[ccp4bb] Senior Developer at LS-CAT sector 21 at the APS

[Joe Brunzelle]

*Apply to Job 43479
*

*Department: *NSRC - LS-CAT
*Salary/Grade: *ITS/80

*Job Summary:*

This position is available at Argonne National Laboratory’s Advanced Photon
Source (APS).

An experienced programmer with X-ray scientific background is required to
support operations at the Life Sciences Collaborative Access Team (LS-CAT).
LS-CAT is a highly productive molecular crystallography beamline requiring
someone to help support the current infrastructure. In addition, this is a
very exciting time with the APS undergoing a major upgrade in April 2023
and LS-CAT needs to implement new equipment and controls to take advantage
of the vastly improved X-ray source.

Participates as technical expert in design, development, coding, testing,
and/or debugging of major new software and/or significant enhancements to
existing software which may include applications over multiple platforms.
Guides and advises junior staff.  Manages complex projects independently
and assists in estimating and planning for future development work.
Performs complex system integration tasks.

*Specific Responsibilities*:

*Strategic Planning*

   - Provides application development leadership for new and existing
   software applications.
   - Partners with users in designing features for technology.
   - Provides recommendations on how to enhance the system for future
   growth.
   - Advises/recommends project and activities as related to
   system/architectural direction and
   strategy.

*Administration*

   - Develops and implements procedures for data security, management and
   compliance
   - Creates and maintains code documentation.
   - Creates ad hoc administrative reports.
   - Delivers system presentations and overviews.
   - Evaluates feature/upgrade/change requests and recommends action.
   - Research new technologies to enhance the current
   system.

*Development*

   - Provides technical leadership on projects.
   - Acts as subject matter expert (SME) in appropriate technologies and
   business domain.
   - Designs, codes, tests, debugs and documents all phases of application
   development.
   - Codes software applications adhering to designs supporting internal
   business requirements or external users.
   - Troubleshoots complex, difficult issues.
   - Designs databases and data structures.
   - Provides recommendations on how to enhance the system to meet full
   business requirements.
   - Determines project feasibility and how to integrate with the current
   system.

*Miscellaneous*

   - Performs other duties as assigned.

*Minimum Qualifications:*

   - Successful completion of a full 4-year course of study in an
   accredited college or university leading to a bachelor's or higher degree
   in a major such as computer science, information technology, or related; OR
   appropriate combination of education and experience.
   - 4 years relevant experience required.

*Minimum Competencies: (Skills, knowledge, and abilities.)*

   - Proven programming capabilities in C, Python or a related language.
   - Database management and experience with SQL/MySQL/Postgres/Redis
   - Knowledge of UNIX/LINUX systems administration.
   - Hardware and software engineering
   - User interface design

*Preferred Qualifications:*

   - 5 years experience with motion control systems
   - Synchrotron X-ray instrumentation.
   - Protein crystallography, X-ray scattering and X-ray spectroscopy.
   - Configuration management systems.

*Preferred Competencies: (Skills, knowledge, and abilities)*

   - EPICS (Experimental Physics and Industrial Control System).
   - Motion control systems (e.g. PMAC, Galil, Newport).
   - Robotics automation and integration (e.g. EPSON, Kuka).
   - Data acquisition systems (e.g. VME, NI).
   - PLC Systems (Schneider, Siemens, Pilz).
   - Networking and IT infrastructure.

*Benefits:*
At Northwestern, we are proud to provide meaningful, competitive,
high-quality health care plans, retirement benefits, tuition discounts and
more! Visit us at https://www.northwestern.edu/hr/benefits/index.html
to learn more.*Work-Life
and Wellness:*
Northwestern offers comprehensive programs and services to help you and
your family navigate life’s challenges and opportunities, and adopt and
maintain healthy lifestyles.
We support flexible work arrangements where possible and programs to help
you locate and pay for quality, affordable childcare and senior/adult care.
Visit us at https://www.northwestern.edu/hr/benefits/work-life/index.html
to learn
more.*Professional Growth & Development:*
Northwestern supports employee career development in all circumstances
whether your workspace is on campus or at home. If you’re interested in
developing your 

[ccp4bb] ACA Abstract requests for Evolution & Impact of Targeted Protein Degradation in Industry

[Joe Patel]

Hi,

Matt Clifton (Novartis) and I (Joe Patel, C4 Therapeutics) are organising a 
session around the impact structural methods have had in the field of targeted 
protein degradation at this year's virtual ACA meeting.

The session is scheduled from 12pm till 3pm EDT on 5th August 2021 and we 
expect to run the talks live rather than pre-recorded.

The abstract submission deadline is fast approaching and we would love to see 
more abstract submissions on this exciting facet of the drug discovery paradigm.

The link to this year's meeting is below and our session is 3.2.3:
https://www.acameeting.com/

Please note that abstract submission does require you to be a current paid up 
member of the ACA.


Looking forward to an opportunity to promote and share the exciting structural 
research this field has delivered.

Regards,

Joe & Matt

C4 Therapeutics, Inc. Confidentiality Notice: This message is private and may 
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[ccp4bb] Research Scientist Position, Structural Biology at C4 Therapeutics in Watertown, MA USA

[Joe Patel]

Dear all,

We are looking for additional members to join and support our structural 
biology efforts here at C4 Therapeutics.  If you are (or soon will be) a recent 
graduate with a solid foundation in structural sciences and wish to be a part 
of an emerging new paradigm in drug discovery, please consider applying for the 
role using the link below.

https://easyapply.co/a/6fa7d973-53ef-443b-9e38-1b63b2968dad

Regards,

Joe Patel


Joe Patel, Ph.D.
Director, Biochemistry,
Biophysics & Crystallography

[C42]
490 Arsenal Way, Suite 200
Watertown, MA, 02472
www.c4therapeutics.com<http://www.c4therapeutics.com/>







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[ccp4bb] PhD positions in structural biology at the Astbury Centre, University of Leeds

[Joe Cockburn]

Dear CCP4BB members,



We invite applications for two PhD positions in structural cell biology
based partly or entirely at the Astbury Centre for Structural Molecular
Biology, University of Leeds:


- *The structure and function of retinal photoreceptor connecting cilium
proteins (**Closing date: 7th January 2019)*

Defects in these proteins are associated with inherited disorders
characterized by blindness and/or kidney failure. This project will
investigate the structure and function of these proteins by X-ray
crystallography, cryo-EM and super-resolution imaging approaches. This is a
BBSRC White Rose DTP project supervised jointly between the Cockburn,
Johnson and Ranson labs at the University of Leeds; please see
https://www.findaphd.com/phds/project/structural-and-functional-studies-on-proteins-required-for-vision/?p59606
for
more details.

-  *The molecular pathogenesis of KIF5A mutations (**Closing date: 21st
January 2019)*

The microtubule motor KIF5A is involved in long-range axonal transport.
KIF5A mutations cause inherited spastic paraplegias. This project will
investigate the effects of pathogenic mutations on the structure and
function of KIF5A. This is an MRC DiMeN DTP project supervised jointly
between the Twelvetrees and De Vos labs at the University of Sheffield and
the Cockburn lab at the Univerisity of Leeds; please see
https://www.findaphd.com/phds/project/mrc-dimen-doctoral-training-partnership-how-do-neurodegenerative-mutations-in-kinesin-1-alter-its-structure-motility-and-cellular-function/?p103997
for
more details.



Information on how to apply can be found in the links above. Please feel
free to get in touch (j.j.b.cockb...@leeds.ac.uk) if you wish to discuss
further or require any additional details.



Kind regards,

Joe


--

Dr Joseph J B Cockburn

Lecturer in X-ray Crystallography

The Astbury Centre for Structural and Molecular Biology

Faculty of Biological Sciences

University of Leeds

Leeds LS2 9JT

UK

+44 (0)113 3430758

j.j.b.cockb...@leeds.ac.uk

http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=JJBC



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[ccp4bb] tenure-track academic fellowships in structural molecular biology at the University of Leeds

[Joe Cockburn]

Hello everyone,

Please see the job advert below regarding tenure-track academic fellowships
in structural molecular biology at the University of Leeds.

Cheers,

Joe


Dear All,



A number of tenure-track academic fellowships in Structural Molecular
Biology are now available at the University of Leeds. We are particularly
looking for researchers who use Cryo-EM, NMR Spectroscopy, X-ray
Crystallography, Single Molecule methods (or combinations of these
techniques with other approaches) to answer cutting-edge research
questions?



The full details of the call are available at:

https://jobs.leeds.ac.uk/Vacancy.aspx?id=8486=1



As a University Academic Fellow in the School of Molecular & Cellular
Biology, you will join the vibrant, interdisciplinary environment of the
Astbury Centre for Structural Molecular Biology which brings together ~60
group leaders with expertise in structural molecular biology, cell biology,
protein chemistry, chemical biology and biological physics with the common
goal of understanding life in molecular detail through investigation of the
structure and function of biological molecules and their complexes.



Leeds invested more than £17 million in 2016 to establish the Astbury
Biostructure Laboratory, a state of the art laboratory for structural
biology providing the Astbury Centre with instruments for NMR spectroscopy
(including 950 MHz with 15N/13C-detect TXO and 3mm TCI Cryo-probes) and
Electron Microscopy (two Titan Krios microscopes, one with Falcon III and
the other with energy-filtered Gatan K2 and VPP), competitive with the very
best infrastructure for structural molecular biology in the world. In
addition, the Centre houses superb facilities for protein production,
protein crystallization, biological mass spectrometry and single molecule
fluorescence and FRET.



You will embark on a five-year development programme, the successful
completion of which will lead to your promotion to Associate Professor. In
the last three years, nine University Academic Fellows have been appointed
within the scientific remit of the Astbury Centre, helping to deliver on
the University’s ambitious plans to grow its research. We are now looking
for outstanding early career structural molecular biologists to join this
growing team. We can offer an excellent mentoring scheme, research
environment and track record of success in developing the careers of young
researchers. Candidates wishing to bring independent fellowships, or apply
for such fellowhips in Leeds, supported by the School, are also welcome to
apply.



To discuss informally, please contact Neil Ranson n.a.ran...@leeds.ac.uk,
or Alex Breeze a.l.bre...@leeds.ac.uk



Kind regards,



Neil Ranson

[ccp4bb] Job opening for a structural biologist

[Joe Chen]

Job opportunity:

Eternity Bioscience is hiring a structure biologist.  Details please see ：
http://www.eternitybioscience.com/contact-us

-- 
Best regards,

Joe

[ccp4bb] Post-doctoral position in Structural Biology of Cilia, Astbury Centre, Leeds, UK

[Joe Cockburn]

Dear All,


A post-doctoral position is available immediately in the laboratory of Dr
Joe Cockburn, at the Astbury Centre, University of Leeds, UK to perform
structure-function studies on ciliary proteins.



The position is funded by The Wellcome Trust and is available immediately
for a period of 24 months. The start date is flexible but must be before
September 2017. The deadline for applications is Monday 16th January 2017.



More information about the position and how to apply can be found here:



https://jobs.leeds.ac.uk/vacancy.aspx?ref=FBSMB1092



Thanks,

Joe



--

Dr Joe Cockburn

The Astbury Centre for Structural and Molecular Biology

University of Leeds

Leeds LS2 9JT

UK

+44 (0)113 3430758

j.j.b.cockb...@leeds.ac.uk

http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=JJBC

[ccp4bb] 3D printing format

[Patel, Joe]

Sorry for the rather random question but has anyone out there used a 3D printer 
to print a protein structure?

If so, what format did you need to convert the PDB into to allow the printer to 
interpret the data?

Many thanks,

Joe P


Joe Patel
FBLG Specialist
_
AstraZeneca
RD | Innovative Medicines | Discovery Sciences
Boston RD, Discovery Sciences
35 Gatehouse Drive, Waltham MA  02451
Tel 1-781-839-4129
joe.pa...@astrazeneca.commailto:steven.kazmir...@astrazeneca.com

 P Please consider the environment before printing this e-mail





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Re: [ccp4bb] Unusual electron density - any guesses??

[Patel, Joe]

Is that a glycine in the sequence next to the Glu/Gln?  Have you tried building 
a 50% occ of the backbone in that region in two conformations, and then a water 
molecule further up into the feature.  The density over the carbonyl looks weak 
and you have some negative density there that might indicate mixed conformation.

Just an idea, hard to tell from still images if my idea would work.

Joe P


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disclosure of the contents of this message is not permitted and may be unlawful.
 
-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jose 
Artur Brito
Sent: Friday, October 25, 2013 1:29 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Unusual electron density - any guesses??

Dear All,
I'm refining an X-ray structure to 1.6A resolution in BUSTER-TNT v2.10.
The model is pretty much finished but I see a strange electron density that I 
can't imagine what it is.

Please take a look at four snapshots in http://www.itqb.unl.pt/~jbrito/ITQB/ . 
Any pointers/guesses are most welcome.

