Re: [ccp4bb] AW: Strange cysteines

2021-12-22 Thread Roger Rowlett
Another possibility to model is a couple of S-hydroxy cysteines. We've seen
that in a Cys-amidase where there was artifactual oxidation damage to the
protein during storage or RT crystallization.

Roger Rowlett

On Wed, Dec 22, 2021, 8:00 AM Andrew Purkiss 
wrote:

> As you say, the Cys are too far apart for it to be a disulphide and it
> looks like oxidation of the Cys. This could be due to radiation
> damage, but other causes are possible.
>
> A former colleague had one in betaB1-crystallin, which is visible in
> pdbcode 1OKI at position 38. The difference density was quite clear
> before it was modeled as Cysteine sulfinic acid (CSD). Of course, it
> was easy to see at 1.4 Angstrom resolution.
>
> Andy
>
> Quoting "Hochberg, Georg" :
>
> > Hi oliver,
> >
> >
> > Thanks! Real space refinment was done in phenix with default
> > settings. The position of these cysteines is quite constrained by
> > where the main chain goes and the Phe you can see just next to the
> > cysteine. To me it seems it would require very odd geometry to pull
> > them into these centroids.
> >
> >
> > All the best,
> >
> > Georg
> >
> > 
> > Von: Weiergräber, Oliver H. 
> > Gesendet: Mittwoch, 22. Dezember 2021 10:51:59
> > An: Hochberg, Georg; CCP4BB@JISCMAIL.AC.UK
> > Betreff: RE: Strange cysteines
> >
> > Hmm, this may indeed be a disulfide bond, but the sulfur atoms do
> > not seem to occupy their density centroids.
> > They could be either _pushed_ apart by the refinement algorithm
> > (which one are you using?) or _pulled_ apart due to incorrect
> > geometry in their neighbourhood.
> >
> > Cheers
> > Oliver
> >
> > ==
> >   PD Dr. Oliver H. Weiergräber
> >   Institut für Biologische Informationsprozesse
> >   IBI-7: Strukturbiochemie
> >   Tel.: +49 2461 61-2028
> >   Fax: +49 2461 61-9540
> > ==
> >
> >
> > 
> > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of
> > Hochberg, Georg [georg.hochb...@mpi-marburg.mpg.de]
> > Sent: 22 December 2021 10:06
> > To: CCP4BB@JISCMAIL.AC.UK
> > Subject: [ccp4bb] Strange cysteines
> >
> > Dear CCP4ers,
> >
> >
> > We have recently solved the structure of an enzyme by cryoEM, which
> > has two cysteines at its dimer interface, one in each monomer. The
> > density around these two cysteines is very odd (see picture). They
> > are too far apart for a disulphide bond, and there is nothing around
> > these two cysteines that could help coordinate a metal. This density
> > is also not a result of symmetry constraints in the density
> > refinement either.  We'd be very grateful for any ideas.
> >
> > [cid:b92417da-97f2-4dc3-9b5c-92278a5318fd]
> >
> >
> > All the best and happy holidays,
> >
> > Georg Hochberg
> >
> > 
> >
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> >
> >
> >
> >
> 
> >
> 
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Re: [ccp4bb] off-topic: pH meters

2021-09-20 Thread Roger Rowlett
The meter doesn't matter so much as the electrode. For the meter, anything
with at least 3 pH multipoint calibration is sufficient for most purposes.
I usually bought something from our preferred university vendor a with very
large LCD displays for my aging eyes.

For protein work, and especially if using anything with Tris buffers, a
refillable, double junction Ag/AgCl reference combination electrode is a
must. Making stock 1M Tris buffer concentrate will wack out single junction
combination electrodes, sometimes permanently. I liked the Fisher
Accutuph/Accuphast electrodes because they were much more difficult for my
students to break, and they last for years. (My students never succeeded in
breaking one.) Gel-filled electrodes are garbage. Refillable double
junction electrodes are the way to go for stable readings, especially in
Tris, and minimizing heavy metal contamination.

