Re: [ccp4bb] PDB to AlphaFold via Uniprot

[Nicholas Larsen]

Or you can go straight to uniprot.  Every available PDB + alphafold model
is linked to the uniprot entry.

On Fri, Jul 29, 2022 at 4:46 PM Robbie Joosten 
wrote:

> Hi Paul,
>
> From the PDB file get the Uniprot primary accession code (see DBREF) and
> use this to get the Alphafold model using 3D-beacons (
> https://www.ebi.ac.uk/pdbe/pdbe-kb/3dbeacons/).
> You can also use 3D-beacons to get the related AlphaFill model (just
> saying...).
>
> Cheers,
> Robbie
>
> > -Original Message-
> > From: CCP4 bulletin board  On Behalf Of Paul
> > Emsley
> > Sent: Friday, July 29, 2022 20:31
> > To: CCP4BB@JISCMAIL.AC.UK
> > Subject: [ccp4bb] PDB to AlphaFold via Uniprot
> >
> > I wonder if others are curious about (or know how to solve) the
> > following problem:
> >
> > I am looking at a PDB file that I've downloaded from a wwPDB site and I
> > would like to see the AlphaFold model(s) overlaid. What the best (or
> > easiest) way of doing that? (let's imagine that I can do a bit of
> > scripting in python (including urllib) and can use the functions in Coot
> > for the superposition).
> >
> > Thanks,
> >
> > Paul.
> >
> > ###
> > #
> >
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-- 
Nick Larsen
Director - Lead Discovery
H3 Biomedicine | an Eisai Oncology Company
300 Technology Sq #5
Cambridge, MA 02139
https://www.h3biomedicine.com/

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[ccp4bb] Structural Biologist opening at H3 Biomedicine (Cambridge - USA)

[Nicholas Larsen]

Hi All,
We have an exciting job opportunity in structural biology!  *Please apply
at:*
https://us.eisai.com/careers-at-eisai/job-detail?searchParam=H3%20Biomedicine=JOB_POSTING-3-9560

H3 is an oncology-focused company leveraging the latest cancer genomics
data to inform drug discovery.  The qualified candidate will have a Ph.D.
and postdoc with 2-4 years of relevant industry experience.  They will be
responsible for leading structure based drug design efforts using x-ray
crystallography and cryo-EM methods, as required.  The candidate will also
manage CRO relationships and possible academic collaborations.

*Basic qualifications:*

Molecular Biology (construct design)

Expression (from E coli, insect & mammalian systems) and purification (AKTA)

Automation (Mosquito, DragonFly, RockMaker, etc)

Co-crystallization, seeding, cross-seeding, soaking, harvesting, freezing,
etc.

Molecular Replacement, CCP4, Coot, Pymol or Chimera, Heavy atom of Se-Met
phasing

Grid preparation & freezing

Relion, cisTEM, CCP-EM, etc

Two or more first author publications.

Team player and excellent communication skills

*Additional desired qualifications:*
Knowledge of drug discovery & oncology.  Experience in biophysics.
Experience with cryo-EM.

-- 
Nick Larsen
Director - Lead Discovery
H3 Biomedicine | an Eisai Oncology Company
300 Technology Sq #5
Cambridge, MA 02139
Mobile: 617-792-4184

https://www.h3biomedicine.com/

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Re: [ccp4bb] Bug in mmCIF handling of UNK residues?

[Nicholas Larsen]

I hope this doesn't confuse the discussion, but my understanding was "UNK"
stood for "unknown" residue and this will cause errors.  UNK naming
convention is the default output of Schrodinger when generating ligand PDB
files.  Coot will display the PDB containing "UNK" as a residue, but if you
try to use the CIF file to real-space refine, the ligand will blow up.   I
found that renaming the residue in the output PDB and regenerating the CIF
file with the corrected RESID name solved the problem.  So in
my experience, the problem is the name "UNK" and this just needs to be
switched to something else.  Has anyone else seen this?
Nick

On Sat, Feb 13, 2021 at 4:29 PM Tristan Croll  wrote:

