Re: [ccp4bb] Assistant Professor in Structural Biology, University of Nebraska-Lincoln

[Mark Wilson]

Hi Gerlind,
To clarify, the position is tenure-leading, where tenure dossiers are typically 
submitted in the 5th year of employment.  If tenure is granted, then the 
position is a continuous appointment.  The 9-month language in the ad refers to 
the academic year-typically faculty can either choose to take their salary over 
either 9 or 12 months.   Faculty can also raise additional funds for salary 
over the summer via grants.  This is common (nearly universal) in the US 
system. Importantly, this is _not_ simply a 9-month contract position, which 
would not make sense for an Assistant Professor.

Best regards,
Mark

Mark A. Wilson (he/him)
Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine St.
Lincoln, NE 68588

From: Gerlind Sulzenbacher 
Date: Sunday, October 15, 2023 at 10:15 AM
To: Mark Wilson , CCP4BB@JISCMAIL.AC.UK 

Subject: Re: [ccp4bb] Assistant Professor in Structural Biology, University of 
Nebraska-Lincoln
Non-NU Email

Dear Mark,

thank you for posting this job offer, probably very interesting for people 
currently unemployed.

Personally, I feel that working conditions and job offers in the scientific 
field have deteriorated considerably in recent years.
I've always been shocked by one-year job offers involving international 
movements.
Now we seem to be down at job offers for 9 months.

>From a human point of view, I find this extremely worrying.
Sorry to pollute the mailing list with my personal opinions.

All the best.
Gerlind


On 15/10/2023 16:46, Mark Wilson wrote:
Dear Colleagues,

The Department of Biochemistry at the University of Nebraska-Lincoln (UNL) 
invites applications for a tenure-track nine-month (academic year) faculty 
position at the rank of Assistant Professor. Areas of particular interest 
include but are not limited to time-resolved approaches to understanding 
macromolecular function, multiscale imaging, and integrated computational and 
experimental approaches to structural biology. Researchers at UNL have 
collaborations with national user facilities pioneering time-resolved X-ray 
diffraction techniques and have established a state-of-the-art cryo-EM facility 
housing a Thermo Scientific Glacios 200 keV Cryo TEM equipped with a Falcon 4i 
direct electron detector camera, a Selectris zero-loss energy filter, and 
capability for micro-electron diffraction of crystalline samples. The new 
cryo-EM facility is part of a strategic growth plan at UNL and complements 
existing strengths in X-ray crystallography, biophysics, and computation at UNL.

Review of applications will begin November 15, 2023 and continue until the 
position is filled or the search is closed. To view details of the position and 
the application, go to https://employment.unl.edu, requisition F_230175 or 
visit https://employment.unl.edu/postings/88408. Click “Apply for this Job” and 
complete the information form. As an EO/AA employer, the University of Nebraska 
considers qualified applicants for employment without regard to race, color, 
ethnicity, national origin, sex, pregnancy, sexual orientation, gender 
identity, religion, disability, age, genetic information, veteran status, 
marital status, and/or political affiliation. See 
https://www.unl.edu/equity/notice-nondiscrimination.


Best regards,
Mark

Mark A. Wilson (he/him)
Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine St.
Lincoln, NE 68588



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--

Gerlind Sulzenbacher

Architecture et Fonction des Macromolécules Biologiques

UMR7257 CNRS, Aix-Marseille Université

Case 932

163 Avenue de Luminy

13288 Marseille cedex 9

France

Tel +33 413 94 95 27

E-mail: 
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[ccp4bb] Assistant Professor in Structural Biology, University of Nebraska-Lincoln

[Mark Wilson]

Dear Colleagues,

The Department of Biochemistry at the University of Nebraska-Lincoln (UNL) 
invites applications for a tenure-track nine-month (academic year) faculty 
position at the rank of Assistant Professor. Areas of particular interest 
include but are not limited to time-resolved approaches to understanding 
macromolecular function, multiscale imaging, and integrated computational and 
experimental approaches to structural biology. Researchers at UNL have 
collaborations with national user facilities pioneering time-resolved X-ray 
diffraction techniques and have established a state-of-the-art cryo-EM facility 
housing a Thermo Scientific Glacios 200 keV Cryo TEM equipped with a Falcon 4i 
direct electron detector camera, a Selectris zero-loss energy filter, and 
capability for micro-electron diffraction of crystalline samples. The new 
cryo-EM facility is part of a strategic growth plan at UNL and complements 
existing strengths in X-ray crystallography, biophysics, and computation at UNL.

Review of applications will begin November 15, 2023 and continue until the 
position is filled or the search is closed. To view details of the position and 
the application, go to https://employment.unl.edu, requisition F_230175 or 
visit https://employment.unl.edu/postings/88408. Click “Apply for this Job” and 
complete the information form. As an EO/AA employer, the University of Nebraska 
considers qualified applicants for employment without regard to race, color, 
ethnicity, national origin, sex, pregnancy, sexual orientation, gender 
identity, religion, disability, age, genetic information, veteran status, 
marital status, and/or political affiliation. See 
https://www.unl.edu/equity/notice-nondiscrimination.


Best regards,
Mark

Mark A. Wilson (he/him)
Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine St.
Lincoln, NE 68588



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[ccp4bb] Session on mix-and-inject serial crystallography at the SSRL/LCLS Users' Meeting

[Mark Wilson]

Dear fellow structural biologists,
Dr. Chris Kupitz (LCLS) and I are co-hosting a session on mix-and-inject serial 
crystallography at XFELS and synchrotrons on Weds, 9/28 as part of this year’s 
SSRL/LCLS Users’ Meeting.  This is an opportunity to hear from leading 
researchers about new methods and results featuring this exciting technique.  
The agenda is here: 
https://events.bizzabo.com/SLAC-UsersMeeting-2022/agenda/session/910608
The meeting is fully remote this year and registration is still open 
(https://events.bizzabo.com/SLAC-UsersMeeting-2022/home).

Best regards,
Mark

Mark A. Wilson (he/him)
Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine St.
Lincoln, NE 68588




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[ccp4bb] Cryo-EM Core Director position, University of Nebraska-Lincoln

[Mark Wilson]

Dear Structural Biologists,
The University of Nebraska is hiring a Core Director (Research Assistant 
Professor) for our Glacios TEM with a Selectris energy filter, Falcon 4i 
detector, and micro-ED capability (delivery expected 12/2022).  This hire is an 
addition to a tenure-leading hire in Cryo-EM that is underway.  Consideration 
of applicants will begin on June 15th and continue until the position is 
filled. Additional information about the position is below the line.

Best regards,
Mark

Mark A. Wilson (he/him)
Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine St.
Lincoln, NE 68588

Research Assistant Professor
Center for Biotechnology
University of Nebraska-Lincoln

We seek to hire an experienced structural biologist with expertise in CryoEM to 
manage a new Cryo-Electron Microscopy core facility that will be part of the 
Nebraska Center for Biotechnology 
(https://biotech.unl.edu/center-biotechnology). The University of 
Nebraska-Lincoln (UNL) purchased a state-of-the-art Glacios CryoEM that will be 
installed in a newly remodeled space on campus in late fall of 2022. This is a 
full-time position and will report to the Director of the Nebraska Center for 
Biotechnology, but the day-to-day operations of the facility will be managed by 
the candidate. The position will oversee the installation of the microscope, 
set up fee-for-service rates, develop a strong customer/user base as well as 
prepare and analyze samples in the CryoEM. In addition, the candidate will work 
with others to oversee and maintain the microscope, train new users and assist 
experienced users. The successful candidate will be responsible for aspects of 
method development, sample preparation, data acquisition, data analysis using 
Cryo-EM software platforms, designing and executing protocols and experiments, 
modifying protocols as needed and may initiate their own research projects. The 
candidate must have previous cryoEM experience and be excited to interact with 
students, postdocs, staff and faculty. In addition to UNL users, the user base 
for this instrument will include external academic scientists and industry 
customers.

For required and preferred qualifications please see: 
https://employment.unl.edu/, requisition F_220070.

This role will be very rewarding in terms of the work and life in Lincoln, 
Nebraska. There will be opportunities to interact with a group of motivated 
core facility managers in the Center for Biotechnology, to do a limited amount 
of teaching, to develop strong professional relationships with faculty, 
students and staff and to work with the newest CryoEM technologies. There will 
be career-development opportunities to expand your skills, enhance your 
expertise, and maximize the potential of your career. In addition, Lincoln has 
extensive cultural and social activities as well as excellent public schools 
and a low cost of living. It is centrally located in the USA with a small 
airport in Lincoln and a larger one in Omaha which is about a 50-minute drive 
from Lincoln.

Review of applications will begin June 15, 2022 and continue until the position 
is filled or the search is closed. To view complete details and to apply, visit 
https://employment.unl.edu/, requisition F_220070. Click “Apply for this Job” 
and complete the information form. Attach a letter of interest, CV and list of 
references.

As an EO/AA employer, qualified applicants are considered for employment 
without regard to race, color, ethnicity, national origin, sex, pregnancy, 
sexual orientation, gender identity, religion, disability, age, genetic 
information, veteran status, marital status, and/or political affiliation. See 
https://www.unl.edu/equity/notice-nondiscrimination.




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[ccp4bb] Fixing input anisotropic scale parameters in Refmac5

[Mark Wilson]

Dear Colleagues,
I would like to run test refinements in Refmac5 where I can provide anisotropic 
scale parameters, fix them, and then proceed with restrained refinement of 
other model parameters.  I could not find how to do this in the 
documentation-is this possible?

Best regards,
Mark

Mark A. Wilson
Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine St.
Lincoln, NE 68588




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Re: [ccp4bb] Covalent Cysteine Aduct

[Mark Wilson]

Hi Richard,
Something that hasn’t been mentioned yet that might help settle the issue
is to calculate an anomalous difference map with your I+, I- unmerged
data.  Even if you collect data far from the S edge, it is often possible
to see positive anomalous difference density for S atoms in maps at this
resolution (and with good phases from the refined model).  In the CME case
that Jack Tanner and other mentioned, you’d expect to see two peaks.
Best regards,
Mark

Mark A. Wilson
Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 





On 3/27/20, 4:53 PM, "CCP4 bulletin board on behalf of Cowan, Richard H.
(Dr.)"  wrote:

>
>Hi to All,
>
>
>Whilst this discussion has been going on, I've been carrying on with the
>work on this, and have now replaced the offending cysteine with CME. It
>seems to refine pretty well, fitting the density well, and the b-factors
>stay similar to those for nearby side chains,
> so I'm reasonably happy with it. Now to figure out where the BME came
>from.
>
>
>Thanks to all who have offered suggestions and help. Now to check another
>structure with a related Fab with the same surface cysteine, this time
>heavily annisotropic, and in complex with the target, at 2.7A at best!
>
>
>Thanks,
>
>
>
>Dr Richard Cowan
>Research Associate
> 
>HWLSB 1/05
>Department of Biochemistry
>University of Leicester
>Lancaster Road
>Leicester, LE1 9HN, U.K.
> 
>Phone +44 (0) 116 229 7077
>
>
>
>
>
>
>
>From: Mark J van Raaij 
>Sent: 27 March 2020 21:46
>To: Cowan, Richard H. (Dr.) 
>Subject: Re: [ccp4bb] Covalent Cysteine Aduct
>
>I agree, but in my opinion the density is quite clear for a disulphide,
>so I would bet beta-mercaptoethanol *was* added at some step (perhaps
>even by accident). Even if it's not considered best practice, you seem to
> have "gotten away with it" and the Fab still crystallised ok.
>
>Mark J van Raaij
>Dpto de Estructura de Macromoleculas
>Centro Nacional de Biotecnologia - CSIC
>calle Darwin 3
>E-28049 Madrid, Spain
>Section Editor Acta Crystallographica F
>https://journals.iucr.org/f/
>ection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fjournals.iucr.org-252Ff-25
>2F-26data-3D02-257C01-257Crc273-2540leicester.ac.uk-257C4d22be0719f0431dc6
>9608d7d298414d-257Caebecd6a31d44b0195ce8274afe853d9-257C0-257C0-257C637209
>423675980222-26sdata-3DQcQSKYwh-252BJ3a86IwEvh6J3UVOplgLRWdfSRvTL8j1sU-253
>D-26reserved-3D0&d=DwMFAg&c=Cu5g146wZdoqVuKpTNsYHeFX_rg6kWhlkLF8Eft-wwo&r=
>pWFylCwRYnT2UZGAgJCqKaJCAAfFi00TZEWmZXJvhsA&m=iYV__iKAZ10TH0IfgFpthVnqpcpX
>ADEZsn1BKx3j4EI&s=Ca2a6GjDTkPqtBnpDQfjcHriOQXlKDuhn2vLvXO6iMI&e=>
>
>
>
>
>
>
>
>
>
>
>
>
>On 27 Mar 2020, at 22:38, Cowan, Richard H. (Dr.) 
>wrote:
>
>Hi,
>
>
>The Fab constructs have a c-terminal cysteines on both the heavy and
>light chains, which should form a disulphide. Adding reducing agent to
>the purification of the protein would potentially reduce this disulphide,
>possible causing issues the stability and heterogeneity?
> At least that's my understanding?
>
>
>
>Thanks,
>
>
>
>Dr Richard Cowan
>Research Associate
> 
>HWLSB 1/05
>Department of Biochemistry
>University of Leicester
>Lancaster Road
>Leicester, LE1 9HN, U.K.
> 
>Phone +44 (0) 116 229 7077
>
>
>
>
>
>
>
>
>From: CCP4 bulletin board  on behalf of Paul
>Emsley 
>Sent: 27 March 2020 21:33
>To: CCP4BB@JISCMAIL.AC.UK 
>Subject: Re: [ccp4bb] Covalent Cysteine Aduct
>
>
>
>On 27/03/2020 21:06, Cowan, Richard H. (Dr.) wrote:
>
>
>
>
>
>
>  although [BME] seems unlikely, since the crystallized protein is a Fab.
>
>
>
>
>
>
>I don't follow.
>
>
>
>
>To unsubscribe from the CCP4BB list, click the following link:
>https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
>ection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.jiscmail.ac.uk-252Fcgi
>-2Dbin-252Fwebadmin-253FSUBED1-253DCCP4BB-2526A-253D1-26data-3D02-257C01-2
>57Crc273-2540leicester.ac.uk-257C4d22be0719f0431dc69608d7d298414d-257Caebe
>cd6a31d44b0195ce8274afe853d9-257C0-257C0-257C637209423675980222-26sdata-3D
>Yky4trFxHWJiSDlMu5Ixtyr2MQf6b4g9Xjiuf36ArWc-253D-26reserved-3D0&d=DwMFAg&c
>=Cu5g146wZdoqVuKpTNsYHeFX_rg6kWhlkLF8Eft-wwo&r=pWFylCwRYnT2UZGAgJCqKaJCAAf
>Fi00TZEWmZXJvhsA&m=iYV__iKAZ10TH0IfgFpthVnqpcpXADEZsn1BKx3j4EI&s=SJBFc6JbO
>Z8aPDgQ4wyx7LZh-C_y9xEnS2okHG24CFE&e=>
>
>
>
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>-2Dbin-252Fwebadmin-253FSUBED1-253DCCP4BB-2526A-2

[ccp4bb] Postdoctoral position in molecular image analysis methods development, University of Nebraska

[Mark Wilson]

Dear Colleagues,
There is a postdoctoral position at the University of Nebraska in methods
development for molecular image analysis. Application review has started
and will remain open until the position is filled. Please see details
below.
Best regards,
Mark

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 

=
Postdoctoral Position in Deep Learning and Molecular Image Analysis

The Department of Statistics and the Department of Biochemistry at the
University of Nebraska-Lincoln are pleased to recruit candidates for a
postdoctoral position in molecular image analysis. This position is
supported by the Quantitative Life Sciences Initiative, a university-wide
program supporting the integration of the data and life sciences. We are
seeking candidates with expertise in data generated by molecular imaging
techniques (e.g. X-ray crystallography, cryo-EM, XFEL microcystallography,
electron diffraction), computer science, statistics and machine learning,
who have demonstrated a high level of skill in image preprocessing,
management, and analysis.

The incumbent will be expected to develop a strong research program in
data science and AI for molecular imaging. Responsibilities will include:
(1) developing methods for processing, segmentation, and analysis of
molecular images derived from diffraction or single particle cryo-EM data,
(2) developing software implementations of novel analytical approaches to
molecular images, and (3) using newly emerging analytical and
computational tools to extract the maximum amount of information from data
produced by modern structural biological imaging modalities (e.g., serial
crystallography, single particle cryo-EM, single particle diffraction).

The Initiative and the Departments of Statistics and Biochemistry will
support successful candidates to establish effective disciplinary and
trans-disciplinary collaborations including integration with existing
research groups; connect with stakeholders, agency, and/or industry
partners; obtain and leverage external and internal support (grants,
fellowships, etc.) for research and teaching activities; publish in
high-quality, high-impact peer-reviewed journals and participate in
scientific meetings and other appropriate activities; and translate
research-based information into learner-centered products.

The successful candidate will be expected to teach at least one regular
course per academic year in molecular imaging and image analysis. In
addition, the successful candidate will participate in program and
curriculum development, including graduate seminars and workshops.

Minimum qualifications: PhD in Statistics, Mathematics, Physics,
Biochemistry, or closely related field. Experience with analysis of data
from molecular imaging experiments, as demonstrated by refereed papers,
presentations, or other completed projects, e.g., PhD thesis. Computing
and methodological skills appropriate to the preprocessing and analysis of
data types with which the candidate has experience.

Preferred qualifications: Demonstrated methodological novelty and creative
ability in one or more area of deep learning and AI applicable to
molecular imaging. This includes, but is not limited to, Bayesian
statistics, Gaussian processes, image analysis, and prediction of optimal
experimental settings and molecular orientation. Collaborative research
experience in structural biology using either X-ray crystallography or
cryo-EM approaches. Proficiency with modern object-oriented programming
languages including C++ and Python. Communication skills, written, verbal
and otherwise, at a level sufficient to interact easily with a broad range
of researchers at UNL, with the academic world more generally, and with
the broader Nebraska scientific community.

To view details of the position and make application, go to
http://employment.unl.edu, requisition F_190163.  Click “Apply to this
job” and complete the information form.  Attach a letter of interest,
curriculum vitae, contact information for three professional references,
and a one-page statement of research interests (attach as “Other
Document”).  Review of applications begins October 31, 2019 and continues
until the position is filled or the search is closed.

As an EO/AA employer, qualified applicants are considered for employment
without regard to race, color, ethnicity, national origin, sex, pregnancy,
sexual orientation, gender identity, religion, disability, age, genetic
information, veteran status, marital status, and/or political affiliation.
See http://www.unl.edu/equity/notice-nondiscrimination.





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Re: [ccp4bb] [EXTERNAL] [ccp4bb] refmac same residue different names

[Mark Wilson]

Hi Diana,
At the risk of thread-jacking Ed’s question, yes, PHENIX handles this
seamlessly for us as well.  However, REFMAC5 doesn’t like this scenario,
with or without alt. confs. or insertion codes.  There are some models
where I’d really like to use REFMAC5 for refining these mixed species
models, and it’s not clear to me how to do so.  It sounds like Ed is
encountering similar issues.
Best regards,
Mark

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 






On 2/6/19, 11:32 AM, "CCP4 bulletin board on behalf of Diana Tomchick"

wrote:

>
>
>
>The scenario I presented earlier works like a charm in refinement with
>the Phenix program suite. For example, I have used it for both mixed
>populations of ligands (e.g., AAMP and BADP) as well as phosphorylation
>(ASER and BSEP).
>
>
>Diana
>
>**
>Diana R. Tomchick
>Professor
>Departments of Biophysics and Biochemistry
>UT Southwestern Medical Center
>5323 Harry Hines Blvd.
>Rm. ND10.214A
>Dallas, TX 75390-8816
>diana.tomch...@utsouthwestern.edu
>(214) 645-6383 (phone)
>(214) 645-6353 (fax)
>
>
>On Feb 6, 2019, at 11:19 AM, Edwin Pozharski 
>wrote:
>
>Herman,
>
>
>thanks - however, it seems like I have poorly worded my question.  I do
>know how to generate alternate conformers, what the PDB ATOM record
>format is etc.  The point was that when I have alternate conformers that
>carry the same residue ID but
> different residue types, Refmac exits with the error.  The question was
>whether there is a "native" solution to this that does not include some
>pdb file acrobatics (i.e. separating the alternative type into a separate
>residue and enforcing connectivity using
> elaborate LINK records).   Based on what I see so far, there likely
>isn't any such native option.  Whether these situations are common enough
>to warrant (possibly elaborate) software changes is a separate question.
>
>
>Cheers,
>
>
>Ed.
>
>
>---
>
>I don't know why the sacrifice didn't work. The science seemed so solid.
>
>Julien XIII, Lord of the Lemurs
>
>
>
>
>On Wed, Feb 6, 2019 at 2:36 AM  wrote:
>
>
>Dear Edwin,
> 
>I do not know whether your question has been answered already, but the
>answer is simple: you have to define alternative conformations. Easiest is
> to generate them in coot with the “add alternate conformation” option in
>the right panel. You may have to delete the original unlabeled
>alternative conformation first though.
>
>Alternatively, if you want to keep the original coordinates, or if the
>alternative residue is different: say a Leu versus a Phe you can open the
>pdb
> file with an editor and generate the alternative conformation yourself:
>One of the residues gets an “A” in front of the residue name, e.g. ALEU,
>and the alternative residue a “B”, say BLEU. You also have to reset the
>occupancies
> to 0.5 for both conformations (or different fractions which add up to
>one).
> 
>Good luck!
>Herman
> 
>Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK]
>Im Auftrag von Edwin Pozharski
>Gesendet: Montag, 4. Februar 2019 22:35
>An: 
>CCP4BB@JISCMAIL.AC.UK 
>Betreff: [EXTERNAL] [ccp4bb] refmac same residue different names
> 
>Belated happy 2019, everyone.
> 
>
>For whatever obscure reason, I need to refine a model that has two
>different residue types as alternate conformers with the same residue ID.
> Presented with a pdb file that has such feature, Refmac fails saying this
>
> 
>
>
> ERROR: in chain A residue: 443
>different residues have the same number
>
>There is an error in the input coordinate file
>At least one the chains has 2 residues with the same number
>Check above to see error
>===> Error: Problem with coordinate file
>
>
>
> 
>
>There are several ways of getting around this I can think of.  Perhaps
>duplicate chain with strict NCS for all but the residue in question could
>work.  Perhaps adding this residue as two separate chains and then adding
>enough LINK records
> to keep things in place could.  Either solution here is inelegant and
>requires reformating pdb file back to sanity prior to deposition.
>
> 
>
>Is there some way to allow different geometries for alternate conformers
>that is native to Refmac?
>
> 
>
>Cheers,
>
> 
>
>Ed.
>
> 
>
>PS.  I know that phenix.refine takes the mixed name pdb file straight up.
> I still want to be able to refine such structure with refmac (and
>buster, actually, but that's a question I already asked in the
>appropriate forum.
>
> 
>
>
>Edwin Pozharski, PhD, Assistant Professor
>University of Maryland, Baltimore
>--
>When the Way is forgotten duty and justice appear;
>Then knowledge and wisdom are born along with hypocrisy.
>When harmonious relationships dissolve then respect and devotion arise;
>When a nation falls to chaos then loyalty and patrioti