In short, I see an oblong piece of density coming straight out of the 
main-chain!! It doesn't refine as a chain of waters and any small piece of 
PEG doesn't refine properly either (actually, not sure if this would make any 
sense but since the crystallization condition is PEG3350 and gave it a try!!).

The crystallization condition is PEG3350, Bis.Tris buffer, (NH4)2SO4 and NaI. 
The protein was purified from recombinant expression in E. coli with trivial 
reagents: Tris and Bis.Tris buffers, NaCl, glycerol, ...

Wishing you all an excellent weekend, best regards, Jose


--

* José Artur Brito, PhD*
*  *
* Post-Doctoral Fellow *
* Membrane Protein Crystallography Lab *
* Instituto de Tecnologia Química e Biológica  *
* Oeiras - Portugal*
*  *
* Tel.: +351.21.446.97.61  *
* Fax:  +351.21.443.36.44  *
*  *
* E-mail: jbr...@itqb.unl.pt   *
* URL: http://mx.itqb.unl.pt   *

[ccp4bb] Supplier for X-ray sensitive paper

[Patel, Joe]

Hi All,

I have done a few searches of the archive and googled a few times but not found 
what I am looking for.

Could someone point me in the direction of a supplier of the X-ray sensitive 
paper I have used in the past to confirm beam position on a home source.  I am 
specifically after this type of paper rather than X-ray film so as not to have 
to go through any developing stage and quickly visualize the location of the 
beam at different points beyond the position of the goniometer towards the 
detector.

A USA supplier would be great but any would do.

Many thanks,

Joe P



--
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and proprietary information. If you have received this message in error, please 
notify us and remove it from your system and note that you must not copy, 
distribute or take any action in reliance on it. Any unauthorized use or 
disclosure of the contents of this message is not permitted and may be unlawful.
 

[ccp4bb] question about CCP4 scripts

[Joe Chen]

Hi All,


I am trying to write a shell script to streamline a few steps, one of which
is Unique, see below.  As you can see, this program requires symmetry and
cell parameters.  In CCP4 GUI Scalepack2mtz, these info are automatically
extracted from .sca file (first two lines).  But I don't know if there is a
way to do this in script, so I don't need to type these values for each
dataset.  Thank you in advance for your help.


#!/bin/sh
# unique.exam
#
# runnnable test script for the program unique - this will use this
# program to generate a reflection list containing null values.
#

set -e

unique hklout ${CCP4_SCR}/unique_out.mtz  eof
labout F=F SIGF=SIGF
symmetry p43212
resolution 1.6
cell 78.1 78.1 55.2 90.0 90.0 90.0
eof


-- 
Best regards,

Joe

Re: [ccp4bb] advices

[Joe Chen]

I guess you may have to decide whether you want to modify your current
conditions or just start over to find new conditions, to get around this
ultra-fast crystallization.  I don't know if you have played around all
parameters of current condition, such as lower temperature, protein
concentration, pH, salt, etc,  or ways to slow down vapor diffusion, i.e.
covering drop or reservoir by silicon oil.  If all these do not help, you
might have to find a new condition under which protein crystallizes in a
different morphology...However, BEFORE you try out things at the
crystallization stage, ask if there is anything that you could improve
before crystallization:  Is your construct good enough?  How is your
protein quality--purity/homogeneity?  Fine-tuning every step from gene to
protein to crystal could eventually lead to good results.

Joe


On Tue, Jan 15, 2013 at 8:30 PM, Mike John perturb-w...@hotmail.com wrote:

  Hello, all,

 My crystal grows very fast even though the protein conc. is now 1.5 mg/ml.
 The shape of the crystal likes a ruler plate with one side very thin. The
 crystal quality has diffraction to about 3.5A in a home source of 1.2 KW
 sealed tube Angilent equipment. But the data can not be indexed due to one
 direction has poor diffraction and the crystal quality. Seeking advices on
 improving the crystal quality. Thank you very much

 Mike




-- 
Best regards,

Joe

Re: [ccp4bb] protein degradation in crystal

[Joe Chen]

Could you identify the cleavage sites by protein sequencing and design new
constructs (truncated versions) accordingly?  It might improve your crystal
quality to get better resolution.


Joe


On Wed, Jan 16, 2013 at 9:22 AM, Tom Murray-Rust
tom.murray.r...@gmail.comwrote:

 Just to add to Herman's suggestions, if you are trying to crystallise a
 protease then you could also try using the S195A variant rather than an
 inhibitor. This would certainly be the case if you ever want to
 co-crystallise in a substrate, as PPACK (or the like) would occupy the
 active site cleft and prevent formation of the protease-substrate complex.

 Tom



 **
 Hi John,

 This is really an amazing wild west story: the man who crystallizes
 faster than his protease! I really must compliment you with how you
 successfully performed these experiments!

 Unfortunately, proteins usually do not crystallize that fast (at least
 not in my hands), so in these cases other methods have to be used. As has
 mentioned before, protease inhibitors are the way to go. Especially with
 autolysis, as one protease cuts another one, the speed of the reaction goes
 with the square of the protease concentration. Whereas in dilute solutions
 not much happens, as soon as you start to concentrate towards
 crystallization conditions, say 10 mg/ml, degradation suddenly goes very
 fast.

 There are 2 cases to consider:
 1) the protein you want to crystallize is a protease and is destroying
 itself. In this case you need to cocrystallize with a potent and specific
 inhibitor. With serine proteases, Wolfram Bode was very successful by using
 chloromethylketone-containing peptides (e.g. PPACK). These compounds would
 make covalent links with both the active site serine and histidine,
 effectively killing any protease activity.

 2) the protein you want to crystallize is not a protease and it is a
 contaminant which is causing the problems. In this case I would add a
 protease inhibitor coctail in an earlier step of the purification to block
 the protease before the final purification steps. I would also add some
 small broad protease inhibitor e.g. PMSF to the protein solution used for
 crystallization.

 Herman

  --
 *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of
 *John Domsic
 *Sent:* Wednesday, January 16, 2013 2:22 PM
 *To:* CCP4BB@JISCMAIL.AC.UK
 *Subject:* Re: [ccp4bb] protein degradation in crystal

 Hi Lisa,

 Speed is definitely a big factor here.  With a protein I work with I can
 get large crystals in myriad conditions that only diffract to about 4-5
 Ang.  What I ended up doing was taking these crystals and seeding entire
 screens.  I found that not only would crystals appear sooner but it
 revealed novel crystallization conditions.  These seeded crystals would
 appear within minutes as Preben described and diffracted to better than 2
 Ang.  Another thought would be to try limited proteolysis to see if you can
 identify a more stable construct.

 -John

 --

 John Domsic
 Postdoctoral Fellow
 Gene Expression and Regulation Program
 The Wistar Institute
 Philadelphia, PA 19104


 On Wed, Jan 16, 2013 at 7:17 AM, jens Preben Morth j.p.mo...@ncmm.uio.no
  wrote:

 Dear Lisa
 It is not uncommon to see breakdown products when you run crystals on
  gel. Espesially if they are older crystals, sometimes you even see higher
 molecular bands, these are probably due to intra molecular cross links
 formed over time.
 If you are worried about stability, try to increase the crystallization
 speed, we have one example where we see a clear difference in both crystal
 quality and even space group depending on when we fish the crystals. The
 crystals appear within 5 min,  the best quality data sets come from
 crystals  we fish after only 30-60 min.
 You may also have a little protease contamination of course, to prevent
 this add protease inhibitor, or DTT, or EDTA to you protein before you set
 it up.
 cheers Preben


 On 1/16/13 12:14 PM, LISA wrote:

 Hi All,
 I have an 36KD protein which can be crystallize in two days. Most of
 the crystals are very big. But all cystals have poor resolution,lower than
 3.8 A. I picked some crystals, washed them in the mother solution and then
 run SDS-PAGE. It is surprised to find that different cystals have different
 components. Some crystals have several samll bands below the band of the
 protein. And in some crysals the bigger size band (as the construct should
 be) almost disappared and have smear. Does the protein was degradated in
 the crystals? Did someone met the similar problem as I? Thanks

 All the best
 lisa


 --
 J. Preben Morth, Ph.D
 Group Leader
 Membrane Transport Group
 Nordic EMBL Partnership
 Centre for Molecular Medicine Norway (NCMM)
 University of Oslo
 P.O.Box 1137 Blindern
 0318 Oslo, Norway

 Email: j.p.mo...@ncmm.uio.no
 Tel: +47 2284 0794 %2B47%202284%200794

 http://www.jpmorth.dk





 --
 Skype: tom.murray.rust
 Twitter: tmurrayrust
 http

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

[Patel, Joe]

Hi Yuri,

If you have access to mogul you can get an understanding of what your geometry 
should be based on the small molecule database.  Of course not everything is 
well represented so if your ligand is unusual this will flag up in lower 
statistical significance.

Mogul will allow you to understand how far you are from ideal.

Not really sure if this is what you might be after

Joe P


--
AstraZeneca UK Limited is a company incorporated in England and Wales with 
registered number: 03674842 and a registered office at 2 Kingdom Street, 
London, W2 6BD.
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security of our computer systems and checking Compliance with our Code of 
Conduct and Policies.
-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Yuri 
Pompeu
Sent: 12 September 2012 19:45
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Ligand geometry obs. vs. ideal

Hi everyone,
I am trying to show that a ligand underwent catalysis during a soaking 
experiment.
One of the things I would like to show is the geometry of the ligand, bond 
angles/lengths, dihedrals, etc...
One of my models has a hi-res of 1.18A and the ligand density is really clear 
and complete.
What is the best way to refine the ligand unrestrained and then generate 
measurements?
Also, the idea is to finally compare to ideal geometry. How should I generate 
these values (any softwares in mind)?
ANy idea is welcome.
Thanks a lot

Re: [ccp4bb] HKL2000 indexing problem

[Joe Watts]

If you have tried all of the other things suggested by others (especially 
beam-center and direction of rotation), you can try the keyword 'weak level' in 
indexing box (see page 29 on HKL manual 
http://hkl-xray.com/sites/default/files/manual_online.pdf  or 
http://www.hkl-xray.com/denzo-keywords-alphabetical-order). 

This has solved that very problem for me more than once.  You will need to play 
with the value for your data try weak level 2, 5, 20, etc...

I assume you don't have the darkest spots in the world, right?


Joseph M. Watts, Ph.D.
Syngenta Biotechnology, Inc.
3054 E. Cornwallis Road
Durham, North Carolina.  27709
USA

Re: [ccp4bb] No diffraction

[Joe Watts]

1. Try room temperature mounts (as suggested by others)
2. Expose the hell out of the crystal (5 min) on home source or go synchrotron 
3. Run your protein through another column (ion exchange) even if it looks pure
4. Try an additive screen
5. Try limited proteolysis or methylation

6. If none of theat works, clone the same protein from another 
organism/strain/variant, ad try again

Re: [ccp4bb] Lithium versus Sodium

[Patel, Joe]

Hi Scott



I may be completely wrong but I worked on a lithium and sodium
inhibited enzyme during my PhD.  At the time, it was considered that
your chances of actually seeing density for a Li+ ion were slim to nil.
Only 2 electrons makes them as tough as hydrogens.  My efforts went into
trying to prove I had a sodium ion bound over magnesium which is the
catalytically active ion



Do you have ultra-high resolution? Something I did not  Are there
many examples in the pdb of proteins with Li+ refined?



Sorry no real help to you, but curious since it brings up old
memories...



Joe P





Dr Joe Patel

_



AstraZeneca

Discovery Sciences, Structure  Biophysics UK

50S38, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG

Tel +44 (0)1625 233543  Fax +44 (0)1625 230164

joe.pa...@astrazeneca.com mailto:name.surn...@astrazeneca.com



 P Please consider the environment before printing this e-mail







From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Scott Pegan
Sent: 12 January 2012 06:10
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Lithium versus Sodium



Hey all,

Does anyone know of a good article that deals with differentiating
between a lithium ion and sodium ion for density in a X-ray structures?

Scott

--
Scott D. Pegan, Ph.D.
Assistant Professor
Chemistry  Biochemistry
University of Denver
Office: 303 871 2533
Fax: 303 871 2254


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[ccp4bb] Permanent Position for NMR spectroscopist at AstraZeneca, Alderley Park, UK

[Patel, Joe]

There is currently a position available within the Structure 
Biophysics group.  The application deadline is 30th January 2012.
Please use the AstraZeneca Careers website to submit your application,
if you are interested.