Roger Rowlett
Gordon & Dorothy Kline Professoe, Emeritus
Department of Chemistry
Colgate University

On Mon, Sep 20, 2021, 4:57 PM Patrick Loll  wrote:

> Fellow protein biochemists:
>
> My ~30 year-old Beckman pH meter is finally showing its age, and I’m
> looking for a high-quality replacement that doesn’t cost insane amounts of
> money. My initial thoughts gravitate toward Mettler (it’s a name I trust,
> and some of their low-end instruments aren’t absurdly expensive); would
> anybody care to recommend other options?
>
> Much obliged for any suggestions (the more obsessive, the better).
>
> Cheers,
>
> Pat
>
>
>
> ---
> Patrick J. Loll, Ph. D.  (he, him, his)
> Professor of Biochemistry & Molecular Biology
> Drexel University College of Medicine
> Room 10-102 New College Building
> 245 N. 15th St., Mailstop 497
> Philadelphia, PA  19102  USA
>
> (215) 762-7706
> pjl...@gmail.com
> pj...@drexel.edu
>
> 
>
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Re: [ccp4bb] Enzyme Vmax and Km

2021-06-18 Thread Roger Rowlett
For sure, you will need to collect data at lower substrate concentration to
determine what is going on for processed substrate. One should also
consider how the "initial rates" are measured. Ideally, the amount of
substrate depleted during the initial rate measurement should be less that
5%. When it is larger than this, or when the product is a strong inhibitor
of the enzyme, complicated or even artifactual results can be obtained. We
have seen similar, time-dependent kinetic behavior for an enzyme that is
strongly allosterically inhibited by its product. For complex kinetic
behavior, it may be necessary to have a finer grid of measurements to allow
for accurate data fitting.

As others have pointed out, linear transforms of the Michaelis-Menten
equation are statistically flawed without applying proper y-weighting. It
is much better to do nonlinear least squares fiting of rate vs. substrate
data. For true product inhibition, there are several possible models that
could be considered, based on the behavior of the data. Consultation with
an enzyme kineticist might be warranted for for complex behavior.

Roger Rowlett


On Fri, Jun 18, 2021, 3:53 AM Harmer, Nicholas 
wrote:

> Dear Prem,
>
>
>
> I agree entirely with Tristan’s conclusion that the processed substrate is
> also acting as an inhibitor at higher concentrations. You would need to run
> an experiment with a wider range of concentrations used (especially lower
> concentrations) to get a better feel for the range of the reaction. There
> is a well described substrate inhibition equation (see e.g.
> https://www.graphpad.com/guides/prism/latest/curve-fitting/reg_substrate_inhibition.htm)
> that you can try. I have a recent chapter on experiment design covering
> such cases (
> https://www.researchgate.net/publication/331806855_Reaction_Chemical_Kinetics_in_Biology)
> that I can share with you if you need.
>
>
>
> I would strongly recommend to avoid the Lineweaver-Burk plot to calculate
> your Km and kcat. There are known issues with this (especially, as in your
> image, overweighting the lowest rate and usually least accurate point).
> Better is to fit directly to the equation with non-linear fitting, for
> example in *R*. This will also give you a better estimate of the error
> and confidence intervals.
>
>
>
> Hope this helps,
>
>
>
> Nic
>
>
>
> *From:* CCP4 bulletin board  *On Behalf Of *Tristan
> Croll
> *Sent:* 18 June 2021 08:19
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] Enzyme Vmax and Km
>
>
>
> Hi Prem,
>
>
>
> The immediate problem here is that the curve for the processed substrate
> simply cannot be described by simple Michaelis-Menten kinetics. Assuming
> the assay has worked as expected, the declining rate with increasing
> substrate concentration suggests to me that this substrate also acts as an
> allosteric inhibitor, so assays at high [substrate] will make it *look*
> like the unprocessed substrate is preferred even though the processed one
> is cleaved faster by the uninhibited enzyme.
>
>
>
> Hope this helps,
>
> Tristan
>
>
> On 18 Jun 2021, at 05:05, Prem Prakash  wrote:
>
> Dear all,
>
> Sorry for this off topic. I am working on an enzyme that has an
> exonuclease activity. The enzyme preferentially cleaves an unprocessed
> substrate at a faster rate than the processed one (known by qualitative
> analysis). Recently, I calculated the Vmax, Km and kcat of the enzyme for
> unprocessed substrate which are 18.2 pmol/min, 182 nM and 7.1 sec-1
> respectively. However, the Processed substrate has apparently a lower range
> of Km (not calculated) as reflected from the curve (because the same
> increasing concentration range which is used for unprocessed, shows a steep
> decline in the initial velocity of the enzyme with processed substrate.
>
> The latter suggests that Km is way lower than expected. In this case, the
> question is, if the Km of processed substrate is way lower than the
> Unprocessed, how can we see a faster rate with the former substrate than
> later. i.e lower Km and slower rate of cleavage. If it's possible please
> give some insights. I have attached the plot comparison between two kinetic
> assays.
>
>
>
> With kind regards,
>
>
>
> Prem
>
>
>
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
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Re: [ccp4bb] Looking for proteins for undergraduate biochemistry lab