> Browsing backwards through a dozen or so of the most recent UNK-containing
> structures, I haven't found a counter-example yet - apart from those where
> the UNK residues are a single contiguous stretch and given their own chain
> ID. So a recent problem?
> --
> *From:* Philip D. Jeffrey 
> *Sent:* 12 February 2021 19:56
> *To:* CCP4BB@JISCMAIL.AC.UK ; Tristan Croll <
> ti...@cam.ac.uk>
> *Subject:* Re: Bug in mmCIF handling of UNK residues?
>
> Doesn't seem to be the case for all instances: that table isn't present in
> 5BV0 despite the N-terminal residues of Nyv1 being modeled as UNK in the
> Vps16:Vps33:Nyv1 complex due to a symmetry overlap.  Nyv1, C165-179 are UNK
> with partial occupancy, which is the N-terminal part of the model for that
> chain, then there's a gap of 3 missing residues, and then there's
> polypeptide model which we've assigned to sequence, all in the same chain.
>
> Not sure if the missing table is something that turned up after the
> deposition date (June 2015), or if it's related to the missing residues
> between the UNK segment and the defined amino acids.
>
> Phil Jeffrey
> Princeton
> --
> *From:* CCP4 bulletin board  on behalf of Tristan
> Croll 
> *Sent:* Friday, February 12, 2021 1:03 PM
> *To:* CCP4BB@JISCMAIL.AC.UK 
> *Subject:* [ccp4bb] Bug in mmCIF handling of UNK residues?
>
> Hi all,
>
> If I open (as far as I can tell) the mmCIF for any structure in the wwPDB
> that contains both defined amino acids and UNK in the same chain, then the
> UNK section is treated as covalently bonded to the flanking sequence. This
> appears to be a bug in the mmCIF generation itself, not in the viewing
> software (ChimeraX, in this case): if I look in 7kzn as a random example, I
> see:
>
> loop_
> _pdbx_validate_polymer_linkage.id
> _pdbx_validate_polymer_linkage.PDB_model_num
> _pdbx_validate_polymer_linkage.auth_atom_id_1
> _pdbx_validate_polymer_linkage.auth_asym_id_1
> _pdbx_validate_polymer_linkage.auth_comp_id_1
> _pdbx_validate_polymer_linkage.auth_seq_id_1
> _pdbx_validate_polymer_linkage.PDB_ins_code_1
> _pdbx_validate_polymer_linkage.label_alt_id_1
> _pdbx_validate_polymer_linkage.auth_atom_id_2
> _pdbx_validate_polymer_linkage.auth_asym_id_2
> _pdbx_validate_polymer_linkage.auth_comp_id_2
> _pdbx_validate_polymer_linkage.auth_seq_id_2
> _pdbx_validate_polymer_linkage.PDB_ins_code_2
> _pdbx_validate_polymer_linkage.label_alt_id_2
> _pdbx_validate_polymer_linkage.dist
> 1 1 C X UNK 345 ? ? N X UNK 348 ? ? 10.08
> 2 1 C X UNK 396 ? ? N X UNK 403 ? ? 28.65
> 3 1 C Y UNK 281 ? ? N Y UNK 284 ? ? 6.72
> 4 1 C Y UNK 387 ? ? N Y UNK 394 ? ? 22.26
> 5 1 C Y UNK 420 ? ? N Y UNK 424 ? ? 12.82
>
> Considering that the coords themselves generally seem fine, I guess this
> is happening post deposition?
>
> Best regards,
>
> Tristan
>
> --
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-- 
Nick Larsen
Director - Lead Discovery
H3 Biomedicine | an Eisai Oncology Company
300 Technology Sq #5
Cambridge, MA 02139
https://www.h3biomedicine.com/

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Re: [ccp4bb] COVID-19 protease inhibitors - 2nd call for designs - Thurs midnight

[Nicholas Larsen]

Hi Frank, I thought we were working quickly, but we only just got all of
your structures uploaded in our Proasis system today and can only now begin
analysis and design ideas.  I don't know if we will have much of anything
by your deadline.  Will there be additional design rounds?  Thanks again
for making all this data available and it really is inspirational what your
team is doing to confront collaboratively this global crisis.
Best,
Nick

On Wed, Apr 1, 2020 at 10:16 AM Frank von Delft 
wrote:

> All - last week's call for compound designs to the CoV-2 main protease (
> https://covid.postera.ai/covid)
> elicited an astonishing response...  (I confess I was quite taken aback.)
>
> I just realised I should let this BB know the second call for designs is
> now open.  *Deadline is tomorrow 23:59 PST (April 2nd).  *(Apologies for
> those that weren't following on twitter.)
>
> Two things:
>
>- we're asking for designs especially focusing on covalent inhibitors (read
>more here
>
> 
>)
>- the most convincing designs will be fast-tracked by spending more
>money to cut several weeks off the testing (read more here
>
> 
>)
>
> Happy designing!
> Frank
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>

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Re: [ccp4bb] COVID-19 - help design protease inhibitors - 1st round Thurs midnight

[Nicholas Larsen]

Hi Frank,
Congratulations on this impressive and valuable contribution to the efforts
against covid and I applaud your transparency and crowdsourcing efforts on
this global problem.  Your team is really an inspiration to us all.  We are
working to get all your structures uploaded for our med chemists and comp
chemist to analyze and hopefully they will be inspired to provide design
ideas.  One question, do you have any biochemical activity data for the
compounds?  Maybe I missed it in your link. Thanks again.
All best,
Nick

On Wed, Mar 25, 2020 at 8:36 PM Frank Von Delft 
wrote:

> To all you structural biologists locked out of your labs, bored with
> answering emails or writing your non-COVID paper, and itching all your
> lives to be medicinal chemists, here's your chance:
>
> https://covid.postera.ai/covid
>
> And you get to do something directly on COVID too.
>
> It's easy:
>
>- stare at the pile of fragments we found bound to the SARS-CoV-2 Main
>Protease in our somewhat epic XChem fragment screen
>
> 
>this month (alluded to by others)
>- see if you can spot cool ways of (especially) merging those hits -
>or anything else
>- draw the compound onto that page
>- hit submit
>
> It's actually a bit harder than it sounds - so we're betting that many
> brains will crack the nut of hitting potency in one go (!).
>
> We will do the rest - well, we'll try, if the Filters Approve and the
> Funds Permit -- and even for that, you can contribute, or tell people that
> could contribute (details on that page).
>
> First collection *closes tomorrow (Thurs) midnight Pacific Time* (but
> there will be more).
>
>
> Yes, it is a huge social experiment and a total crapshoot - but we must do *
> something*!  So thank you for all and any help...
>
> *** Do forward to anybody relevant, especially experienced medicinal
> chemists.
>
> *** If you know virology labs set up to do SARS-CoV-2 assays, email the
> address on that page.
>
> *** If you have help to offer or wisdom to share, do so on the forums on
> that page
>
> *** If you want to use the data for anything else - PLEASE DO - we were
> anxious to push it out ahead of PDB release cycle or getting preprint
> written.
>
>
> Frank
>
>
> --
> Prof Frank von Delft
> Professor for Structural Chemical Biology
>
> Principal Beamline Scientist: I04-1 and XChem
> Diamond Light Source
> +44 1235 778997 (office: M,T,T)
> +44 7471 026103 (mobile)
>
> Principal Investigator: Protein Crystallography
> Structural Genomics Consortium
> Oxford University
> +44 1865 617583 (office: W,F)
>
>
>
> --
>
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[ccp4bb] Structure biologist opening at H3 Biomedicine (Cambridge, USA)

[Nicholas Larsen]

Dear All, we have an opening for a permanent hire in my group so please
send me your resumes if interested and/or please forward to anyone you know
who may be looking for a terrific opportunity in biotech.

Thanks!

Nick


*Scientist – Structural Biology*

*Contact: nicholas_lar...@h3biomedicine.com
*

*Contact: john_ry...@h3biomedicine.com *



LOCATION: Cambridge,MA

We are looking for an exemplary structural biologist to join H3 Biomedicine
who enjoys working in a team environment. The candidate will assist with a
range of early to late stage projects in our oncology portfolio. The
candidate will be integrated into project teams to (1) enable new and
support ongoing structure-based drug design efforts and (2) support our
understanding of how disease mutations impact target biology in cancer. We
desire an individual with passion, motivation, and ambition to further H3’s
mission to improve patient lives.

*Principal Duties and Responsibilities*

- As an integral member of project teams, the candidate is expected to
design the best overall structural biology strategy to support each project.

- Design constructs for protein expression and participate in the
optimization of protein purification protocols for the generation of
crystallization-grade and cryo-EM-grade proteins.

- Perform various crystal screens and follow-up optimization. Solve/interpret
novel structures and provide detailed structure analysis.

- Provide regular team updates and participate in dynamic chemistry design
meetings to help guide structure-based drug design efforts.

- Participate in the management of CRO relationships and possible academic
collaborations.

*Basic qualifications:*

- Ph.D. and 3-4 years of relevant postdoctoral experience in structural
biology with a strong publication track record showing innovation and
scientific excellence.

-Demonstrated competencies in protein engineering, including construct
design, expression (E coli, insect cell, and mammalian), and protein
purification. Experience with multi-protein complexes is a plus.

- Extensive experience in crystallization with automation,
co-crystallization, seeding, cross-seeding, soaking, harvesting, freezing,
etc.

- Extensive experience with structure determination using both molecular
replacement and de novo phasing techniques, and refinement.  Experience
with de novo phasing at low resolution (< 3.0 A) is a plus.  Experience
with multi-crystal data processing and scaling is a plus.

- Competence with CCP4, Coot, Pymol, and Chimera are essential. Experience
with Phenix and Shelx is a plus.

- Collaborative and proactive attitude with excellent written and oral
communication skills.

- Flexibility to accommodate rapidly changing priorities and deadlines.

- Ability to work in a team-based environment.

*Additional desired qualifications:*

Knowledge of drug discovery, oncology, splicing biology.  Experience in
biophysics, specifically with SPR and ITC.  Experience with cryo-EM is a
plus.  Experience with relevant software, Sparx, Relion, cisTEM, etc, is
also a plus.

*About H3 Biomedicine Inc.*

H3 Biomedicine Inc. is a privately-held, uniquely-structured oncology
discovery enterprise whose sole mission is to become a prolific source of
new drugs that treat more human cancers with greater success. H3
Biomedicine is applying the expertise of leading scientists to the
integration of insights from cancer genomics with innovative capabilities
in synthetic chemistry and tumor biology to pursue the most promising
current opportunity in cancer therapeutics: patient-based, genomics-driven,
small molecule drugs.