The link to the job, with a role description can be found using the link
below.



http://gs.globalsuccessor.com/fe/tpl_astrazenecav2.asp?newms=jjid=58285
newlang=1





Thanks,



Joe P





Dr Joe Patel

_



AstraZeneca

Discovery Sciences, Structure  Biophysics UK

50S38, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG

Tel +44 (0)1625 233543  Fax +44 (0)1625 230164

joe.pa...@astrazeneca.com mailto:name.surn...@astrazeneca.com



 P Please consider the environment before printing this e-mail






--
AstraZeneca UK Limited is a company incorporated in England and Wales with 
registered number: 03674842 and a registered office at 2 Kingdom Street, 
London, W2 6BD.
Confidentiality Notice: This message is private and may contain confidential, 
proprietary and legally privileged information. If you have received this 
message in error, please notify us and remove it from your system and note that 
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Re: [ccp4bb] MR question

[Joe Watts]

If it is of moderate resolution (3 ish), uncheck automatic weighting in RefMac 
and constrain to 0.007-0.01.  You're probably over-fitting your data.

-Joe


Joseph M. Watts, Ph.D.
Research Scientist
Syngenta Biotechnology, Inc.

Re: [ccp4bb] strange density

[Patel, Joe]

Hi Alex,

Was it purified via a Ni2+ resin?  Is the protein oligomeric in
solution?  Could it have stripped an ion out during purification and
brought it all the way through to crystallisation?

Have you tried refining a Ni2+ in the location?

JP


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AstraZeneca UK Limited is a company incorporated in England and Wales with 
registered number: 03674842 and a registered office at 2 Kingdom Street, 
London, W2 6BD.
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message in error, please notify us and remove it from your system and note that 
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and may be unlawful.
Disclaimer: Email messages may be subject to delays, interception, non-delivery 
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-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Alex Singer
Sent: 24 February 2011 00:35
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] strange density

Hi -- I have a high resolution structure (1.6 A) where I'm ready to
deposit except I have some very strange density, shown in the two
pictures here -- sort of a sphere with a split cresent around it,
falling between molecule A and B His 138 imidazole rings.  The sphere is
modeled as a Cl atom, more for kicks because resulting 2Fo-Fc maps
still have considerable positive difference density throughout the
sphere.  There are 4 molecules in the AU, the imidazole ring of H138 in
molecules C and D point into a solvent channel.
Crystallization conditions are 0.2M Mg Chloride, 0.1M Bis-Tris pH6.5,
25% PEG3350, cocrystallized in 2.5mM Glycero-3Phosphocholine and
cryoprotected by dipping in Paratone_N oil.

Let me know what you're thoughts are and thank you for your help.

Alex Singer


--
Dr. Alex Singer
C.H. Best Institute
112 College St. Room 70
University of Toronto
Toronto, Canada, M5G 1L6
416-978-4033

[ccp4bb] Your suggestions needed: Difficulties in reproducing HT crystallization conditions. Thanks!

[Joe]

Hi all,

I recently have problems reproducing some conditions identified from high
throughput screenings.

The initial screening (10 mg/ml protein, 0.5 ul well+ 0.6 ul protein, 23 C)
gave rise to at least three hits from different screen kits.
 The follow-up grid optimization (1.5 ul well + 1.5 ul protein) varying
precipitant concentrations and pHs did not produce any crystals for all
three conditions. Instead, the drops have heavier precipitation
background.  The following experiments have been done in order to get
crystals back.

1. Seeding, with various precipitant concentrations
2. Varying volume ratios between well solution and protein (from 2: 1 to 1:
2 v/v).
3. Using original solutions from screen kits.
4. Hanging drops and sitting drops,
5. Dispensing protein first or well solutions first.
6. Using robot to set up drops on 96-well plate to see if I can reproduce
the original hits.

But none of them has worked so far.  This is the first time I encountered
such a scale-up issue.  I am running out of ideas, so hope you could give me
some suggestions.  Thank you in advance.

-- 
Best regards,

Joe

[ccp4bb] Your suggestions needed: Difficulties in reproducing HT crystallization conditions. Thanks!

[Joe]

Hi all,

I recently have problems reproducing some conditions identified from high
throughput screenings.

The initial screening (10 mg/ml protein, 0.5 ul well+ 0.6 ul protein, 23 C)
gave rise to at least three hits from different screen kits.
 The follow-up grid optimization (1.5 ul well + 1.5 ul protein) varying
precipitant concentrations and pHs did not produce any crystals for all
three conditions. Instead, the drops have heavier precipitation
background.  The following experiments have been done in order to get
crystals back.

1. Seeding, with various precipitant concentrations
2. Varying volume ratios between well solution and protein (from 2: 1 to 1:
2 v/v).
3. Using original solutions from screen kits.
4. Hanging drops and sitting drops,
5. Dispensing protein first or well solutions first.
6. Using robot to set up drops on 96-well plate to see if I can reproduce
the original hits.

But none of them has worked so far.  This is the first time I encountered
such a scale-up issue.  I am running out of ideas, so hope you could give me
some suggestions.  Thank you in advance.

-- 
Best regards,

Joe

[ccp4bb] Pittsburgh Diffraction Conference Oct 27-29

[Joe Ng]

This is a reminder that the 64th Annual Pittsburgh Diffraction Conference
will be in Pittsburgh Oct 27-29 at the Holiday Inn University Center.  

Registration and program information can be found at:

www.pdc2010.net




Joseph D. Ng
Associate Professor
University of Alabama in Huntsville
Huntsville, AL 35899
256-824-3715 (office), 256-824-3204 (fax)
Visit our updated website: www.uah.edu/nglab

[ccp4bb] Accuracy of the position of coordinates

[Joe Yap]

Dear CCP4 user,

 

I have a set of pdb coordinates and I would like to know the accuracy (error) 
of the position of the coordinates.

 

I understand that this is dependent on the variation of Rfree with respect to 
resolution.
  
I would appreciate it if you can help me with this, like what is the 
appropriate program to use.
 
Thank you so much.
 
Best regards,
 
Joe Yap


  

[ccp4bb] Estimation of coordinate errors

[Joe Yap]

Dear CCP4 users,

 

I have two sets of coordinates that are of similar structure and I would like 
to know is there a program I can use to calculate the coordinate errors between 
these two sets of coordinates?

 

I would appreciate it if you can help me with this.

 

Thank you so much.

 

Best regards,

 

Joe Yap
  

Re: [ccp4bb] question about the zinc binding protein

[Patel, Joe]

BTWBy the way  I think, not a buffer abbreviation


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W1K 1LN.
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message in error, please notify us and remove it from your system and note that 
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and may be unlawful.
Disclaimer: Email messages may be subject to delays, interception, non-delivery 
and unauthorised alterations. Therefore, information expressed in this message 
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Monitoring: AstraZeneca UK Limited may monitor email traffic data and content 
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Conduct and Policies.
-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Enrico Stura
Sent: 01 April 2010 11:02
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] question about the zinc binding protein

What is 1mM BTW? I am not familiar with this abbreviation.

PBS phoshate buffered saline (Phosphate + NaCl) not suitable for zinc  
binding proteins
BBS borate buffered saline (Borate + NaCl) wrong pH for zinc binding.
BTW ? (? ? ?) No idea what this is. Charles, do you know if it includes

HEPES or Phosphate?

Enrico.

On Thu, 01 Apr 2010 11:06:10 +0200, Charles Allerston  
charles.allers...@sgc.ox.ac.uk wrote:

 Hi,

 are you sure it is your protein precipitating?  You will get a cloudy

 precipitate appearing in HEPES and Phosphate buffers on addition of  
 ZnCl, without protein.


 cheers

 charlie

 dengzq1987 dengzq1...@gmail.com 3/31/2010 5:08 pm 
 hello everyone, recently i purify  a protein conteining zinc binding  
 domain,and i want to determine its structure.i get the crystal,but
poor  
 diffraction.so i try to adding zinc into the protein to optimize the  
 crystal,but the protein precipitate immidiately  even the znic is 1  
 mM.BTW,we use the protein to do zinc scan,we don't find the zinc. does

 anyone have some advice?

 2010-03-31



 dengzq1987


-- 
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,  Tel: 33 (0)1 69 08 9449  Lab
LTMB, SIMOPRO, IBiTec-S, CEA Saclay,  91191 Gif-sur-Yvette
Cedex FRANCE  http://www-dsv.cea.fr/en/ibitecs/82
 http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90
71

[ccp4bb] DMMULTI question

[Joe Cockburn]

Dear BB,
We are trying to solve the structure of a complex between two proteins. We
have two crystal forms of the complex, and a partial MR solution in each.
We now want to average the density between these two forms to improve the
maps, using DMMULTI. However, there are a couple of things I don't
understand.

1. You input F, SIGF, the initial best phase estimate and the Figure of
Merit. But how does DM calculate a map from this? Where does it get the
Fcalc from?

2. The program outputs the modified phases and their weights - but again,
how do you calculate a map from this?

Any help/commente would be appreciated!
Thanks
Joe

Re: [ccp4bb] FW: pdb-l: Retraction of 12 Structures

[Joe Cockburn]

I agree with Randy Read though - it's a mistake to get too carried away
with the foul-play aspect of this. There were clearly very serious
problems with those structures anyway. What it shows is that you can
desposit just about anything in the PDB, which is quite worrying when you
consider how much more accessible crystallography has become to
non-specialists. If it is to continue to be a valuable resource, then
everyone needs to know that structures in the PDB meet some basic quality
criteria.


 Yes, totally agree that the good thing about it all is that the problems
 have been identified and the wrong data will hopefully be eliminated.
 Shame
 it took almost 10 years since the first fabricated structure was published
 and more then 2 since questions were first risen about this work...

 Paula

 2009/12/11 Dale Tronrud det...@uoxray.uoregon.edu



 Paula Salgado wrote:
 
  Actually, I don't think that should be any consolation at all... As
  scientists, from whatever field, we should be appalled by this kind of
  mischief from anyone that calls themselves scientists. Not only it has
  effects on further research, delaying science sometimes by years, but
 it
  just  gives an appalling image of science and scientists. And of
 course,
  is unethical and wrong...
 
  Today is a sad moment for crystallography and science.
 

   There are reasons for optimism.  The self-correcting nature of
 the scientific method worked in this case.  Kudos to the University
 of Alabama at Birmingham for facing this problem and following
 through with their investigation of earlier allegations.

 Dale Tronrud

 
  =
 
   Dr Paula Salgado
 
   Division of Molecular Biosciences
   Department of Life Sciences
   Faculty of Natural Sciences
   Biochemistry Building, 5th Floor
   Imperial College London
   South Kensington Campus
   SW7 2AZ
   London
 
  Tel: 02075945464
 
  2009/12/10 Boaz Shaanan bshaa...@bgu.ac.il
 mailto:bshaa...@bgu.ac.il
 
  If that's of any consolation for us crystallographers, this
  situations arise in other fields too. Here is another example.
 See
  this link:
 
 
 http://www.biotechniques.com/news/Glycosylation-methods-paper-retracted/biotechniques-182060.html
 
 
Boaz
 
  - Original Message -
  From: Roger Rowlett rrowl...@colgate.edu mailto:
 rrowl...@colgate.edu
  Date: Thursday, December 10, 2009 21:07
  Subject: Re: [ccp4bb] FW: pdb-l: Retraction of 12 Structures
  To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK
 
  This kind of unfortunate situation only reinforces the notion that
  there must be some sort of laboratory
  oversight/communication/mentoring/documentation procedures in
 place.
  In my research lab (populated by a postdoc and a bunch of
  undergraduates) raw images and data processing log files are
 visible
  to everyone on the central XRD server, there is a lot of
  intra-laboratory communication about every structure that is
  processed, and lots of required documentation that must go onto
 our
  electronic laboratory notebook/wiki. While a determined individual
  could still find a way to perpetrate fraud, it is a lot more
  difficult when there are a lot of eyes looking at every structure,
  and raw data and documentation is widely visible within the lab.
  This is not a bad thing for co-authorship purposes, also.
 
  Nathaniel Echols wrote:
 
  On Thu, Dec 10, 2009 at 5:59 PM, Jacob Keller
  j-kell...@md.northwestern.edu wrote:
 
  I assume this is the denouement of the Ajees et al debacle a
  while back? Does this mean all authors on all of those papers
  were complicit? Otherwise, how would one author alone
  perpetrate this kind of thing? He pretends to go to the
  synchrotron, comes back with the hkl file, and goes from
  there? What about the crystals? Grows some lysozyme crystals,
  labels as protein x, proceeds to go to the synchrotron and
  then...? This whole thing is really hard to imagine--is there
  an initiation procedure in that lab, when the noble lie
 is
  revealed to all would-be authors?
 
 
  I'm curious about this too, but it is actually very likely that
  some (perhaps the majority) of the co-authors were unaware of the
  fraud, especially those whose name is only present on a single
  paper.  I didn't look closely, but I recognized one name of
  someone who certainly doesn't need to fake anything at this point
  in his career; I would be shocked if he had any clue what was
  going on.  Likewise, if there were co-authors from entirely
  different fields, I'm sure they wouldn't know what a Wilson plot
  is supposed to look like.  Many excellent scientists have been
  burned like this before; wouldn't you assume that your
  collaborators are acting in good 

[ccp4bb] How to compare the binding affinity between two domains structurally?

