MAD data is a
slightly different procedure than what you would do to get the total
heavy atom contrtibution (FH) you would use to solve the structure by
MAD,SIRAS, etc.  Specifically, you are not just interested in getting
the maximum anomalous signal, you are trying to isolate the signal
coming from a particular element.  You must have more than one
wavelength to do this!  For example, if you have "peak", "inflection"
and "remote", then you can obtain a map of "f'' from Zn" by computing
a phased anomalous difference Fourier map from the "peak", and the
"inflection" and then subtracting the "inflection" map from the "peak"
map.  This will add a bit of noize, but it has the nice property of
eliminating peaks from S, Cl and any other anomalous scatterers you
might have.  Another way to get "element contrast" is to use f'.  Here
you would make a "regular" isomorphous difference map between two
wavelengths where the f' of the element of interest changed a lot.
This only happens near the edge.  Once you have both kinds of "Zn
density" maps, you can then add them together (perhaps scaling them as
described by Matthews 1966, or using something like REVISE).


As for the rest of the discussion, when it comes to maximizing phasing
signal per unit dose, I prefer two wavelengths: split the difference
between the "peak" and "inflection" as "wavelength 1", and then use a
high remote as "wavelength 2".  I flippantly call this "DAD" for
"dual-wavelength anomalous diffraction", but I suppose you could also
call it "Bifrucate Absorptive and Dispersive Anomalous Scattering".
Anyway, this gives you both kinds of anomalous difference (f" =
"absorptive", f' = "dispersive") for the price of two wavelengths.
The compromise from not being exactly "on" the optima isn't so bad in
practice.  This actually makes sense when you consider that the "peak"
and "inflection" for Se are generally only ~3 eV apart and the
bandpass of a Si(111) monochromator is ~1.8 eV at 0.98 A wavelength.

In general, however, I cannot over-stress how important it is to
collect quasi-simultaneous wavelengths!  If you just collect one
wavelength after another (OAA) and there is even a smidge of radiation
damage, then your f' will be completely lost in the non-isomorphism
generated by the damage.  By changing the wavelength often (every
image is preferable, unless your monochromator is incapable of doing
this), all your f' differences will at least be comparing "apples to
apples" and "oranges to oranges" (in this analogy, X-rays turn apples
into oranges).

-James Holton
MAD Scientist

On Wed, Jul 6, 2011 at 12:03 PM, Pete Meyer  wrote:
> It'll depend on your data, but you'd probably be better off using the
> inflection (rather than peak) and remote datasets for dispersive difference
> maps.  This signal is usually fairly weak to begin with, and not using the
> infection datasets weakens it further.
>
>
> Pete
>
> FWIW - my understanding is that "dispersive" is often used to distinguish
> differences in f' from Bijvoet differences (in f'') during MAD, at least
> when treating MAD as MIRAS.
>
> Jacob Keller wrote:
>>
>> Dear Crystallographers,
>>
>> it seems to me that for clearly identifying/characterising anomalous
>> scatterers for a solved structure, one could make a map using two
>> datasets: one at the f" peak, one low energy remote. One would then
>> use the signal both from the Bijvoet differences in the peak dataset
>> plus the differences between the peak and low-energy datasets, which I
>> think I have seen called "dispersive" differences. I guess this would
>> be like a MAD map, but using pre-existing model phases--is there such
>> an animal in the software, or would it even be helpful?
>>
>> Jacob Keller
>>
>

Re: [ccp4bb] Off topic - transformation problems

[aaleshin]

I never used pET20b, but  I found on the Internet that it expresses inserts 
constitutively. Since the cloned protease should be toxic to cells, its 
constitutive expression might prevent the formation of colonies.  But there 
might be millions of other reasons.  Why did you use pET20b?

http://www.biovisualtech.com/bvplasmid/pET-20b%28+%29.htm

Alex




On Jul 13, 2011, at 5:54 PM, Wonjin Bae wrote:

> Hi, all 
> 
> Recently, some bactrial source enzyme(sialidase) was subcloned pET20b and 
> pGEX4T3 vector. 
> Then, these two construct were transformed to BL21(DE3) expression host. 
> DNA sequencing results were accurate. 
> 
> In case of pGEX, many colony was formed and shown the high level of 
> expression. 
> But, colony was not shown in pET20b case. (no one colony) 
> In case of mock-pET20a vector transformed very well. 
> 
> What's the problems? I never experieced transfomation problems. 
> Does anyone know how to solve the this problems? 
> 
> Thanks a lot !! 
> Genie,

Re: [ccp4bb] Off topic - transformation problems

[JinSoo.Bae]

Thank you for your kind advices.

In this case, pGEX4T3 vector also expressed inserts constitutively.
http://www.biovisualtech.com/bvplasmid/pGEX-4T-3.htm

I also thought that target protein may have tocicity on  host strain.
But, why pGEX4T3-target protein was not show the same phenomena.
Now, we are trying to change the host BL21(DE3) to Rosetta(DE3) and
BL21(DE3)pLysS.
Is this try may helpful to solve the this kind of problems?

Thanks for your valuable comments.

Genie

2011/7/14 aaleshin 

> I never used pET20b, but  I found on the Internet that it expresses inserts
> constitutively. Since the cloned protease should be toxic to cells, its
> constitutive expression might prevent the formation of colonies.  But there
> might be millions of other reasons.  Why did you use pET20b?
>
> http://www.biovisualtech.com/bvplasmid/pET-20b%28+%29.htm
>
> Alex
>
>
>
>
>
>  On Jul 13, 2011, at 5:54 PM, Wonjin Bae wrote:
>
> Hi, all
>
> Recently, some bactrial source enzyme(sialidase) was subcloned pET20b and
> pGEX4T3 vector.
> Then, these two construct were transformed to BL21(DE3) expression host.
> DNA sequencing results were accurate.
>
> In case of pGEX, many colony was formed and shown the high level of
> expression.
> But, colony was not shown in pET20b case. (no one colony)
> In case of mock-pET20a vector transformed very well.
>
> What's the problems? I never experieced transfomation problems.
> Does anyone know how to solve the this problems?
>
> Thanks a lot !!
> Genie,
>
>
>


-- 
Jin Soo Bae
790-784
room 204, Dept of Life Science,
POSTECH, san31, Hyoja-dong,
Nam-gu, Pohang, Gyungbuk, Korea
tel:82-54-279-8627 fax:82-54-279-8111

Re: [ccp4bb] Off topic - transformation problems

[Dima Klenchin]

In this case, pGEX4T3 vector also expressed inserts constitutively.
http://www.biovisualtech.com/bvplasmid/pGEX-4T-3.htm


Not quite. pGEX vectors carry a copy of lacI^q, resulting in very low leak 
expression from PTAC promoter. Definitely lower than the relatively leaky 
T7 promoter in pET20, which does not express laqI^q. Switching to a plasmid 
from higher pET series that do have laqI^q (e.g. pET31) should reduce the 
leakiness.



 I also thought that target protein may have tocicity on  host strain.
But, why pGEX4T3-target protein was not show the same phenomena.
Now, we are trying to change the host BL21(DE3) to Rosetta(DE3) and 
BL21(DE3)pLysS.


pLysS should help with repression under non-inducing conditions.

If it is true toxicity issue, you might need to switch to plates with 
synthetic medium that does not contain any traces of lactose.


- Dima