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[ccp4bb] HDX data representation

[Nicholas Larsen]

Dear Colleagues,
We recently acquired HDX data on a target, which basically gives us %change
per residue over the entire primary sequence.  Is there a simple way to
reassign the B-factor column of my PDB with those %change numbers so I can
then easily color the molecular surface?  I hope the question is clear.
Also open to any other suggestions on how to map this kind of data to
molecular surface.
Thanks for any suggestion!
Nick

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Re: [ccp4bb] PDB based Fold recognition

[Nicholas Larsen]

You use the Dali server for that :)

http://ekhidna.biocenter.helsinki.fi/dali_server

On Mon, Sep 18, 2017 at 12:21 PM, Chetan Arya 
wrote:

> Hi all,
>   I want to know if there is any server/software which does the PDB based
> (structure based) fold recognition. All I got for fold prediction are
> sequence based. I am asking this because what if I have a novel sequence
> and I got the crystal structure of that protein but I don't know what
> kind of fold this protein has. Will it be novel fold or not?
> Thanks in advance.
>
> Cheers
> Chetan Arya
> Instem, Bengaluru
>

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Re: [ccp4bb] Protein rapidly precipitates when off ice

[Nicholas Larsen]

Did anyone suggest adding any known ligand?  If your protein happens to
bind some compound/peptide/DNA/RNA/whatever, including that other partner
could dramatically change it's solution properties and be an easy fix for
your handling/formulation issues.
Good luck!

On Fri, Jul 14, 2017 at 6:33 AM, Chris Fage  wrote:

> Dear All,
>
> Thank you for the many suggestions. After sending my first message to the
> BB, I tried exchanging the sample into buffer containing 5 mM EDTA and also
> into buffer at pH 9.0 (using BICINE). Neither of these appear to have
> helped the instability--precipitation still occurred within ~1 min of
> removal of the tube from ice. The theoretical pI is ~6.1, which is far from
> my working pH, although as Mark indicated the calculated pI may be
> inaccurate, and I may even need to try a more acidic pH. I will test the
> ideas provided by everyone over the next week and leave some feedback. It
> may be that I need to prepare a different truncation, as this domain is
> excised from a larger covalent assembly. I have purified homologs trimmed
> at a similar position in the past, but of course that doesn't guarantee
> good behavior in my current system.
>
> Best,
> Chris
>
> On Fri, Jul 14, 2017 at 10:20 AM, Eugene Osipov 
> wrote:
>
>>  Hi, try to add 1-5 mM EDTA, because trace amounts of metals cause
>> precipitation of tagged proteins.
>>
>> 14 июля 2017 г. 1:40 пользователь "Chris Fage" 
>> написал:
>>
>> Dear CCP4BB Community,
>>>
>>> This week, I purified a nicely overexpressing protein by Ni-NTA followed
>>> by gel filtration. In a 4 C centrifuge, I concentrated my gel filtration
>>> fractions to ~1 mL, transferred the spin filter to ice, and then collected
>>> 2 uL for measurement on the Nanodrop. Sadly, the protein precipitated
>>> heavily in the pipet tip before I could dispense it onto the Nanodrop
>>> pedestal, directly adjacent to my ice box. This effect seems to be abated
>>> at 4 C, as the protein remained stable in cold room-chilled pipet tips.
>>> However, the protein also precipitated heavily when overnight at 4 C in 1
>>> mL gel filtration buffer (150 mM NaCl, 10 mM HEPES pH 7.5), but not
>>> overnight at 4 C in 10 mL Ni-NTA buffer (500 mM NaCl, 30 mM HEPES pH 7.5,
>>> 10% glycerol) prior to gel filtration. Has anyone experienced and resolved
>>> a similar issue before? Do any useful additives come to mind?
>>>
>>> Things I have tried with the gel filtration sample:
>>> -Exchanging buffer to restore the salt concentration to Ni-NTA levels
>>> (e.g. 500 mM).
>>> -Exchanging buffer to add 10% glycerol.
>>> -Simply diluting the protein in gel filtration buffer to rule out
>>> concentration dependence.
>>>
>>> In each case, the protein precipitates to a milky solution within about
>>> a minute of removal from ice (I am working with 20-50 uL volumes in PCR
>>> tubes).
>>>
>>> Many thanks for any suggestions!
>>>
>>> Best,
>>> Chris
>>>
>>
>

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Re: [ccp4bb] off-topic: negative thermal shift upon ligand binding

[Nicholas Larsen]

In theory, what you say is quite sensible.  But there is one interesting
counter example I am aware of.The fragment tool compound that
eventually gave rise to the clinical compound indeglitazar (
http://www.pnas.org/content/106/1/262.full.pdf) gives a negative shift by
DSF (in our hands):
[image: Inline image 1]

Therefore, this experience taught us to bin compounds that are negative and
positive and follow up on both, prioritizing depending on this and any
other data we might have.In the end, we don't overthink it and just put
them in other assays and crystallography if appropriate.