2021-06-16 Thread Roger Rowlett
Human carbonic anhdydrase II is very expressible in *E. coli,* and
purifiable in one step via affinity chromatography with
para-aminobenzenesulfonamide affinity resin (which is relatively easy to
make, and reusable for many years.) It can be assayed by stopped-flow
spectrophotometry for CO2 hydration, by a timed colorimetric assay, or you
can investigate it's esterase activity with p-nitrophenylacetate. This
enzyme, as well as most other carbonic anhdydrases, is also easy to purify
by a classical combination of anion exchange (Q-sepharose), hydrophobic
interaction (butylsepharose), and gel exclusion polishing. The latter would
be a good exercise for students in general protein purification
optimization, which is an increasingly lost art. (Just had a conversation
with one of the protein chemists ast BioGen who pretty much observed the
same thing.) We routinely did classical purifications on tagless
overexpressed proteins for crystallography work. The time saved in His-Tag
purification is sometimes lost in cleaving the tag to make tagless protein
for crystallography.

A paper describing the purification procedure can be found in J. Chem. Ed. (
https://doi.org/10.1021/ed075p1021) for the similar bovine carbonic
anhydrase. An fun long-term undergraduate research training project might
involve improving the esterase activity through student-initiated point
mutations.

I did this kind of parallel protein mutation project with my students in a
biochemistry research training studio course I taught, often  with one of
my research target proteins. Teaching lab students can do all sorts of
crazy things you might never prioritize in your funded research. Some of
these crazy things turn out to be fun and interesting. One of my students
insisted on making a mutation in a protein that seemed to have low chances
of leading to a successful publication based on prior work. Lo and behold,
that mutation turned out to be gold, and he was published within the year.
I still can't believe it not only worked, but crystallized easily from the
first screen and optimization for structure determination. Go figure.

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

email: rrowl...@colgate.edu

On Wed, Jun 16, 2021 at 7:09 PM Chun Luo  wrote:

> Many phosphatases, such as lambda phosphatase, have good soluble
> expression in E. coli. Their activity can be shown by simply colorimetric
> assay.
>
>
>
> *From:* CCP4 bulletin board  *On Behalf Of *P. H
> *Sent:* Wednesday, June 16, 2021 3:19 PM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] Looking for proteins for undergraduate biochemistry
> lab
>
>
>
> Hello All,
>
>
>
> We are looking for some candidate proteins for an undergraduate level
> advanced biochemistry lab. They should be expressed in bacteria, simple
> enough to purify and it will be nice to perform some simple
> characterization experiments(binding assays, enzymatic assays).
>
> Any suggestions?
>
>
>
> Thank you in advance.
>
> Prerna gupta
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
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Re: [ccp4bb] To solve the problem of an extremely asymmetric peak shape obtained from gel filtration chromatography

2020-12-09 Thread Roger Rowlett
Salt concentrations less than 100 mM can lead to nonspecific adsorption to
the gel exclusion media, potentially leading to band broadening, and
delayed elution.  Overloading gel exclusion columns (more than 2-4% Vt) can
also lead to elution band artifacts. Check these issues first.