Re: [ccp4bb] AW: [EXTERNAL] [ccp4bb] refmac same residue different names

[Mark Wilson]

I agree. We have encountered refinement problems similar to those Ed
describes for models with mixtures of modified cysteine species, which is
a fairly common occurrence.
Best regards,
Mark

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 






On 2/6/19, 11:19 AM, "CCP4 bulletin board on behalf of Palm, Gottfried"
 wrote:

>The situation might not be so rare, if you consider 50% Lys and 50%
>Acetyl-Lys or other post-translational modifications.
>
>Gottfried
>
>On Wednesday, 06-02-2019 at 18:05 Diana Tomchick wrote:
>
>If you have the odd case where one residue (of the same number in the
>polypeptide chain) is a Leu and the alternative residue is a Phe, then it
>would be ALEU and BPHE, both residues would have the same residue number,
>and reset the occupancies to fractions
> that sum to 1.0.
>
>
>Diana
>
>**
>Diana R. Tomchick
>Professor
>Departments of Biophysics and Biochemistry
>UT Southwestern Medical Center
>5323 Harry Hines Blvd.
>Rm. ND10.214A
>Dallas, TX 75390-8816
>diana.tomch...@utsouthwestern.edu
>(214) 645-6383 (phone)
>(214) 645-6353 (fax)
>
>
>On Feb 6, 2019, at 1:36 AM,
>herman.schreu...@sanofi.com  wrote:
>
>Dear Edwin,
> 
>I do not know whether your question has been answered already, but the
>answer is simple: you have to define alternative conformations. Easiest
>is to generate
> them in coot with the “add alternate conformation” option in the right
>panel. You may have to delete the original unlabeled alternative
>conformation first though.
>Alternatively, if you want to keep the original coordinates, or if the
>alternative residue is different: say a Leu versus a Phe you can open the
>pdb file with
> an editor and generate the alternative conformation yourself:
>One of the residues gets an “A” in front of the residue name, e.g. ALEU,
>and the alternative residue a “B”, say BLEU. You also have to reset the
>occupancies to
> 0.5 for both conformations (or different fractions which add up to one).
> 
>Good luck!
>Herman
> 
>Von: CCP4 bulletin board
> [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Edwin
> Pozharski
>Gesendet: Montag, 4. Februar 2019 22:35
>An: CCP4BB@JISCMAIL.AC.UK
>Betreff: [EXTERNAL] [ccp4bb] refmac same residue different names
> 
>Belated happy 2019, everyone.
> 
>
>For whatever obscure reason, I need to refine a model that has two
>different residue types as alternate conformers with the same residue ID.
> Presented with a pdb file that has such feature, Refmac fails saying this
>
> 
>
>
> ERROR: in chain A residue: 443
>different residues have the same number
>
>There is an error in the input coordinate file
>At least one the chains has 2 residues with the same number
>Check above to see error
>===> Error: Problem with coordinate file
>
>
>
> 
>
>There are several ways of getting around this I can think of.  Perhaps
>duplicate chain with strict NCS for all but the residue in question could
>work.  Perhaps adding this residue as two separate chains and then adding
>enough LINK records to keep things in
> place could.  Either solution here is inelegant and requires reformating
>pdb file back to sanity prior to deposition.
>
> 
>
>Is there some way to allow different geometries for alternate conformers
>that is native to Refmac?
>
> 
>
>Cheers,
>
> 
>
>Ed.
>
> 
>
>PS.  I know that phenix.refine takes the mixed name pdb file straight up.
> I still want to be able to refine such structure with refmac (and
>buster, actually, but that's a question I already asked in the
>appropriate forum.
>
> 
>
>
>Edwin Pozharski, PhD, Assistant Professor
>University of Maryland, Baltimore
>--
>When the Way is forgotten duty and justice appear;
>Then knowledge and wisdom are born along with hypocrisy.
>When harmonious relationships dissolve then respect and devotion arise;
>When a nation falls to chaos then loyalty and patriotism are born.
>-- / Lao Tse /
>
>
>
>
> 
>
>
>To unsubscribe from the CCP4BB list, click the following link:
>https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
>gi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1&d=DwMFaQ&c=Dbf9zoswcQ-CRvvI7VX
>5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=5
>33Wexf2P2dxZ7xNYAOkGTAhejw65fhzx7fdVjiaqR0&s=3CLcdo1WJ40rHm6l4rs8gd7nqHBgf
>_cZbvJjRgUfgHg&e=>
>
>
>
>To unsubscribe from the CCP4BB list, click the following link:
>https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
>gi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1&d=DwMFaQ&c=Cu5g146wZdoqVuKpTNs
>YHeFX_rg6kWhlk

Re: [ccp4bb] collective term for hydrogen bonds and salt bridges

[Mark Wilson]

Just to put a finer point on some of this, the Lennard-Jones (6-12)
potential is short-range, containing an attractive component arising from
London dispersion forces (quantum mechanical induced dipole-induced dipole
interactions) that decay as 1/r^6 and a repulsive component arising from
the Pauli exclusion principle that rises as 1/r^12.  The minimum of this
potential for a given homoatomic interaction vs. interatomic distance
corresponds to the van der Waals radius for that atom.  Also, while
hydrogen bonds are predominantly electrostatic, it is worth noting that
there are some unresolved issues with the exact physical nature of these
interactions, particularly at short donor-acceptor distances.
Best regards,
Mark

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 





On 9/20/18, 10:13 AM, "CCP4 bulletin board on behalf of Daniel M. Himmel,
Ph. D."  wrote:

>Hello again.  I just want to add that hydrogen bonds by convention are
>usually
>considered to be a type of electrostatic interaction (please see the
>review by
>E. N. Baker in the International Tables of Crystallography Volume F,
>second edition, 
>p. 721) and are generally grouped with other "short range" electrostatic
>interactions
>along with dipole-dipole, dipole-ionic, and ionic-ionic interactions.
>This is by
>contrast to long term interactions that are approximated by the Lennard
>Jones potential, 
>which includes van der Waals forces.  My understanding is that van der
>Waals forces
>result from weak overall attractions between the protons in the nucleus
>of one atom
>and the electron cloud of another atom and therefore increase as the
>atomic number
>of the atoms increase.
>
>
>-Daniel
>
>
>
>
>On Thu, Sep 20, 2018 at 10:04 AM Daniel M. Himmel, Ph. D.
> wrote:
>
>
>Stefano is correct that hydrophobic interactions are chiefly entropically
>driven.  Thank you for your
>input, Stefano.  I disagree with Matthew, however.  It is true that van
>der Waals forces are always
>present and therefore form a small contribution to even hydrogen bonds.
>However, since the major contributions
>to hydrogen bonds are various types of electronic components, it is proper
>to group hydrogen bonds with electrostatic interactions.  When using
>computational
>software, one must look under the hood (or read the user manual) to see
>how different
>force components are being grouped for calculations.
>
>
>I would say to Sheila that, when you write up your analysis, just be sure
>to define how you are
>using terms such as "electrostatic" or else specifically list the
>individual types of interatomic
>attractive forces that you are surveying  (which I know you prefer to
>avoid).  
>
>
>If any computational chemists are following this discussion, perhaps you
>can pipe in and
>share your perspective and expertise.
>
>
>-Daniel
>
>
>
>
>On Wed, Sep 19, 2018 at 3:51 PM Sheila Boreiko
> wrote:
>
>
>Interesting discussion is coming out of this question. I thank all that
>have provided input.
>
>
>Let me go a bit further concerning Daniel's considerations. What
>other dipole interaction might be distinctively ascribed by programs out
>of hydrogen bonds (and of course, they use to describe salt bridges in
>addition, as an ionic interaction)? Possibly
> difficult for programs that use only atom positions, distances and
>angles (excluding the question of pH dependence, let us suppose neutral
>pH)?
>
>I might here be specific with program PISA, which lists Hydrogen
>Bonds and Salt Bridges. They seem to use these bonds to estimate a Gibbs
>energy for the formation of the interface.
>
>
>Sheila
>
>
>
>
>De: Daniel M. Himmel, Ph. D. 
>Enviado: terça-feira, 18 de setembro de 2018 15:10
>Para: sheila_bore...@hotmail.com
>Cc: CCP4BB@jiscmail.ac.uk
>Assunto: Re: [ccp4bb] collective term for hydrogen bonds and salt bridges
>
>Sorry.  I may have been unclear.  H-bonds are actually a subset of dipole
>interactions.
>
>
>On Tue, Sep 18, 2018 at 1:57 PM Daniel M. Himmel, Ph. D.
>
> wrote:
>
>
>By the way, distinguishing between dipole and ionic (salt bridge)
>interactions could
>be a slippery slope, because which one you have sometimes depends on the
>protonation 
>state of the protein(s), which is pH dependent.
>
>
>-Daniel
>
>
>
>
>On Tue, Sep 18, 2018 at 1:31 PM Daniel M. Himmel, Ph. D.
>
> wrote:
>
>
>
>
>
>
>Sheila,
>
>
>Hydrogen bonds, ionic (i.e. salt bridge), and polar (dipole) interactions
>are often collectively called
>electrostatic interactions.  Note that dipole interactions involve
>partial charges.  If you want to exclude
>dipole interactions, you have say so specifically in your manuscript.
>Non-bonded interactions include
>both electrostatic and van der Waals contacts (where hydrophobic
>interactions result from van der Waals
>forces in an aqueous environment).  Water can also interact

Re: [ccp4bb] [ccp4bb] protein precipitation reg

[Mark Wilson]

I heartily concur with Craig.  Tris can be a dangerous buffer for many
reasons, including those listed below.  In addition, as a primary amine,
it can complicate work with metalloproteins and has moderate
nucleophilicity.  There is almost always a better buffer choice than Tris.
Best regards,
Mark

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 






On 3/29/17 2:53 PM, "CCP4 bulletin board on behalf of CRAIG A BINGMAN"
 wrote:

>
>
>
>There are almost always better choices than Tris buffer.
>
>
>Mo Cleland used to call it “Trash” buffer.  He is no longer with us, but
>today I will happily carry that flag in his honor.
>
>
>Tris may show up in your crystal structure, especially at carbohydrate
>binding sites.
>Tris may be a surprisingly strong competitive inhibitor in your enzyme
>assays, especially as above.
>Tris has an absolutely miserably bad change in pKa vs. temperature.  It
>is larger than -0.03 pKa/dT(C).  It can be a catastrophically bad choice
>for flash-freezing protein aliquots.
>
>
>If you taken the time and incurred the expense of preparing a
>macromolecular sample for crystallization studies, and you are worried
>about the price difference between Tris and HEPES, in my opinion you are
>absolutely worried about the wrong things.
>
>
>Why are people substantially concerned about the buffering capacity of a
>buffer for final sample preparation?  You have a purified protein,
>presumably without substrate present.  Exactly what do you think is
>generating or absorbing hydrogen ions
> in that solution?  Oxidation of reducing agent should be about the only
>thing that is taxing the buffer.  From the example below, oxidation of 5
>mM BME will put some pressure on the buffer, but unfortunately Tris
>accelerates the oxidation of BME relative,
> to, say, HEPES. And surely you aren’t just letting the protein sit and
>oxidize in the refrigerator? Oh you might be since when you tried to snap
>freeze it in Tris, it turned into cooked egg white because the pH went to
>over 10 before it vitrified.
>(http://www.sciencedirect.com/science/article/pii/S0031942200801429)
>
>
>Isn’t the whole point to use a small amount of buffer so you can easily
>push the pH around in crystallization screens? (At which point the sample
>is usually in 100+ mM buffer.)
>
>
>On Mar 29, 2017, at 2:03 PM, Hughes, Jon
> wrote:
>
>...it's just a wonderful tradition! there's an interesting description of
>the history of tris in maniatis
>cheers
>jon
> 
>Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im
> Auftrag von David Briggs
>Gesendet: Mittwoch, 29. März 2017 17:53
>An: CCP4BB@JISCMAIL.AC.UK
>Betreff: Re: [ccp4bb] protein precipitation reg
> 
>It doesn't cost as much as HEPES, iirc.
>On Wed, 29 Mar 2017, 16:36 Keller, Jacob, 
>wrote:
>
>
>A bit off topic, but I’ve always wondered how TRIS got so popular what
>with it’s pKa of 8.3—does anyone know?
> 
>JPK
> 
>From: CCP4
> bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger
> Rowlett
>Sent: Wednesday, March 29, 2017 11:10 AM
>To: CCP4BB@JISCMAIL.AC.UK
>Subject: Re: [ccp4bb] protein precipitation reg
>
>
>
>
> 
>What are you dialyzing against? Your storage solution should typically be
>buffered away from the pI and contain at least a small amount of
>kosmotropic salt, e.g. NaCl. Some proteins will require additional
>stabilizing/solubilizing
> agents such as glycerol or reducing agents. FYI, Tris-Cl, pH 7.5 has
>very little buffer capacity (about 15% of the total concentration in the
>acid direction). We typically use Tris-Cl pH 8.0, which is closer to the
>Tris pKa and has good buffer capacity for
> both acid and base. For pH 7.5 we would typically use HEPES as the
>storage buffer.
>
>___
>Roger S. Rowlett
>Gordon & Dorothy Kline Professor
>Department of Chemistry
>Colgate University
>13 Oak Drive
>Hamilton, NY 13346
>
>tel: (315)-228-7245
>ofc: (315)-228-7395
>fax: (315)-228-7935
>email: rrowl...@colgate.edu
>On 3/29/2017 9:38 AM, Akilandeswari Gopalan wrote:
>
>
>Dear all,
>I am a PhD student doing structural studies on a few proteins from
>Mycobacterium tuberculosis. The gene encoding the proteins I work on are
>cloned into pet22b with c terminal His tag. the proteins are expressing
>well. upon purification
> I am getting good yield of protein but during dialysis, the proteins
>precipitate. Kindly suggest some solutions to avoid aggregation. pI of
>one protein is 9.7 and that of the other is 5.6
>
>I am using 25mM Tris pH 7.5 and 100 mM NaCl buffer with 5mM
>beta-mercaptoethanol and 0.5% triton x 100 for lysis, the same buffer
>with 20-30mM imidazole for washing and 300mM imidazole for eluting the
>proteins.
>
> 
>
>Thank you
>
>Regards
>
>Akila  
>
> 
>
>--
>Akilandeswari G
>
>
>
>
>
> 
>
>
>
>
>
>-- 
>
> 
>Fehler! Es wurde kein Dateiname angegeben.
>
>
> 
>David Briggs PhD

Re: [ccp4bb] clash crash

[Mark Wilson]

I second Bernhard’s frustration with this, and have dealt with this many
times.  I note that SHELX uses an occupancy-based clash criterion that
will not generate a clash for atoms whose occupancies sum to <1.1.  It
seems to me that this could be a more generally adopted criterion.
Furthermore, it would be nice to move away from strictly altloc-based
clash criteria for correlated disorder and always use occupancies for this
purpose, since occupancies report information in the data and altlocs are
essentially a notation issue.  I had mentioned this when comments were
solicited for the PDB validation task force, and perhaps more frustrated
voices will help advance the cause.
Best regards,
Mark

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 





On 2/1/16, 3:47 AM, "CCP4 bulletin board on behalf of Bernhard Rupp
(Hofkristallrat a.D.)"  wrote:

>Dear Developers/PDBe,
> 
>I just finished an otherwise unremarkable structure to the point of my
>personal level
>of tedium, which includes a few correlated partial group occupancies*).
>
>(a)   
>Upon clash checking via coot/probe, the only 5 significant red porcupines
>involve those
>
>partial occupancies, say a side chain at 0.56 and a close water at 0.44,
>the idea that the water is only there when the side chain is in its
>alternate position.
>
>(b)  
>running the coordinates through
>http://wwpdb-validation.wwpdb.org/validservice/
>lists exactly and only these 5 as worst ahem ‘errors’ **)
> 
>I think it should be reasonably easy to fix the program so that clashes
>between atoms
>
>with sum(occ) =< 1 are not reported as errors. This *IS* relevant,
>because remembering the
>
>‘lively discussion’ there was the opinion that the PDB reports should
>suffice for a reviewer to
>judge the quality of a structure model***). As these ‘errors’ also show
>up in the PDB report, I
>see no reason why a reviewer should not simply tell me to clean up that
>mess (which one really
>should – these spikey porcupines usually DO mean something is wrong &
>fixable).
>
> 
>(c)   
>looking at the next non-severe pinkish clashes, I notice that most
>involve methyl groups,
>and if I eyeball them they are precisely staggered @60 deg around the
>H3C-X torsion. But when 2 Leu
>for example are involved, I doubt that they face off at precisely that
>torsion angle, because
>energetically this is a periodic potential. I think each methyl group
>torsion would give a little
>
>bit, and voila, pink gone.
>In this case, I worry less about the pink, but how is this treated when
>riding Hs are added in
>
>refinement, for the purpose of improved VdW restraints?  Is this torsion
>potential used (as
>in MD) or to we assume the ‘hard’ 60 deg stagger or nothing conformation?
>Because if the
>torsion gives, also the C position might change slightly, ultimately
>resulting in a more realistic
>
>model. Has anyone played with that?
> 
>Thx, BR
> 
>*) which is best done at the very very end, because if you renumber
>residues or sort waters,
>the extra occupancy keyword file needs to be changed as well; a process
>whose tediousness
>
>is better not described. Indicative of errors in this file is a refmac
>infanticide message
>child killed: SIGABRT
> 
>**) or is there a more current version of the validation server already
>online?
> 
>***) Ceterum censeo coordinatae densitaeque providati eunt.
> 
>--
>--
>CVMO
>Vista, CA 92084
>001 (925) 209-7429
>b...@ruppweb.org
>b...@hofkristallamt.org
>http://www.ruppweb.org/
>---
> 
> 
>

Re: [ccp4bb] AW: [ccp4bb] Translational NCS with one molecule in ASU

[Mark Wilson]

Hi Eleanor,
I agree that experimental phases may be needed, although I may take the
lazy way out and just aggressively screen for a less problematic crystal
form.
Best regards,
Mark

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 






On 9/3/15 4:06 PM, "CCP4 bulletin board on behalf of Eleanor Dodson"
 wrote:

>How similar is this data set to the previously crystallised one?
>I think you said there is no translation vector in the previous one so
>they can't be very similar.
>Maybe it is time for experimental phases!
>Eleanor
>
>
>  
>
>
>On 3 September 2015 at 21:47, Adrian Goldman
> wrote:
>
>This would be my feeling too - one real 21, a twin axis and
>pseudosymmetry. The standard perfect storm.
>
>Sent from my iPhone
>
>> On 3 Sep 2015, at 21:07, Mark Wilson  wrote:
>>
>> Hi Eleanor,
>> Yes, of course you are correct about the beta~90° requirement for
>>possible
>> twinning here-I was mistakenly thinking about pseudomerohedry in higher
>> symmetry space groups. The L plot looks like well-behaved data, with a
>> straight line that closely tracks the model untwinned one.  Pointless,
>> provided with data integrated in P1, choses P212121 with high
>>confidence
>> (0.95-0-.99), which is similar to the results of analysis using xtriage
>>in
>> PHENIX.  Unsymmetrized cell angles are within 0.02-0.05° of 90°.
>>Finally,
>> the protein we crystallized is (to-be-tested wrong protein scenario
>>aside)
>> identical to the previously crystallized one, and our unit cell axes are
>> the same within 5% or so.
>> Best regards,
>> Mark
>>
>> Mark A. Wilson
>> Associate Professor
>> Department of Biochemistry/Redox Biology Center
>> University of Nebraska
>> N118 Beadle Center
>> 1901 Vine Street
>> Lincoln, NE 68588
>> (402) 472-3626
>> mwilso...@unl.edu
>>
>>
>>
>>
>>
>>
>>> On 9/3/15 2:45 PM, "Eleanor Dodson"  wrote:
>>>
>>> Disorder almost always produces streaked spots, but I guess it isn't
>>> compulsory!
>>>
>>> By the way, you can have twinning in Monoclinic if B ~ 90. without
>>>having
>>> a = c.
>>>
>>>
>>> . What does the L plot look like? Have you used pointless which gives
>>>the
>>> CC for each symmetry operator separately - sometimes that shows say the
>>> 00 l axis is more 2-fold ish than the 0k0 axis..
>>>
>>>
>>>
>>> Eleanor
>>> PS - If the related protein fits into a similar cell with the same SG
>>>is
>>> yours bigger?
>>>
>>>
>>>
>>>
>>> On 3 September 2015 at 18:56, Mark Wilson
>>>  wrote:
>>>
>>> Dear Remy,
>>> Indeed, I think you may be correct and we're pursuing this now.  A
>>>perfect
>>> 0.5 lattice translocation along b in P212121 would (I think) result in
>>>the
>>> pathology we observe.  We do not see zones of streaked reflections in
>>>the
>>> images, but my thinking is that if the lattice defect is coincident
>>>with a
>>> crystallographic translation operator, perhaps we wouldn't expect to.
>>> Best regards,
>>> Mark
>>>
>>> Mark A. Wilson
>>> Associate Professor
>>> Department of Biochemistry/Redox Biology Center
>>> University of Nebraska
>>> N118 Beadle Center
>>> 1901 Vine Street
>>> Lincoln, NE 68588
>>> (402) 472-3626  
>>> mwilso...@unl.edu
>>>
>>>
>>>
>>>
>>>
>>>
>>>> On 9/3/15 12:49 PM, "Remy Loris"  wrote:
>>>>
>>>> Dear Mark,
>>>>
>>>> My suspicion is that what you observe here is a lattice disorder,
>>>> possibly related to what is described in
>>>>
>>>> Jimin Wang, Satwik Kamtekar, Andrea J. Berman and Thomas A. Steitz
>>>> (2005) Correction of X-ray intensities from single crystals containing
>>>> lattice-translocation defects Acta Cryst D61,  67-74.
>>>>
>>>> If your unit cell is offset statistically by 0.5 in the b-direction,
>>>> this should provide such a strong non-origin peak as you observe. In
>>>>the
>>>> cases that have been described until now, this type of disorder also
>>>> involves zones of nice sharp reflections and o