Waving my hands around, you might imagine a scenario where the dye itself
binds and stabilizes a folded form of the protein.  If the fragment also
binds AND displaces the dye AND the fragment stabilizes the protein LESS
effectively than the dye, THEN I believe you could have a true binder that
gives a negative shift.

Nick


On Sat, Jul 8, 2017 at 8:30 PM, megha abbey  wrote:

> Hello,
>
> I am working on DSF to verify if some compounds bind to my protein. I see
> a negative shift of about 3-4 degrees upon ligand addition (dose-response)
> in comparison to the protein alone. I assume that this might be due to the
> binding of compound to the unfolded stated rather than folded protein.
>
> In such a situation where compounds are to be screened with the aim of
> drug discovery, are these negative thermal shift compounds relevant and how
> can they be followed upon, or they should simply be discarded?
>
> Thank you.
>

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Re: [ccp4bb] Large number of outliers in the dataset

[Nicholas Larsen]

One thing that sometimes helps me in this situation is reprocess and refine
in lower symmetry like P21.  It could be you have pseudosymmetry and need
to model more molecules in the AU to better reflect your data.  Sometimes
this can help.  If that doesn't work, then you may have to stick with your
2.6 A cutoff.  You are right that some reviewers might raise objections if
the R-factors are too high.

Good luck,
Nick

On Wed, Mar 29, 2017 at 11:56 AM, Mark J van Raaij 
wrote:

> To be really convinced I think you should also compare the maps at 2.6 and
> 2.3 Å. If the 2.3 Å map looks better, go for it. If it doesn’t look better,
> perhaps you are adding noise, but the I/sigma and CC1/2 values suggest you
> aren’t.
> Perhaps try 2.5 and 2.4 Å also.
> And perhaps remove a well-ordered aa from the input model, refine at
> different resolutions and compare the difference maps for that aa. Or
> calculate omit maps at different resolutions and compare those.
>
> Mark J van Raaij
> Dpto de Estructura de Macromoleculas
> Centro Nacional de Biotecnologia - CSIC
> calle Darwin 3
> E-28049 Madrid, Spain
> tel. (+34) 91 585 4616 <+34%20915%2085%2046%2016>
> http://wwwuser.cnb.csic.es/~mjvanraaij
>
> On 29 Mar 2017, at 17:44, Phil Evans  wrote:
>
> It is not clear to me why you believe that cutting the resolution of the
> data would improve your model (which after all is the aim of refinement).
> At the edge CC(1/2) and I/sigI are perfectly respectable, and there doesn’t
> seem to be anything wrong with the Wilson plot. Th R-factor will of course
> be higher if you include more weak data, but minimising R is _not_ the aim
> of refinement. You should keep all the data
>
> I don’t know what xtriage means by “large number of outliers”: perhaps
> someone else can explain
>
> Phil
>
>
>
> On 29 Mar 2017, at 14:54, Juliana Ferreira de Oliveira <
> juliana.olive...@lnbio.cnpem.br> wrote:
>
> Hello,
> I have one dataset at 2.3 Å (probably it can be better, I/σ = 2.1 and
> CC1/2 = 0.779, the summary data is below), but when I perform Xtriage
> analysis it says that “There are a large number of outliers in the data”.
> The space group is P212121. When I refine the MR solution the Rfree stops
> around 30% and it doesn´t decrease (in fact if I continue refining it
> starts to increase).
> The Wilson plot graph is not fitting very well between 2.3 and 2.6 Å:
>
> 
>
> So I decided to cut the data at 2.6A and Xtriage analysis doesn’t notify
> about outliers anymore. I could refine the MR solution very well, the final
> Rwork is 0.2427 and Rfree = 0.2730 and validation on Phenix results in a
> good structure.
> I run Zanuda to confirm the space group and it says that the space group
> assignment seems to be correct.
> Do you think that I can improve my structure and solve it at 2.3 Å or
> better? Or I can finish it with 2.6 Å? To publish at 2.6 Å I need to
> justify the resolution cut, right? What should I say?
> Thank you for your help!
> Regards,
> Juliana
>
> Summary data:
> OverallInnerShell  OuterShell
> Low resolution limit  51.51  51.51
>   2.42
> High resolution limit  2.30 7.27
>2.30
> Rmerge   0.147
>   0.054   0.487
> Rmerge in top intensity bin0.080   -
>  -
> Rmeas (within I+/I-)  0.155   0.057
>   0.516
> Rmeas (all I+ & I-)0.155   0.057
>   0.516
> Rpim (within I+/I-)0.048   0.017
>   0.164
> Rpim (all I+ & I-)  0.048   0.017
>   0.164
> Fractional partial bias-0.006 -0.003
> 0.146
> Total number of observations83988 2907
>11885
> Total number unique  8145307
>  1167
> Mean((I)/sd(I))   9.3
>   23.9 2.1
> Mn(I) half-set correlation CC(1/2)0.991   0.998
>   0.779
> Completeness 99.9
> 99.5 100.0
> Multiplicity10.3
> 9.5   10.2
>
> Average unit cell: 37.57 51.51 88.75 90.00 90.00 90.00
> Space group: P212121
> Average mosaicity: 1.90
>
>
> Juliana Ferreira de Oliveira
> Brazilian Laboratory of Biosciences - LNBio
> Brazilian Center for Research in Energy and Materials - CNPEM
> Campinas-SP, Brazil
>
>
>