[Joe]

Hi,

Recently we determined two structures of the same protein in complex with
different molecules.  The protein contains two domains (called domain A and
B here).  In the two structures, domain A and B have different arrangements
relative to each other, resulting different interaction interface.  I want
to know which inter-domain interaction is stronger.   Is there a way to
quantatively compare the interaction energy or intensity between the two
domains?  I have calculated the buried surface area.  However, just
comparing the buried surface does not provide definitive answer, given that
the interacting residues on the interface are also different.

BTW, we were not able to purify individual domains, so we cannot measure the
binding affinity by wet lab approaches (so far).

Thank you in advance for your inputs.

-- 
Best regards,

Joe

Re: [ccp4bb] negative density peaks where there is no model.

[Joe Cockburn]

Hi Andrew,
If there is no *protein* model built into this region of the map, then it
will be modelled by the bulk solvent correction - therefore, the negative
peaks are telling you that the mean electron density there is lower than
in the bulk solvent. Probably.
Joe


 Hello everyone I have a question for the experts.

  I am in the final stages of refining my model
 placing waters and whatnot. However when I refine
 my model against the pure scalepack output, I see
 some rather signifigant negative difference
 density peaks (3sigma) in a marginally important
 region of the protein. The strange thing is that
 there is no model built into this region. How can
 there be negative difference density when there
 is no model built there? It is obviously not a
 weighting problem because the structure factors
 are directly from the raw reflection list. Has
 anyone else ecperienced this phenomenon and what
 if any actions would you suggest. I have attached
 also a screenshot. Thanks for your advice
 everyone I have learned so much from reading this
 BB everyday.

   
 Drew

 
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Re: [ccp4bb] problems of co-crystallization of protein-DNA complex

[Joe]

Hi ruheng,

Since you synthesized the oligos, you probably already know if there is
any residual salt or buffer in your oligos. I don't know if that
caused the problem. Sometimes people purify and desalt the oligos
before the annealing step.

Joe


ruheng wrote:

  
Dear CCP4bbers,

I am now working ona DNA binding protein and the purity of the protein
is quite good, however the results of DLS showed that the protein
aggregates terribly in quite a lot of different buffer conditions I
tried and still no crystals can be obtained. So I am going to
co-crystallize the protein in complex with DNA. I synthesized the
oligonucleotides varying differentnumbers of basepairs to determine
the optimal length which can bound to my protein by EMSA. I dissoved
the oligos in the buffer containing 50mM Tris-HCl, 100mM NaCl, 10mM
MgCl2 and1mM DTT, pH 7.9 and then annealed the DNA into the double
stranded form at a final concentration of 50uM. When I performed the
EMSA experiment, I mixed the purified protein with the dsDNA at the
molecular ratio approximately 1:1, but white precipitate was generated
as I mixed them.

Does anyone have this kinds of experience when working on DNA binding
proteins and co-crystallizing theprotein-DNA complex? Any suggestions
from yours will be appreciated.

Thank you all.


Ru Heng


  
  搜索本应是快乐的，不是么? 快乐搜索，有问必应！微软隆重推出! 立即试用！

[ccp4bb] Pictures of DDM crystals?

[Joe]

Hi all,


I am doing initial crystallization screening for a membrane protein purified
in DDM.  The actual concentration of DDM in the protein solution is  2 CMC
after the protein concentration step (Amicon MWCO 50 kD used).  I just found
over 50 conditions out of 1000 give me crystals.  Having worked on soluble
proteins, frankly they all look ugly to me to different degrees.  I have
selected a few conditions to go after, hoping that they will grow big enough
for me to verify the crystal identity by gels.  I wonder if there is a quick
way to tell if they are detergent crystals.  I know a microscope with UV
capacity would do the job, but we probably will not get one very soon.  I
have read through all threads on CCP4BBS regarding detergent crystals.  I
have an impression that it is actually not easy to crystallize
detergents--please correct me if I am wrong.  Your thoughts and comments are
helpful.

Thank you for your attention.

-- 
Best regards,

Joe

Re: [ccp4bb] Pictures of DDM crystals?

[Joe]

I forgot the mention this:  does anyone happen to have some pictures of DDM
crystals?  It would be very very helpful for me to take a look and get a
sense how they look like.  I appreciate your inputs and help.

Thanks,

Joe

On Mon, Aug 10, 2009 at 4:47 PM, Joe gch...@gmail.com wrote:

 Hi all,


 I am doing initial crystallization screening for a membrane protein
 purified in DDM.  The actual concentration of DDM in the protein solution is
  2 CMC after the protein concentration step (Amicon MWCO 50 kD used).  I
 just found over 50 conditions out of 1000 give me crystals.  Having worked
 on soluble proteins, frankly they all look ugly to me to different degrees.
 I have selected a few conditions to go after, hoping that they will grow big
 enough for me to verify the crystal identity by gels.  I wonder if there is
 a quick way to tell if they are detergent crystals.  I know a microscope
 with UV capacity would do the job, but we probably will not get one very
 soon.  I have read through all threads on CCP4BBS regarding detergent
 crystals.  I have an impression that it is actually not easy to crystallize
 detergents--please correct me if I am wrong.  Your thoughts and comments are
 helpful.

 Thank you for your attention.

 --
 Best regards,

 Joe




-- 
Best regards,

Joe

Re: [ccp4bb] Scalepack error model?

[Joe Cockburn]

Dear Richard,
I *think* it works like this, don't know if it's detailed or rigourous
enough for you!

If I(l,h,i) is the intensity of the ith observation of reflection h on
frame l, with error sig(l,h,i), and S(l) is the (inverse) scale factor to
be applied to frame l, then the error in the scaled intensity
I(l,h,l)/S(l) is parameterised in terms of the error scale factor (E1) and
estimated error (E2) as

sqrt( (E1*sig(l,h,i))**2  +  (E2*I(h)*S(l))**2  )

where I(h) is the weighted mean of the scaled intensity values for index h
(i.e. the merged, scaled intensity).

The reasoning behind this that the errors in the intensities of strong and
weak reflections generally arise from different sources. Weak data are
noisy, whilst very strong data can often be systematically badly measured,
especially in DENZO, which assumes that all your spots are the same size
and shape, or overloaded. E1 and E2 thus tend to dominate the error model
at high and low resolutions, respectively.
Hope that helps,
Joe


 Does anyone know of a detailed rigorous discussion of how the
 scalepack error model/Bayesian reasoning works? The scalepack manual
 has no equations for this.

 Richard Gillilan
 MacCHESS

Re: [ccp4bb] Scalepack error model?

[Joe Cockburn]

Dear Richard,
I *think* it works like this, don't know if it's detailed or rigourous
enough for you!

If I(l,h,i) is the intensity of the ith observation of reflection h on
frame l, with error sig(l,h,i), and S(l) is the (inverse) scale factor to
be applied to frame l, then the error in the scaled intensity
I(l,h,l)/S(l) is parameterised in terms of the error scale factor (E1) and
estimated error (E2) as

sqrt( (E1*sig(l,h,i))**2  +  (E2*I(h)*S(l))**2  )

where I(h) is the weighted mean of the scaled intensity values for index h
(i.e. the merged, scaled intensity).

The reasoning behind this that the errors in the intensities of strong and
weak reflections generally arise from different sources. Weak data are
noisy, whilst very strong data can often be systematically badly measured
(especially in DENZO, which assumes that all your spots are the same size
and shape) or overloaded. E1 and E2 thus tend to dominate the error model
at high and low resolutions, respectively.
Hope that helps,
Joe



 Does anyone know of a detailed rigorous discussion of how the
 scalepack error model/Bayesian reasoning works? The scalepack manual
 has no equations for this.

 Richard Gillilan
 MacCHESS

Re: [ccp4bb] can I try crystallization in high salt?

[Joe]

You can try including some salt to the reservoir after mixing protein 
and well solution.


Joe


rui wrote:

Dear All,

I have a peri domain protein that is stable in high salt 
concentration(500 mM), if I dialysis to a lower salt buffer and then 
concentrate, it'll preticipate out. If I need to crystallize it, can I 
use the high salt buffer? Is there any optimization kits that could 
help to increase the solubility? Thanks.

[ccp4bb] Rapid Access slot available at LS-CAT at the APS

[Joe Brunzelle]

The following slots are available at LS-CAT (sector-21) at the APS

Available Rapid Access Data Collection for the Next 21 days Station Start
Time Duration (Hours)  21-ID-D Jun 12, 2009 at 10:00 AM CDT 24  21-ID-D Jun
21, 2009 at 10:00 AM CDT 22  21-ID-D Jun 25, 2009 at 10:00 AM CDT 24
21-ID-F Jun 07, 2009 at 10:00 AM CDT 24  21-ID-F Jun 11, 2009 at 10:00 AM
CDT 24  21-ID-F Jun 15, 2009 at 10:00 AM CDT 22  21-ID-F Jun 18, 2009 at
10:00 AM CDT 24  21-ID-G Jun 11, 2009 at 10:00 AM CDT 24  21-ID-G Jun 12,
2009 at 10:00 AM CDT 24  21-ID-G Jun 15, 2009 at 10:00 AM CDT 22  21-ID-G Jun
21, 2009 at 10:00 AM CDT 22

Re: [ccp4bb] Find water in coot

[Joe]

It should be under "Calculate  Other modeling tools  Find
waters"

Joe

Raja Dey wrote:

  
  Hi,
   Is there any "Find Water..." button in the
"MOdel/Fit/Refine" window in coot? I did not find it in version
0.6-pre.
   
  
Raja
  
  
  

   Explore your hobbies and interests. Click
here to begin.

Re: [ccp4bb] Lost my protein

[Joe]

Hi Yanming,

I usually keep the follow through before I know nothing is in it. Also
you may want to suspend your protein solution every 5-10 min to avoid
precipitation because the concentration gradient forms rapidly during
centrifugation. If all of these do not help, just try concentrators
with different types of membrane.

Joe



Kornelius Zeth wrote:

  Hi Yanming,

you can try adding 1 M of urea, 1% detergent (OG,DM) that often helps to keep proteins in solution.

Best wishes

Kornelius

On Tue, 5 May 2009 09:06:09 -0700
 yanming Zhang shanma...@yahoo.com wrote:
  
  
Hi all,

during concentrating my protein, using Amicon Ultra centrifugal filter devices, 5000g, 4C, I lost large amount of my protein (75%). I heard the same story from one of my colleagues too. It seems the membrane of the Amicon tube ate my protein. 
Why this happen and how to recover my protein? 

Thank you very much.

Yan



  

  
  
 --
 Kornelius Zeth
 Max Planck Institute for Developmental Biology
 Dept. Protein Evolution
 Spemannstr. 35
 72076 Tuebingen, Germany
 kornelius.z...@tuebingen.mpg.de
 Tel -49 7071 601 323
 Fax -49 7071 601 349
  

Re: [ccp4bb] Hanging vs. Sitting

[Joe]

Sure, there are differences between these two methods, but no systematic 
study has been reported showing one is better than the other in terms of 
getting initial hits. Since we have a crystallization robot, I routinely 
set up sitting drops for initial screens and hanging drops (manually) 
for optimization thereafter. As long as I have no problem getting 
reproducible conditions, I will stick to the one I found most efficient 
and convenient for myself.


Joe


Frank von Delft wrote:
Sorry, disagree again:  with the right plate type (e.g. SwissCi 
plates), it's far far easier from sitting drop, because:
1. you don't have to muck around with flipping over the cover slip, 
instead just cut the seal
2. you have more time, because your drop does not evaporate as quickly 
(see earlier mail)
3. if the crystal sticks, just poke an acupuncture needle into the 
plastic below it:  off it pops.

phx



Simon Kolstoe wrote:
It's also easier to fish the crystals out of the solution with a 
hanging drop.


Simon



On 1 May 2009, at 06:35, Debajyoti Dutta wrote:



Hi,

From the experiance of mine I can tell you that the crystal size 
sometimes matters between these two methods. Hanging drop may yield 
bigger crystals than sitting drop, that may be due to the 
evaporation rate(surface area). Hanging drop allow us to set 
different protocols also like free interface diffusion, area covered 
by the drp etc.


These informations are gained purely by experiance.

cheers
Deb


On Thu, 30 Apr 2009 20:40:35 +0530 wrote
I have noticed that a significant majority of crystallizations are 
done in

hanging- rather than sitting-drop configuration, and considering the
significant extra labor involved in hanging drops, can only 
understand this
preference as a historical bias. I understand that sometimes one 
technique
works and not the other, but all things being equal, why is hanging 
drop

still hanging around? Any insights appreciated...

Jacob Keller

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: j-kell...@northwestern.edu mailto:j-kell...@northwestern.edu
***

http://sigads.rediff.com/RealMedia/ads/click_nx.ads/www.rediffmail.com/signatureline@middle? 

