[ccp4bb] Job vacancy: structural analyst/programmer

[Gary Battle]

The Protein Data Bank in Europe (PDBe) is seeking to recruit a 
structural analyst/programmer to join the team at the European 
Bioinformatics Institute located on the Wellcome Trust Genome Campus 
near Cambridge in the UK.


For further information and to apply, please see the full EMBL-EBI job 
listing at http://bit.ly/1jvNjIO
To discuss this post informally, please contact PDBe's Dr Sameer 
Velankar (sam...@ebi.ac.uk).


About the position:

Established in 1994, CATH and SCOP are the world’s most comprehensive 
resources classifying protein-domain structures into evolutionary 
superfamilies. They are currently being combined in a collaborative 
project - Genome3D - that aims to provide predicted 3D structures for 
sequences assigned to SCOP and CATH superfamilies. Combining SCOP and 
CATH based predictions allows us to identify more accurately regions 
that agree between the two methods. We aim to provide these 
Genome3D-predicted structures via the PDBe resource. To improve the 
assessment of the reliability of the predictions it is necessary to 
develop a mapping between SCOP and CATH and to remove any conflicts.


We are now seeking to recruit a structural analyst/programmer to assist 
with this task. The structural analyst/programmer will also build an 
automatic pipeline to generate putative domain assignments (from CATH) 
for new PDB structures, prior to classification in SCOP or CATH. The 
structural analyst/programmer will be based at PDBe, and will be jointly 
supervised by Prof. Christine Orengo (UCL, London) and Dr Alexey Muzin 
(MRC-LMB, Cambridge), together with Prof. Gerard Kleywegt and Dr Sameer 
Velankar at PDBe.


About PDBe:

The Protein Data Bank in Europe (PDBe; pdbe.org) is part of the 
Worldwide Protein Data Bank organisation (wwPDB; wwpdb.org), which 
maintains the global archive of 3D structural data on biomacromolecules. 
The PDBe team also maintains a number of databases that support 
deposition and advanced search services for structural biologists and 
the wider scientific community. The team consists of an international 
and inter-disciplinary mix of professionals (scientists and IT specialists).


Best regards,
Gary.

--
Gary Battle
Protein Data Bank in Europe (PDBe)
EMBL-EBI
Wellcome Trust Genome Campus
Hinxton, Cambridge
CB10 1SD

http://www.facebook.com/proteindatabank
http://twitter.com/PDBeurope

Re: [ccp4bb] Sister CCPs

[Eleanor Dodson]

I agree with Frank - it keeps crystallographers modest to know how
challenging wet lab stuff still is..
Eleanor

On 12 February 2014 19:23, Robbie Joosten  wrote:
> It's not an e-mail bulletin board, but Researchgate seems to be quite
> popular for wet lab questions. IMO the Q&A section of the social network is
> a bit messy. That said, the quality seems to improve gradually.
>
> Cheers,
> Robbie
>
> Sent from my Windows Phone
> 
> Van: Paul Emsley
> Verzonden: 12-2-2014 19:23
> Aan: CCP4BB@JISCMAIL.AC.UK
> Onderwerp: Re: [ccp4bb] Sister CCPs
>
>
> On 12/02/14 15:59, George Sheldrick wrote:
>> It would be so nice to have a 'sister CCP' for questions aboud wet-lab
>> problems that have nothing to do with CCP4 or crystallographic
>> computing, The is clearly a big need for it, and those of us who try
>> to keep out of wet-labs would not have to wade though it all.
>
>
> FWIW, the remit of CCP4BB, held at jiscmail-central, is describes as:
>
> /The CCP4BB mailing list is for discussions on the use of the CCP4
> suite, and macromolecular crystallography in general./
>
>
>
> Thus wet-lab questions are not off-topic (not that anyone recently
> described them as such).
>
> Having said that, Jiscmail mailing lists are easy to set-up (providing
> that you can reasonably expect that the mailing list will improve
> knowledge sharing within the UK centered academic community) and
> relatively low maintenance. I, for one, would not be entirely unhappy to
> miss out on questions about lysis.
>
> Paul.

[ccp4bb] Senior Scientist / Group leader Position (tenure-track); Institute of Biochemistry, Biocenter, Goethe-University Frankfurt

[Martin Pos]

Dear all, 

I am posting an advertisement for a tenure-track group leader position for
structural biology on membrane proteins on behalf of Prof. Dr. Robert Tampé,
Institute of Biochemistry, Biocenter, Goethe-University Frankfurt.

Please reply to  
applicat...@biochem.uni-frankfurt.de for questions and/or application.
Deadline is March 16th, 2014.

With kind regards,

Martin Pos

 

Senior Scientist / Group leader Position (tenure-track)

Structural Biology on Membrane Proteins

 

Institute of Biochemistry, Biocenter, Goethe-University Frankfurt

 

A senior scientist position is available to join a research group interested
in investigating

the structure and mechanism of membrane transport complexes involved in
immunity,

viral and tumor immune evasion. The position funded for two years (with a
renewal for

long-term carrier perspective as tenured group leader) is embedded in a
lively and

international competitive research environment, including the Cluster of
Excellence on

Macromolecular Complexes (www.cef-mc.de) and the Collaborative Research
Center

on Membrane Transport and Communication (www.sfb807.de).

 

Candidates having proved their outstanding commitment to science by a prior

experimental thesis in biochemistry, molecular biology, structural biology,
or a closely

related field are encouraged to apply. The successful candidate should have
significant

experience in x-ray crystallography and present a strong publication record
in quality

peer-reviewed journals.

 

The position also includes radiation survey and supervision of the central
isotope lab as

well as of the central gene technical security S1/S2 lab. Parallel to
previously mentioned

scientific tasks, the successful candidate will be given opportunity to
promote her/his

own scientific career and to acquire further qualifications.

 

Very good IT skills, teaching experience, creative thinking, the ability to
work

independently and collaboratively so as to lead a small team of
(under)graduate

students are required.

 

Interested applicants are to send till March 16th 2014 an comprehensive and

enlightening application (CV, summary of past research experience and

accomplishments together with name/contact information of two references)
to:

Robert Tampé, Institute of Biochemistry, Goethe-University Frankfurt,
Max-von-Laue-Str. 9,

60438 (applicat...@biochem.uni-frankfurt.de).

 

Reference:

Hulpke S &Tampé R (2013) Trends Biochem Sci 38, 412-20.

Parcej D & Tampé R (2010) Nature Chem Biol 6, 572-580.

 

 

 

 

 

 

 



Klaas Martinus Pos, Ph.D.

Professor of Membrane Transport Machineries

Institute of Biochemistry N200/1.09

Goethe-University Frankfurt am Main

Max-von-Laue-Str. 9

D-60438 Frankfurt am Main

Germany

Tel: +49-69-798 29251

Fax: +49-69-798 29201

E-Mail:   p...@em.uni-frankfurt.de

Website:  
www.biochem.uni-frankfurt.de



 

[ccp4bb] Call for MX beamtime proposals at HZB, BESSY II, deadline March 1, 2014 is approaching

[Müller , Uwe]

Next MX-proposal application deadline: March 1, 2014 is approaching
BAG proposal applications are now possible
Single 8h-shift beamtime bookings are now possible

We kindly invite new MX-proposals for beamtime applications for the next
beamtime period.

In order to apply for beamtime, please register at the HZB on-line
access tool "GATE" (https://www.helmholtz-berlin.de/pubbin/hzbgate) and submit 
a new
beamtime application proposal.

Please note:
Old BOAT accounts are not valid for the new system GATE. Proposals cannot be 
submitted via BOAT (GATE-Photons) anymore

HZB provides beamtime at the MX-beamlines 14.1, 14.2 and 14.3. The requested 
beamtime is
granted based on the reviewed proposal and reports from previous
research activities. Reported results from previous beamtimes stated in the
"Experimental Reports" form will affect the chances for future beamtimes 
significantly.
Please make sure to include them if available.

Experimental setup:
BL14.1 setup:
- Photon flux: 1.4x10¹¹ Phot/sx100mAx0.05%BW at sample position (0.1-1 sec 
exposure time per frame)
- User defined beam shaping from 30µm-100µm diameter possible
- Pilatus 6M detector, 138mm-680mm max. distance from the sample
- Microdiffractometer (MD2) with Mini-kappa goniometer MK3 (STAC controlled)
- Automatic sample changer (CATS), 90 sample storage capacity (SPINE-Pin & EMBL 
sample magazine compatibility)
- 96-well crystallization plate scanning operational upon request
- 32-core XEON-CPU server, with 10Gb uplink to Pilatus 6M
- EDNA installed and available
- Common MX-software installed including XDS, IMOSFLM, CCP4, Phenix, SHELXC-E, 
etc.
- Automated data processing with XDSAPP available
- Remotely controlled cryo-shutter for crystal annealing
- Bruker AXS X-Flash XRF detector

We are offering the hard- and software environment for carrying out:
- UVRIP experiments at BL14.1. For further information, please visit:
  
http://www.helmholtz-berlin.de/forschung/funkma/soft-matter/forschung/bessy-mx/ancillary-facilities/uvrip_en.html

BL14.2 setup:
- Photon flux: 1.9x10¹¹ Phot/sx100mAx0.05%BW at sample position (3-20 sec 
exposure time per frame)
- MX-225 X-ray detector, 45mm-380mm distance from the sample, 30 deg 2-Theta 
possible
- mardtb goniometer installed
- 40-core XEON-CPU server, with fibre channel SAN up-link data processing 
environment
- EDNA installed and available
- Common MX-software installed including XDS, IMOSFLM, CCP4, Phenix, SHELXC-E, 
etc.
- Automated data processing with XDSAPP available
- Remotely controlled cryo-shutter for crystal annealing
- Amptek XRF detector
- Pressure chamber for noble gas derivatization (Xe, Kr available upon request)
- Ultra high performance stereo microscope Leica M205A, 20-255x zoom, 8 Mpixel 
CCD-camera


BL14.3 setup:
- Photon flux: 4x10exp10 Phot/sx100mAx0.05%BW at sample position (3-20 sec 
exposure time per frame)
- MX-225 X-ray detector, 45mm-380mm distance from the sample, 30 deg 2-Theta 
possible
- mardtb goniometer installed
- 40-core XEON-CPU server, with fibre channel SAN up-link data processing 
environment
- EDNA installed and available
- Common MX software installed including XDS, IMOSFLM, CCP4, Phenix, SHELXC-E, 
etc.
- Automated data processing with ixds and xdsi available
- Remotely controlled cryo-shutter for crystal annealing
- HC-1c dehydration device installed, HC1-beamtime upon request
- Pressure chamber for noble gas derivatization (Xe, Kr available upon request)
- Ultra high performance stereo microscope Leica M205A, 20-255x zoom, 8 Mpixel 
CCD-camera

S1-biolab facilities:
- Protein production and purification
- Nanoliter 96 well crystallization plate formulation and storage at 5 °C and 
20 °C
- Biophysical characterization with real time PCR (thermofluor assay)

Please visit our web page 
www.helmholtz-berlin.de/bessy-mx to 
gain updated
information about our experimental setup and other requirements.


Uwe Mueller, Manfred Weiss and the HZB BESSY-MX group



Dr. Uwe Mueller
Soft Matter and Functional Materials
Macromolecular Crystallography (BESSY-MX) | Group leader
Elektronenspeicherring BESSY II
Albert-Einstein-Str. 15, D-12489 Berlin, Germany

Fon: +49 30 8062 14974
Fax: +49 30 8062 14975
url: www.helmholtz-berlin.de/bessy-mx
email:u...@helmholtz-berlin.de




Helmholtz-Zentrum Berlin für Materialien und Energie GmbH

Mitglied der Hermann von Helmholtz-Gemeinschaft Deutscher Forschungszentren e.V.