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[ccp4bb] ITC question

[Nicholas Larsen]

Dear colleagues,
We have a target where people have measured Kd's for ligands using
radioligand binding assays.  Several publications report Kd's of single
digit nanomolar and we are able to reproduce that data using this assay
format.  When we try to do the same measurement using ITC, we generate
beautiful data, but the Kd's from ITC are at least 100-fold weaker.  Does
anyone have a suggestion how to reconcile this huge difference?

SPR studies show the ligands have a very long residence time, so one thing
I wondered is if ITC can underestimate a Kd if the off-rate is on the order
of minutes-hours.  Is this a reasonable explanation?

Please, any other ideas are welcome.
Best,
Nick

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Re: [ccp4bb] AW: [ccp4bb] Composit omit map vs. ligand

[Nicholas Larsen]

I agree with Herman about shorter soak.  In addition, if your crystals can
tolerate any change in pH, you can slow your reaction down by tweaking this
parameter.  Another thing you should think about is the kinetics of the
reaction, especially the rate limiting step.  If the rate limiting step is
product release, for example, then it may be challenging to capture the
initial substrate bound form.

On Wed, Feb 8, 2017 at 7:54 AM,  wrote:

> Dear Petr,
>
> Another possibility is that your very good substrate got turned over by
> the enzyme and that the 5 atoms with good electron density you see is all
> that is left. When you know the enzymatic reaction, this is easy to check.
> If this is the case, you should try a short soak (5-30 minutes) and
> immediately freeze the crystals.
>
> On the other hand, if your difference map shows reasonable density at the
> 1.5 to 2-sigma level I would not worry too much.
>
> Best,
> Herman
>
>
> -Ursprüngliche Nachricht-
> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von
> Kay Diederichs
> Gesendet: Mittwoch, 8. Februar 2017 11:43
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: Re: [ccp4bb] Composit omit map vs. ligand
>
> Dear Petr,
>
> if I understand correcty, the mFo-DFc density (1)  shows almost nothing,
> but the 2mFo-DFc  (2) as well as the composite omit map (3) show the ligand?
>
> As you say, the apparent contradiction between (1) versus (2)&(3) is
> unexpected. One explanation could be that the Fc are simply too bad, i.e.
> the model not good enough to result in useful signal in the difference
> map.  OTOH, that you see the ligand in (2) may be simply model bias, so is
> not meaningful. (3) is hopeful since there is no model bias.
>
> I would suggest to
> - refine the occupancy, to find out why the density is so weak
> - calculate a  (Fo,soak - Fo,native) (4) map with phases from a model
> unbiased by the ligand
> - try a Polder map (5)
>
> - If the occupancy is around 0.5 or higher, that would be a good sign.
> - but if you don't see density in (4) and (5), then your ligand is
> probably not there in any useful amount
>
> I consider (4) as the most sensible method to show presence of the ligand,
> and it should convince reviewers.
>
> HTH,
>
> Kay
>
>
>
> On Wed, 8 Feb 2017 09:05:50 +0100, Petr Kolenko 
> wrote:
>
> >Dear colleagues,
> >
> >we have a dataset with potential enzyme:ligand complex at 2.2 AA
> >resolution. The ligand is very good substrate for the enzyme, we used
> >soaking. We do not see the ligand in the regular difference electron
> >density, only five out of twenty atoms. However, the ligand placed at
> >the active site (model used from structure of a mutant variant) is
> >refined well, giving no negative peaks in difference electron density
> >map and nice observed electron density. I have calculated composit omit
> >map with annealing in Phenix (input model did not contain the ligand)
> >and the electron density for the ligand is there.
> >
> >I have my own opinion, but we are desperate to obtain such data (more
> >than 40 crystals already tested). My question is, would this be proof
> >of presence of the ligand with reduced occupancy? Will this map
> >convince the reviewers? Is there any other way to validate presence of
> the ligand?
> >
> >Best regards,
> >Petr
>

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Re: [ccp4bb] How to determine the concentration of biotinylated peptide?