Re: [ccp4bb] Cryo-protectant

[Joe]

Title: Re: [ccp4bb] Cryo-protectant





First of all, you need to find out if crystals you grew have similar
quality before you conclude poor diffraction is due to cryoprotectant
solutions. As others have suggested, you can test crystals using
capillary mounting method. Second, there are a lot more cryoprotectant
agents out there you can try. Not sure why you sticked to MPD, which is
not even present in your crystallization condition. 

One of protein crystallized in almost the same condition as yours. What
I did is just simply increasing % PEG3350, in addition to 10%-15%
increment of every other ingredients (your protein buffer + well
solution). I also introduced 5% glycerol to bring down % PEG3350. You
can play around % PEG3350 and % glycerol to find a fine combination
that is cryo-clear and makes you crystals happy. 

Joe

Liew Chong Wai wrote:

  
  
  
  
  Hi all
  
  Thanks for your
precioussuggestions and ideas. 
  The crystallization buffer
condition is 0.1M BIS-TRIS pH 5.5, 0.2M MgCl.6H2O, 35% PEG3350
  Now, my crystal seem ok in
20% ethylene glycol, but only after a couple minutes of dehydration at
room temperature. For sure, i will try other cryoprotectant that was
suggested here. 
  I just wondering why MPD
kills the crystal.
  Many thanks
  
  
  
  LIEW
  
  
  

  
  
  
  
  
  

[ccp4bb] pI for protein-detergent complexes

[Joe]

Hi,

Is there a way to estimate pI for protein-detergent complexes? Thanks.

Joe

Re: [ccp4bb] Refmac refinement with two ligands

[Joe]

Alex,

The CCP4i 6.1.1 refmac allows you to combine multiple .cif using Merge 
LIBINs. Have you tried that?


Joe

aber...@mrc-lmb.cam.ac.uk wrote:

Dear CCP4 community,

I would like to refine a structure with two bound ligands using Refmac.
However, the Rafmac GUI allows only one library to be read in. How can I
combine or synthesise two .cif/.lib files (originating from the Dundee DRG
server) and circumvent this problem?


Many thanks in advance,
Alex
  

[ccp4bb] how generate maps only to cover atoms in pdb

[JOE CRYSTAL]

Hi,

I want to generate maps only covering atoms in the pdb file. I tried
fft-creat map in ccp4i (output map to cover all atoms in pdb), but it
actually also generated map in places other than pdb atoms. Are there any
other ways to do this? Thank you.

-- 
Best regards,

Joe

Re: [ccp4bb] How to generate maps only to cover atoms in pdb

[JOE]

Thanks a lot for the replies I have received so far!

I have one homodimer in each ASU. What I intended to do is to show the
electron density for each monomer in different colors  (*only for
presentation purpose*). I guess the only way to achieve this is to
individually create (not sure it is a right word) the map for each monomer
and then display them in pymol or coot. Any better solutions?

Thanks,

Joe


On Sun, Apr 19, 2009 at 11:06 AM, JOE CRYSTAL gch...@gmail.com wrote:

 Hi,

 I want to generate maps only covering atoms in the pdb file. I tried
 fft-creat map in ccp4i (output map to cover all atoms in pdb), but it
 actually also generated map in places other than pdb atoms. Are there any
 other ways to do this? Thank you.

 --
 Best regards,

 Joe




-- 
Best regards,

Joe

[ccp4bb] Beam Time at LS-CAT Sector-21 at APS

[Joe Brunzelle]

I would like to take the time to welcome all general users to the newest
beam lines at the Advance Photon Source, Life Sciences CAT Sector 21.

http://ls-cat.org http://protein.nsls.bnl.gov/

There is time available for rapid access, the dates are listed on the
website and users can subscribe for upcoming times too.

LS-CAT current;y operates three ID end stations, one station (21ID-D) is
fully tunable with an MD2, MX-300 and CATS robotic sample changer.  21ID-F 
G are fixed wavelength at 0.978A with MX-225 and MX-300 detectors, both
stations have MD2s and CATS robots too.

To schedule time visit the LS-CAT website.

If you like to know more

send an email:

k-bris...@ls-cat.org x6an...@bnl.gov

or call:

+1 (630) 252 0626

Joseph S Brunzelle, PhD
Assistant Research Professor
Dept. Mol Pharm  Bio Chem
Life Sciences Collaborative Access Team
Synchrotron Research Center, Northwestern University
Ph. 630-252-0629 Fax. 630-252-4664
j-brunze...@northwestern.edu
http://www.ls-cat.org

Re: [ccp4bb] Interaction between the domains

[Joe Crystal]

Contact in CCP4i can calculate the distance between specified 
chains/residues. Pymol can display hydrogen bonds too.


Joe


peter hudson wrote:

Hello all

I have a very quick question. Is there any programme, which can 
calculate the H-bond pattern or residues involved in H-bonds formation 
between the interface of monomers or the between the domains of the 
same monomer. I will appreciate your suggesstions.


Thanks in advance

Peter

[ccp4bb] Refmac failed at the end of run

[JOE CRYSTAL]

 Hi all,

I am using refmac in ccp4i 6.1.1 (installed in windows). It runs well when
using automatic weight, but failed when using user-specified weight (0.12 in
this case). I attach the error message as follow. Your help is very much
appreciated.



..
 18   0.2064   0.2348   0.846407628.   21897.0   0.0075  0.318
1.170  0.502   0.076
 19   0.2065   0.2343   0.846407625.   21896.4   0.0075  0.318
1.169  0.501   0.076
 20   0.2065   0.2347   0.846407629.   21897.3   0.0075  0.318
1.167  0.501   0.076
$$

$TEXT:Warning: $$ comment $$
WARNING: CCPOPN Logical name
C:/Chen/Xtal/cvir/472cr8_c10/work2/refmac/DepositFiles/472cr8nsls/unknown211008:17:49:34.refmac
has no asso
$$
Open failed: Unit:   9, File:
C:/Chen/Xtal/cvir/472cr8_c10/work2/refmac/DepositFiles/472cr8nsls/unknown211008:17:49:34.refmac
(logical:
C:/Chen/Xtal/cvir/472cr8_c10/work2/refmac/DepositFiles/472cr8nsls/unknown211008:17:49:34.refmac)

Refmac_5.5.0072:   Open failed: File:
C:/Chen/Xtal/cvir/472cr8_c10/work2/refmac/DepositFiles/472cr8nsls/unknown211008:17:49:34.refmac

Times: User:   0.0s System:0.0s Elapsed:27:38 /pre
/html
***
* Information from CCP4Interface script
***
The program run with command: refmac5 XYZIN
C:/Chen/Xtal/cvir/472cr8_c10/work2/refmac/472cr8c10_v1-coot-4_refmac1.pdb
XYZOUT C:/Ccp4Temp/47crc10_3_2_pdb_1.tmp HKLIN
C:/Chen/Xtal/cvir/472cr8_c10/work2/refmac/472cr8nsls_refmac1.mtz HKLOUT
C:/Ccp4Temp/47crc10_3_3_mtz_1.tmp LIBIN
C:/Chen/Xtal/cvir/472cr8_c10/work2/refmac/libcheck_C10.cif TLSIN
C:/Chen/Xtal/cvir/472cr8_c10/work2/refmac/472cr8c10_v1-coot-4_edit_tls1_refmac1.tls
TLSOUT C:/Ccp4Temp/47crc10_3_4_tls_1.tmp LIBOUT
C:/Chen/Xtal/cvir/472cr8_c10/work2/refmac/47crc10_3_lib.cif
has failed with error message
Last system error message: Unknown error
Refmac_5.5.0072:   Open failed: File:
C:/Chen/Xtal/cvir/472cr8_c10/work2/refmac/DepositFiles/472cr8nsls/unknown211008:17:49:34.refmac

***


#CCP4I TERMINATION STATUS 0 Last system error message: Unknown error
Refmac_5.5.0072:   Open failed: File:
C:/Chen/Xtal/cvir/472cr8_c10/work2/refmac/DepositFiles/472cr8nsls/unknown211008:17:49:34.refmac

#CCP4I TERMINATION TIME 20 Mar 2009  13:23:28
#CCP4I MESSAGE Task failed





-- 
Best regards,

Joe

[ccp4bb] Please recommend screen kits for membrane proteins

[JOE CRYSTAL]

Sorry for the off-topic subject. I am new to membrane proteins. We already
have Qiagen MB class I and II, Sigma Membrane, and Membrane Gold kits, but I
want to know if there other kits out there you would recommend. Thanks a
lot,

Joe

[ccp4bb] Broken chain in Pymol display

[Joe Xtal]

Hi all,

I tried to display a refined structure (final steps in phenix.refine) in 
Pymol, but several places are not connected. BTW, the structure displays 
normally in Coot and bond angle and length deviation are below 1.0 and 
0.006, respectively.


Thank you,

Joe

[ccp4bb] Questions regarding Peptidoglycan-binding protein

[JOE CRYSTAL]

Dear all,

Thank you in advance for your expert opinions.

I am working on a peptidoglycan (PG)-binding protein, which is composed of
two functionally identical domains. Interestingly, when subjected to gel
filtration chromatography (Superdex 200), both full-length protein and the
single domains migrate as a major peak tailed with a broad shoulder.
SDS-PAGE indicates the expected MW for both samples from the major peak and
the shoulder, however, with the presence of smears at high molecular weight.
Also, after all fractions from both the major peak and the shoulder were
combined and incubated at 4 C over 48 hours, the sample was applied to gel
filtration again; this time the shoulder actually disappeared (or shifted),
resulting in a nice symmetric peak (with the same elution volume as the
major peak).

My guess is that the protein expressed in E. coli may already specifically
or unspecifically associate with PG fragments from cell wall. My question
is: *how to detect PG fragments in my protein sample*? I have tried
denaturation-wash-renaturation steps, but I want to know if I have
completely got rid of the possible contaminants.

Thank you for suggestions.

Joe

[ccp4bb] Poor electron density - polyAla or PolyGly?

[Joe Smith]

Hello,
I have been building a protein model (resolution 2.2A) which has one
small loop of 6 residues having poor density. I cannot see any side
chain in this region but I can see relatively poor main-chain density
which at least clearly indicate the loop conformation. I am trying my
best to build polyAla chain in this region. But 3-4 residues end up
coming in disallowed region of Ramachandran plot. I think the problem
is - in this region i can not even build the main chain atoms with
high confidence.
Can I build polyGly in this region? or should I just leave this
region? I just don't feel like leaving this region empty.
Any suggestions in this regards is highly appreciated!
regards
Joe

Re: [ccp4bb] Protein Color

[Joe Cockburn]

Hi Matt,
I sometimes see a similar thing with my proteins, which definitely don't
possess metal co-factors or prosthetic groups. I found that gel filtration
got rid of it - the browny-yellow stuff came out in the void fraction so I
figured it was aggregated protein. I think it was aggregation via the
his-tags around traces of copper in my sample, which could explain the
brown-ish colour.
What happens if you concentrate the protein in the presence of EDTA?
Joe

 Hello.

 I am working with a protein that turns a yellowish-brown color when it is
 concentrated to around 2 mg/ml or higher in a small volume (a few hundred
 uL).  I was wondering if the protein bound a metal or other prosthetic
 group that would give it this color?  The protein's color somewhat
 resembles iron binding proteins, but there is no peak in the 400 nm range
 that would suggest heme, and an iron sulfur cluster is not that likely
 since there are only five cysteines in the protein.  Proteins with
 structures homologous to the one I am studying bind magnesium, but are not
 know to bind other metals.  Any information about what this color might
 suggest about the protein or how I could analyze possible bound metals or
 prosthetic groups using only a small amount of protein would be helpful.

 Matt

Re: [ccp4bb] Question re: ice rings in diffraction data

[Joe Cockburn]

Dear Mona,
Yes. You can get Denzo to reject reflections on or close to ice rings, or
any other features with highly irregular background variations, by
increasing the value of the REJECT keyword (see p. 40 of the HKL2000
manual by D. Gerwith,
http://www.hkl-xray.com/hkl_web1/hkl/manual_online.pdf).
HTH,
Joe



 Hello all,

 I have a query re: processing data.  I have some lovely diffraction data
 that has been marred by ice rings (so...not as lovely as it could be).  I
 was told it may be possible to mask the regions of the ice rings
 (obviously sacrificing completeness) during processing.  I have been using
 HKL2000 to process data.  Is this feature available with this program?
 Otherwise, we also have Mosfilm.  Any advice would be greatly appreciated.

 Thank you.