Roger Rowlett
Gordon & Dorothy Kline Professor, Emeritus
Department of Chemistry
Colgate University

On Wed, Dec 9, 2020, 9:17 AM   wrote:

> Dear All
> There is a 43kd protein purified via Ni-chelating affinity
> chromatography, anion exchange chromatography and gel filtration
> chromatography in sequence. However the chromatogram obtained showed an
> extremely asymmetric peak shape. The aggregation forms of proteins are
> mainly in the range of monomers and dimers(Hepes and low concentration of
> salt were used as buffers for gel filtration chromatography). 5% glycerinum
> and 1mM Benzamidine hydrochloride had been added in order to maintain the
> stability of the protein and prevent it from degrading. But well, all the
> efforts seem to be useless. We wonder if there are any effective measures
> can be taken to radically solve this problem. We would be much indebted for
> the suggestions you offer.
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] electron density close Histidine side chain

2020-07-20 Thread Roger Rowlett
Almost certainly a metal ion, possibly Ni(2+) if a Ni-NTA column was used
for purification. Ni-N bond lengths are typically around 2.0 A. Additional
density is probably coordinated water molecules, which should have similar
Ni-O bond distances around 1.9 A. It is fairly common to find adventitious
metal ions (zinc, copper, nickel) bound to His residues.

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

email: rrowl...@colgate.edu

On Mon, Jul 20, 2020 at 12:17 PM samer halabi <
30c2162795b2-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hello all,
> I have few blobs in an MHC II structure I am working on, especially
> opposite to Histidine as in the accompanying screenshot, that I am confused
> about.
>
> In the crystal conditions, I have Tris, Imidazole, Acetate, PEG and
> Glycerol.
> Whatever ligand I am fitting in I am getting a clash (overlap -1.029),
> which makes me think whether there is a covalent bond forming between
> Histidine and other molecule. Perhaps by oxidation.
>
> I would greatly appreciate if you can advice me about it, whether there is
> some kind of ligand I can try to fit and if this is something that occurs
> in some structures.
> Thank you.
> Best regards,
> Samer
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] insertion of a chemical

2020-06-30 Thread Roger Rowlett
To verify the presence of a non protein ligand, you need to come at this
from an unbiased perspective.

1. Remove what you think is not there (e.g. disulfide bond) by truncating
the Cys residues
2. Examine the difference density map to see if it conforms to your
expectations of what is proposed to be there (or does not)
3. If appropriate, build and attach ligand and re-refine. I am assuming
that you are proposing a thioketal adduct, which is not the same as
inserting an acetone molecule in the structure.

You can find generic instructions for these types of tasks on my
XRD-protocols site, https://sites.google.com/colgate.edu/xrd-protocols.

Roger Rowlett

On Tue, Jun 30, 2020, 7:25 AM Daniele Veggi  wrote:

> Hi Yadav,
> yes, this is only a representation of what I would like to do. If during
> the refinement will continue to appear density, probably I'm on the good
> track..but currently my big issue is inserting the ACN
> covalently linked to both cys.
>
> many thanks
>
> Daniele Veggi
> GSK Vaccines Siena Italy
>
>
>
>
>
> Il giorno mar 30 giu 2020 alle ore 12:52 Lumbini Yadav <
> lumbin...@gmail.com> ha scritto:
>
>> Hi Daniele,
>> Do you see Fo-Fc density  near disuphide bond where you are trying to fit
>> acetone?
>>
>> On Tue, Jun 30, 2020 at 3:28 PM Daniele Veggi 
>> wrote:
>>
>>> Dear CCP4bb,
>>>
>>> I'm trying to insert an Acetone molecule between two cysteine residues
>>> in coot or modifying the pdb. I'm working on this modified molecule where
>>> the disulfide bridge was chemichally opened and inserted an acetone
>>> molecule between the two cysteine.
>>>
>>> MS data confirm this modification (98% of molecules modified) but I can
>>> not insert the acetone covalently linked to the S of both cysteine
>>> (S-ACN-S).
>>> Maybe what I think and what I am doing is not the right solution (the
>>> disulfide bridge is still alive?) anyway i would like to know how to link
>>> the acetone to the two cysteine
>>> To better understand what I mean in attach you find a screenshot
>>>
>>> I thank in advance all those who will have the courtesy to make a
>>> suggestion
>>>
>>> Daniele Veggi
>>>
>>> --
>>>
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>>>
>>
> --
>
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Re: [ccp4bb] insertion of a chemical