Aufsichtsrat: Vorsitzender Prof. Dr. Dr. h.c. mult. Joachim Treusch, stv. 
Vorsitzende Dr. Beatrix Vierkorn-Rudolph
Geschäftsführung: Prof. Dr. Anke Rita Kaysser-Pyzalla, Thomas Frederking

Sitz Berlin, AG Charlottenburg, 89 HRB 5583

Postadresse:
Hahn-Meitner-Platz 1
D-14109 Berlin

http://www.helmholtz-berlin.de

Re: [ccp4bb] Sister CCPs

[Mike S]

I'm sorry, but did you just use the words "crystallographers" and "modest"
in the same sentence?  :-)

On Thu, Feb 13, 2014 at 6:41 AM, Eleanor Dodson
wrote:

> I agree with Frank - it keeps crystallographers modest to know how
> challenging wet lab stuff still is..
> Eleanor
>
> On 12 February 2014 19:23, Robbie Joosten 
> wrote:
> > It's not an e-mail bulletin board, but Researchgate seems to be quite
> > popular for wet lab questions. IMO the Q&A section of the social network
> is
> > a bit messy. That said, the quality seems to improve gradually.
> >
> > Cheers,
> > Robbie
> >
> > Sent from my Windows Phone
> > 
> > Van: Paul Emsley
> > Verzonden: 12-2-2014 19:23
> > Aan: CCP4BB@JISCMAIL.AC.UK
> > Onderwerp: Re: [ccp4bb] Sister CCPs
> >
> >
> > On 12/02/14 15:59, George Sheldrick wrote:
> >> It would be so nice to have a 'sister CCP' for questions aboud wet-lab
> >> problems that have nothing to do with CCP4 or crystallographic
> >> computing, The is clearly a big need for it, and those of us who try
> >> to keep out of wet-labs would not have to wade though it all.
> >
> >
> > FWIW, the remit of CCP4BB, held at jiscmail-central, is describes as:
> >
> > /The CCP4BB mailing list is for discussions on the use of the CCP4
> > suite, and macromolecular crystallography in general./
> >
> >
> >
> > Thus wet-lab questions are not off-topic (not that anyone recently
> > described them as such).
> >
> > Having said that, Jiscmail mailing lists are easy to set-up (providing
> > that you can reasonably expect that the mailing list will improve
> > knowledge sharing within the UK centered academic community) and
> > relatively low maintenance. I, for one, would not be entirely unhappy to
> > miss out on questions about lysis.
> >
> > Paul.
>

Re: [ccp4bb] Sister CCPs

[Eleanor Dodson]

Well - we ought to be! There is an awful lot of unpredictable slog
goes on before we have the fun!
  Eleanor

On 13 February 2014 12:50, Mike S  wrote:
> I'm sorry, but did you just use the words "crystallographers" and "modest"
> in the same sentence?  :-)
>
>
> On Thu, Feb 13, 2014 at 6:41 AM, Eleanor Dodson 
> wrote:
>>
>> I agree with Frank - it keeps crystallographers modest to know how
>> challenging wet lab stuff still is..
>> Eleanor
>>
>> On 12 February 2014 19:23, Robbie Joosten 
>> wrote:
>> > It's not an e-mail bulletin board, but Researchgate seems to be quite
>> > popular for wet lab questions. IMO the Q&A section of the social network
>> > is
>> > a bit messy. That said, the quality seems to improve gradually.
>> >
>> > Cheers,
>> > Robbie
>> >
>> > Sent from my Windows Phone
>> > 
>> > Van: Paul Emsley
>> > Verzonden: 12-2-2014 19:23
>> > Aan: CCP4BB@JISCMAIL.AC.UK
>> > Onderwerp: Re: [ccp4bb] Sister CCPs
>> >
>> >
>> > On 12/02/14 15:59, George Sheldrick wrote:
>> >> It would be so nice to have a 'sister CCP' for questions aboud wet-lab
>> >> problems that have nothing to do with CCP4 or crystallographic
>> >> computing, The is clearly a big need for it, and those of us who try
>> >> to keep out of wet-labs would not have to wade though it all.
>> >
>> >
>> > FWIW, the remit of CCP4BB, held at jiscmail-central, is describes as:
>> >
>> > /The CCP4BB mailing list is for discussions on the use of the CCP4
>> > suite, and macromolecular crystallography in general./
>> >
>> >
>> >
>> > Thus wet-lab questions are not off-topic (not that anyone recently
>> > described them as such).
>> >
>> > Having said that, Jiscmail mailing lists are easy to set-up (providing
>> > that you can reasonably expect that the mailing list will improve
>> > knowledge sharing within the UK centered academic community) and
>> > relatively low maintenance. I, for one, would not be entirely unhappy to
>> > miss out on questions about lysis.
>> >
>> > Paul.
>
>

Re: [ccp4bb] Sister CCPs

[Bosch, Juergen]

Let me pick up Eleanor’s comment:
is there something like a crystallographer today ? I mean in the true sense ?
I think as a “crystallographer” you won’t be able to survive the next decade, 
you need to diversify your toolset of techniques as pointed out in this article
http://www.nature.com/naturejobs/science/articles/10.1038/nj7485-711a

And I’m not quite sure how software developers see themselves, as I would argue 
they are typically maybe not doing so much wet lab stuff related to 
crystallography (I may be wrong here) but rather code these days.

What “type” of crystallographer is a software developer ?

I think like our beloved crystals “we” come in different flavors. And we need 
to train the next generation of students with that perspective in mind.

Just my two cents on a snowy day (>30cm over night)

Jürgen
..
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

On Feb 13, 2014, at 6:41 AM, Eleanor Dodson 
mailto:eleanor.dod...@york.ac.uk>> wrote:

I agree with Frank - it keeps crystallographers modest to know how
challenging wet lab stuff still is..
Eleanor

On 12 February 2014 19:23, Robbie Joosten 
mailto:robbie_joos...@hotmail.com>> wrote:
It's not an e-mail bulletin board, but Researchgate seems to be quite
popular for wet lab questions. IMO the Q&A section of the social network is
a bit messy. That said, the quality seems to improve gradually.

Cheers,
Robbie

Sent from my Windows Phone

Van: Paul Emsley
Verzonden: 12-2-2014 19:23
Aan: CCP4BB@JISCMAIL.AC.UK
Onderwerp: Re: [ccp4bb] Sister CCPs


On 12/02/14 15:59, George Sheldrick wrote:
It would be so nice to have a 'sister CCP' for questions aboud wet-lab
problems that have nothing to do with CCP4 or crystallographic
computing, The is clearly a big need for it, and those of us who try
to keep out of wet-labs would not have to wade though it all.


FWIW, the remit of CCP4BB, held at jiscmail-central, is describes as:

/The CCP4BB mailing list is for discussions on the use of the CCP4
suite, and macromolecular crystallography in general./



Thus wet-lab questions are not off-topic (not that anyone recently
described them as such).

Having said that, Jiscmail mailing lists are easy to set-up (providing
that you can reasonably expect that the mailing list will improve
knowledge sharing within the UK centered academic community) and
relatively low maintenance. I, for one, would not be entirely unhappy to
miss out on questions about lysis.

Paul.

[ccp4bb] create a lower resolution data set by truncating a high resolution data

[Mooers, Blaine H.M. (HSC)]

For some simulated phasing experiments, I want to create a lower resolution 
diffraction data set by truncating a high resolution data set. I would like to 
avoid Fourier ripples due to the truncation of the high resolution data by 
downscaling the data  such that =2.0 in the highest resolution shell 
of the truncated data. What is the best way to do this?

Blaine Mooers
Assistant Professor
Department of Biochemistry and Molecular Biology
University of Oklahoma Health Sciences Center
S.L. Young Biomedical Research Center Rm. 466

Shipping address:
975 NE 10th Street, BRC 466
Oklahoma City, OK 73104-5419

Letter address:  
P.O. Box 26901, BRC 466  
Oklahoma City, OK 73190 

office: (405) 271-8300   lab: (405) 271-8313  fax:  (405) 271-3910
e-mail:  blaine-moo...@ouhsc.edu

Faculty webpage: 
http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/faculty/blaine-mooers-ph-d-

X-ray lab webpage: 
http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/department-facilities/macromolecular-crystallography-laboratory

Small Angle Scattering webpage: 
http://www.oumedicine.com/docs/default-source/ad-biochemistry-workfiles/small-angle-scattering-links.html?sfvrsn=0

[ccp4bb] Determining concentration of membrane protein

[Raji Edayathumangalam]

Dear CC4BBers,

I am trying to figure out what is the best way to determine the protein
concentration of my membrane protein. My purified membrane protein is in
20mM Tris pH 7, 150mM NaCl and 0.02% DDM (CMC of DDM=0.0076%).

After reading the friendly manuals and searching online, I've learned that
detergents interferes with assays like Bradford but can't find good
descriptions of what works best. For now, I am trying to estimate
concentration from absorbance at 280nm and using molar extinction
coefficients based on aromatic amino acids, but again suspect detergent
interference. I would like to know what other folks working on membrane
proteins are doing.

Thanks very much.
Raji

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

[ccp4bb] Postdoctoral position at Pasteur Institute, Paris

[Nicolas Reyes]

A Postdoctoral position is available at the Pasteur Institute (Paris) in
the group of Nicolas Reyes. The lab is part of the Department of Structural
Biology and Chemistry, and focuses on the molecular mechanism of membrane
transport proteins using both structural and functional approaches. The
researcher will join an international and dynamic team, founded by the
European Research Council, and will have access to state-of-the-art
equipment to perform structural, biophysical and biochemical studies on
membrane transport proteins, including: X-ray crystallography, Electron
Microscopy, Fluorescence Spectroscopy, Calorimetry and Electrophysiology.

Candidates with a Ph.D. in Structural Biology, Biochemistry, Biophysics or
a related discipline, obtained no more than 2 years before the application
date are highly encouraged to apply. Hands on experience and knowledge in
molecular biology, protein expression and purification, are essential for
this position. Experience on X-ray crystallography or Cryo-EM data
collection and analysis are highly desirable.
To apply, please send an email to nre...@pasteur.fr with subject *postdoc
application*, and a single pdf file including: your C.V. (please include
graduation date and list of publications), research interest and
experience, and the name and contact information of three references.

Nicolas Reyes, Ph.D
Molecular Mechanisms of Membrane Transport Laboratory
Dept. Structural Biology and Chemistry
Institut Pasteur
25,28 rue du Dr. Roux
75724 Paris CEDEX 15
France

Re: [ccp4bb] Determining concentration of membrane protein

[Xavier Brazzolotto]

I think that BCA assay is what you are looking for.

Le 13 févr. 2014 à 16:06, Raji Edayathumangalam  a écrit :

> Dear CC4BBers,
> 
> I am trying to figure out what is the best way to determine the protein 
> concentration of my membrane protein. My purified membrane protein is in 20mM 
> Tris pH 7, 150mM NaCl and 0.02% DDM (CMC of DDM=0.0076%).
> 
> After reading the friendly manuals and searching online, I've learned that 
> detergents interferes with assays like Bradford but can't find good 
> descriptions of what works best. For now, I am trying to estimate 
> concentration from absorbance at 280nm and using molar extinction 
> coefficients based on aromatic amino acids, but again suspect detergent 
> interference. I would like to know what other folks working on membrane 
> proteins are doing.
> 
> Thanks very much.
> Raji
> 
> -- 
> Raji Edayathumangalam
> Instructor in Neurology, Harvard Medical School
> Research Associate, Brigham and Women's Hospital
> Visiting Research Scholar, Brandeis University
> 

Xavier Brazzolotto, PhD
Département de Toxicologie et Risque Chimique
Institut de Recherche Biomédicale des Armées
BP 73
91223 Brétigny sur Orge
France

Cell: +33 (0) 6 58 36 39 09

Actual Street Adress

Institut de Biologie Structurale
Equipe DYNAMOP
6 Rue Jules Horowitz
38027 Grenoble Cedex 1
France

Phone: +33 (0) 4 57 42 87 19

The information in this e-mail may be privileged and confidential, intended 
only for the use of the addressee(s) above. Any unauthorized use or disclosure 
of this information is prohibited. If you have received this e-mail by mistake, 
please delete it and immediately contact the sender.

Re: [ccp4bb] Sister CCPs

[Pierre Rizkallah]

>Let me pick up Eleanor’s comment:is there something like a 
>crystallographer today ?

Yes, James Holton and countless others.

> I mean in the true sense ?I think as a “crystallographer” you 
> won’t be able to survive the next decade, you need to diversify your 
> toolset of techniques as pointed out in this 
> articlehttp://www.nature.com/naturejobs/science/articles/10.1038/nj7485-711a

Will a spectroscopist survive? Will a biochemist? 

>And I’m not quite sure how software developers see themselves, as I 
>would argue they are typically maybe not doing so much wet lab stuff related 
>to crystallography (I may be wrong here) but rather code these days.
What “type” of crystallographer is a software developer ?

Perhaps someone like Randy Read and team, or George Sheldrick who started this 
thread, or Eleanor Dodson whom you are responding to.

>I think like our beloved crystals “we” come in different flavors. 
>And we need to train the next generation of students with that perspective in 
>mind.

Well said.

>Just my two cents on a snowy day (>30cm over night)
Jürgen

At least you get the snow. We only get the stormy wind and the rain.


Pierre Rizkallah (from the windswept Cardiff in Wales)

..
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


On Feb 13, 2014, at 6:41 AM, Eleanor Dodson  wrote:
I agree with Frank - it keeps crystallographers modest to know how
challenging wet lab stuff still is..
Eleanor

On 12 February 2014 19:23, Robbie Joosten  wrote:
It's not an e-mail bulletin board, but Researchgate seems to be quite
popular for wet lab questions. IMO the Q&A section of the social network is
a bit messy. That said, the quality seems to improve gradually.

Cheers,
Robbie

Sent from my Windows Phone

Van: Paul Emsley
Verzonden: 12-2-2014 19:23
Aan: CCP4BB@JISCMAIL.AC.UK
Onderwerp: Re: [ccp4bb] Sister CCPs


On 12/02/14 15:59, George Sheldrick wrote:
It would be so nice to have a 'sister CCP' for questions aboud wet-lab
problems that have nothing to do with CCP4 or crystallographic
computing, The is clearly a big need for it, and those of us who try
to keep out of wet-labs would not have to wade though it all.