 Sincerely in anticipation
 Mona Rahman

 ---
 Mona N. Rahman, Ph.D.
 Dept. of Biochemistry/Pharmacology  Toxicology
 Botterell Hall, Rooms 623 and 634 (lab)
 Queen's University, Kingston, ON, K7L 3N6
 Phone:  613-533-2993, 613-533-6293 (lab)
 E-mail:  [EMAIL PROTECTED]

[ccp4bb] coordinates of TCEP

[Joe]

Hi there,
I suspect my reducing agent TCEP-HCl is bound to my protein in the
crystal. I am trying to put a TCEP molecule into the bulky density,
but could not find any available coordinates of TCEP.
Do you know any where I can download the TCEP coordinates or any where
I can build one starting from its chemical formula?
thanks alot.
-Joe

[ccp4bb] Problem with crystallization of Se-Met labeled protein

[Joe Smith]

Dear all,
Sorry for an off-topic query.
I have been unable to crystallize a Se-met containing protein (8 Met
in 206 amino acids) in the native crystallization condition ( 0.1 M
Tris pH 8.5, 1.2 M K-Na-tartrate; Theoretical pI of protein is 8.4).
As expected, solubility of Se-Met containing protein is little less
than the wild type. Other than seeding, i don't know what else I
should try for obtaining a Se-met crystal for phasing. Can I mutate
some of the exposed Met  (based on secondary structure prediction and
homologous structure) to Ala as I feel I don't really need 8 Se for
phasing 208 aa long polypeptide. I want to know what generally one
should do when Se-Met containing proteins fail to crystallize.
Thanks in advance.
Joe
PS: Since, protein contains 3 Cys residues.. I am also planning to try
my luck with heavy atom compounds containing Hg.

Re: [ccp4bb] Fwd: [ccp4bb] crystallisation robot

[JOE CRYSTAL]

Hi,


Does anyone have information about how long it takes to set up a 96-well
tray for the crystallization robots available?  Besides cost per tray and
maintenance cost, another important feature we consider is the time for
setting up a 96-well tray.  It is an important factor since we are talking
about sub-microliter drops.


Best,


Joe

On Fri, Jan 18, 2008 at 12:28 PM, Lisa A Nagy [EMAIL PROTECTED] wrote:

 Al's Oil on the plates:
 What a nightmare!!!
 The oil creeps up the plate and over the sides. It dissolves adhesives.
 It makes me say bad words in multiple languages.
 Bigger drops + no oil = fewer bad words.

 Lisa
 --
 Lisa A. Nagy, Ph.D.
 University of Alabama-Birmingham
 [EMAIL PROTECTED]

 -Original Message-
 From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
 Patrick Shaw Stewart
 Sent: Friday, January 18, 2008 2:20 AM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] Fwd: [ccp4bb] crystallisation robot

 One thing that people often overlook is that quite a lot of protein
 can be lost by denaturation on the surface of the drop.  This is more
 significant for smaller drops.  Two suggestions: (1) increase the
 proportion of protein in the - technical term - teeny drop to say two
 thirds and (2) cover the drops with oil eg Al's oils
 (silicone/paraffin).  You still get vapor diffusion though the oil ,
 and you'd like to slow up equilibration.  of course (2) slows up the
 robotics a little, but both should be trivial to set up..

[ccp4bb] Characterization of common salt crystal forms?

[Joe Krahn]

Salt crystals are common in macromolecular crystallography. Has anyone
tried to tabulate salt crystal forms that commonly occur?

I just identified a salt crystal as Mirabilite, made of Na2SO4·10H2O.
The high water content makes them rather soft, and may not be recognized
as salt right away. In this case, it probably happened because the
buffer was made with Na·Citrate + HCl instead of citric acid, while
trying to optimize conditions. So, characterization of salt crystals can
help to avoid the conditions that cause them.

There is probably a reasonably small number of salt crystal forms that
are very common in crystallization trials. Maybe it would be useful to
tabulate common salt crystals to help guide optimization experiments.
Has anyone else tried to use salt crystal information beyond ensuring
that it is not protein?

Joe Krahn

Re: [ccp4bb] DTT sensitive?

[Joe]

The other possibility is 400 mM imidazole in the buffer. The
precipitate looks like silk.

On 11/13/07, Bryan W. Lepore [EMAIL PROTECTED] wrote:
 you didn't say how you know its protein - is it?

 interesting though.

Re: [ccp4bb] DTT sensitive?

[Joe]

It did not contain DTT when frozen.

On 11/13/07, deena [EMAIL PROTECTED] wrote:
 Are you sure its 1mM DTT, because DTT itself precipitates in the freezer.

 Deena


 On Nov 13, 2007, at 3:30 PM, Joe wrote:

 Hi there,
 I see enormous precipitate of my receptor protein when I take it out
 of freezer. But all the precipitate dissolved quickly after I added
 1mM DTT to the solution. Does this mean that some surface Cys are
 causing problem? Or why is the protein so sensitive to DTT?
 Anybody experienced this kind?
 Any advice is appreciated.
 -Joe

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

Re: [ccp4bb] DTT sensitive?

[Joe]

The precipitate does not disappear automaticly if I don't add DTT.
If it's the precipitate of imidazole, then why DTT can dissolve it?
thanks

On 11/13/07, Sanishvili, Ruslan [EMAIL PROTECTED] wrote:
 This may not apply in your case but it is not uncommon for a protein to
 precipitate in a microcrystalline shower when put in cold. Once it
 worms up, the crystals dissolve and the precipitate clears up. It is
 easy to check under a high magnification microscope.
 Cheers,
 N.


 Ruslan Sanishvili (Nukri), Ph.D.

 GM/CA-CAT, Bld. 436, D007
 Biosciences Division, ANL
 9700 S. Cass Ave.
 Argonne, IL 60439

 Tel: (630)252-0665
 Fax: (630)252-0667
 [EMAIL PROTECTED]




 -Original Message-
 From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
 Joe
 Sent: Tuesday, November 13, 2007 2:30 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] DTT sensitive?

 Hi there,
 I see enormous precipitate of my receptor protein when I take it out of
 freezer. But all the precipitate dissolved quickly after I added 1mM DTT
 to the solution. Does this mean that some surface Cys are causing
 problem? Or why is the protein so sensitive to DTT?
 Anybody experienced this kind?
 Any advice is appreciated.
 -Joe

[ccp4bb] problems with map quality, refinement and R/Rfree

[Joe Smith]

Hi all,

We have data sets for a protein-RNA complex at 3.2 A resolution. . The
data belong to the space group I4122 and contains one molecule of
Protein-RNA complex (300AA and 16nt; 66% solvent content) in the asym
unit. We have managed to get phases using Se-SAD and the present model
contains 60% of the protein atoms (250 residues many of them build as
Ala) and almost complete RNA (16nt).  We could not locate ~20 % of the
residues (present in various loops as well as at N- and C-terminal
ends). First 120 residues present at N-terminal end seem to have poor
relatively main-chain density and almost no side chain density. RNA
and 130 residues present at the C-terminal end have good electron
density for main-chain as well as side chain.

The solution seems to be correct as molecular replacement trials with
this model gave same solution in Phaser, Molrep and AMoRe. Solution
seems to be also correct because as expected one RNA strand present in
asym unit pairs with another one coming from symmetry related
molecule.

We have checked data in space group I4122 with CCP4 (SFCHECK,
cumulative intensity distribution), Yeates server as well as
Phenix.xtriage. In all the cases it indicates no twinning. However if
I process the data in lower symmetry space group it does indicate
presence of almost perfect twin. We realize that this could be just
because of processing data in lower symmetry space group and data in
all probability may still be fine.

The problem is now with refinement and model building. Refinement with
CNS and Refamc gave considerably higher value of R and R free. In
CNS1.1, refinement with small changes in the model some time leads to
large shift in R and R-free value.
 Refmac: R=36; Rfree=47
CNS1.1:  R=40-59; R-free=45-63
However in both the cases map looks more or less same (with reasonably
good density for the main-chain as well as RNA)

I tried refining with phenix.refine and there is some improvement in
the R(34%) and Rfree(40%) values. I think may be robust bulk solvent
correction incorporated in phenix.refine has helped in this case.
However, I still see no improvement in the map quality for the first
120 residues.

In the absence of any clear density I am unable to build any further.
I feel N-terminal domain is bit flexible and may have overall poor
density. I have used Se position as well as predicted secondary
structure in assigning the amino acid in the map but due to few breaks
in the N-terminal domain as well as poor density I am unable to assign
any amino acid into the poly ala main chain.

Frankly speaking, I do not know how to proceed further. I welcome any
kind of suggestions which could help us in this case.

Regards
Joe

[ccp4bb] Nearly perfect twinned data????

[Joe Smith]

  Hi all,

We have collected few X-ray data sets for a protein-RNA complex to
resolutions of 3.2-3.5A. While processing the data using HKL2000, we
have obtained following distortion index consistently:

 primitive cubic 19.16% 127.98  74.67 130.85  74.57  85.00  73.91
   111.17 111.17 111.17
90.00  90.00  90.00

  I centred cubic 24.26% 132.28 189.90 127.98 120.33  85.97 120.47
150.05 150.05 150.05
90.00  90.00  90.00

 F centred cubic 22.17% 177.47 190.41 189.90  46.24  90.79  91.90
 185.93 185.93 185.93
90.00  90.00  90.00

 primitive rhombohedral  16.32%  130.85 129.06 174.88 131.21 133.20  86.12
  144.93 144.93 144.93
116.85 116.85 116.85
  229.30 229.30  74.67
 90.00  90.00 120.00

 primitive hexagonal 15.40%130.85 127.98  74.67 106.09  74.57  95.00
 129.41 129.41  74.67
90.00  90.00 120.00

 primitive tetragonal 9.40%   127.98 130.85  74.67  74.57 106.09  95.00
  129.41 129.41  74.67
 90.00  90.00  90.00

 I centred tetragonal 0.91%  177.47 174.88  74.67  90.21  88.59  91.47
 176.18 176.18  74.67
90.00  90.00  90.00

 primitive orthorhombic   9.38% 74.67 127.98 130.85  85.00 105.43 106.09
  74.67 127.98 130.85
90.00  90.00  90.00

   C centred orthorhombic   6.53%   74.67 245.95 130.85  89.44 105.43  89.13
   74.67 245.95 130.85
 90.00  90.00  90.00

 I centred orthorhombic   0.84%74.67 174.88 177.47  88.53  91.41  90.21
  74.67 174.88 177.47
90.00  90.00  90.00

 F centred orthorhombic   0.68%74.67 245.95 252.32  89.16  88.86  89.13
 74.67 245.95 252.32
90.00  90.00  90.00

 primitive monoclinic 6.52% 74.67 130.85 127.98  95.00 106.09  74.57
  74.67 130.85 127.98
90.00 106.09  90.00

 C centred monoclinic 0.49% 74.67 245.95 130.85  89.44 105.43  89.13
  74.67 245.95 130.85
90.00 105.43  90.00

 primitive triclinic   0.00%  74.67 127.98 130.85  85.00
74.57  73.91


As you see, distortion index table indicates I centered tetragonal, I
centered orthorhombic, F centered orthorhombic, C centered monoclinic
and triclinic as possible Bravais lattices.

 Data processed in I centered tetragonal gives low Rmerge in all the
possible space groups namely I4, I41, I422 and even I4122.  Other
space groups in lower symmetry lattices also gave low R merge values
(around 6% in most of the cases).

Since we have not been able to obtain a solution in any of the space
group from I centered tetragonal to triclinic (I4, I4122, I222, C2 and
even P1) using Se-SAD, we decided to check the data for any intrinsic
problem such as twinning.

Cumulative intensity distribution calculated using scalepack2mtz shows
no sign of twinning. However, data processed in I4 shows nearly a
perfect twin (twin fraction=0.489 with twin operator 100 0-10 00-1) in
Yeates server whereas SFcheck indicates a twin fraction of 0.431 with
twin operator –h,+k,-l. Data processed in I4122, I222, C2 and P1
doesn't show any twinning due to absence of any twin laws for these
space groups.

Now my question is:
-  Are data showing low Rmerge value in I4122 due to nearly perfect
twin in space group I4?

-  Why cumulative intensity distribution shows a normal pattern for
the data where as Yeates server and SFcheck indicates nearly a perfect
twin?  Why Yeates server and SFCheck shows different twin fraction and
twin operator?

-  Is it possible to detwin this data and use it for structure solution?

Thank you for reading till this line and I am sorry for such a long
mail. I hope I haven't made any mistake at any stage. I really need
your valuable suggestions to solve this problem.

Regards
Joe

Re: [ccp4bb] Covalently bound drug molecule

[Joe Krahn]

I was using X-PLOR at the time. I later switched to a PRESidue patch. I
was not sure how many atoms must be added to a residue before a modified
amino acid becomes a linked amino acid. The PDB seems a bit adverse to
non-standard links. For example, why is NADP not two residues with a
LINK? There are no well-defined rules, so I thought that the big
nonstandard residue would better fit the usual pattern of modified
residues. Before submitting, I switched to two residues because it made
more sense to me.

However, a modified residue requires a MODRES entry, which was deemed
ugly, at least for the way that nucleic acids handled them. I think
that many modified nucleic residues were converted to non-standard
nucleic residues. I think it would be good to have some well-defined
rules about what modifications produces a non-standard amino acid versus
a linked residue.