Re: [ccp4bb] AW: [ccp4bb] Translational NCS with one molecule in ASU

[Mark Wilson]

Hi Eleanor,
Yes, of course you are correct about the beta~90° requirement for possible
twinning here-I was mistakenly thinking about pseudomerohedry in higher
symmetry space groups. The L plot looks like well-behaved data, with a
straight line that closely tracks the model untwinned one.  Pointless,
provided with data integrated in P1, choses P212121 with high  confidence
(0.95-0-.99), which is similar to the results of analysis using xtriage in
PHENIX.  Unsymmetrized cell angles are within 0.02-0.05° of 90°.  Finally,
the protein we crystallized is (to-be-tested wrong protein scenario aside)
identical to the previously crystallized one, and our unit cell axes are
the same within 5% or so.
Best regards,
Mark

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 






On 9/3/15 2:45 PM, "Eleanor Dodson"  wrote:

>Disorder almost always produces streaked spots, but I guess it isn't
>compulsory!
>
>By the way, you can have twinning in Monoclinic if B ~ 90. without having
>a = c.
>
>
>. What does the L plot look like? Have you used pointless which gives the
>CC for each symmetry operator separately - sometimes that shows say the
>00 l axis is more 2-fold ish than the 0k0 axis..
>
>
>
>Eleanor
>PS - If the related protein fits into a similar cell with the same SG is
>yours bigger? 
>
>
>
>
>On 3 September 2015 at 18:56, Mark Wilson
> wrote:
>
>Dear Remy,
>Indeed, I think you may be correct and we're pursuing this now.  A perfect
>0.5 lattice translocation along b in P212121 would (I think) result in the
>pathology we observe.  We do not see zones of streaked reflections in the
>images, but my thinking is that if the lattice defect is coincident with a
>crystallographic translation operator, perhaps we wouldn't expect to.
>Best regards,
>Mark
>
>Mark A. Wilson
>Associate Professor
>Department of Biochemistry/Redox Biology Center
>University of Nebraska
>N118 Beadle Center
>1901 Vine Street
>Lincoln, NE 68588
>(402) 472-3626 
>mwilso...@unl.edu
>
>
>
>
>
>
>On 9/3/15 12:49 PM, "Remy Loris"  wrote:
>
>>Dear Mark,
>>
>>My suspicion is that what you observe here is a lattice disorder,
>>possibly related to what is described in
>>
>>Jimin Wang, Satwik Kamtekar, Andrea J. Berman and Thomas A. Steitz
>>(2005) Correction of X-ray intensities from single crystals containing
>>lattice-translocation defects Acta Cryst D61,  67-74.
>>
>>If your unit cell is offset statistically by 0.5 in the b-direction,
>>this should provide such a strong non-origin peak as you observe. In the
>>cases that have been described until now, this type of disorder also
>>involves zones of nice sharp reflections and other zones with more
>>streaky reflections. Do you see something similar?
>>In order to use such data, they have to be corrected as described in the
>>paper above.
>>
>>Possibly, the crystals with the 10% non-origin peak also have the
>>disorder, but much less pronounced so that omitting the required
>>correction did not prevent structure determination and refinement
>>(similar to let say a small fraction of merohedral twinning that is
>>overlooked).
>>
>>Remy Loris
>>Vrije Universiteit Brussel and VIB
>>
>>DOI: 10.1107/S0907444904026721
>>
>>On 03/09/15 18:13, George Sheldrick wrote:
>>> Dear Mark,
>>>
>>> Since your resolution is good enough, perhaps you should try to solve
>>> it ab initio with Arcimboldo Lite. This has already solved a number of
>>> structures that turned out to be unexpected.
>>>
>>> Best wishes, George
>>>
>>>
>>> On 09/03/2015 05:44 PM, Mark Wilson wrote:
>>>> Hi Herman,
>>>> A fair point-the odds of "depressing coincidence" do seem to be
>>>> climbing!
>>>> We did inspect the deposited data for a similar peak and, while one is
>>>> present, it is only ~10% of the origin and at a different location.
>>>> We'll
>>>> do some due diligence on our end by re-dissolving crystals and
>>>> performing
>>>> mass spec.  As there seems to be some interest in this, I'll update
>>>>once
>>>> we've figured it out, even if it's an embarrassing case of wrong
>>>> protein,
>>>> same cell.
>>>> Best regards,
>>>> Mark
>>>>
>>>> Mark A. Wilson
>>>> Associate Professor
>>>> Department of Biochemistry

Re: [ccp4bb] AW: [ccp4bb] Translational NCS with one molecule in ASU

[Mark Wilson]

Dear Remy,
Indeed, I think you may be correct and we're pursuing this now.  A perfect
0.5 lattice translocation along b in P212121 would (I think) result in the
pathology we observe.  We do not see zones of streaked reflections in the
images, but my thinking is that if the lattice defect is coincident with a
crystallographic translation operator, perhaps we wouldn't expect to.
Best regards,
Mark

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 






On 9/3/15 12:49 PM, "Remy Loris"  wrote:

>Dear Mark,
>
>My suspicion is that what you observe here is a lattice disorder,
>possibly related to what is described in
>
>Jimin Wang, Satwik Kamtekar, Andrea J. Berman and Thomas A. Steitz
>(2005) Correction of X-ray intensities from single crystals containing
>lattice-translocation defects Acta Cryst D61,  67-74.
>
>If your unit cell is offset statistically by 0.5 in the b-direction,
>this should provide such a strong non-origin peak as you observe. In the
>cases that have been described until now, this type of disorder also
>involves zones of nice sharp reflections and other zones with more
>streaky reflections. Do you see something similar?
>In order to use such data, they have to be corrected as described in the
>paper above.
>
>Possibly, the crystals with the 10% non-origin peak also have the
>disorder, but much less pronounced so that omitting the required
>correction did not prevent structure determination and refinement
>(similar to let say a small fraction of merohedral twinning that is
>overlooked).
>
>Remy Loris
>Vrije Universiteit Brussel and VIB
>
>DOI: 10.1107/S0907444904026721
>
>On 03/09/15 18:13, George Sheldrick wrote:
>> Dear Mark,
>>
>> Since your resolution is good enough, perhaps you should try to solve
>> it ab initio with Arcimboldo Lite. This has already solved a number of
>> structures that turned out to be unexpected.
>>
>> Best wishes, George
>>
>>
>> On 09/03/2015 05:44 PM, Mark Wilson wrote:
>>> Hi Herman,
>>> A fair point-the odds of "depressing coincidence" do seem to be
>>> climbing!
>>> We did inspect the deposited data for a similar peak and, while one is
>>> present, it is only ~10% of the origin and at a different location.
>>> We'll
>>> do some due diligence on our end by re-dissolving crystals and
>>> performing
>>> mass spec.  As there seems to be some interest in this, I'll update
>>>once
>>> we've figured it out, even if it's an embarrassing case of wrong
>>> protein,
>>> same cell.
>>> Best regards,
>>> Mark
>>>
>>> Mark A. Wilson
>>> Associate Professor
>>> Department of Biochemistry/Redox Biology Center
>>> University of Nebraska
>>> N118 Beadle Center
>>> 1901 Vine Street
>>> Lincoln, NE 68588
>>> (402) 472-3626
>>> mwilso...@unl.edu
>>>
>>>
>>>
>>>
>>>
>>>
>>> On 9/3/15 10:25 AM, "CCP4 bulletin board on behalf of
>>> herman.schreu...@sanofi.com">> herman.schreu...@sanofi.com>  wrote:
>>>
>>>> Dear Mark,
>>>>
>>>> In this case you will have to apply Baysian statistics: given the
>>>> prior:
>>>> same protein, same space group same cell dimensions and molecular
>>>> replacement fails completely, the likelihood of having some depressing
>>>> coincidence somewhere is approaches 100%!
>>>>
>>>> What I would do in addition to excellent suggestions you already
>>>> got, is
>>>> to try to download the Fobs from the pdb for the structures with the
>>>> same
>>>> protein, space group and cell dimensions, and calculate pattersons
>>>>with
>>>> those. Sometimes strong peaks appear in pattersons for no obvious
>>>> reasons.
>>>> I would also consider statistical disorder, which will not show up in
>>>> twinning statistics since in this case F's (including phases) are
>>>>added
>>>> instead of I's. Anyways, it will be an interesting puzzle to solve!
>>>>
>>>> Good luck,
>>>> Herman
>>>>
>>>>
>>>> -Ursprüngliche Nachricht-
>>>> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von
>>>> Mark Wilson
>>>> Gesendet: Donnerstag, 3. September 2015 02:06
>

Re: [ccp4bb] AW: [ccp4bb] Translational NCS with one molecule in ASU

[Mark Wilson]

Hi Herman,
A fair point-the odds of "depressing coincidence" do seem to be climbing!
We did inspect the deposited data for a similar peak and, while one is
present, it is only ~10% of the origin and at a different location.  We'll
do some due diligence on our end by re-dissolving crystals and performing
mass spec.  As there seems to be some interest in this, I'll update once
we've figured it out, even if it's an embarrassing case of wrong protein,
same cell.
Best regards,
Mark

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 






On 9/3/15 10:25 AM, "CCP4 bulletin board on behalf of
herman.schreu...@sanofi.com"  wrote:

>Dear Mark,
>
>In this case you will have to apply Baysian statistics: given the prior:
>same protein, same space group same cell dimensions and molecular
>replacement fails completely, the likelihood of having some depressing
>coincidence somewhere is approaches 100%!
>
>What I would do in addition to excellent suggestions you already got, is
>to try to download the Fobs from the pdb for the structures with the same
>protein, space group and cell dimensions, and calculate pattersons with
>those. Sometimes strong peaks appear in pattersons for no obvious reasons.
>I would also consider statistical disorder, which will not show up in
>twinning statistics since in this case F's (including phases) are added
>instead of I's. Anyways, it will be an interesting puzzle to solve!
>
>Good luck,
>Herman
>
>
>-----Ursprüngliche Nachricht-
>Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von
>Mark Wilson
>Gesendet: Donnerstag, 3. September 2015 02:06
>An: CCP4BB@JISCMAIL.AC.UK
>Betreff: Re: [ccp4bb] Translational NCS with one molecule in ASU
>
>Dear CCP4 Community,
>I've had a number of helpful responses (on- and off-list) that I will
>briefly summarize via response, including information that I probably
>should have included in the original post.  Many have suggested a wrong
>space group, which I agree seems probable.  MR was attempted in PHASER
>with all possible choices of space group for a primitive orthorhombic
>lattice, and in all cases failed with no rotation or translation peaks
>above a Z-score of 5.
>   I've not yet tried monoclinic lattices and will, but this still wouldn't
>explain (to me anyway) an apparently impossible combination of
>translational NCS in P212121 with a cell that can't accommodate a second
>molecule unless twinning was also present, which may be the case (as
>Eleanor suggested).  Others have asked about evidence of missed weak
>reflections indicating a larger true cell, which I looked for but didn't
>see in these images.  The crystal that was used was mounted at room
>temperature, so there is no opportunity for cryo artifacts to have done
>something strange to the cell.
>   Other suggestions included the presence of strong internal symmetry in
>the molecule, which is present, but as a pseudo-threefold, which seems
>incompatible with my NCS centering operation.  One respondent suggested
>that we've crystallized the wrong molecule, which is something I also
>worried about a bit.  Although possible, the space group and cell for our
>crystal are both as previously reported for this protein by another
>group, so it would be a depressing coincidence if we crystallized the
>wrong protein in the same cell. I'll be happy to update if/when we figure
>this out should it be of interest to the board. Thank you all for your
>thoughtful responses, which arrived in impressive number in the time it
>took me to drive home.
>Best regards,
>Mark
>
>Mark A. Wilson
>Associate Professor
>Department of Biochemistry/Redox Biology Center University of Nebraska
>N118 Beadle Center
>1901 Vine Street
>Lincoln, NE 68588
>(402) 472-3626
>mwilso...@unl.edu 
>
>
>
>
>
>On 9/2/15 5:30 PM, "CCP4 bulletin board on behalf of Eleanor Dodson"
> wrote:
>
>>Well -  a translation of 0 0.5 0 would generate absences along b so
>>that the SG could be P212121 or P 21 2 21Š
>>
>>
>>I would suspect twinning and a monoclinic SG .
>>Or as we found sadly - half the protein had disappeared in the
>>crystallisation trials..
>>
>>
>>But such a translation must mean you almost have a halved unit cell?
>>Another way of saying there isn't enough room for your molecule..
>>
>>
>>
>>On 2 September 2015 at 22:38, Shane Caldwell
>> wrote:
>>
>>Are you certain it's actually P212121? One possibility is you're at
>>lower sym