FWIW, the remit of CCP4BB, held at jiscmail-central, is describes as:

/The CCP4BB mailing list is for discussions on the use of the CCP4
suite, and macromolecular crystallography in general./



Thus wet-lab questions are not off-topic (not that anyone recently
described them as such).

Having said that, Jiscmail mailing lists are easy to set-up (providing
that you can reasonably expect that the mailing list will improve
knowledge sharing within the UK centered academic community) and
relatively low maintenance. I, for one, would not be entirely unhappy to
miss out on questions about lysis.

Paul.

Re: [ccp4bb] Sister CCPs

[Eugene Valkov]

I absolutely agree with Juergen.

Leaving aside methods developers, who are a completely different breed,
there is no such thing as a "crystallographer" sitting in a dark room
solving structures all day. If there are, these are anachronisms destined
for evolutionary demise.

More and more cell biologists, immunologists and all other kinds of
biologists are having a go at doing structural work with their molecules of
interest themselves without involving the "professionals". Typically, they
learn on the job and they need advice with all kinds of things ranging from
cloning and protein preps through to issues with tetartohedrally-twinned
data and interpreting their structures.

So, a modern structural biologist is one who is equipped for the wet lab
and has some idea of how to go about solving structures. CCP4BB is a
wonderful resource that is great for both the quality of the advice offered
to those that seek it and for the variety of topics that are addressed in
the scope of structural biology. I have learnt greatly from reading posts
from very skilled and knowledgeable scientists at this forum and then
implemented these insights into my own research. I am very grateful for
this.

In short, please do not discourage your colleagues, particularly very
junior ones, from posting to the CCP4BB. Some of the questions may appear
quaint or irrelevant but it is easy to simply ignore topics that are of no
interest!

Eugene


On 13 February 2014 14:41, Bosch, Juergen  wrote:

> Let me pick up Eleanor's comment:
> is there something like a crystallographer today ? I mean in the true
> sense ?
> I think as a "crystallographer" you won't be able to survive the next
> decade, you need to diversify your toolset of techniques as pointed out in
> this article
> http://www.nature.com/naturejobs/science/articles/10.1038/nj7485-711a
>
> And I'm not quite sure how software developers see themselves, as I would
> argue they are typically maybe not doing so much wet lab stuff related to
> crystallography (I may be wrong here) but rather code these days.
>
> What "type" of crystallographer is a software developer ?
>
> I think like our beloved crystals "we" come in different flavors. And we
> need to train the next generation of students with that perspective in mind.
>
> Just my two cents on a snowy day (>30cm over night)
>
> Jürgen
> ..
> 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
>
> On Feb 13, 2014, at 6:41 AM, Eleanor Dodson 
> wrote:
>
> I agree with Frank - it keeps crystallographers modest to know how
> challenging wet lab stuff still is..
> Eleanor
>
> On 12 February 2014 19:23, Robbie Joosten 
> wrote:
>
> It's not an e-mail bulletin board, but Researchgate seems to be quite
> popular for wet lab questions. IMO the Q&A section of the social network is
> a bit messy. That said, the quality seems to improve gradually.
>
> Cheers,
> Robbie
>
> Sent from my Windows Phone
> 
> Van: Paul Emsley
> Verzonden: 12-2-2014 19:23
> Aan: CCP4BB@JISCMAIL.AC.UK
> Onderwerp: Re: [ccp4bb] Sister CCPs
>
>
> On 12/02/14 15:59, George Sheldrick wrote:
>
> It would be so nice to have a 'sister CCP' for questions aboud wet-lab
> problems that have nothing to do with CCP4 or crystallographic
> computing, The is clearly a big need for it, and those of us who try
> to keep out of wet-labs would not have to wade though it all.
>
>
>
> FWIW, the remit of CCP4BB, held at jiscmail-central, is describes as:
>
> /The CCP4BB mailing list is for discussions on the use of the CCP4
> suite, and macromolecular crystallography in general./
>
>
>
> Thus wet-lab questions are not off-topic (not that anyone recently
> described them as such).
>
> Having said that, Jiscmail mailing lists are easy to set-up (providing
> that you can reasonably expect that the mailing list will improve
> knowledge sharing within the UK centered academic community) and
> relatively low maintenance. I, for one, would not be entirely unhappy to
> miss out on questions about lysis.
>
> Paul.
>
>
>


-- 
Dr Eugene Valkov

Room 3N049
Division of Structural Studies

MRC Laboratory of Molecular Biology
Francis Crick Avenue
Cambridge Biomedical Campus
Cambridge CB2 0QH, U.K.

Email: eval...@mrc-lmb.cam.ac.uk
Tel: +44 (0) 1223 267358

Re: [ccp4bb] Sister CCPs

[Nat Echols]

One comment (not a complaint) on all this: it seems like the same questions
get asked over and over again.  If there is a good place for a general
crystallography FAQ list it is well past time for one to be put together -
or maybe it just needs to be better advertised?  At a minimum, for instance:

- what cryoprotectant should I use?
- how do I get big single crystals?
- how do I improve diffraction?
- how can I tell if I've solved my structure?
- why is my R-free stuck?
- is  suitable for publication?

Some of the other common queries ("name my blob!") still need to be handled
on a case-by-case basis, but it would be much more efficient for everyone
if the standard answers were collected somewhere permanent.

-Nat



On Thu, Feb 13, 2014 at 7:05 AM, Eugene Valkov wrote:

> I absolutely agree with Juergen.
>
> Leaving aside methods developers, who are a completely different breed,
> there is no such thing as a "crystallographer" sitting in a dark room
> solving structures all day. If there are, these are anachronisms destined
> for evolutionary demise.
>
> More and more cell biologists, immunologists and all other kinds of
> biologists are having a go at doing structural work with their molecules of
> interest themselves without involving the "professionals". Typically, they
> learn on the job and they need advice with all kinds of things ranging from
> cloning and protein preps through to issues with tetartohedrally-twinned
> data and interpreting their structures.
>
> So, a modern structural biologist is one who is equipped for the wet lab
> and has some idea of how to go about solving structures. CCP4BB is a
> wonderful resource that is great for both the quality of the advice offered
> to those that seek it and for the variety of topics that are addressed in
> the scope of structural biology. I have learnt greatly from reading posts
> from very skilled and knowledgeable scientists at this forum and then
> implemented these insights into my own research. I am very grateful for
> this.
>
> In short, please do not discourage your colleagues, particularly very
> junior ones, from posting to the CCP4BB. Some of the questions may appear
> quaint or irrelevant but it is easy to simply ignore topics that are of no
> interest!
>
> Eugene
>
>
> On 13 February 2014 14:41, Bosch, Juergen  wrote:
>
>> Let me pick up Eleanor's comment:
>> is there something like a crystallographer today ? I mean in the true
>> sense ?
>> I think as a "crystallographer" you won't be able to survive the next
>> decade, you need to diversify your toolset of techniques as pointed out in
>> this article
>> http://www.nature.com/naturejobs/science/articles/10.1038/nj7485-711a
>>
>> And I'm not quite sure how software developers see themselves, as I would
>> argue they are typically maybe not doing so much wet lab stuff related to
>> crystallography (I may be wrong here) but rather code these days.
>>
>> What "type" of crystallographer is a software developer ?
>>
>> I think like our beloved crystals "we" come in different flavors. And we
>> need to train the next generation of students with that perspective in mind.
>>
>> Just my two cents on a snowy day (>30cm over night)
>>
>> Jürgen
>> ..
>> 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
>>
>> On Feb 13, 2014, at 6:41 AM, Eleanor Dodson 
>> wrote:
>>
>> I agree with Frank - it keeps crystallographers modest to know how
>> challenging wet lab stuff still is..
>> Eleanor
>>
>> On 12 February 2014 19:23, Robbie Joosten 
>> wrote:
>>
>> It's not an e-mail bulletin board, but Researchgate seems to be quite
>> popular for wet lab questions. IMO the Q&A section of the social network
>> is
>> a bit messy. That said, the quality seems to improve gradually.
>>
>> Cheers,
>> Robbie
>>
>> Sent from my Windows Phone
>> 
>> Van: Paul Emsley
>> Verzonden: 12-2-2014 19:23
>> Aan: CCP4BB@JISCMAIL.AC.UK
>> Onderwerp: Re: [ccp4bb] Sister CCPs
>>
>>
>> On 12/02/14 15:59, George Sheldrick wrote:
>>
>> It would be so nice to have a 'sister CCP' for questions aboud wet-lab
>> problems that have nothing to do with CCP4 or crystallographic
>> computing, The is clearly a big need for it, and those of us who try
>> to keep out of wet-labs would not have to wade though it all.
>>
>>
>>
>> FWIW, the remit of CCP4BB, held at jiscmail-central, is describes as:
>>
>> /The CCP4BB mailing list is for discussions on the use of the CCP4
>> suite, and macromolecular crystallography in general./
>>
>>
>>
>> Thus wet-lab questions are not off-topic (not that anyone recently
>> described them as such).
>>
>> Having said that, Jiscmail mailing lists are easy to set-up (providing
>> that yo

Re: [ccp4bb] create a lower resolution data set by truncating a high resolution data

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Blaine Mooers,

I don't think that you avoid Fourier ripples with this method. You
may, however, increase the noise level so that the Fourier ripples
drown in the noise.

You don't really generate a low resolution data set by truncation for
that very reason. Crystals that only diffract to 3.5A, say, usually do
this because of a high degree of disorder with leeds to more noise.

Of course you could add noise to the sigma-values, but this is merely
cosmetics.

There are repositories for frames (JCSG?), where you might want to get
real data that diffract to the desired resolution, reprocess and then
carry out the phasing experiments.

Best,
Tim

On 02/13/2014 03:54 PM, Mooers, Blaine H.M. (HSC) wrote:
> For some simulated phasing experiments, I want to create a lower
> resolution diffraction data set by truncating a high resolution
> data set. I would like to avoid Fourier ripples due to the
> truncation of the high resolution data by downscaling the data
> such that =2.0 in the highest resolution shell of the
> truncated data. What is the best way to do this?
> 
> Blaine Mooers Assistant Professor Department of Biochemistry and
> Molecular Biology University of Oklahoma Health Sciences Center 
> S.L. Young Biomedical Research Center Rm. 466
> 
> Shipping address: 975 NE 10th Street, BRC 466 Oklahoma City, OK
> 73104-5419
> 
> Letter address: P.O. Box 26901, BRC 466 Oklahoma City, OK 73190
> 
> 
> office: (405) 271-8300   lab: (405) 271-8313  fax:  (405) 271-3910 
> e-mail:  blaine-moo...@ouhsc.edu
> 
> Faculty webpage:
> http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/faculty/blaine-mooers-ph-d-
>
>  X-ray lab webpage:
> http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/department-facilities/macromolecular-crystallography-laboratory
>
>  Small Angle Scattering webpage:
> http://www.oumedicine.com/docs/default-source/ad-biochemistry-workfiles/small-angle-scattering-links.html?sfvrsn=0
>
> 
- -- 
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A

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Version: GnuPG v1.4.12 (GNU/Linux)
Comment: Using GnuPG with Icedove - http://www.enigmail.net/

iD8DBQFS/OMOUxlJ7aRr7hoRAmShAJ9ML9rKT1IiW8FGa+j748FYmAegHwCgmDGK
zmPCnjjlQ2vVbXSh4FMPvG4=
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Re: [ccp4bb] Rmerge of Images

[Jim Pflugrath]

I think you are talking about the R of the reflections in an image to the set 
of unique reflections resulting from scaling.

Here is a definition of Rmerge from a dtscaleaverage log file:

In the tables below Rmerge is defined as:
Rmerge = Sum Sum |Ihi - | / Sum Sum 
  h   i  h   i
where Ihi is the ith used observation for unique hkl h,
and   is the mean intensity for unique hkl h.

Here is a definition of Rmeas from a dtscaleaverage log file:

In the tables below the redundancy-independent merging
R factor Rmeas (or Rrim) is defined as:
Rmeas = Sum [N/(N-1)]^(1/2) Sum |Ihi - | / Sum Sum 
 h   i  h   i
where Ihi is the ith used observation for unique hkl h,
and   is the mean intensity for unique hkl h.

Sorry, but that's what "text only" gives you.

 is calculated from the entire scaled data set and is the unique hkl.  If a 
batch or image does not have that unique reflection, then it would not appear 
in the above equations.

Also if only 1 reflection was used to calculate , then it is not included 
in the sums.  Quiz time: Can you tell me why?

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Keller, Jacob 
[kell...@janelia.hhmi.org]
Sent: Wednesday, February 12, 2014 10:46 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Rmerge of Images

Dear Crystallographers,

Where can I find the definition of the R vs batch reported in scaling? 
Specifically I am wondering whether it is cumulative (each new frame versus all 
previous ones pooled together) or something else, and also how this metric can 
have any meaning on early frames when one has measured each reflection only 
once (in p1, this would be 180 degrees). I am wondering about its efficacy as a 
radiation-damage metric.