Marius Schmidt wrote:
 What refinement program are you using? I assume cns.
 Ihe idea with the non-standard residue works
 very good, but the usual way is to PATCH the covalently
 linked molecule to its respective covalently bound atom
 in cns or xplor.
 So, you do not need to alter your pdb-file.
 Define a PRESI entry in topology file and set
 parameters in param file (see how it is done
 for standard patches in the existing top and param
 files).

Re: [ccp4bb] Covalently bound drug molecule

[Joe Krahn]

In general, it requires a LINK record to add the required bond. I don't
know how to set up refinement parameters. I had a similar case once (see
1ECC), and created a big non-standard residue containing both the amino
acid and ligand as one residue. (But, that was done in X-PLOR.) If
setting up link paramaters become a hassle, you might try the
single-residue approach.

Joe

Hall Gareth wrote:
 Dear ccp4bb users,
  
 I am currently refining a crystal structure of a protein with a
 drug molecule in the active site.  The drug molecule is seen, as
 expected, to covalently bind to the active site. Therefore, can anyone
 tell me what I need to do to alter the pdb file so as to form a covalent
 bond between the protein and my drug molecule?
  
 Cheers in advance,
  
 Gareth Hall
 
 This message has been checked for viruses but the contents of an
 attachment may still contain software viruses, which could damage your
 computer system: you are advised to perform your own checks. Email
 communications with the University of Nottingham may be monitored as
 permitted by UK legislation.
 

Re: [ccp4bb] MSE

[Joe Krahn]

One more comment on MSE: Does anyone know why MSE was defined without a
'delta' on the selenium? Isn't that obviously wrong? Selenium-delta
should be SED , just like S-delta is  SD , so it can't be left off
just because selenium is not a carbon.

Phil Evans wrote:
 flame
 As an aside, does anyone understand why MSE is not an amino-acid?
 Phil
 /flame
 
 On 30 Aug 2007, at 15:31, Clemens Vonrhein wrote:
 
 If your PDB file conforms to standard

   http://www.wwpdb.org/documentation/format23/sect9.html#ATOM

 you could do

   % egrep ^CRYST|^SCALE|^ATOM your.pdb  standard_residues_only.pdb

 You'll miss the 'non-standard' Se-MET residue 'MSE' ;-)

Re: [ccp4bb] PDB format survey?

[Joe Krahn]

Edward A. Berry wrote:

Ethan A Merritt wrote:

On Wednesday 08 August 2007 20:47, Ralf W. Grosse-Kunstleve wrote:

Implementations to generate intuitive, maximally backward compatible
numbers can be found here:

  http://cci.lbl.gov/hybrid_36/


From that URL:

ATOM  8  SD  MET L  48.231 -64.383  -9.257  1.00 
11.54   S
ATOM  9  CE  MET L  49.398 -63.242 -10.211  1.00 
14.60   C
ATOM  A  N   VAL LA000  52.228 -67.689 -12.196  1.00  
8.76   N
ATOM  A0001  CA  VAL LA000  53.657 -67.774 -12.458  1.00  
3.40   C


Could you please clarify this example?
Is that A a hexidecimal number, or is it a decimal number
that just happens to have an A in front of it?
[A-Z][0-] gives a larger range of values than 5 bytes of hexadecimal,
so I'm guessing it's the former.  But the example is not clear.


I'm guessing the former also. A 5-digit hex number would not be
backwards compatible. With this system legacy programs can still
read the files with 9 atoms or less, and anything more than
that they couldn't have handled anyway. Very nice!

Ed
I still prefer the idea of just truncating serial numbers, and using an 
alternative to CONECT for large structures. Almost nobody uses 
atomSerial, but it still may be parsed as an integer, where the above 
idea could cause errors. Furthermore, non-digit encoding still results 
in another maximum, whereas truncating the numbers has no limit. The 
truncated serial number is ambiguous only if taken out of context of the 
complete PDB file, but PDB files are by design sequential.


Another alternative is to define an atom-serial offset record. It can 
define a number which is added to all subsequently parsed atom serial 
numbers. Every ATOM/HETATM record is then perfectly valid to an older 
program, but may only be able to handle one chunk of atoms at once.


Likewise, I like the idea of a ChainID map record, which maps 
single-letter chainID's to larger named ID's. Each existing PDB record 
can then be used unchanged, but files can then support very long ChainID 
strings. The only disadvantage is that old PDB readers will get 
confused, but at least the individual record formats are not changed in 
a way that makes them crash.


I think that keeping the old record definitions completely unchanged are 
an important feature to any PDB format revisions. Even if we continue to 
use it for another 20 years, it's primary advantage is that it is a 
well-established legacy format. If we change existing records, we 
break that one useful feature. Therefore, I think that any changes to 
existing records should be limited to using characters positions that 
are currently. (The one exception is that we need to make the HEADER Y2K 
compatible by using a 4-digit year, which means the existing decade+year 
characters have to be moved.)


Of course, the more important issue is that the final decision needs 
community involvement, and not just a decision by a small group of RCSB 
or wwPDB administrators.


Maybe it would be useful to set up a PDB format Wiki where 
alternatives can be defined, along with advantages and disadvantages. If 
there was sufficient agreement, it could be used as a community tool to 
put together a draft revision of the next PDB format. With any luck, 
some RCSB or wwPDB people would participate as well.


Joe Krahn

[ccp4bb] PDB format survey?

[Joe Krahn]

So, I am thinking about putting up a survey somewhere to get a measure
of the user-communities interests, because RCSB and wwPDB seem
uninterested in doing so. Maybe a group result would be more useful in
influencing the standards. I am hoping that the wwPDB can become a
better place for format standards instead of RCSB which keeps busy
handling new data.

In addition to questions about the PDB standard, it is probably
important to consider mmCIF. One thing I don't like about it is that
columns can be randomized (i.e. X, Y, and Z can be in any column), but
the mmCIF standards people have no interest in defining a more strict
standard that would require files to be as human readable as RCSB's
mmCIF files.

Does this sound useful, or have most people given up on having any
influence on standards? Or, should the structural biology software
developers get together and just make our own OpenPDB format?

Joe Krahn

Re: [ccp4bb] PDB format survey?

[Joe Krahn]

Ethan Merritt wrote:
 Examples include:
 - very large structures, for which the current 80 column PDB format
  runs out of space for atom numbers (4 columns - max )
   or for chain ids (1 column - single char A-Z 0-9)
   [don't ask my why they don't want lower case]
 - new classes of experiment (SAXS, EM)
 - new classes of model (TLS or normal-mode displacements,
   ensemble models, envelope representations)
It would be trivial to update the PDB format to handle large structures.
In fact, such extensions are already being planned. Atom numbers can
simply be handled by truncating them; the serial design of PDB files
makes it redundant.

As for other experiments, like SAX or EM, I only think that the PDB
format should continue to be used for atomic coordinates. Using them as
a complete data reference has never been good.

...
 Currently-maintained programs should move to mmCIF or XML, whichever
 is convenient.  These formats are intrinsically open-ended, and can
 handle the problematic structures mentioned above so long as the
 corresponding mmCIF dictionaries are updated to define the relevant
 entities.
Being intrinsically open-ended is an advantage for parsing, but it still
takes a lot of work to actually make use of new data. The software still
has to be updated to handle the data. Formats like mmCIF and XML only
handle part of the 'file format' issue. One problem is that mmCIF can be
too open-ended, depending on how the schema is managed.

I would be much more willing to work toward switching to mmCIF if RCSB
showed more interest in collaborating with the user community. If we
can't even get involvement in something as simple as the PDB format, why
should we think working with mmCIF will be any better?

Joe Krahn

[ccp4bb] ---solved---Fwd: [ccp4bb] problem in running DM (NCS averaging)

[JOE CRYSTAL]

Dear all,


Thank you so much for your valuable thoughts.


The problem is gone after I used rotation matrix instead of O matrix.  Now
DM is running fine.  Thanks.



Best regards,


Joe



-- Forwarded message --
From: Stein, ND (Norman) [EMAIL PROTECTED]
Date: Jul 31, 2007 8:13 AM
Subject: RE: [ccp4bb] problem in running DM (NCS averaging)
To: JOE CRYSTAL [EMAIL PROTECTED]
Cc: [EMAIL PROTECTED]

 Hi

Your first error seems to have been caused by invalid input. The program
seems to have read the lines

AVERAGE REFI

and

LABOUT ...

correctly, so the problem input is in the lines between these two. It looks
as if there might be two such lines; one of these is presumably LABIN
Are there any typos in these input lines?

Regrading your second error, have you supplied all the necessary data in the
CCP4i gui? in particular, have you specified a rotation and translation
defining the NCS operator. (the Define NCS Symmetry section in the gui; you
may have to scroll down to see all of it).

Norman Stein
CCP4

 --
*From:* CCP4 bulletin board [mailto:[EMAIL PROTECTED] *On Behalf Of *JOE
CRYSTAL
*Sent:* 30 July 2007 17:09
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* [ccp4bb] problem in running DM (NCS averaging)

Dear all,


I tried to run DM for NCS averaging (DM module in CCP4i 6.0.2 installed
under Windows XP), but the running failed at different stages with various
error message as listed below.   I was told it could be an installation
problem, but I don't know how to fix it.  I am wondering if you have
encountered  such problems before.  I am very thankful for any  help and
comments.   Thanks  in advance.



Error Message 1:

*

#CCP4I JOB_ID 69
#CCP4I SCRATCH C:/Ccp4Temp/
#CCP4I PID 772
/pre

BFONT COLOR=#FF!--SUMMARY_BEGIN--
html !-- CCP4 HTML LOGFILE --
hr
!--SUMMARY_END--/FONT/B

a name=dmdmh1dm 2.1/h1/a
BFONT COLOR=#FF!--SUMMARY_BEGIN--
pre

 ###
 ###
 ###
 ### CCP4 6.0 : dm version 6.0   : ##
 ###
 User: Chen  Run date: 30/ 7/2007 Run time: 10:27:58


 Please reference: Collaborative Computational Project, Number 4. 1994.
 The CCP4 Suite: Programs for Protein Crystallography. Acta Cryst. D50,
760-763.
 as well as any specific reference in the program write-up.

!--SUMMARY_END--/FONT/B
/pre

a 
href=http://www.yorvic.york.ac.uk/~cowtan/dm/refs.htmlhttp://www.yorvic.york.ac.uk/%7Ecowtan/dm/refs.htmldm
reference:/a
blockquote
K. Cowtan (1994),
  dm: An automated procedure for phase improvement by density modification.
  Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography, 31,
p34-38.
/blockquotep


a name=tocdmh2Contents/h2/a
ul
lia href=#commanddmCommand input/a
lia href=#commentsdmComments/a
lia href=#mtzindmMTZ input/a
lia href=#datachkdmData Checking/a
lia href=#datascldmData Scaling/a
lia href=#solmskdmSolvent Mask/a
lia href=#cyc0001dmFirst Cycle/a
lia href=#dataoutdmOutput/a
/ul

a name=commanddmh2Command Input/h2/a
pre
 Data line--- a href=C:\CCP4-Packages\ccp4-6.0.2\html/dm.html#modeMODE
/a SOLV AVER
 Data line--- a
href=C:\CCP4-Packages\ccp4-6.0.2\html/dm.html#combineCOMBINE
/a PERT
 Data line--- a
href=C:\CCP4-Packages\ccp4-6.0.2\html/dm.html#ncsmaskNCSMASK
/a SIZE 10
 Data line--- a href=C:\CCP4-Packages\ccp4-6.0.2\html/dm.html#schemeSCHEME
/a ALL
 Data line--- a href=C:\CCP4-Packages\ccp4-6.0.2\html/dm.html#ncycleNCYCLE
/a AUTO
 Data line--- a href=C:\CCP4-Packages\ccp4-6.0.2\html/dm.html#solcSOLCONT
/a 0.5
 Data line--- a
href=C:\CCP4-Packages\ccp4-6.0.2\html/dm.html#averageAVERAGE
/a REFI

 ***  Warning
 Invalid keyword


 ***  Warning
 Invalid sub-keyword in position  2


 ***  Warning
 Invalid keyword


 ***  Warning
 Invalid sub-keyword in position  2


 ***  Warning
 Invalid keyword


 ***  Warning
 Invalid sub-keyword in position  2


 ***  Warning
 Invalid keyword


 ***  Warning
 Invalid sub-keyword in position  2


 ***  Warning
 Invalid keyword


 ***  Warning
 Invalid sub-keyword in position  2


 ***  Warning
 Invalid keyword


 ***  Warning
 Invalid sub-keyword in position  2


 ***  Warning
 Invalid sub-keyword in position  3


 ***  Warning
 Invalid sub-keyword in position  4


 ***  Warning
 Invalid sub-keyword in position  5


 ***  Warning
 Invalid sub-keyword in position  6


 ***  Warning
 Invalid sub-keyword in position  7


 ***  Warning
 Invalid sub-keyword in position  8


 ***  Warning
 Invalid sub-keyword in position  9

 Data line--- a href=C:\CCP4-Packages\ccp4-6.0.2\html/dm.html#laboutLABOUT
/a FDM=FDM PHIDM=PHIDM FOMDM=FOMDM
BFONT COLOR=#FF!--SUMMARY_BEGIN--
 dm:  Input error (see above)
Times: User:   0.0s System

[ccp4bb] problem in running DM (NCS averaging)

[JOE CRYSTAL]

Dear all,


I tried to run DM for NCS averaging (DM module in CCP4i 6.0.2 installed
under Windows XP), but the running failed at different stages with various
error message as listed below.   I was told it could be an installation
problem, but I don't know how to fix it.  I am wondering if you have
encountered  such problems before.  I am very thankful for any  help and
comments.   Thanks  in advance.