Re: [ccp4bb] Translational NCS with one molecule in ASU

[Mark Wilson]

Hi All,
An important point is that the cell dimensions are: 54.98 58.45 66.89 90
90 90.  While a and b are similar, they are (in my opinion) not similar
enough to support pseudo-merohedral twinning.  The absences are indeed
absent, with I/sigma(I) between ~ -1 and 2.  All three axes have similarly
convincing absences in P212121, included below.
Best regards,
Mark
==

 Intensities of systematic absences
  h   k   l  Intensity Sigma   I/Sigma

  0   0   5   1.8   2.5   0.7
  0   0   7  -4.1   3.5  -1.2
  0   0   9   9.9   4.6   2.2
  0   0  11  -1.1   6.5  -0.2
  0   0  13  12.9  10.0   1.3
  0   0  15  -7.2   9.9  -0.7
  0   0  17 -11.2   8.8  -1.3
  0   0  19   1.1  10.0   0.1
  0   0  21  -1.4   9.3  -0.2
  0   0  23   0.8   9.3   0.1
  0   0  25   1.5   8.1   0.2
  0   0  27   7.4   9.3   0.8
  0   3   0  -1.6   4.5  -0.4
  0   5   0  -4.7   5.5  -0.9
  0   7   0 -18.1  12.2  -1.5
  0   9   0  13.0  13.6   1.0
  0  11   0  19.3  18.6   1.0
  0  13   0  51.6  29.8   1.7
  0  15   0  38.6  24.4   1.6
  0  17   0  -9.5  30.2  -0.3
  0  19   0 -42.8  36.1  -1.2
  0  21   0 -12.5  22.4  -0.6
  0  23   0  -3.2  24.9  -0.1
  0  25   0  54.8  38.7   1.4
  0  27   0 -28.2  36.2  -0.8
  0  29   0  -0.1  30.6   0.0
  0  31   0 -25.7  39.8  -0.6
  0  33   0 -11.6  38.1  -0.3
  3   0   0   1.6   3.3   0.5
  5   0   0   9.2   5.2   1.8
  7   0   0  -0.4   9.6   0.0
  9   0   0  -2.7  11.2  -0.2
 11   0   0  65.1  21.1   3.1
 13   0   0  19.8  19.0   1.0
 15   0   0 -24.5  20.9  -1.2
 17   0   0  29.9  31.8   0.9
 19   0   0  -3.3  24.3  -0.1
 21   0   0 -33.4  33.0  -1.0
 23   0   0  -4.8  29.4  -0.2
 25   0   0  -6.7  33.3  -0.2
 27   0   0  55.3  37.6   1.5
 29   0   0  11.0  38.6   0.3
 31   0   0 -36.4  46.6  -0.8




Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 






On 9/3/15 1:51 AM, "CCP4 bulletin board on behalf of Adrian Goldman"
 wrote:

>I agree. This was essentially Eleanor's viewpoint too.  So p21
>merohedrally twinned to orthorhombic with the 0.47 translational ncs.. I
>would look at the h00, k00, l00 odd peaks (the ones systematically
>disallowed) to look for evidence of some intensity
> along one of the axes in the disallowed spots.  That will help establish
>which axis has the true 21 screw. My guess is that at high Bragg angle
>there is some intensity in the k odd spots at least.
>
>
>Adrian
>
>Sent from my iPad
>
>On 3 Sep 2015, at 6:40 am, Sudipta Bhattacharyya
> wrote:
>
>
>
>Hi Mark,
>
>
>One strategy that worked for me is to reprocess/expand the data to P1 and
>try to do MR in that SG, do initial one cycle of rigid body and
>restrained refinements and then you can feed the P1 data and the model to
>zanuda to get correct assessment of SG.
> Then you have to reprocess the data accordingly. Anyway, twining tests
>are most of the time obscured when tNCS is present. So, I think, the
>presence of twining can't be simply overruled in this case. reprocessing
>the data in monoclinic SG, followed by doing
> MR could be the key to this problem.
>
>
>Good luck..!!!
>Sudipta  
>
>
>On Wed, Sep 2, 2015 at 6:05 PM, Mark Wilson
> wrote:
>
>Dear CCP4 Community,
>I've had a number of helpful responses (on- and off-list) that I will
>briefly summarize via response, including information that I probably
>should have included in the original post.  Many have suggested a wrong
>space group, which I agree seems probable.  MR was attempted in PHASER
>with all possible choices of space group for a primitive orthorhombic
>lattice, and in all cases failed with no rotation or translation peaks
>above a Z-score of 5.
>I've not yet tried monoclinic lattices and will, but this still
>wouldn't
>explain (to me anyway) an apparently impossible combination of
>translational NCS in P212121 with a cell that can't accommodate a second
>molecule unless twinning was also present, which may be the case (as
>Eleanor suggested).  Others have asked about evidence of missed weak
>reflect

[ccp4bb] Segmentation fault in Pirate cmakereference

[Mark Wilson]

Dear CCP4 community,
I am running the "make Pirate reference file" utility through CCP4i using
downloaded coordinates and data.  It consistently fails with:

#CCP4I TERMINATION STATUS 0 "child killed: segmentation violation"
#CCP4I TERMINATION TIME 26 May 2015  17:41:21
#CCP4I MESSAGE Task failed

No other information about the cause of the segmentation fault is
provided.  Any insight would be appreciated.
Best regards,
Mark



Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 

Re: [ccp4bb] Strategies to bring out over-expressed protein from inclusion bodies to soluble fraction!!!

[Mark Wilson]

Hi Gloria,
Yes, chloramphenicol-resistant cells are presumably a problem for this
method, so other ribosome-targeting antibiotics (such as tetracycline, as
you mention) may work in those cases. We've not tried this, but it would
be interesting to see if it works.  It also would serve to test the
mechanism a bit, as similar behavior would indicate that halting protein
translation is the critical aspect.  I will add that, in my experience,
this method works best when there is some small amount of soluble material
detectable on SDS-PAGE.  For totally insoluble material, we've not much
success, but we also have not explored all of the options discussed in the
FEBS Letters reference.
Best regards,
Mark

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 





On 10/17/14 10:05 AM, "Gloria Borgstahl"  wrote:

>Thanks Mark, this is a good tip
>what would you use for Rosetta cells though?
>Tetracyclin?
>
>
>On Fri, Oct 17, 2014 at 8:31 AM, Mark Wilson
> wrote:
>
>Hi Ivan,
>We've had good luck with the addition of chloramphenicol ~1 hour prior to
>harvest, as described in:
>Carrió, M. M., and Villaverde, A. (2001) Protein aggregation as bacterial
>inclusion bodies is reversible. FEBS Lett. 489, 29­33.
>We usually combine this with lower temperature growth (20 C).
>Best regards,
>Mark
>
>Mark A. Wilson
>Associate Professor
>Department of Biochemistry/Redox Biology Center
>University of Nebraska
>N118 Beadle Center
>1901 Vine Street
>Lincoln, NE 68588
>(402) 472-3626
>mwilso...@unl.edu
>
>
>
>
>
>On 10/17/14 7:51 AM, "xaravich ivan"  wrote:
>
>>Dear cc4bb enthusiasts,
>>
>>This is slightly off topic but many protein crystallographers might be
>>familiar with this problem.
>>
>>
>>I have been trying to over-express a bacterial (non-E.Coli) protein  in
>>E.Coli and more than 80% goest to inclusion bodies.
>>
>>
>>
>>I tried the following
>>
>>
>>Lowering the IPTG concentration to 0.1mM (range test from 0.075 mM - 0.5
>>mM)
>>
>>
>>Cold shock for 30 minutes in ice before induction
>>
>>
>>Slow rotation speed at 18 degrees O/N after induction
>>
>>
>>While these steps helped a bit I still get about 50-60% of my protein in
>>inclusion bodies.
>>
>>
>>I would like to know what other steps do you suggest to enhance the yield
>>in the soluble fraction (without changing the host strain or manipulating
>>the DNA)
>>
>>
>>Thanks in advance
>>
>>Ivan
>>
>>
>
>
>
>
>
>
>

Re: [ccp4bb] Strategies to bring out over-expressed protein from inclusion bodies to soluble fraction!!!

[Mark Wilson]

Hi Ivan,
We've had good luck with the addition of chloramphenicol ~1 hour prior to
harvest, as described in:
Carrió, M. M., and Villaverde, A. (2001) Protein aggregation as bacterial
inclusion bodies is reversible. FEBS Lett. 489, 29­33.
We usually combine this with lower temperature growth (20 C).
Best regards,
Mark

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 





On 10/17/14 7:51 AM, "xaravich ivan"  wrote:

>Dear cc4bb enthusiasts,
>
>This is slightly off topic but many protein crystallographers might be
>familiar with this problem.
>
>
>I have been trying to over-express a bacterial (non-E.Coli) protein  in
>E.Coli and more than 80% goest to inclusion bodies.
>
>
>
>I tried the following
>
>
>Lowering the IPTG concentration to 0.1mM (range test from 0.075 mM - 0.5
>mM)
>
>
>Cold shock for 30 minutes in ice before induction
>
>
>Slow rotation speed at 18 degrees O/N after induction
>
>
>While these steps helped a bit I still get about 50-60% of my protein in
>inclusion bodies.
>
>
>I would like to know what other steps do you suggest to enhance the yield
>in the soluble fraction (without changing the host strain or manipulating
>the DNA)
>
>
>Thanks in advance
>
>Ivan
>
>

Re: [ccp4bb] correlated alternate confs - validation?