JPK

***
Jacob Pearson Keller, PhD
Looger Lab/HHMI Janelia Farms Research Campus
19700 Helix Dr, Ashburn, VA 20147
email: kell...@janelia.hhmi.org
***

[ccp4bb] AW: [ccp4bb] create a lower resolution data set by truncating a high resolution data

[Herman . Schreuder]

Dear Blaine,

The way to do this is not to downscale, but to apply a sufficiently high 
temperature factor. This can be done with e.g. the program CAD. However, I 
guess that the Bfactor will be applied to the sigma's as well, so you may want 
not to apply the Bfactor to the sigma's, or if this is not possible, generate a 
dummy sigma column and have that scaled. 

Best,
Herman

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Mooers, 
Blaine H.M. (HSC)
Gesendet: Donnerstag, 13. Februar 2014 15:54
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] create a lower resolution data set by truncating a high 
resolution data

For some simulated phasing experiments, I want to create a lower resolution 
diffraction data set by truncating a high resolution data set. I would like to 
avoid Fourier ripples due to the truncation of the high resolution data by 
downscaling the data  such that =2.0 in the highest resolution shell 
of the truncated data. What is the best way to do this?

Blaine Mooers
Assistant Professor
Department of Biochemistry and Molecular Biology University of Oklahoma Health 
Sciences Center S.L. Young Biomedical Research Center Rm. 466

Shipping address:
975 NE 10th Street, BRC 466
Oklahoma City, OK 73104-5419

Letter address:  
P.O. Box 26901, BRC 466  
Oklahoma City, OK 73190 

office: (405) 271-8300   lab: (405) 271-8313  fax:  (405) 271-3910
e-mail:  blaine-moo...@ouhsc.edu

Faculty webpage: 
http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/faculty/blaine-mooers-ph-d-

X-ray lab webpage: 
http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/department-facilities/macromolecular-crystallography-laboratory

Small Angle Scattering webpage: 
http://www.oumedicine.com/docs/default-source/ad-biochemistry-workfiles/small-angle-scattering-links.html?sfvrsn=0

Re: [ccp4bb] Sister CCPs

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1



On 02/13/2014 04:21 PM, Nat Echols wrote:
> Some of the other common queries ("name my blob!") still need to be
> handled on a case-by-case basis, but it would be much more
> efficient for everyone if the standard answers were collected
> somewhere permanent.
> 
> -Nat
Some call it 'Jiscmail archive', others call it google or lmgtfy.com,
and having yet another 'somewhere' won't make people use more search
facilities. And since answers are not permanent overall I think it is
a good idea questions get asked again and again. It is my choice (as
putative person answering a question) to decide how much time I spent
with the question.

Cheers,
Tim

> 
> 
> 
> On Thu, Feb 13, 2014 at 7:05 AM, Eugene Valkov
> wrote:
> 
>> I absolutely agree with Juergen.
>> 
>> Leaving aside methods developers, who are a completely different
>> breed, there is no such thing as a "crystallographer" sitting in
>> a dark room solving structures all day. If there are, these are
>> anachronisms destined for evolutionary demise.
>> 
>> More and more cell biologists, immunologists and all other kinds
>> of biologists are having a go at doing structural work with their
>> molecules of interest themselves without involving the
>> "professionals". Typically, they learn on the job and they need
>> advice with all kinds of things ranging from cloning and protein
>> preps through to issues with tetartohedrally-twinned data and
>> interpreting their structures.
>> 
>> So, a modern structural biologist is one who is equipped for the
>> wet lab and has some idea of how to go about solving structures.
>> CCP4BB is a wonderful resource that is great for both the quality
>> of the advice offered to those that seek it and for the variety
>> of topics that are addressed in the scope of structural biology.
>> I have learnt greatly from reading posts from very skilled and
>> knowledgeable scientists at this forum and then implemented these
>> insights into my own research. I am very grateful for this.
>> 
>> In short, please do not discourage your colleagues, particularly
>> very junior ones, from posting to the CCP4BB. Some of the
>> questions may appear quaint or irrelevant but it is easy to
>> simply ignore topics that are of no interest!
>> 
>> Eugene
>> 
>> 
>> On 13 February 2014 14:41, Bosch, Juergen 
>> wrote:
>> 
>>> Let me pick up Eleanor's comment: is there something like a
>>> crystallographer today ? I mean in the true sense ? I think as
>>> a "crystallographer" you won't be able to survive the next 
>>> decade, you need to diversify your toolset of techniques as
>>> pointed out in this article 
>>> http://www.nature.com/naturejobs/science/articles/10.1038/nj7485-711a
>>>
>>>
>>> 
And I'm not quite sure how software developers see themselves, as I would
>>> argue they are typically maybe not doing so much wet lab stuff
>>> related to crystallography (I may be wrong here) but rather
>>> code these days.
>>> 
>>> What "type" of crystallographer is a software developer ?
>>> 
>>> I think like our beloved crystals "we" come in different
>>> flavors. And we need to train the next generation of students
>>> with that perspective in mind.
>>> 
>>> Just my two cents on a snowy day (>30cm over night)
>>> 
>>> Jrgen .. Jrgen 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
>>> 
>>> On Feb 13, 2014, at 6:41 AM, Eleanor Dodson
>>>  wrote:
>>> 
>>> I agree with Frank - it keeps crystallographers modest to know
>>> how challenging wet lab stuff still is.. Eleanor
>>> 
>>> On 12 February 2014 19:23, Robbie Joosten
>>>  wrote:
>>> 
>>> It's not an e-mail bulletin board, but Researchgate seems to be
>>> quite popular for wet lab questions. IMO the Q&A section of the
>>> social network is a bit messy. That said, the quality seems to
>>> improve gradually.
>>> 
>>> Cheers, Robbie
>>> 
>>> Sent from my Windows Phone  
>>> Van: Paul Emsley Verzonden: 12-2-2014 19:23 Aan:
>>> CCP4BB@JISCMAIL.AC.UK Onderwerp: Re: [ccp4bb] Sister CCPs
>>> 
>>> 
>>> On 12/02/14 15:59, George Sheldrick wrote:
>>> 
>>> It would be so nice to have a 'sister CCP' for questions aboud
>>> wet-lab problems that have nothing to do with CCP4 or
>>> crystallographic computing, The is clearly a big need for it,
>>> and those of us who try to keep out of wet-labs would not have
>>> to wade though it all.
>>> 
>>> 
>>> 
>>> FWIW, the remit of CCP4BB, held at jiscmail-central, is
>>> describes as:
>>> 
>>> /The CCP4BB mailing list is for discussions on the use of the
>>> CCP4 suite, and macromolecular crystallography in general./
>>> 
>>> 
>>> 
>>> Thus wet-lab questions are not off-topic (not that anyone
>>> recently described the

Re: [ccp4bb] Sister CCPs

[Eugene Valkov]

Nat - two such places already exist! They are the official ccp4 wiki at
http://ccp4wiki.org/ and the unofficial one (but a very useful one!) at
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/

Eugene


On 13 February 2014 15:21, Nat Echols  wrote:

> One comment (not a complaint) on all this: it seems like the same
> questions get asked over and over again.  If there is a good place for a
> general crystallography FAQ list it is well past time for one to be put
> together - or maybe it just needs to be better advertised?  At a minimum,
> for instance:
>
> - what cryoprotectant should I use?
> - how do I get big single crystals?
> - how do I improve diffraction?
> - how can I tell if I've solved my structure?
> - why is my R-free stuck?
> - is  suitable for publication?
>
> Some of the other common queries ("name my blob!") still need to be
> handled on a case-by-case basis, but it would be much more efficient for
> everyone if the standard answers were collected somewhere permanent.
>
> -Nat
>
>
>
> On Thu, Feb 13, 2014 at 7:05 AM, Eugene Valkov wrote:
>
>> I absolutely agree with Juergen.
>>
>> Leaving aside methods developers, who are a completely different breed,
>> there is no such thing as a "crystallographer" sitting in a dark room
>> solving structures all day. If there are, these are anachronisms destined
>> for evolutionary demise.
>>
>> More and more cell biologists, immunologists and all other kinds of
>> biologists are having a go at doing structural work with their molecules of
>> interest themselves without involving the "professionals". Typically, they
>> learn on the job and they need advice with all kinds of things ranging from
>> cloning and protein preps through to issues with tetartohedrally-twinned
>> data and interpreting their structures.
>>
>> So, a modern structural biologist is one who is equipped for the wet lab
>> and has some idea of how to go about solving structures. CCP4BB is a
>> wonderful resource that is great for both the quality of the advice offered
>> to those that seek it and for the variety of topics that are addressed in
>> the scope of structural biology. I have learnt greatly from reading posts
>> from very skilled and knowledgeable scientists at this forum and then
>> implemented these insights into my own research. I am very grateful for
>> this.
>>
>> In short, please do not discourage your colleagues, particularly very
>> junior ones, from posting to the CCP4BB. Some of the questions may appear
>> quaint or irrelevant but it is easy to simply ignore topics that are of no
>> interest!
>>
>> Eugene
>>
>>
>> On 13 February 2014 14:41, Bosch, Juergen  wrote:
>>
>>> Let me pick up Eleanor's comment:
>>> is there something like a crystallographer today ? I mean in the true
>>> sense ?
>>> I think as a "crystallographer" you won't be able to survive the next
>>> decade, you need to diversify your toolset of techniques as pointed out in
>>> this article
>>> http://www.nature.com/naturejobs/science/articles/10.1038/nj7485-711a
>>>
>>> And I'm not quite sure how software developers see themselves, as I
>>> would argue they are typically maybe not doing so much wet lab stuff
>>> related to crystallography (I may be wrong here) but rather code these days.
>>>
>>> What "type" of crystallographer is a software developer ?
>>>
>>> I think like our beloved crystals "we" come in different flavors. And we
>>> need to train the next generation of students with that perspective in mind.
>>>
>>> Just my two cents on a snowy day (>30cm over night)
>>>
>>> Jürgen
>>> ..
>>> 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
>>>
>>> On Feb 13, 2014, at 6:41 AM, Eleanor Dodson 
>>> wrote:
>>>
>>> I agree with Frank - it keeps crystallographers modest to know how
>>> challenging wet lab stuff still is..
>>> Eleanor
>>>
>>> On 12 February 2014 19:23, Robbie Joosten 
>>> wrote:
>>>
>>> It's not an e-mail bulletin board, but Researchgate seems to be quite
>>> popular for wet lab questions. IMO the Q&A section of the social network
>>> is
>>> a bit messy. That said, the quality seems to improve gradually.
>>>
>>> Cheers,
>>> Robbie
>>>
>>> Sent from my Windows Phone
>>> 
>>> Van: Paul Emsley
>>> Verzonden: 12-2-2014 19:23
>>> Aan: CCP4BB@JISCMAIL.AC.UK
>>> Onderwerp: Re: [ccp4bb] Sister CCPs
>>>
>>>
>>> On 12/02/14 15:59, George Sheldrick wrote:
>>>
>>> It would be so nice to have a 'sister CCP' for questions aboud wet-lab
>>> problems that have nothing to do with CCP4 or crystallographic
>>> computing, The is clearly a big need for it, and those of us who try
>>> to keep out of wet-labs would not have to wade though it all.
>>>
>>>
>>>
>>> FW

Re: [ccp4bb] Sister CCPs

[Bosch, Juergen]

I guess somebody could add it here perhaps ?
http://ccp4wiki.org/~ccp4wiki/wiki/index.php?title=Special:Allpages

or start their own wiki on "Tips & tricks in crystallography" and why you 
should always have olive oil when traveling to a synchrotron (well nowadays 
it’s becoming more difficult to actually be physically present at such a 
facility, then the olive oil won’t help either for cryo protection)

Jürgen

..
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

On Feb 13, 2014, at 10:21 AM, Nat Echols 
mailto:nathaniel.ech...@gmail.com>> wrote:

One comment (not a complaint) on all this: it seems like the same questions get 
asked over and over again.  If there is a good place for a general 
crystallography FAQ list it is well past time for one to be put together - or 
maybe it just needs to be better advertised?  At a minimum, for instance:

- what cryoprotectant should I use?
- how do I get big single crystals?
- how do I improve diffraction?
- how can I tell if I've solved my structure?
- why is my R-free stuck?
- is  suitable for publication?

Some of the other common queries ("name my blob!") still need to be handled on 
a case-by-case basis, but it would be much more efficient for everyone if the 
standard answers were collected somewhere permanent.

-Nat



On Thu, Feb 13, 2014 at 7:05 AM, Eugene Valkov 
mailto:eugene.val...@gmail.com>> wrote:
I absolutely agree with Juergen.

Leaving aside methods developers, who are a completely different breed, there 
is no such thing as a "crystallographer" sitting in a dark room solving 
structures all day. If there are, these are anachronisms destined for 
evolutionary demise.