Error Message 1:

*

#CCP4I JOB_ID 69
#CCP4I SCRATCH C:/Ccp4Temp/
#CCP4I PID 772
/pre

BFONT COLOR=#FF!--SUMMARY_BEGIN--
html !-- CCP4 HTML LOGFILE --
hr
!--SUMMARY_END--/FONT/B

a name=dmdmh1dm 2.1/h1/a
BFONT COLOR=#FF!--SUMMARY_BEGIN--
pre

 ###
 ###
 ###
 ### CCP4 6.0: dm version 6.0   : ##
 ###
 User: Chen  Run date: 30/ 7/2007 Run time: 10:27:58


 Please reference: Collaborative Computational Project, Number 4. 1994.
 The CCP4 Suite: Programs for Protein Crystallography. Acta Cryst. D50,
760-763.
 as well as any specific reference in the program write-up.

!--SUMMARY_END--/FONT/B
/pre

a href=http://www.yorvic.york.ac.uk/~cowtan/dm/refs.html;dm
reference:/a
blockquote
K. Cowtan (1994),
  dm: An automated procedure for phase improvement by density modification.
  Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography, 31,
p34-38.
/blockquotep


a name=tocdmh2Contents/h2/a
ul
lia href=#commanddmCommand input/a
lia href=#commentsdmComments/a
lia href=#mtzindmMTZ input/a
lia href=#datachkdmData Checking/a
lia href=#datascldmData Scaling/a
lia href=#solmskdmSolvent Mask/a
lia href=#cyc0001dmFirst Cycle/a
lia href=#dataoutdmOutput/a
/ul

a name=commanddmh2Command Input/h2/a
pre
 Data line--- a href=C:\CCP4-Packages\ccp4-6.0.2\html/dm.html#modeMODE
/a SOLV AVER
 Data line--- a
href=C:\CCP4-Packages\ccp4-6.0.2\html/dm.html#combineCOMBINE
/a PERT
 Data line--- a
href=C:\CCP4-Packages\ccp4-6.0.2\html/dm.html#ncsmaskNCSMASK
/a SIZE 10
 Data line--- a href=C:\CCP4-Packages\ccp4-6.0.2\html/dm.html#schemeSCHEME
/a ALL
 Data line--- a href=C:\CCP4-Packages\ccp4-6.0.2\html/dm.html#ncycleNCYCLE
/a AUTO
 Data line--- a href=C:\CCP4-Packages\ccp4-6.0.2\html/dm.html#solcSOLCONT
/a 0.5
 Data line--- a
href=C:\CCP4-Packages\ccp4-6.0.2\html/dm.html#averageAVERAGE
/a REFI

 ***  Warning
 Invalid keyword


 ***  Warning
 Invalid sub-keyword in position  2


 ***  Warning
 Invalid keyword


 ***  Warning
 Invalid sub-keyword in position  2


 ***  Warning
 Invalid keyword


 ***  Warning
 Invalid sub-keyword in position  2


 ***  Warning
 Invalid keyword


 ***  Warning
 Invalid sub-keyword in position  2


 ***  Warning
 Invalid keyword


 ***  Warning
 Invalid sub-keyword in position  2


 ***  Warning
 Invalid keyword


 ***  Warning
 Invalid sub-keyword in position  2


 ***  Warning
 Invalid sub-keyword in position  3


 ***  Warning
 Invalid sub-keyword in position  4


 ***  Warning
 Invalid sub-keyword in position  5


 ***  Warning
 Invalid sub-keyword in position  6


 ***  Warning
 Invalid sub-keyword in position  7


 ***  Warning
 Invalid sub-keyword in position  8


 ***  Warning
 Invalid sub-keyword in position  9

 Data line--- a href=C:\CCP4-Packages\ccp4-6.0.2\html/dm.html#laboutLABOUT
/a FDM=FDM PHIDM=PHIDM FOMDM=FOMDM
BFONT COLOR=#FF!--SUMMARY_BEGIN--
 dm:  Input error (see above)
Times: User:   0.0s System:0.0s Elapsed: 0:00
/pre
/html
!--SUMMARY_END--/FONT/B
***
* Information from CCP4Interface script
***
The program run with command: dm HKLIN
D:/xtalwork/DM21/delM_arpwarp/work1/7_solvent_refmac7.mtz HKLOUT
D:/xtalwork/DM21/delM_arpwarp/work1/7_solvent_dm4.mtz SOLOUT
D:/xtalwork/DM21/delM_arpwarp/work1/7_solvent_dm2.msk NCSIN1
D:/xtalwork/DM21/delM_arpwarp/work1/A.msk VUOUT
D:/xtalwork/DM21/delM_arpwarp/work1/delM_69_ncs.odat
has failed with error message
 dm:  Input error (see above)
***


#CCP4I TERMINATION STATUS 0  dm:  Input error (see above)
#CCP4I TERMINATION TIME 30 Jul 2007  10:27:58
#CCP4I MESSAGE Task failed


Error Message 2:

+++
a name=ncsmskdmh3Initial Averaging Mask/h3/a
BFONT COLOR=#FF!--SUMMARY_BEGIN--
***
* Information from CCP4Interface script
***
The program run with command: dm HKLIN
D:/xtalwork/DM21/delM_arpwarp/work1/7_solvent_refmac7.mtz 

[ccp4bb] Stop the new PDB format!

[Joe Krahn]

The new PDB format (version 3) has a lot of very useful improvements,
and an update is long overdue. However, I am irate that RCSB chose NOT
to use the ACA meeting to discuss the changes. Instead, the format is
being put into production at the same time as the ACA meeting. It is
essentially stating that opinions expressed at the ACA do not count.
Their was a lot of conflict at their last attempt at an update. Instead
of working to better involve the structural biologist community, I feel
that they are intentionally discounting our interests because working
with the user community is too much effort.

Unfortunately, structural biologists generally do not want to spend time
arguing about file formats, while computer scientists can carry on for
weeks over minor details. This change is going to affect all of us. If
you have concerns about the new format that have not been addressed, it
is important to take action now. The PDB format is not just their
personal database format (that's what mmCIF is for), but the format that
we all use in our daily research. They don't even want to keep the PDB
format at all. It's primary purpose now is for structural biologists. It
is essential that we be part of the decision making process.

I just sent the following letter to the wwPDB, which is where
comments about the new format are supposed to go. If you will be at the
ACA meeting, I encourage you to complain loudly.

Joe Krahn

---
To: [EMAIL PROTECTED]
Subject: The new PDB format is WRONG.

It seems obvious to me that the RCSB and wwPDB worked on the new format
to consider database users needs, but has intentionally ignored the rest
of the user community. RCSB manages mmCIF for database purposes, and has
declared a lack of interest in even keeping the PDB format. Obviously,
the primary purpose of the PDB format is for structural biologists
working with individual structures, and not database users.

Most of the updates are quite positive and beneficial, but I think that
some changes are detrimental. My only serious complaint is that RCSB,
and now wwPDB, seem to be ignoring the interests of much of the
scientific community which they are supposed to be serving. All that I
ask for is appropriate inclusion of all of the user community. This is a
big change that will affect thousands of people. We should ensure that
it is the best possible format update before we all have to expend a
huge effort to deal with it.

I have seen many comments about the format by well known
crystallographers ignored. One example is the use of SegID. Most
structural biologists have favored it for years, but RCSB continued to
deny us, on grounds that it is not well defined. It would be better to
make a better definition, and allow it to be used to group together
non-covalent groups, such as waters with a specific protein molecule.
This is important because the use of ChainID for non-polymers has been
banned, which also goes against the wishes of most users.

The latest atom alignment rule changes is also detrimental. RCSB has
totally broken the element alignment rules, on baseless grounds that it
was too hard to follow. The new change convolutes this rule even
further, and essentially follows an earlier attempt at IUPAC hydrogen
names that the community strongly rejected. At this point, the best
solution is probably to make it completely left justified. Again, my
main concern is not to follow my idea, but to ensure that the user
community gets a fair chance to participate in the final decision.

Another problem is that the original meaning of HET groups continues to
be corrupted. ATOM records are for commonly occurring residues from a
list of standard residues. Water is obviously common, and should not
have been converted to a HET group. HET groups have NO relation ship to
polymeric state. With water as a HET group, a proper PDB file for a
modeller with bulk solvent would require CONECT entries for every single
water. It is also important to emphasize that the HETNAM is the actual
unique ID, not the 3-letter code. The current hack is to treat
everything as an ATOM, which has a pre-determined connectivity. This
cannot continue forever, and we are already stuck with meaningless
3-letter codes instead of useful 3-letter abbreviations. The unique
3-letter code should be continued for now, but there should be an
emphasis on beginning to use the full HETNAM so that the inevitable
switch top non-unique 3-letter codes will not have a big impact.

Thank you,
Joe Krahn

[ccp4bb] Combining MR and MAD phases

[Joe Batchelor]

Hi,

I have a 1.7 A native dataset, a good MR solution for 2/3 of the
protein, and MAD phases to 3 A. How should I combine the MR phases
with the MAD phases?

Thanks,
Joe

Re: [ccp4bb] Protein-DNA complex for crystallization

[JOE CRYSTAL]

Hi Kumar,

I also have the same issue.  If you get any helpful response, could you
forward me a copy?  Thank you.

P.S. Could anyone who has any comments or suggestions on this issue also
forward the response to ccp4bb?  Thank you in advance.

Best,


Joe



On 7/16/07, bputcha [EMAIL PROTECTED] wrote:


Hi,
I am trying to crystallize a protein-DNA complex. I purify the protein
finally
using gel filtration. I purchase
single stranded complementary oligos (desalting from idtdna.com), mix them
up
and make DNA duplex by
heating to 95 degree C and cooling to room temperature. I mix protein and
DNA,
concentrate and use it
for crystallization.
I am geting small crystals consistently under a specific condition. These
crystals take up IZIT dye but are
not well shaped. I am not able to improve the size and shape of the
crystals
substantially even after
screening with additives (Hampton research).
I suspect that purity of the duplex DNA (presence of unpaired oligos) is
limiting the chances of obtaining
better crystals.

How can I purify the duplex DNA further?

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

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

Thank you
Kumar

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

[ccp4bb] extend resolution in refmac

[JOE CRYSTAL]

Dear all,


I am refining a structure in Refmac at 2.3 A and I set resolution range to
2.2 A to extend the resolution.  However, Refmac seemed to ignore my setting
and still refined structure at 2.3 A.  Thank you in advance for your any
helpful suggestions.


Best,


Joe

[ccp4bb] Solving structure of protein-protein complexes using MR

[Joe Smith]

Hi all,

We have been trying to solve a structure of protein-protein complexes using
3.1A data (one of the proteins is of 120kDa whereas the other is of 20kDa).
The structure of smaller protein is known (individually-100% sequence
identity) whereas the bigger protein does not share more than 10% sequence
identity with the similar proteins solved from other sources.

Due to some problem in getting Seleno-labelled protein, we have also been
trying to use molecular replacement (MR) to solve the structure. We want to
find out the correct position of smaller protein using MR and then plan to
extend the phases to the whole asymmetric unit (we hope it could be done but
not sure). We are more or less sure about the fold of bigger protein and
expect it to be similar to the other known related structures.
In one of the solution obtained using phaser, the map looks really good, but
this solution doesn't provide good packing of the complex inside the unit
cell. Due to low scattering contribution of the smaller protein, we are
unable to refine any possible solutions using REFMAC.

We welcome any kind of suggestions in this regard.

Thanking you in advance.

Joe

[ccp4bb] How to determine ligand binding from diffraction pattern?

[Joe Chen]

Dear all,


Is there a simple way to determine whether ligand is bound or not by
comparing the diffraction patterns between ligand-free (structure known) and
ligand-soaked protein crystals?  I would like to solve the ligand bound
protein structure, but before I do so, I have to find out if the ligand is
actually bound.  Thank you very much!


Best,


Joe