[Nicholas Larsen]

These suggestions are all possible, but why not simply lyophilize it into a
tared tube and weigh it out?

On Mon, Feb 6, 2017 at 12:28 PM, Alex Lee  wrote:

> Thank you all for your suggestions!
>
> On Mon, Feb 6, 2017 at 5:53 AM, Artem Evdokimov  > wrote:
>
>> Hi,
>>
>> In addition to HABA dye assay (which will work great but will also be
>> fooled by any biotin that is not conjugated) you can do:
>>
>> * quantitative MS
>> * TLC
>> * HPLC
>> * elemental analysis
>> * https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3614710/ biotin catalysis
>> of the N3- + I3- reaction (also fooled by free biotin of course)
>> * UV (but beware, biotin only absorbs strongly below 240nm so you're not
>> super well off there
>>
>> Artem
>> www.harkerbio.com
>> "all of our Biotin comes only from free-range gummy vitamin bears..."
>>
>> - Cosmic Cats approve of this message
>>
>> On Mon, Feb 6, 2017 at 2:03 AM, Debasish Kumar Ghosh > > wrote:
>>
>>> Hi Alex,
>>>
>>> In addition to Mirella's suggestion I would like to make an addition
>>> which might be specifically useful for you. Since your peptide has biotin
>>> tag, You may use HABA dye assay for the exact quatifiation of biotin (and
>>> thus biotinylated peptide). As far I recall, Thermo scientific provide a
>>> kit for this assay. The assay is simple and gives accurate results.
>>>
>>> Best !!!
>>>
>>>
>>>
>>> Debasish
>>>
>>> CSIR- Senior Research Fellow (PhD Scholar)
>>> C/o: Dr. Akash Ranjan
>>> Computational and Functional Genomics Group
>>> Centre for DNA Fingerprinting and Diagnostics
>>> Hyderabad, INDIA
>>>
>>> Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
>>> Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab)
>>> Lab URL: http://www.cdfd.org.in/labpages/computational_functional_gen
>>> omics.html
>>>
>>>
>>>
>>> - Original Message -
>>> From: Alex Lee 
>>> To: CCP4BB@JISCMAIL.AC.UK
>>> Sent: Mon, 06 Feb 2017 03:02:07 +0530 (IST)
>>> Subject: [ccp4bb] How to determine the concentration of biotinylated
>>> peptide?
>>>
>>> Dear All,
>>>
>>> Sorry for the off-topic question, I'd like to do Biacore SPR assay with
>>> N-terminal biotinylated peptide as ligand (to Biacore SA chip) and my
>>> protein as analyte. I have a question of how to determine the
>>> concentration
>>> of biotinylated peptide （synthetic peptide), if the peptide has no Tyr
>>> and
>>> no Trp residue, I guess amino acid analysis may not work because the
>>> N-terminal of the peptide is biotinylated.
>>>
>>> I'd appreciate if anyone share his/her experience on this.
>>>
>>
>>
>

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[ccp4bb] Glass etching of structure

[Nicholas Larsen]

Dear All,
I'm sorry if this is off topic, but it might be of interest to general
audience.  I want to get one of our crystal structures etched in glass.
Can anyone help recommend a company that can provide this service?  BioEtch
had come highly recommended to me, but unfortunately their website
currently shows they are experiencing equipment malfunction and it's
unclear when they would be up and running again.

Thanks for any suggestion!
Nick

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[ccp4bb] jligand help

[Nicholas Larsen]

Dear All,
I have CCP4 6.5.0 installed on my laptop PC.  Everything runs fine, to my
knowledge, except JLigand.  When I click this program from the program
list, absolutely nothing happens.  No error message, nothing.  It doesn't
launch.  Do I need to reinstall cpp4 or is JLigand a separate installation?

Thanks for any suggestion,
Nick

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Re: [ccp4bb] The phase problem

[Nicholas Larsen]

Screen for heavy atoms with native gel.  Run protein with variety of heavy
atoms.  If you see a discreet shift in the native gel, then you will get
derivative.  If you don't see a shift, don't bother soaking.  It's worked
for me every time.  Also, ammonium sulfate will mess up your heavy atoms,
so if that's in your condition, you need to substitute AmSulf with LiSO4 in
your soak.

Nick

On Wed, Nov 5, 2014 at 2:20 PM, Giulliana Rangel giulliana.ran...@gmail.com
 wrote:

 Dear all,


 I would like to known if someone could help me with some idea about the
 phase problem.

 I am a beginner in crystallography and the first time I tryed to solve the
 structure by Molrep, amore, mr. Bump and I didnt find anything for
 molecular replacement.

 Thus, currently I've tried to soak heavy-atom ( Hg, Pt, I, Pb) and the
 diffraction was good, the results in XDS didnt show so good. I didnt look
 the heavy atom in density.

 I appreciate any suggestion and ideas what I could do.