More and more cell biologists, immunologists and all other kinds of biologists 
are having a go at doing structural work with their molecules of interest 
themselves without involving the "professionals". Typically, they learn on the 
job and they need advice with all kinds of things ranging from cloning and 
protein preps through to issues with tetartohedrally-twinned data and 
interpreting their structures.

So, a modern structural biologist is one who is equipped for the wet lab and 
has some idea of how to go about solving structures. CCP4BB is a wonderful 
resource that is great for both the quality of the advice offered to those that 
seek it and for the variety of topics that are addressed in the scope of 
structural biology. I have learnt greatly from reading posts from very skilled 
and knowledgeable scientists at this forum and then implemented these insights 
into my own research. I am very grateful for this.

In short, please do not discourage your colleagues, particularly very junior 
ones, from posting to the CCP4BB. Some of the questions may appear quaint or 
irrelevant but it is easy to simply ignore topics that are of no interest!

Eugene


On 13 February 2014 14:41, Bosch, Juergen 
mailto:jubo...@jhsph.edu>> wrote:
Let me pick up Eleanor’s comment:
is there something like a crystallographer today ? I mean in the true sense ?
I think as a “crystallographer” you won’t be able to survive the next decade, 
you need to diversify your toolset of techniques as pointed out in this article
http://www.nature.com/naturejobs/science/articles/10.1038/nj7485-711a

And I’m not quite sure how software developers see themselves, as I would argue 
they are typically maybe not doing so much wet lab stuff related to 
crystallography (I may be wrong here) but rather code these days.

What “type” of crystallographer is a software developer ?

I think like our beloved crystals “we” come in different flavors. And we need 
to train the next generation of students with that perspective in mind.

Just my two cents on a snowy day (>30cm over night)

Jürgen
..
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

On Feb 13, 2014, at 6:41 AM, Eleanor Dodson 
mailto:eleanor.dod...@york.ac.uk>> wrote:

I agree with Frank - it keeps crystallographers modest to know how
challenging wet lab stuff still is..
Eleanor

On 12 February 2014 19:23, Robbie Joosten 
mailto:robbie_joos...@hotmail.com>> wrote:
It's not an e-mail bulletin board, but Researchgate seems to be quite
popular for wet lab questions. IMO the Q&A section of the social network is
a bit messy. That said, the quality seems to improve gradually.

Cheers,
Robbie

Sent from my Windows Phone

Van: Paul Emsley
Verzonden: 12-2-2014 19:23
Aan: CC

[ccp4bb] AW: [ccp4bb] create a lower resolution data set by truncating a high resolution data

[Herman . Schreuder]

By applying a high temperature factor, one should not suffer Fourier ripples, 
since the "missing" high resolution reflections have negligible intensities, or 
put differently, one simulates a poorly diffracting crystal.

Best,
Herman

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Tim 
Gruene
Gesendet: Donnerstag, 13. Februar 2014 16:22
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] create a lower resolution data set by truncating a high 
resolution data

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Blaine Mooers,

I don't think that you avoid Fourier ripples with this method. You may, 
however, increase the noise level so that the Fourier ripples drown in the 
noise.

You don't really generate a low resolution data set by truncation for that very 
reason. Crystals that only diffract to 3.5A, say, usually do this because of a 
high degree of disorder with leeds to more noise.

Of course you could add noise to the sigma-values, but this is merely cosmetics.

There are repositories for frames (JCSG?), where you might want to get real 
data that diffract to the desired resolution, reprocess and then carry out the 
phasing experiments.

Best,
Tim

On 02/13/2014 03:54 PM, Mooers, Blaine H.M. (HSC) wrote:
> For some simulated phasing experiments, I want to create a lower 
> resolution diffraction data set by truncating a high resolution data 
> set. I would like to avoid Fourier ripples due to the truncation of 
> the high resolution data by downscaling the data such that 
> =2.0 in the highest resolution shell of the truncated data. 
> What is the best way to do this?
> 
> Blaine Mooers Assistant Professor Department of Biochemistry and 
> Molecular Biology University of Oklahoma Health Sciences Center S.L. 
> Young Biomedical Research Center Rm. 466
> 
> Shipping address: 975 NE 10th Street, BRC 466 Oklahoma City, OK
> 73104-5419
> 
> Letter address: P.O. Box 26901, BRC 466 Oklahoma City, OK 73190
> 
> 
> office: (405) 271-8300   lab: (405) 271-8313  fax:  (405) 271-3910 
> e-mail:  blaine-moo...@ouhsc.edu
> 
> Faculty webpage:
> http://www.oumedicine.com/department-of-biochemistry-and-molecular-bio
> logy/faculty/blaine-mooers-ph-d-
>
>  X-ray lab webpage:
> http://www.oumedicine.com/department-of-biochemistry-and-molecular-bio
> logy/department-facilities/macromolecular-crystallography-laboratory
>
>  Small Angle Scattering webpage:
> http://www.oumedicine.com/docs/default-source/ad-biochemistry-workfile
> s/small-angle-scattering-links.html?sfvrsn=0
>
> 
- --
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A

-BEGIN PGP SIGNATURE-
Version: GnuPG v1.4.12 (GNU/Linux)
Comment: Using GnuPG with Icedove - http://www.enigmail.net/

iD8DBQFS/OMOUxlJ7aRr7hoRAmShAJ9ML9rKT1IiW8FGa+j748FYmAegHwCgmDGK
zmPCnjjlQ2vVbXSh4FMPvG4=
=RXur
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Re: [ccp4bb] Determining concentration of membrane protein

[Roger Rowlett]

Your basic choices for protein assays are:

1. Alkaline copper methods (e.g., Biuret and micro-biuret)
2. alkaline copper + molybdate methods (e.g., Lowry, BCA assays)
3. Hydrophobic dye methods (e.g. Bradford)
4. UV methods (e.g., A280, A230, A210, etc.)

Method 1 is least sensitive to amino acid composition, but is also has 
highest detection limits. Thiols interfere. Method 2 is very 
idiosyncratic with amino acid composition, and also subject to 
interference by thiols. Method 3 is not usable in detergent solutions. 
Method 4 has many inteferences as most everything absorbs in the far UV 
region.


If you have some special protein cofactors, metals, chromophores, etc. 
these can be exploited for better measurements. For ecample 
metalloproteins are easy to quantify by ICP-OES or TXRF if they are 
reasonably pure.


Cheers,

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

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

On 2/13/2014 10:06 AM, Raji Edayathumangalam wrote:

Dear CC4BBers,

I am trying to figure out what is the best way to determine the 
protein concentration of my membrane protein. My purified membrane 
protein is in 20mM Tris pH 7, 150mM NaCl and 0.02% DDM (CMC of 
DDM=0.0076%).


After reading the friendly manuals and searching online, I've learned 
that detergents interferes with assays like Bradford but can't find 
good descriptions of what works best. For now, I am trying to estimate 
concentration from absorbance at 280nm and using molar extinction 
coefficients based on aromatic amino acids, but again suspect 
detergent interference. I would like to know what other folks working 
on membrane proteins are doing.


Thanks very much.
Raji

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

Re: [ccp4bb] Sister CCPs

[Raji Edayathumangalam]

Hi Folks,

Like many other folks who read, benefit and hopefully contribute to this
bulletin board, I am required to stay abreast of the latest cloning,
heterologous membrane protein expression,
purification, biochemistry, crystallization, and structure solution
techniques, as a matter of sheer necessity. So much of my time in the
lab everyday is spent banging against one of these walls, so to speak, that
it only makes sense to reach out to this extremely diverse and
interdisciplinary community in times of genuine need. As is clear from many
of the posts (but maybe not all), a lot of people seem to have done their
homework (RTFM, tried several things, etc.) and are clearly stuck at the
time of posting to the bulletin board.

Consider my case, which I'm sure is also the predicament of many many folks
who post here. I am the sole "structural biologist" in a lab in a
department of Neurology where I am surrounded by biochemists and cell
biologists whose idea of a large-scale culture is 250mL. I can read all I
want, use my common sense and brain of limited capacity all I want, and can
talk to myself all I want, but in the end, when I am badly stuck, this is
not an approach I find scientifically healthy or productive.

A couple of other things: Many rules of thumbs for soluble protein
constructs, expression, purification and crystallization do not
automatically carry over to membrane proteins or large macromolecular
complexes or the remaining unsolved mysteries of challenging soluble
proteins.

I'd rather just delete emails where the subject line clearly indicates my
lack of immediate interest in the topic than run the risk of splitting this
bulletin boards into many smaller parts. The learning impact from the
collective experience of the diverse members of this single historic
bulletin board will always be orders of magnitude larger the sum of its
individual superspecialized parts.

To me, it seems natural that the ccp4bb has, over time, morphed in good
ways not originally conceived or intended. If anything, this tide speaks to
the collective scientific impact that a global and diverse group of
researchers from various interrelated disciplines can have on one other.

Many thanks to the amazing members of the ccp4bb!
Raji

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





On Thu, Feb 13, 2014 at 10:05 AM, Eugene Valkov wrote:

> I absolutely agree with Juergen.
>
> Leaving aside methods developers, who are a completely different breed,
> there is no such thing as a "crystallographer" sitting in a dark room
> solving structures all day. If there are, these are anachronisms destined
> for evolutionary demise.
>
> More and more cell biologists, immunologists and all other kinds of
> biologists are having a go at doing structural work with their molecules of
> interest themselves without involving the "professionals". Typically, they
> learn on the job and they need advice with all kinds of things ranging from
> cloning and protein preps through to issues with tetartohedrally-twinned
> data and interpreting their structures.
>
> So, a modern structural biologist is one who is equipped for the wet lab
> and has some idea of how to go about solving structures. CCP4BB is a
> wonderful resource that is great for both the quality of the advice offered
> to those that seek it and for the variety of topics that are addressed in
> the scope of structural biology. I have learnt greatly from reading posts
> from very skilled and knowledgeable scientists at this forum and then
> implemented these insights into my own research. I am very grateful for
> this.
>
> In short, please do not discourage your colleagues, particularly very
> junior ones, from posting to the CCP4BB. Some of the questions may appear
> quaint or irrelevant but it is easy to simply ignore topics that are of no
> interest!
>
> Eugene
>
>
> On 13 February 2014 14:41, Bosch, Juergen  wrote:
>
>> Let me pick up Eleanor's comment:
>> is there something like a crystallographer today ? I mean in the true
>> sense ?
>> I think as a "crystallographer" you won't be able to survive the next
>> decade, you need to diversify your toolset of techniques as pointed out in
>> this article
>> http://www.nature.com/naturejobs/science/articles/10.1038/nj7485-711a
>>
>> And I'm not quite sure how software developers see themselves, as I would
>> argue they are typically maybe not doing so much wet lab stuff related to
>> crystallography (I may be wrong here) but rather code these days.
>>
>> What "type" of crystallographer is a software developer ?
>>
>> I think like our beloved crystals "we" come in different flavors. And we
>> need to train the next generation of students with that perspective in mind.
>>
>> Just my two cents on a snowy day (>30cm over night)
>>
>> Jürgen
>> ..
>> Jürgen Bosch
>> Johns Hopkins University
>> Bloomberg

Re: [ccp4bb] Sister CCPs

[Francis Reyes]

The CCP4bb is great.. it truly is. 

The access to experts and their experience  (probably the most valuable) is 
unparalleled. 

However, mailing lists to organize discussions and disseminate new ideas is 
just so ... 90s. 

Wikis? maybe you've just crossed into the new millenium.

These days, if the questions/answers of the ccp4bb moved into something like 
Quora,
It would save me a lot of time.