[Mark Wilson]

As an additional comment on the issue of spatially correlated disorder,
SHELX (and possibly other programs) has an elegant way of handling bump
generation between disordered atoms.  SHELX will only generate a bump if
two atoms are in van der Waals conflict AND their occupancies sum to 1.1
or greater.  This is on top of an exclusion for atoms from different
altloc (non-zero part) numbers, and has the attraction of enforcing a
physically reasonable model in any event.  This approach requires no
information about the correlated group membership of the atoms, although
as George Sheldrick pointed out, this can be handled using free variables
in SHELX.  I bring this up because several current validation programs do
not take correlated disorder into account when generating clashes, and I
wish that an occupancy check would be more widely implemented.
Best regards,
Mark

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 





On 7/25/14 2:51 AM, "Thomas Lütteke"
 wrote:

>Just a quick thought that might be a workaround solution within the PDB
>format restrictions: In NMR structures there are often multiple
>conformations stored in different MODEL sections - this should in
>principle be possible for x-ray structures as well, shouldn't it?
>
>Of course this is a waste of space, as you have to duplicate all atoms
>and not only the ones that are included in multiple conformations, and
>not all features might be handled by validation tools, but with the
>limitations of the current PDB format there are not many ways to encode
>this anyway.
>
>Best regards,
>Thomas

Re: [ccp4bb] PDB passes 100,000 structure milestone

[Mark Wilson]

Hi Nat,
I agree that journals should be doing the heavy lifting here, for the
reasons that you note. I also want to be clear that I believe the PDB is a
crowning achievement of transparency and open access in the sciences,
which is one reason that I am so concerned about this issue.  I am in no
way trying to impugn the hard and superb work that they have done over
many decades.  I still contend, however, that having models whose
integrity is highly suspect lurking in the PDB with no indications of
problems beyond a dodgy validation report is a non-optimal outcome.  As
for the meaning of integrity, I'm using this word in place of others that
might be considered more legally actionable.  A franker conversation would
likely more clearly draw the line that we're wrestling with here.
Best regards,
Mark
 
Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 






On 5/14/14 12:41 PM, "Nat Echols"  wrote:

>On Wed, May 14, 2014 at 10:26 AM, Mark Wilson  wrote:
>
>Getting to Eric's point about an impasse, if the PDB will not claim the
>authority to safeguard the integrity of their holdings (as per their
>quoted statement in Bernhard's message below), then who can?
>
>
>
>I think this may in part boil down to a semantic dispute over the meaning
>of "integrity".  I interpreted it to mean "integrity (and public
>availability) of the data as deposited by the authors", which by itself
>is quite a lot of work.  Safeguarding
> the integrity of the peer-review process is supposed to be the job of
>the journals, some of which - unlike the PDB - are making a tidy profit
>from our efforts.  Since they justify this profit based on the value they
>supposedly add as gatekeepers, I don't think
> it's unreasonable for us to expect them to do their job, rather than
>leave it to the PDB annotators, who surely have enough to deal with.
>
>
>I do share some of the concern about 2hr0, but I am curious where the
>line should be drawn.  This is an extraordinary case where the
>researcher's institution requested retraction, but I think everyone who's
>been in this field for a while has
> a list of dodgy structures that they think should be retracted - not
>always with justification.
>
>
>-Nat
>
>
>
>

Re: [ccp4bb] PDB passes 100,000 structure milestone

[Mark Wilson]

Hi Tim,
Getting to Eric's point about an impasse, if the PDB will not claim the
authority to safeguard the integrity of their holdings (as per their
quoted statement in Bernhard's message below), then who can?  I understand
that there are many potential complications to the PDB claiming some
plenary authority to prune out structures that they don't like for
whatever reason and agree that they should not claim such authority.
Furthermore, I sympathize with the difficult situation that the curators
must confront in the (hopefully) very rare cases of models whose integrity
is suspect.  However, dealing with these in some manner surely falls
squarely within a mission "to safeguard the integrity and improve the
quality of the PDB archive." Strict neutrality on the part of the PDB in
these cases is not working well in my opinion, as evidenced by the absence
of any indication of the dark history of 2HR0 on its PDB page.  There are
many possible ways of indicating something is seriously amiss with these
entries, and I wish that the community wasn't in the position of having
PDB entries that some users know are deeply suspect but that other, less
informed users do not.
Best regards,
Mark

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 






On 5/14/14 12:06 PM, "Tim Gruene"  wrote:

>Hi Mark,
>
>I understand the discussion, yet as far as I understand the PDB does not
>claim to be the authority to decide about the integrity of an entry (or
>maybe better said, the PDB claims not to be this authority), and I find
>it very honorable that the PDB have not abused their power. I don't mean
>such an authority should not exist, but I think it is a good think it is
>not the PDB. It is a form of separation of powers.
>
>Best,
>Tim
>
>On 05/14/2014 06:47 PM, Mark Wilson wrote:
>> Hi Tim,
>> I agree with everything you've said about the importance of validation,
>> but aren't we really talking about something different here?  Users of
>> structural information should of course be keeping a careful eye on
>> validation reports. On the other hand, what possible reason is there for
>> the PDB to continue to archive and offer for public use models whose
>> fundamental integrity (rather than quality or reliability) are highly
>> suspect?  I hope that I'm not the only one who is frustrated that the
>>page
>> for 2HR0 is still available and unblemished by warnings.
>> Best regards,
>> Mark
>> 
>> Mark A. Wilson
>> Associate Professor
>> Department of Biochemistry/Redox Biology Center
>> University of Nebraska
>> N118 Beadle Center
>> 1901 Vine Street
>> Lincoln, NE 68588
>> (402) 472-3626
>> mwilso...@unl.edu
>> 
>> 
>> 
>> 
>> 
>> 
>> On 5/14/14 11:35 AM, "Tim Gruene"  wrote:
>> 
>>> Dear Eric,
>>>
>>> On 05/14/2014 06:05 PM, Eric Williams wrote:
>>>> [...]
>>>> We seem to be at an impasse. The PDB won't evict highly suspect
>>>> structure
>>>> models unless journals retract them, and the journals in question have
>>>> shown no indication of desiring to retract them. Is there anything
>>>>that
>>>> can
>>>> be done? [...]
>>>>
>>>> What's the appropriate course of action for conscientious consumers of
>>>> PDB
>>>> data? Is there a way to petition journals to issue retractions? I
>>>>wonder
>>>> what the gents at Retraction Watch (http://retractionwatch.com) would
>>>> recommend.
>>>>
>>>> Eric
>>>>
>>>
>>> you can teach the consumers how to help themselves - you are welcome to
>>> join my session MS-84 at the IUCr 2014 :-) because I believe that one
>>>of
>>> the New Paradigms in Crystallography is the requirement to how to
>>> correctly interpret crystallographic models, and validation is becoming
>>> more and more important as subject.
>>>
>>> Best,
>>> Tim
>>>
>>>>
>>>> On Wed, May 14, 2014 at 10:04 AM, Bernhard Rupp
>>>>
>>>> 
>>>>https://mail.google.com/mail/?view=cm&fs=1&tf
>>>>=1
>>>> &to=hofkristall...@gmail.com>
>>>>> wrote:
>>>>
>>>>>> which structure ended up as number 100.000?
>>>>> I guess that depends if we still count the Murthy corpses like 2a01
>>>>> This
>>>&g

Re: [ccp4bb] PDB passes 100,000 structure milestone

[Mark Wilson]

Hi Tim,
I agree with everything you've said about the importance of validation,
but aren't we really talking about something different here?  Users of
structural information should of course be keeping a careful eye on
validation reports. On the other hand, what possible reason is there for
the PDB to continue to archive and offer for public use models whose
fundamental integrity (rather than quality or reliability) are highly
suspect?  I hope that I'm not the only one who is frustrated that the page
for 2HR0 is still available and unblemished by warnings.
Best regards,
Mark

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 






On 5/14/14 11:35 AM, "Tim Gruene"  wrote:

>Dear Eric,
>
>On 05/14/2014 06:05 PM, Eric Williams wrote:
>> [...]
>> We seem to be at an impasse. The PDB won't evict highly suspect
>>structure
>> models unless journals retract them, and the journals in question have
>> shown no indication of desiring to retract them. Is there anything that
>>can
>> be done? [...]
>> 
>> What's the appropriate course of action for conscientious consumers of
>>PDB
>> data? Is there a way to petition journals to issue retractions? I wonder
>> what the gents at Retraction Watch (http://retractionwatch.com) would
>> recommend.
>> 
>> Eric
>> 
>
>you can teach the consumers how to help themselves - you are welcome to
>join my session MS-84 at the IUCr 2014 :-) because I believe that one of
>the New Paradigms in Crystallography is the requirement to how to
>correctly interpret crystallographic models, and validation is becoming
>more and more important as subject.
>
>Best,
>Tim
>
>> 
>> On Wed, May 14, 2014 at 10:04 AM, Bernhard Rupp
>> 
>>https://mail.google.com/mail/?view=cm&fs=1&tf=1
>>&to=hofkristall...@gmail.com>
>>> wrote:
>> 
 which structure ended up as number 100.000?
>>> I guess that depends if we still count the Murthy corpses like 2a01
>>>This
>>> 3-armed Swastika for example still does not come with a single warning
>>> short of a poor quality report
>>> http://www.ebi.ac.uk/pdbe-srv/view/entry/2a01/summary_details.html So,
>>> sorry, 0 (or lessŠ.) valid entries only at the time of
>>>announcement.
>>>
>>> Cheers, BR
>>>
>>>
>>>
>>> Supplemental material:
>>>
>>>
>>>
>>> ³The PDB says it will remove the other ten structures only when
>>>editors at
>>> the journals in which they were originally published or the authors
>>> themselves retract them²
>>>
>>> *http://www.nature.com/news/2009/091222/full/462970a.html
>>> *
>>>
>>>
>>>
>>>
>>>
>>> ³With the support of the structural-biology community, the mission of
>>>the
>>> wwPDB is to safeguard the integrity and improve the quality of the PDB
>>> archive.²
>>>
>>> http://www.nature.com/nature/journal/v463/n7280/full/463425c.html
>>>
>>>
>>>
>>> Not to be overly cynical, but
>>>
>>>
>>>
>>> http://tinyurl.com/pmupalt
>>>
>>>
>>>
>>>
>>>
>>> *From:* CCP4 bulletin board
>>>[mailto:CCP4BB@JISCMAIL.AC.UK>>&tf=1&to=CCP4BB@JISCMAIL.AC.UK>]
>>> *On Behalf Of *mesters
>>> *Sent:* Mittwoch, 14. Mai 2014 14:42
>>> *To:* 
>>>CCP4BB@JISCMAIL.AC.UK>>=CCP4BB@JISCMAIL.AC.UK>
>>>
>>> *Subject:* Re: [ccp4bb] PDB passes 100,000 structure milestone
>>>
>>>
>>>
>>> Amazing, great!
>>>
>>> And, which structure ended up as number 100.000?
>>>
>>> - J. -
>>>
>>>
>>> Am 14.05.14 10:42, schrieb battle:
>>>
>>> The Worldwide Protein Data Bank (wwPDB) organization is proud to
>>>announce
>>> that the Protein Data Bank archive now contains more than 100,000
>>>entries.
>>>
>>> Established in 1971, this central, public archive of
>>> experimentally-determined protein and nucleic acid structures has
>>>reached a
>>> critical milestone thanks to the efforts of structural biologists
>>> throughout the world.
>>>
>>> Read the full story at:
>>> http://www.wwpdb.org/news/news_2014.html#13-May-2014
>>>
>>> --
>>> Gary Battle
>>> on behalf on the wwPDB
>>>
>>>
>>>
>>> --
>>> Dr. Jeroen R. Mesters
>>> Deputy, Senior Researcher & Lecturer
>>>
>>> Institute of Biochemistry, University of Lübeck
>>> Ratzeburger Allee 160, 23538 Lübeck, Germany
>>>
>>> phone: +49-451-5004065 (secretariate 5004061)
>>> fax: +49-451-5004068
>>>
>>> http://www.biochem.uni-luebeck.de 
>>> http://www.iobcr.org 
>>>
>>>
>>> --
>>> If you can look into the seeds of time and tell which grain will grow
>>>and
>>> which will not, speak then to me who neither beg nor fear
>>>(Shakespeare's
>>> Macbeth, Act I, Scene 3)
>>> --
>>>
>>>
>>>
>>>
>>> *Disclaimer * This message contains confidential information and is
>>> intended only for the individual named. If you are not the named
>>>addressee
>>> you should not disseminate, distribute or copy this e-mail. Please
>>>notify
>>> the sender immediately by e-mail if y