 Best regards,



 --
 Giulliana Rangel
 Mestranda PPG-Biotecnologia UNIFESP
 Laboratório de Biologia Estrutural
 Tel.: (12) 3309-9698
 Rua Talim 330, Vila Nair
 CEP 12231-280
 São José dos Campos - SP



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[ccp4bb] KRAS maps

[Nicholas Larsen]

Dear All,
I frequently use the Coot feature Fetch PDB and MAP using EDS... with
great success when peer reviewing literature reports.  However, when I try
this for the recent KRAS structures deposited by Kevan Shokat and Jim Wells
(Nature 2013), the Coot generated maps are garbage, although the resolution
is better than 1.5 A.  Does anyone have an explanation?   I also checked
with one kind colleague at another institute and she confirmed my problem
using Linux platform (I am using Windows).

See PDBs, 4LUC and 4LV6, for example.

Cheers,
Nick

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Re: [ccp4bb] How to store PEG screens

[Nicholas Larsen]

I don't think storage matters.  I doubt Hampton stores their PEG stock
solutions at -80 before they ship out to customers.
I've solved tons of structures leaving my PEGS and PEG screens at RT in the
light.

Nick


On Mon, Jul 14, 2014 at 12:32 PM, Chris Fage cdf...@gmail.com wrote:

 Hi Jerome,

 -I have heard that PEG solutions can become unstable in light. We usually
 store our block in the fridge, where photons are scant anyway. For any
 stocks that I prepare, I wrap the tube/bottle in aluminum foil. I'm not
 sure about freezing them.
 -Some labs (not ours) evidently prepare buffered stocks of PEG solutions,
 as their pHs tend to shift with time. This is something I've been meaning
 to try. Of course, you may need to worry about buffer components that are
 incompatible with crystal hits.

 Hope this helps,
 Chris


 On Mon, Jul 14, 2014 at 9:33 AM, Jerome Nwachukwu jnwac...@scripps.edu
 wrote:

 Dear all,
 I have 3 short questions about PEG solutions:
 Does anyone know the best way to store crystallization screening blocks
 that contain PEG 3350?
 Is it a good idea to freeze the PEG solutions at -80°C and thaw them
 before use?
 Would the freeze-thaw process considerably alter the PEG chain lengths?

 Thank you,
 -Jerome

 Jerome Nwachukwu




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Re: [ccp4bb] Unidentified density in coot

[Nicholas Larsen]

Looks like you're on the twofold axis, which will make interpretation
challenging.  Anything you put in may end up being too close to itself in
the neighboring AU.  What happens if you put in a water and display the
symmetry?


On Mon, Jun 23, 2014 at 8:17 AM, Shanti Pal Gangwar gangwar...@gmail.com
wrote:

 Dear All

 I have solved a structure of my protein at 3.0 A. The crystallization
 condition is consisting of PEG400, NaCl, MgCl2 and Sodium citrate. The
 protein was purified in HEPES buffer.
 I can see an unidentified electron density blob in coot and I am not able
 to figure out what it could be?

 I have attached the snapshot of that blob with this mail. I request
 everyone to please help me in identification of this blob.

 Thanking you in advance.







 Shanti Pal


 
 Best regards
 Shanti Pal Gangwar, Ph.D
 School of Life Sciences
 Jawaharlal Nehru University
 New Delhi-110067
 India
 Email:gangwar...@gmail.com




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[ccp4bb] Industry Postdoc Position - Boston

[Nicholas Larsen]

H3 Biomedicine seeks to recruit an outstanding postdoctoral candidate in
structural biology.  Our major area of research is RNA splicing in cancer.
We have developed tool molecules targeting aberrant splicing in SF3B1
mutant cancers. Our goal is to understand the structural basis for aberrant
splicing in this mutant background and use the tool molecules as a platform
for structure based drug design.


Relevant Review:

http://www.nature.com/nrd/journal/v11/n11/full/nrd3823.html


Qualifications

The applicant must have a Ph.D. with a strong background in recombinant
protein expression, crystallization, and structure determination.
 Experience in biophysics is also desirable.  Candidate should be highly
motivated and results driven.   Strong organizational and interpersonal
skills are desired.   Funding is for three years with competitive salary
and benefits.  The laboratory is newly equipped.



The candidate will be integrated into a dynamic and collaborative work
environment with:

* Regular interactions with mentor and potential for external academic
advisor as appropriate

* Regular presentations at Scientific Founders Meeting to the H3
Biomedicine senior scientists and our academic founders Stuart Schreiber,
PhD and Todd Golub, MD.

* Presentation/publication of research at major scientific
conferences/journals

* Potential for transition into full-time employment should appropriate
positions be available at the end of the postdoctoral position


To apply for this position, submit your resume at:

http://www.h3biomedicine.com/career/postdoctoral-fellows-oncology-research


Or e-mail: nicholas_lar...@h3biomedicine.com

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