F


  
On Feb 13, 2014, at 10:21 AM, Nat Echols  wrote:

> One comment (not a complaint) on all this: it seems like the same questions 
> get asked over and over again.  If there is a good place for a general 
> crystallography FAQ list it is well past time for one to be put together - or 
> maybe it just needs to be better advertised?  At a minimum, for instance:
> 
> - what cryoprotectant should I use?
> - how do I get big single crystals?
> - how do I improve diffraction?
> - how can I tell if I've solved my structure?
> - why is my R-free stuck?
> - is  suitable for publication?
> 
> Some of the other common queries ("name my blob!") still need to be handled 
> on a case-by-case basis, but it would be much more efficient for everyone if 
> the standard answers were collected somewhere permanent.
> 
> -Nat
> 
> 
> 
> On Thu, Feb 13, 2014 at 7:05 AM, Eugene Valkov  
> wrote:
> I absolutely agree with Juergen.
> 
> Leaving aside methods developers, who are a completely different breed, there 
> is no such thing as a "crystallographer" sitting in a dark room solving 
> structures all day. If there are, these are anachronisms destined for 
> evolutionary demise.
> 
> More and more cell biologists, immunologists and all other kinds of 
> biologists are having a go at doing structural work with their molecules of 
> interest themselves without involving the "professionals". Typically, they 
> learn on the job and they need advice with all kinds of things ranging from 
> cloning and protein preps through to issues with tetartohedrally-twinned data 
> and interpreting their structures.
> 
> So, a modern structural biologist is one who is equipped for the wet lab and 
> has some idea of how to go about solving structures. CCP4BB is a wonderful 
> resource that is great for both the quality of the advice offered to those 
> that seek it and for the variety of topics that are addressed in the scope of 
> structural biology. I have learnt greatly from reading posts from very 
> skilled and knowledgeable scientists at this forum and then implemented these 
> insights into my own research. I am very grateful for this.
> 
> In short, please do not discourage your colleagues, particularly very junior 
> ones, from posting to the CCP4BB. Some of the questions may appear quaint or 
> irrelevant but it is easy to simply ignore topics that are of no interest! 
> 
> Eugene 
> 
> 
> On 13 February 2014 14:41, Bosch, Juergen  wrote:
> Let me pick up Eleanor’s comment:
> is there something like a crystallographer today ? I mean in the true sense ?
> I think as a “crystallographer” you won’t be able to survive the next decade, 
> you need to diversify your toolset of techniques as pointed out in this 
> article
> http://www.nature.com/naturejobs/science/articles/10.1038/nj7485-711a
> 
> And I’m not quite sure how software developers see themselves, as I would 
> argue they are typically maybe not doing so much wet lab stuff related to 
> crystallography (I may be wrong here) but rather code these days.
> 
> What “type” of crystallographer is a software developer ?
> 
> I think like our beloved crystals “we” come in different flavors. And we need 
> to train the next generation of students with that perspective in mind.
> 
> Just my two cents on a snowy day (>30cm over night)
> 
> Jürgen
> ..
> 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
> 
> On Feb 13, 2014, at 6:41 AM, Eleanor Dodson  wrote:
> 
>> I agree with Frank - it keeps crystallographers modest to know how
>> challenging wet lab stuff still is..
>> Eleanor
>> 
>> On 12 February 2014 19:23, Robbie Joosten  wrote:
>>> It's not an e-mail bulletin board, but Researchgate seems to be quite
>>> popular for wet lab questions. IMO the Q&A section of the social network is
>>> a bit messy. That said, the quality seems to improve gradually.
>>> 
>>> Cheers,
>>> Robbie
>>> 
>>> Sent from my Windows Phone
>>> 
>>> Van: Paul Emsley
>>> Verzonden: 12-2-2014 19:23
>>> Aan: CCP4BB@JISCMAIL.AC.UK
>>> Onderwerp: Re: [ccp4bb] Sister CCPs
>>> 
>>> 
>>> On 12/02/14 15:59, George Sheldrick wrote:
 It would be so nice to have a 'sister CCP' for questions aboud wet-lab
 problems that have nothing to do with CCP4 or crystallographic
 computing, The is clearly 

[ccp4bb] X6A Workbench - Registration is Open

[Stojanoff, Vivian]

X6A Workbench: Hands-on Synchrotron Structural Biology
February 25 - 28, 2014

The NIGMS facility at the National Synchrotron Light Source is offering 
comprehensive hands-on training in synchrotron data collection and analysis for 
biophysicists, biochemists and molecular biologists. The goal is to provide 
participants an insight into Structural Biology methods available at a 
synchrotron radiation facility. Four to six participants are invited to follow 
a four day experiment at the X6A beamline. Most of the program will concentrate 
in the development of experimental skills. Introductory lecture will be 
presented by local experts.

Participants are invited to bring their samples for the experiments but samples 
will be provided for training.

http://workshops.ps.bnl.gov/default.aspx?w=X6AFeb2014

For more information, please contact:

Mercy Baez
Photon Sciences User Administration Office
Telephone No.: 631.344.5769
Email Address: b...@bnl.gov

Re: [ccp4bb] Determining concentration of membrane protein

[R. M. Garavito]

Roger,

While I agree with your list, the BCA assay does not use molybdate (as we make 
it from scratch with bicinchoninic acid, sodium carbonate, sodium bicarbonate, 
sodium tartrate, and cupric sulfate pentahydrate).  For membrane proteins, I 
prefer the BCA assay until the protein is pure enough to use A280.

Cheers,

Michael


R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
603 Wilson Rd., Rm. 513   
Michigan State University  
East Lansing, MI 48824-1319
Office:  (517) 355-9724 Lab:  (517) 353-9125
FAX:  (517) 353-9334Email:  rmgarav...@gmail.com





On Feb 13, 2014, at 10:39 AM, Roger Rowlett  wrote:

> Your basic choices for protein assays are:
> Alkaline copper methods (e.g., Biuret and micro-biuret)
> alkaline copper + molybdate methods (e.g., Lowry, BCA assays)
> Hydrophobic dye methods (e.g. Bradford)
> UV methods (e.g., A280, A230, A210, etc.)
> Method 1 is least sensitive to amino acid composition, but is also has 
> highest detection limits. Thiols interfere. Method 2 is very idiosyncratic 
> with amino acid composition, and also subject to interference by thiols. 
> Method 3 is not usable in detergent solutions. Method 4 has many inteferences 
> as most everything absorbs in the far UV region.
> If you have some special protein cofactors, metals, chromophores, etc. these 
> can be exploited for better measurements. For ecample metalloproteins are 
> easy to quantify by ICP-OES or TXRF if they are reasonably pure.
> Cheers,
> ___
> Roger S. Rowlett
> Gordon & Dorothy Kline Professor
> Department of Chemistry
> Colgate University
> 13 Oak Drive
> Hamilton, NY 13346
> 
> tel: (315)-228-7245
> ofc: (315)-228-7395
> fax: (315)-228-7935
> email: rrowl...@colgate.edu
> On 2/13/2014 10:06 AM, Raji Edayathumangalam wrote:
>> Dear CC4BBers,
>> 
>> I am trying to figure out what is the best way to determine the protein 
>> concentration of my membrane protein. My purified membrane protein is in 
>> 20mM Tris pH 7, 150mM NaCl and 0.02% DDM (CMC of DDM=0.0076%).
>> 
>> After reading the friendly manuals and searching online, I've learned that 
>> detergents interferes with assays like Bradford but can't find good 
>> descriptions of what works best. For now, I am trying to estimate 
>> concentration from absorbance at 280nm and using molar extinction 
>> coefficients based on aromatic amino acids, but again suspect detergent 
>> interference. I would like to know what other folks working on membrane 
>> proteins are doing.
>> 
>> Thanks very much.
>> Raji
>> 
>> -- 
>> Raji Edayathumangalam
>> Instructor in Neurology, Harvard Medical School
>> Research Associate, Brigham and Women's Hospital
>> Visiting Research Scholar, Brandeis University
>> 
> 

Re: [ccp4bb] Sister CCPs

[Tim Gruene]

Dear Francis,

could you elaborate how something like Quora (what is it) would save you
time compared to en email based? From what I gathered at wikipedia,
Quora is web based, and in my experience web based services usually
follow Wirth's law: they are slow.

Cheers,
Tim

On 02/13/2014 05:47 PM, Francis Reyes wrote:
> The CCP4bb is great.. it truly is. 
> 
> The access to experts and their experience  (probably the most valuable) is 
> unparalleled. 
> 
> However, mailing lists to organize discussions and disseminate new ideas is 
> just so ... 90s. 
> 
> Wikis? maybe you've just crossed into the new millenium.
> 
> These days, if the questions/answers of the ccp4bb moved into something like 
> Quora,
> It would save me a lot of time.
> 
> F
> 
> 
>   
> On Feb 13, 2014, at 10:21 AM, Nat Echols  wrote:
> 
>> One comment (not a complaint) on all this: it seems like the same questions 
>> get asked over and over again.  If there is a good place for a general 
>> crystallography FAQ list it is well past time for one to be put together - 
>> or maybe it just needs to be better advertised?  At a minimum, for instance:
>>
>> - what cryoprotectant should I use?
>> - how do I get big single crystals?
>> - how do I improve diffraction?
>> - how can I tell if I've solved my structure?
>> - why is my R-free stuck?
>> - is  suitable for publication?
>>
>> Some of the other common queries ("name my blob!") still need to be handled 
>> on a case-by-case basis, but it would be much more efficient for everyone if 
>> the standard answers were collected somewhere permanent.
>>
>> -Nat
>>
>>
>>
>> On Thu, Feb 13, 2014 at 7:05 AM, Eugene Valkov  
>> wrote:
>> I absolutely agree with Juergen.
>>
>> Leaving aside methods developers, who are a completely different breed, 
>> there is no such thing as a "crystallographer" sitting in a dark room 
>> solving structures all day. If there are, these are anachronisms destined 
>> for evolutionary demise.
>>
>> More and more cell biologists, immunologists and all other kinds of 
>> biologists are having a go at doing structural work with their molecules of 
>> interest themselves without involving the "professionals". Typically, they 
>> learn on the job and they need advice with all kinds of things ranging from 
>> cloning and protein preps through to issues with tetartohedrally-twinned 
>> data and interpreting their structures.
>>
>> So, a modern structural biologist is one who is equipped for the wet lab and 
>> has some idea of how to go about solving structures. CCP4BB is a wonderful 
>> resource that is great for both the quality of the advice offered to those 
>> that seek it and for the variety of topics that are addressed in the scope 
>> of structural biology. I have learnt greatly from reading posts from very 
>> skilled and knowledgeable scientists at this forum and then implemented 
>> these insights into my own research. I am very grateful for this.
>>
>> In short, please do not discourage your colleagues, particularly very junior 
>> ones, from posting to the CCP4BB. Some of the questions may appear quaint or 
>> irrelevant but it is easy to simply ignore topics that are of no interest! 
>>
>> Eugene 
>>
>>
>> On 13 February 2014 14:41, Bosch, Juergen  wrote:
>> Let me pick up Eleanor’s comment:
>> is there something like a crystallographer today ? I mean in the true sense ?
>> I think as a “crystallographer” you won’t be able to survive the next 
>> decade, you need to diversify your toolset of techniques as pointed out in 
>> this article
>> http://www.nature.com/naturejobs/science/articles/10.1038/nj7485-711a
>>
>> And I’m not quite sure how software developers see themselves, as I would 
>> argue they are typically maybe not doing so much wet lab stuff related to 
>> crystallography (I may be wrong here) but rather code these days.
>>
>> What “type” of crystallographer is a software developer ?
>>
>> I think like our beloved crystals “we” come in different flavors. And we 
>> need to train the next generation of students with that perspective in mind.
>>
>> Just my two cents on a snowy day (>30cm over night)
>>
>> Jürgen
>> ..
>> 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
>>
>> On Feb 13, 2014, at 6:41 AM, Eleanor Dodson  
>> wrote:
>>
>>> I agree with Frank - it keeps crystallographers modest to know how
>>> challenging wet lab stuff still is..
>>> Eleanor
>>>
>>> On 12 February 2014 19:23, Robbie Joosten  
>>> wrote:
 It's not an e-mail bulletin board, but Researchgate seems to be quite
 popular for wet lab questions. IMO the Q&A section of the social network is
 a bit messy. That said, the quality seems to improve gradually.

 Ch

Re: [ccp4bb] AW: [ccp4bb] create a lower resolution data set by truncating a high resolution data

[Edward A. Berry]

I agree with raising the B-factor as the way to simulate a poorly diffracting 
crystal.
By how much, to simulate a given resolution? Until  in the new "last shell" 
is
some multiple of (sigma before sharpening)? Until "optical resolution" as 
calculated
by sfcheck is what would be expected for the desired resolution?
http://sb20.lbl.gov/eytung/optical/opticalresolution.gif

herman.schreu...@sanofi.com wrote:

By applying a high temperature factor, one should not suffer Fourier ripples, since the 
"missing" high resolution reflections have negligible intensities, or put 
differently, one simulates a poorly diffracting crystal.

Best,
Herman

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Tim 
Gruene
Gesendet: Donnerstag, 13. Februar 2014 16:22
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] create a lower resolution data set by truncating a high 
resolution data

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Blaine Mooers,

I don't think that you avoid Fourier ripples with this method. You may, 
however, increase the noise level so that the Fourier ripples drown in the 
noise.

You don't really generate a low resolution data set by truncation for that very 
reason. Crystals that only diffract to 3.5A, say, usually do this because of a 
high degree of disorder with leeds to more noise.

Of course you could add noise to the sigma-values, but this is merely cosmetics.

There are repositories for frames (JCSG?), where you might want to get real 
data that diffract to the desired resolution, reprocess and then carry out the 
phasing experiments.

Best,
Tim

On 02/13/2014 03:54 PM, Mooers, Blaine H.M. (HSC) wrote:

For some simulated phasing experiments, I want to create a lower
resolution diffraction data set by truncating a high resolution data
set. I would like to avoid Fourier ripples due to the truncation of
the high resolution data by downscaling the data such that
=2.0 in the highest resolution shell of the truncated data.
What is the best way to do this?