Re: [ccp4bb] Powder Rings in Single Crystals

[Mark Wilson]

Perhaps I misunderstood Jacob's original question, but it seems like two 
different phenomena are being discussed here.  My read of Jacob's original 
question was, roughly, shouldn't we observe non-Bragg, powder-like 
scattering from a well-ordered macromolecular crystal due to the abundance 
of ~ 1 Å interatomic distances?  In my opinion, the answer is "no" unless 
translational periodicity is violated.  When translational periodicity is 
violated (as it is to some extent in all real crystals), then the 
non-Bragg (diffuse) scatter can contain many features, as it is the 
Fourier transform of the variance-covariance function for all disorder in 
the unit cell.  If I misunderstood Jacob's original question, my 
apologies.
Best regards,
Mark

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu



Jacob Keller  
Sent by: CCP4 bulletin board 
05/09/2012 12:22 PM
Please respond to
Jacob Keller 


To
CCP4BB@JISCMAIL.AC.UK
cc

Subject
Re: [ccp4bb] Powder Rings in Single Crystals







Yes, I just looked up the paper--seems right on topic--a powder-type ring 
at ~4.2 Ang, corresponding to Calpha-Calpha distances! But no 1.2-1.5 Ang 
ring, from what I saw. Maybe it gets swamped out by other things. I am 
thinking that the variety/distribution of bonds/distances of length 1-3 
Ang in the crystal/mother liquor combo is so high/broad that you can't see 
them anymore. I wonder whether when people soak in various heavy atom 
clusters, they see powder rings for the HA-HA distances in the unbound 
clusters?

JPK

On Wed, May 9, 2012 at 11:51 AM, Philip Kiser  wrote:
Hey Jacob,

There was a paper by Robert M. Blessing et al (Acta Cryst D 1996) that
at least partially attributed the diffuse ring that one sees around
3-4 A to something similar to what you are describing (scattering
between amide oxygen and nitrogen  for example).

Philip

-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] Crystal Structures as Snapshots

[Mark Wilson]

Hi Jacob,
For Ca2+-CaM, and flexible proteins in general, the average conformation 
in solution may differ from the most crystallizable conformation. However, 
any crystallized conformation had to be sampled in solution at some point 
in order to form a crystal, and thus the crystal structure tells us 
something about the range of conformations accessible to the protein under 
the crystallization conditions.  In Ca2+-CaM, the presence of MPD is 
probably more responsible for the continuous central helix than the pH, 
but early analysis of the thermal factors in that region of the crystal 
structure predicted flexibility in the center of this helix that was 
subsequently observed by NMR to be  a flexible linker region.  More 
generally, I'd argue that crystal disorder is a subset of solution motion: 
i.e. disorder observed in crystalline protein almost certainly corresponds 
to motions that occur in solution (perhaps with altered amplitude), but 
not all solution motions are observed as disorder in the crystal.
Best regards,
Mark


Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu



Jacob Keller  
Sent by: CCP4 bulletin board 
02/10/2012 03:37 PM
Please respond to
Jacob Keller 


To
CCP4BB@JISCMAIL.AC.UK
cc

Subject
Re: [ccp4bb] Crystal Structures as Snapshots






Isn't calcium-calmodulin one of the archetypical examples of the
crystal structure probably not representing the solution structure
(perhaps because the crystallization pH = 4.5)? Look at that linker
helix--how stable can that be in solution? I don't think a single one
of the NMR ca-calmodulin structures/conformers has the central helix
like that.

Jacob



On Fri, Feb 10, 2012 at 3:31 PM, Nat Echols  
wrote:
> Just to clarify - I actually think the original assumption that Jacob
> posted is generally reasonable.  But it needn't necessarily follow
> that the conformation we see in crystal structures is always
> representative of the solution state; given the extreme range of
> conditions in which crystals grow, I would be surprised if there
> weren't counter-examples.  I'm not familiar enough with the literature
> on domain swapping (e.g. diptheria toxin) to know if any of those
> structures are crystal packing artifacts.
>
> On Fri, Feb 10, 2012 at 1:04 PM, George  wrote:
>>>Packing billions of copies into a compact lattice
>> Not so compact there is 40-80% water
>>>freezing it to 100K
>> We have frozen many times protein solutions in liquid nitrogen and then 
thaw
>> and were working OK
>>> non-physiological amounts of salt and various organics
>> What is the amount of salt and osmotic pressure in the cell??
>>>non-physiological pH too
>> What is the non-physiological pH too? I am sure that some enzymes they 
are
>> not working in pH 7. Also most of the proteins they have crystallized 
in pH
>> close to 7 so I would not say non-physiological.
>>
>> George
>>
>> PS There are lots of solution NMR structures as well supporting the
>> physiological crystal structures
>>
>>
>> -Original Message-
>> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Nat
>> Echols
>> Sent: Friday, February 10, 2012 10:35 PM
>> To: CCP4BB@JISCMAIL.AC.UK
>> Subject: Re: [ccp4bb] Crystal Structures as Snapshots
>>
>> On Fri, Feb 10, 2012 at 12:29 PM, James Stroud  
wrote:
>>> How could they not be snapshots of conformations adopted in solution?
>>
>> Packing billions of copies of an irregularly-shaped protein into a
>> compact lattice and freezing it to 100K isn't necessarily
>> representative of "solution", especially when your solution contains
>> non-physiological amounts of salt and various organics (and possibly
>> non-physiological pH too).
>>
>> -Nat
>>



-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] Transferring a Free R set.

[Mark Wilson]

Alun,
I agree completely with previously stated opinions (and your practice) 
that preservation of a single test set is very important for the 
refinement of similar structures.  One complication that for spacegroups 
in which there is a potential axis-indexing ambiguity (e.g. certain 
trigonal and tetragonal cells), the same set of indices can refer to 
reflections with different intensities in the different datasets.  That 
would be immediately apparent as a very high Rmerge if multiple, 
differently indexed dataset are merged, and may complicate transfer of a 
single test set among multiple, inconsistently indexed datasets for those 
spacegroups.
Best regards,
Mark

Mark A. Wilson
Assistant Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N164 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu



"Borhani, David"  
Sent by: CCP4 bulletin board 
12/18/08 02:38 PM
Please respond to
"Borhani, David" 


To
CCP4BB@JISCMAIL.AC.UK
cc

Subject
Re: [ccp4bb] Transferring a Free R set.






Alun,

Kay is right on target; I think there is no debate about that you must
use one FreeR set for all (even marginally*) isomorphous crystals. Your
colleagues who are not following this practice are not doing themselves
any favors.

If you don't keep the same FreeR set, R & Rfree for that 2nd crystal
will be almost identical --- and low!, like the R from crystal 1. Best
practice is to create an initial, master FreeR set that extends well
beyond your current (first crystal) resolution...crystals usually get
better as time spent on a project goes on, and extending the set
correctly takes some careful neuronal work (admittedly, now a bit easier
with the CCP4i GUI).

Dave 

* I've not seen any arguments for why one *shouldn't* keep the same set,
even as isomorphism fades away, due, e.g., to  a slightly variable unit
cell. Keeping the same set does no harm; calculating the potential harm
due to switching sets seems not worth the bother (though I guess one of
our more theoretically-inclined contributors will see an opportunity
there!).

> -Original Message-
> From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On 
> Behalf Of Kay Diederichs
> Sent: Thursday, December 18, 2008 3:05 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Transferring a Free R set.
> 
> Alun R. Coker schrieb:
> > Hi All,
> > 
> > I have been in the habit of transferring my initial free R 
> assignments 
> > to any new data sets or to isomorphous data sets such as substrate 
> > complexes.  Although theoretically this is necessary to 
> obtain a valid 
> > free R many of my colleagues maintain that this is completely 
> > unnecessary in practice.  Does anyone on the list have a 
> view on this or 
> > has anyone tested to see if it makes any difference.
> > 
> > Alun.
> > 
> 
> Alun,
> 
> I completely agree with you about the way how to treat R-free 
> reflections. If you want to have an unbiased R-free then you 
> need to set 
> these reflections aside for all refinement calculations of a project, 
> and this applies to all datasets you collect, as long as they are 
> isomorphous.
> This is especially important for low resolution work where 
> the danger of 
> overfitting is highest.
> 
> Kay
> 

[ccp4bb] Postdoctoral Position Available at the University of Nebraska

[Mark Wilson]

Postdoctoral Position in the Structural Biology of Neurodegeneration at 
the University of Nebraska

A postdoctoral position is available immediately in the laboratory of Dr. 
Mark Wilson in the Department of Biochemistry, University of 
Nebraska-Lincoln.  The position is for two years and involves a 
multidisciplinary structural, biophysical, and biochemical study of 
multiple redox-active proteins involved in neurodegeneration. A Ph.D. in 
Biochemistry or an allied discipline and previous research experience in 
protein biochemistry is required. Preference will be given to applicants 
with experience in either X-ray crystallography or NMR spectroscopy. 
Salary and benefits will be provided according to current NIH guidelines. 
Interested applicants should send a C.V. and the contact information for 
three references to:

Dr. Mark Wilson
N164 Beadle Center
University of Nebraska
Lincoln, NE 68516
[EMAIL PROTECTED]

The University of Nebraska is committed to a pluralistic campus community 
through affirmative action and equal opportunity.  We assure reasonable 
accommodation under the Americans with Disabilities Act. 

Mark A. Wilson
Assistant Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N164 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
[EMAIL PROTECTED]

[ccp4bb] ACA 2008 New Structures Session

[Mark Wilson]

This is an additional reminder to the community that January 12 (Saturday) 
is the deadline for abstract submissions for the New Structures session at 
the 2008 ACA meeting in Knoxville, TN.
The session will feature a balance of talks from both the protein and 
nucleic acid crystallographic communities. Abstract submissions from 
students interested in the Etter Student Lecturer Award are strongly 
encouraged.

01.01   New Structures
Sunday, June 1st
Organizers: David Giedroc  and Mark Wilson
Description: Recent structures from both the protein and nucleic acid 
structural communities that are of general interest will be featured, some 
of which will be discussed prior to publication.  In addition to invited 
talks, some presentations will be chosen from the submitted abstracts.  As 
in previous years, the talks will span a broad range of biological topics 
and will integrate the discussion of crystallographic methodology with the 
broader context of biological function


Mark A. Wilson
Assistant Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N164 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
[EMAIL PROTECTED]