2020-06-30 Thread Roger Rowlett
The first step would be to verify consistent, convincing electron density
for the proposed thioketal adduct. A good way to start might be an "omit"
map of the region by mutating the Cys residues to Ala, truncating them to
the beta-carbon. The resulting difference density may be suggestive of a
thioketal adduct or a simple disulfide bridge. Building the thioketal, if
actually present, will require addition skill with ligand building and
Coot. You are essentially attaching an isopropyl group to the two Cys
sulfur atoms.

Roger Rowlett
Gordon & Dorothy Kline Professor, Emeritus
Dept. Of Chemisty
Colgate University

On Tue, Jun 30, 2020, 5:58 AM Daniele Veggi  wrote:

> Dear CCP4bb,
>
> I'm trying to insert an Acetone molecule between two cysteine residues in
> coot or modifying the pdb. I'm working on this modified molecule where the
> disulfide bridge was chemichally opened and inserted an acetone molecule
> between the two cysteine.
>
> MS data confirm this modification (98% of molecules modified) but I can
> not insert the acetone covalently linked to the S of both cysteine
> (S-ACN-S).
> Maybe what I think and what I am doing is not the right solution (the
> disulfide bridge is still alive?) anyway i would like to know how to link
> the acetone to the two cysteine
> To better understand what I mean in attach you find a screenshot
>
> I thank in advance all those who will have the courtesy to make a
> suggestion
>
> Daniele Veggi
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Molecular replacement problem

2020-06-18 Thread Roger Rowlett
I managed to solve a structure by MR at 2.4 A with a 27% identity model.
Like you, I had to use a dimer search model to make any headway. To get
usable maps and an initial model, I used Chainsaw to truncate the search
model, Phaser (MR), Parrot (DM) with NCS averaging, then auto building with
Buccaneer. I had the advantage of 8 chains in the ASU, which greatly
improved the NCS averaging. NCS helps as the square root of the chains
averaged.

Before going down this road, all potential MR solutions were carefully
checked by examining crystal lattice packing to ensure the MR solution was
reasonable and space group was likely correct, and that I had the correct
number of chains per ASU. My initial MR attempts, based on Matthew's number
estimates, were 2 chains short, which was obvious from crystal packing.

Cheers, and good luck!

Roger Rowlett
Dorothy & Gordon Kline Professor, Emeritus
Colgate University

On Thu, Jun 18, 2020, 9:01 AM Robert S Phillips  wrote:

> I've been pulling out my hair with this for a few months now.  I have data
> sets to 2.6 A for a new enzyme in the aminotransferase superfamily.
> Unfortunately, the closest structure is only 25% identity.  MR with PHASER
> using the monomer was a complete failure.  Since the minimum structure of
> enzymes in the family is a dimer (the active site is formed at the
> monomer-monomer interface), I used dimers for MR with PHASER.  Most of the
> results were marginal, but one looks good.  However, it will not refine.
> Everything I have done with this solution has failed, simulated annealing,
> morphing, etc., it will not refine; AUTOBUILD and BUCCANEER with it do not
> give any useable models, since they have poor statistics and low
> completeness.  The output from PHASER is below.
>
> ** SINGLE solution
>
> ** Solution written to PDB file:  DGL_phaser.1.pdb
> ** Solution written to MTZ file:  DGL_phaser.1.mtz
>Solution annotation (history):
>SOLU SET  RFZ=18.9 TFZ=23.0 PAK=0 LLG=193 TFZ==8.3 LLG=994 TFZ==20.1
> PAK=0 LLG=994 TFZ==20.1
>SOLU SPAC P 2 2 21
>SOLU 6DIM ENSE ense_1 EULER  360.00.00.0 FRAC -0.00 -0.00 -0.00
> BFAC -0.12 MULT 2 #TFZ==20.1
>SOLU ENSEMBLE ense_1 VRMS DELTA -3.5403 #RMSD  2.08 #VRMS  0.89
>
> With LLG = 994 and TFZ = 20.1, isn't this a real solution?
>
> Robert S. Phillips
> Professor of Chemistry and of Biochemistry and Molecular Biology
> University of Georgia
> Athens, GA 30602
> Phone: (706) 542-1996
> Fax: (706) 542-9454
> E-mail: rsphill...@chem.uga.edu
> Web:  http://tryptophan.net
> <https://pod51004.outlook.com/owa/redir.aspx?C=ccbf42ffea5f48b1bf8e9bb950454bab=http%3a%2f%2ftryptophan.net>
>
> --
>
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Re: [ccp4bb] Covalent Cysteine Aduct