Blaine Mooers Assistant Professor Department of Biochemistry and
Molecular Biology University of Oklahoma Health Sciences Center S.L.
Young Biomedical Research Center Rm. 466

Shipping address: 975 NE 10th Street, BRC 466 Oklahoma City, OK
73104-5419

Letter address: P.O. Box 26901, BRC 466 Oklahoma City, OK 73190


office: (405) 271-8300   lab: (405) 271-8313  fax:  (405) 271-3910
e-mail:  blaine-moo...@ouhsc.edu

Faculty webpage:
http://www.oumedicine.com/department-of-biochemistry-and-molecular-bio
logy/faculty/blaine-mooers-ph-d-

  X-ray lab webpage:
http://www.oumedicine.com/department-of-biochemistry-and-molecular-bio
logy/department-facilities/macromolecular-crystallography-laboratory

  Small Angle Scattering webpage:
http://www.oumedicine.com/docs/default-source/ad-biochemistry-workfile
s/small-angle-scattering-links.html?sfvrsn=0



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

GPG Key ID = A46BEE1A

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Version: GnuPG v1.4.12 (GNU/Linux)
Comment: Using GnuPG with Icedove - http://www.enigmail.net/

iD8DBQFS/OMOUxlJ7aRr7hoRAmShAJ9ML9rKT1IiW8FGa+j748FYmAegHwCgmDGK
zmPCnjjlQ2vVbXSh4FMPvG4=
=RXur
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Re: [ccp4bb] AW: [ccp4bb] create a lower resolution data set by truncating a high resolution data

[Tim Gruene]

Dear Herman,

this might work. To get more realistic scaling/ merging statistics, one
should expand to P1, add independent noise to U's and coordinates,
create the hkl and force it back into the original symmetry.

Best,
Tim

On 02/13/2014 04:38 PM, herman.schreu...@sanofi.com wrote:
> By applying a high temperature factor, one should not suffer Fourier ripples, 
> since the "missing" high resolution reflections have negligible intensities, 
> or put differently, one simulates a poorly diffracting crystal.
> 
> Best,
> Herman
> 
> -Ursprüngliche Nachricht-
> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Tim 
> Gruene
> Gesendet: Donnerstag, 13. Februar 2014 16:22
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: Re: [ccp4bb] create a lower resolution data set by truncating a high 
> resolution data
> 
> Dear Blaine Mooers,
> 
> I don't think that you avoid Fourier ripples with this method. You may, 
> however, increase the noise level so that the Fourier ripples drown in the 
> noise.
> 
> You don't really generate a low resolution data set by truncation for that 
> very reason. Crystals that only diffract to 3.5A, say, usually do this 
> because of a high degree of disorder with leeds to more noise.
> 
> Of course you could add noise to the sigma-values, but this is merely 
> cosmetics.
> 
> There are repositories for frames (JCSG?), where you might want to get real 
> data that diffract to the desired resolution, reprocess and then carry out 
> the phasing experiments.
> 
> Best,
> Tim
> 
> On 02/13/2014 03:54 PM, Mooers, Blaine H.M. (HSC) wrote:
>> For some simulated phasing experiments, I want to create a lower 
>> resolution diffraction data set by truncating a high resolution data 
>> set. I would like to avoid Fourier ripples due to the truncation of 
>> the high resolution data by downscaling the data such that 
>> =2.0 in the highest resolution shell of the truncated data. 
>> What is the best way to do this?
> 
>> Blaine Mooers Assistant Professor Department of Biochemistry and 
>> Molecular Biology University of Oklahoma Health Sciences Center S.L. 
>> Young Biomedical Research Center Rm. 466
> 
>> Shipping address: 975 NE 10th Street, BRC 466 Oklahoma City, OK
>> 73104-5419
> 
>> Letter address: P.O. Box 26901, BRC 466 Oklahoma City, OK 73190
> 
> 
>> office: (405) 271-8300   lab: (405) 271-8313  fax:  (405) 271-3910 
>> e-mail:  blaine-moo...@ouhsc.edu
> 
>> Faculty webpage:
>> http://www.oumedicine.com/department-of-biochemistry-and-molecular-bio
>> logy/faculty/blaine-mooers-ph-d-
> 
>>  X-ray lab webpage:
>> http://www.oumedicine.com/department-of-biochemistry-and-molecular-bio
>> logy/department-facilities/macromolecular-crystallography-laboratory
> 
>>  Small Angle Scattering webpage:
>> http://www.oumedicine.com/docs/default-source/ad-biochemistry-workfile
>> s/small-angle-scattering-links.html?sfvrsn=0
> 
> 
> 

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

GPG Key ID = A46BEE1A



signature.asc
Description: OpenPGP digital signature

Re: [ccp4bb] Determining concentration of membrane protein

[Raji Edayathumangalam]

Hi Everyone,

Thanks very much for your helpful responses and suggestions. I will use the
BCA assay.

Cheers,
Raji


On Thu, Feb 13, 2014 at 11:27 AM, R. M. Garavito wrote:

> Roger,
>
> While I agree with your list, the BCA assay does not use molybdate (as we
> make it from scratch with bicinchoninic acid, sodium carbonate, sodium
> bicarbonate, sodium tartrate, and cupric sulfate pentahydrate).  For
> membrane proteins, I prefer the BCA assay until the protein is pure enough
> to use A280.
>
> Cheers,
>
> Michael
>
> **
> *R. Michael Garavito, Ph.D.*
> *Professor of Biochemistry & Molecular Biology*
> *603 Wilson Rd., Rm. 513*
> *Michigan State University  *
> *East Lansing, MI 48824-1319*
> *Office:*  *(517) 355-9724 <%28517%29%20355-9724> Lab:  (517)
> 353-9125 <%28517%29%20353-9125>*
> *FAX:  (517) 353-9334 <%28517%29%20353-9334>
>  Email:  rmgarav...@gmail.com *
> **
>
>
>
>
> On Feb 13, 2014, at 10:39 AM, Roger Rowlett  wrote:
>
>  Your basic choices for protein assays are:
>
>1. Alkaline copper methods (e.g., Biuret and micro-biuret)
>2. alkaline copper + molybdate methods (e.g., Lowry, BCA assays)
>3. Hydrophobic dye methods (e.g. Bradford)
>4. UV methods (e.g., A280, A230, A210, etc.)
>
> Method 1 is least sensitive to amino acid composition, but is also has
> highest detection limits. Thiols interfere. Method 2 is very idiosyncratic
> with amino acid composition, and also subject to interference by thiols.
> Method 3 is not usable in detergent solutions. Method 4 has many
> inteferences as most everything absorbs in the far UV region.
>
> If you have some special protein cofactors, metals, chromophores, etc.
> these can be exploited for better measurements. For ecample metalloproteins
> are easy to quantify by ICP-OES or TXRF if they are reasonably pure.
>
> Cheers,
>
> ___
> Roger S. Rowlett
> Gordon & Dorothy Kline Professor
> Department of Chemistry
> Colgate University
> 13 Oak Drive
> Hamilton, NY 13346
>
> tel: (315)-228-7245
> ofc: (315)-228-7395
> fax: (315)-228-7935
> email: rrowl...@colgate.edu
>  On 2/13/2014 10:06 AM, Raji Edayathumangalam wrote:
>
> Dear CC4BBers,
>
>  I am trying to figure out what is the best way to determine the protein
> concentration of my membrane protein. My purified membrane protein is in
> 20mM Tris pH 7, 150mM NaCl and 0.02% DDM (CMC of DDM=0.0076%).
>
>  After reading the friendly manuals and searching online, I've learned
> that detergents interferes with assays like Bradford but can't find good
> descriptions of what works best. For now, I am trying to estimate
> concentration from absorbance at 280nm and using molar extinction
> coefficients based on aromatic amino acids, but again suspect detergent
> interference. I would like to know what other folks working on membrane
> proteins are doing.
>
>  Thanks very much.
> Raji
>
>  --
> Raji Edayathumangalam
> Instructor in Neurology, Harvard Medical School
> Research Associate, Brigham and Women's Hospital
> Visiting Research Scholar, Brandeis University
>
>
>
>


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

Re: [ccp4bb] Sister CCPs

[Francis Reyes]

Hi Tim

Yes it is web-based. (Their iOS app works pretty well).

Is it slow ?  Well it depends. 

If you consider the situation where a ccp4bb poster has a specific question... 

"How do I determine the resolution of my data..."

On the CCP4BB you get a number of posts of the following types in response:
answers (some good, some blatantly incorrect).
some arguing
some debating
somehow a topic about his-tag purification sneaks in with the same subject 
title, 
somehow a topic about someone's plans to visit a conference 

We can all agree that sifting through this information on a mail program is 
inconvenient, frustrating though sometimes amusing.  

Wiki-approach:  The advantages of the wiki approach is that you don't get 
redundant information, you can be as comprehensive as you want, you can use 
hyperlinks to reference for more content, multiple users can edit so there's 
some content curation. 

However, the wiki style is not really oriented to  questions and answers.. it's 
subject oriented. 
If someone were to ask you a question about resolution at a seminar, you don't 
start with a diatribe of bragg's law and breaking waves on the beach. You start 
with a simple but comprehensive answer.  As the questioner, I have a specific 
question and I want an answer, hopefully a reliable one. If I want 
backstory/supporting information, that's fine, but it's secondary to getting a 
reliable answer. 

Quora (in its basic sense) focuses on people asking questions and other people 
providing answers.  It uses the 'crowd'  (ideally more knowledgeable peers) to 
determine what answers are 'good' and should be 'upvoted'.  You can invite 
experts (also other 'quorans') to answer specific questions by spending quora 
credits (which you earn by getting followers to the questions you ask, by 
providing answers which garner upvotes from other quorans, by answering a 
question that someone asked you to answer). 

These mechanisms are used by Quora to curate content, determining what are 
interesting questions to put on people's feed (those with the most followers), 
and what are 'good' answers (those with the most upvotes written by reputable 
people). 

Can this system be manipulated? Absolutely, but provided you have answers from 
trusted/known peers, you can have some idea on how good a particular answer is. 
 However, I assume most of us are scientists and are skeptical of any answer 
given to us. 

Is this the best solution? Of course not, however, I think Quora might be a 
competitive solution. 

Surprisingly, there are NO ADS on quora which tells me that they're early stage 
or have yet to succumb to investor's expectations, i.e. it's a fun time to be a 
quoran. 

(Disclaimer) I don't work for Quora and they don't give me money. 

Cheers,

F


On Feb 13, 2014, at 1:19 PM, Tim Gruene  wrote:

> Dear Francis,
> 
> could you elaborate how something like Quora (what is it) would save you
> time compared to en email based? From what I gathered at wikipedia,
> Quora is web based, and in my experience web based services usually
> follow Wirth's law: they are slow.
> 
> Cheers,
> Tim

-
Francis E. Reyes PhD
215 UCB
University of Colorado at Boulder

--

[ccp4bb] 3letter code

[Faisal Tarique]

Hi everyone

Can anyone please tell me the three letter code for D-Ribulose
1,5-bisphosphate..

Thanks in advance

-- 
Regards

Faisal
School of Life Sciences
JNU

Re: [ccp4bb] 3letter code

[Mario Sanches]

It is "RUB".
http://xray.bmc.uu.se/hicup/RUB/index.html




On Thu, Feb 13, 2014 at 1:07 PM, Faisal Tarique wrote:

> Hi everyone
>
> Can anyone please tell me the three letter code for D-Ribulose
> 1,5-bisphosphate..
>
> Thanks in advance
>
> --
> Regards
>
> Faisal
> School of Life Sciences
> JNU
>



-- 
Mario Sanches
http://ca.linkedin.com/in/mariosanches

[ccp4bb] How to find the unfound part of a big protein

[Sun Bingfa]

Dear Crystallographers,

I'm working on a ~90KDa membrane protein, with big extracellular part,
probably function as dimer.
Now we have dataset to ~4.2 Angstrom and using extracellular homolog
structure we can find a solution for this part(~45% of the whole molecule
MW) through molecular replacement, and the molecules are packed as layers,
and the other part are presumably between these layers. However, we are
having trouble to fit the rest of the protein even though there're some
density between the solved part. Rfree is at 40% now.

We're trying to do heavy atom soaking, such as TaBr. We collected data for
MIR but it's not helping so far. (Can I combine these MIR data with the
native dataset because the MIR set is only at ~6.5 Angstrom)?

Other information: this protein is expressed in Sf9 cells (so very hard to
do Se-Met derivatives). The crystals is nice and big and cubic.

Any suggestions or examples? Thanks a lot.

Bingfa

Re: [ccp4bb] How to find the unfound part of a big protein

[Bosch, Juergen]

Hi Bingfa,

first suggestion would be to process your data with the anomalous flag turned 
on - just in case you happen to have some  metal bound coincidentally and you 
happen to have collected at a decent wavelength to pickup some anomalous 
scattering. ~30% of proteins in the PDB have a metal bound just FYI and not all 
of them were soaked in on purpose.

In case you should be that lucky, then you should be able to use the HA phases 
+ your MR model to help building.

Jürgen

..
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

On Feb 13, 2014, at 4:53 PM, Sun Bingfa 
mailto:sunbin...@gmail.com>> wrote:

Dear Crystallographers,

I'm working on a ~90KDa membrane protein, with big extracellular part, probably 
function as dimer.
Now we have dataset to ~4.2 Angstrom and using extracellular homolog structure 
we can find a solution for this part(~45% of the whole molecule MW) through 
molecular replacement, and the molecules are packed as layers, and the other 
part are presumably between these layers. However, we are having trouble to fit 
the rest of the protein even though there're some density between the solved 
part. Rfree is at 40% now.

We're trying to do heavy atom soaking, such as TaBr. We collected data for MIR 
but it's not helping so far. (Can I combine these MIR data with the native 
dataset because the MIR set is only at ~6.5 Angstrom)?

Other information: this protein is expressed in Sf9 cells (so very hard to do 
Se-Met derivatives). The crystals is nice and big and cubic.

Any suggestions or examples? Thanks a lot.

Bingfa

[ccp4bb] Acetyl link problems.

[Ian Tickle]

All, I'm having problems refining a structure with an N-terminal acetylated
MET residue.  I'm trying it with both Refmac & Buster.  Buster works fine &
gives perfect planar geometry for the ACE-MET linkage.  Refmac gives a
pyramidal acetyl group after refinement which to my eyes is wrong (sp2 C
atom?).

I have this line in my input PDB:

LINKRC   ACE A   0 N   MET A   1
ACE_C-N

which as I understand it should solve the problem.  However, looking at the
CIF entry for the ACE_C-N link I see restraints defined for bonds, angles &
torsion angles but not for the CC(=O)N plane.  So the problem seems to be
that the planar restraints for this link group are missing - or are they
defined elsewhere?  Anyway I added planar restraints to the ACE_C-N link
entry & it solves the problem, at least for regularisation - I still have
the same problem with refinement.  Refmac in regularisation mode now gives
the correct (planar) geometry for the ACE-MET linkage.  I'm just puzzled
why no-one has noticed this, after all post-translational acetylation is
surely not that uncommon (according to Wikipedia > 80% of human proteins
are N-term acetylated!).

Further, looking at the entry for ACE I see:

ACE  ACE 'ACETYL GROUP' non-polymer 7
3 .

 ACE   O  OO 0.000  0.0000.0000.000
 ACE   C  CC10.000 -1.044   -0.6060.000
 ACE   H  HH 0.000 -1.978   -0.0690.000
 ACE   CH3CCH3   0.000 -1.041   -2.1130.000
 ACE   H3 HH 0.000 -0.541   -2.4640.865
 ACE   H2 HH 0.000 -2.038   -2.4680.000
 ACE   H1 HH 0.000 -0.540   -2.464   -0.864

Where did the extra H atom (3rd atom) come from?  Acetyl is CH3C=O: the
extra H atom would make it acetaldehyde which of course has nothing
whatsoever to do with acetylation!  Is this the reason for the lack of
planar link restraints (though that wouldn't explain why the other link
restraints are present)?

Any insights appreciated!

Cheers

-- Ian

Re: [ccp4bb] How to find the unfound part of a big protein

[Francis Reyes]

On Feb 13, 2014, at 4:53 PM, Sun Bingfa  wrote:

> Now we have dataset to ~4.2 Angstrom and using extracellular homolog 
> structure we can find a solution for this part(~45% of the whole molecule MW) 
> through molecular replacement, and the molecules are packed as layers, and 
> the other part are presumably between these layers. However, we are having 
> trouble to fit the rest of the protein even though there're some density 
> between the solved part. 

You have an incomplete MR problem.

> Rfree is at 40% now. 
> 


This is fairly typical.

> We're trying to do heavy atom soaking, such as TaBr. We collected data for 
> MIR but it's not helping so far.

This is also fairly typical.

> (Can I combine these MIR data with the native dataset because the MIR set is 
> only at ~6.5 Angstrom)? 
> 

Yes, but you need to be careful in how you do this.  The bad news is that 
jumping from 6.5 -> 4A is not trivial. 

The good news is that it can be done. 

See for example,
http://www.nature.com/nature/journal/v492/n7427/full/nature11607.html

Cheers,

F

-
Francis E. Reyes PhD
215 UCB
University of Colorado at Boulder

--

[ccp4bb] Postbac position in membrane protein structure and function at the NIH

[Anirban Banerjee]

Dear all,

I am looking for a postbac for a membrane protein structure-function
project in my lab at the NIH. The NIH Postbac IRTA program provides
recent college graduates who are planning to apply to graduate or
professional (medical/dental/pharmacy) school an opportunity to spend
one or two years performing full-time research at the NIH.

For this position I am looking for someone who has experience with
protein purification, has good organizational skills and work ethics,
and the right mindset to pay attention to nitty-gritty details that
are essential for working with membrane proteins. The candidate should
apply to the NIH Postbac Program
(https://www.training.nih.gov/programs/postbac_irta)  and get in touch
with me as well. Postbac candidates have to be US citizens or
permanent residents.

Thanks very much.

Anirban Banerjee

Re: [ccp4bb] Determining concentration of membrane protein

[Ho Leung Ng]

Hi Raji,

 There are also some proprietary stains such as the "660 nm" (can't
they think of a better product name?) stain from Pierce that are detergent
compatible. I used this briefly with success when comparing against Abs 280
nm.


Ho

Ho Leung Ng
University of Hawaii at Manoa
Assistant Professor, Department of Chemistry
h...@hawaii.edu


Date:Thu, 13 Feb 2014 10:06:12 -0500
> From:Raji Edayathumangalam 
> Subject: Determining concentration of membrane protein
>
> Dear CC4BBers,
>
> I am trying to figure out what is the best way to determine the protein
> concentration of my membrane protein. My purified membrane protein is in
> 20mM Tris pH 7, 150mM NaCl and 0.02% DDM (CMC of DDM=0.0076%).
>
> After reading the friendly manuals and searching online, I've learned that
> detergents interferes with assays like Bradford but can't find good
> descriptions of what works best. For now, I am trying to estimate
> concentration from absorbance at 280nm and using molar extinction
> coefficients based on aromatic amino acids, but again suspect detergent
> interference. I would like to know what other folks working on membrane
> proteins are doing.
>
> Thanks very much.
> Raji
>

Re: [ccp4bb] Determining concentration of membrane protein

[Niks]

Dear All,
May be a stupid question. But if we take buffer with detergent as control
(Blank), would not the difference in ODs using any of the methods used e.g.
Bradford assay, gives protein concentration?

Regards
Nishant


On Thu, Feb 13, 2014 at 9:09 PM, Roger Rowlett  wrote:

>  Your basic choices for protein assays are:
>
>1. Alkaline copper methods (e.g., Biuret and micro-biuret)
>2. alkaline copper + molybdate methods (e.g., Lowry, BCA assays)
>3. Hydrophobic dye methods (e.g. Bradford)
>4. UV methods (e.g., A280, A230, A210, etc.)
>
> Method 1 is least sensitive to amino acid composition, but is also has
> highest detection limits. Thiols interfere. Method 2 is very idiosyncratic
> with amino acid composition, and also subject to interference by thiols.
> Method 3 is not usable in detergent solutions. Method 4 has many
> inteferences as most everything absorbs in the far UV region.
>
> If you have some special protein cofactors, metals, chromophores, etc.
> these can be exploited for better measurements. For ecample metalloproteins
> are easy to quantify by ICP-OES or TXRF if they are reasonably pure.
>
> Cheers,
>
> ___
> Roger S. Rowlett
> Gordon & Dorothy Kline Professor
> Department of Chemistry
> Colgate University
> 13 Oak Drive
> Hamilton, NY 13346
>
> tel: (315)-228-7245
> ofc: (315)-228-7395
> fax: (315)-228-7935
> email: rrowl...@colgate.edu
>  On 2/13/2014 10:06 AM, Raji Edayathumangalam wrote:
>
> Dear CC4BBers,
>
>  I am trying to figure out what is the best way to determine the protein
> concentration of my membrane protein. My purified membrane protein is in
> 20mM Tris pH 7, 150mM NaCl and 0.02% DDM (CMC of DDM=0.0076%).
>
>  After reading the friendly manuals and searching online, I've learned
> that detergents interferes with assays like Bradford but can't find good
> descriptions of what works best. For now, I am trying to estimate
> concentration from absorbance at 280nm and using molar extinction
> coefficients based on aromatic amino acids, but again suspect detergent
> interference. I would like to know what other folks working on membrane
> proteins are doing.
>
>  Thanks very much.
> Raji
>
>  --
> Raji Edayathumangalam
> Instructor in Neurology, Harvard Medical School
> Research Associate, Brigham and Women's Hospital
> Visiting Research Scholar, Brandeis University
>
>
>


-- 
"The most difficult phase of  life is not when No one understands you;It is
when you don't understand yourself"

Re: [ccp4bb] Sister CCPs

[Kay Diederichs]

Nat,

that's why I set up the CCP4 wiki at 
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Main_Page ! The 
idea is that everybody benefits: experienced crystallographers/biologists can 
concentrate on the new and difficult questions coming up on the bulletin board, 
and novices find answers to those ever-recurring questions. Everybody can 
contribute answers, or improve existing ones!

But the wiki can only be useful in the long run if there are contributors. Why 
are there (almost) no contributors? It cannot be due to technical difficulty; 
it's very easy to contribute to a wiki. One guess is that a posting on a BB is 
more socially rewarding, because the interaction via emails is more immediate. 

Re-vitalize the wiki!

Kay

On Thu, 13 Feb 2014 07:21:14 -0800, Nat Echols  
wrote:

>One comment (not a complaint) on all this: it seems like the same questions
>get asked over and over again.  If there is a good place for a general
>crystallography FAQ list it is well past time for one to be put together -
>or maybe it just needs to be better advertised?  At a minimum, for instance:
>
>- what cryoprotectant should I use?
>- how do I get big single crystals?
>- how do I improve diffraction?
>- how can I tell if I've solved my structure?
>- why is my R-free stuck?
>- is  suitable for publication?
>
>Some of the other common queries ("name my blob!") still need to be handled
>on a case-by-case basis, but it would be much more efficient for everyone
>if the standard answers were collected somewhere permanent.
>
>-Nat
>
>
>
>On Thu, Feb 13, 2014 at 7:05 AM, Eugene Valkov wrote:
>
>> I absolutely agree with Juergen.
>>
>> Leaving aside methods developers, who are a completely different breed,
>> there is no such thing as a "crystallographer" sitting in a dark room
>> solving structures all day. If there are, these are anachronisms destined
>> for evolutionary demise.
>>
>> More and more cell biologists, immunologists and all other kinds of
>> biologists are having a go at doing structural work with their molecules of
>> interest themselves without involving the "professionals". Typically, they
>> learn on the job and they need advice with all kinds of things ranging from
>> cloning and protein preps through to issues with tetartohedrally-twinned
>> data and interpreting their structures.
>>
>> So, a modern structural biologist is one who is equipped for the wet lab
>> and has some idea of how to go about solving structures. CCP4BB is a
>> wonderful resource that is great for both the quality of the advice offered
>> to those that seek it and for the variety of topics that are addressed in
>> the scope of structural biology. I have learnt greatly from reading posts
>> from very skilled and knowledgeable scientists at this forum and then
>> implemented these insights into my own research. I am very grateful for
>> this.
>>
>> In short, please do not discourage your colleagues, particularly very
>> junior ones, from posting to the CCP4BB. Some of the questions may appear
>> quaint or irrelevant but it is easy to simply ignore topics that are of no
>> interest!
>>
>> Eugene
>>
>>
>> On 13 February 2014 14:41, Bosch, Juergen  wrote:
>>
>>> Let me pick up Eleanor's comment:
>>> is there something like a crystallographer today ? I mean in the true
>>> sense ?
>>> I think as a "crystallographer" you won't be able to survive the next
>>> decade, you need to diversify your toolset of techniques as pointed out in
>>> this article
>>> http://www.nature.com/naturejobs/science/articles/10.1038/nj7485-711a
>>>
>>> And I'm not quite sure how software developers see themselves, as I would
>>> argue they are typically maybe not doing so much wet lab stuff related to
>>> crystallography (I may be wrong here) but rather code these days.
>>>
>>> What "type" of crystallographer is a software developer ?
>>>
>>> I think like our beloved crystals "we" come in different flavors. And we
>>> need to train the next generation of students with that perspective in mind.
>>>
>>> Just my two cents on a snowy day (>30cm over night)
>>>
>>> J�rgen
>>> ..
>>> J�rgen 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
>>>
>>> On Feb 13, 2014, at 6:41 AM, Eleanor Dodson 
>>> wrote:
>>>
>>> I agree with Frank - it keeps crystallographers modest to know how
>>> challenging wet lab stuff still is..
>>> Eleanor
>>>
>>> On 12 February 2014 19:23, Robbie Joosten 
>>> wrote:
>>>
>>> It's not an e-mail bulletin board, but Researchgate seems to be quite
>>> popular for wet lab questions. IMO the Q&A section of the social network
>>> is
>>> a bit messy. That said, the quality seems to improve gradually.
>>>
>>> Cheers,
>>> Robbie
>>>
>>> Sent f