2020-03-27 Thread Roger Rowlett
S-hydroxycysteine can cartainly show up under nonreducing storage or
crystallization conditions, as an artifact.

Cheers,

Roger Rowlett

On Fri, Mar 27, 2020, 5:45 PM Chris Fage  wrote:

> Hi Richard,
>
> I recently observed the sulfenic acid derivative of a cysteine residue
> (S-hydroxy-Cys, ligand ID CSO) in one of my high-resolution maps. Could it
> be possibly be this, H-bonded to a nearby water molecule?
>
> Best wishes,
> Chris
>
>
> On Fri, Mar 27, 2020 at 21:33 Paul Emsley 
> wrote:
>
>>
>> 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.
>>
>>
>>
>> --
>>
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Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Wincoot middle-mouse button does not work

2020-03-23 Thread Roger Rowlett
For mice with a pressable scroll wheel it should be possible to go into the
configuration settings to enable the scroll wheel press to be a middle
click. It is typically not set to that function by default.

For two button mice, it is usually possible to configure a L-R press to
emulate middle-click. Again, you have to go into the mouse configuration
settings.

I have two mice (a 3-button and a 2-button) configured to work properly
with Wincoot in Win10.


Roger Rowlett
Gordon & Dorothy Kline Professor, Emeritus
Colgate University

On Mon, Mar 23, 2020, 11:56 AM Schreuder, Herman /DE <
herman.schreu...@sanofi.com> wrote:

> Hi Arne (and others),
>
>
>
> I am using Windows 10 und just downloaded WinCoot 0.8.9.2 EL. Since
> everybody else’s mouses seem to be perfectly fine, I will try another
> mouse. Maybe the one I am using is having a problem.
>
>
>
> Best,
>
> Herman
>
>
>
> *Von:* EchelonIV . 
> *Gesendet:* Montag, 23. März 2020 16:34
> *An:* Schreuder, Herman /DE 
> *Cc:* CCP4BB@jiscmail.ac.uk
> *Betreff:* [EXTERNAL] Re: [ccp4bb] Wincoot middle-mouse button does not
> work
>
>
>
> *EXTERNAL : *Real sender is arne.raasa...@gmail.com
>
>
>
> Hi Herman,
>
>
>
> which version, OS and mouse are you using? I can confirm that middle mouse
> button works perfectly fine in version 0.8.6.2 (yes I know I'm outdated)
> win Win10 with a generic Logitech plug-and-play mouse with the third mouse
> button being the wheel. I also quickly checked the latest version in
> Windows 7 ultimate (yikes!) with the same generic mouse as well as a fancy
> Razer mouse with cloud-based button profiles and it seems to work equally
> well. Have you checked your mouse settings and drivers?
>
>
>
> Stay healthy,
>
> Arne
>
>
>
> On Mon, Mar 23, 2020 at 3:48 PM Schreuder, Herman /DE <
> herman.schreu...@sanofi.com> wrote:
>
> Dear community,
>
>
>
> Courtesy to the Corona crisis, I am now separated from my Linux
> workstation and I am trying to get everything working from home. Running
> coot remotely via a low-speed internet connection was no option so I had
> Wincoot installed on my laptop. Everything appears to work fine with one
> exception: The middle-mouse button is not working, so I cannot center on an
> atom.
>
>
>
> Does anyone know how to solve this problem?
>
>
>
> Thank you for your help and stay healthy!
>
> Herman
>
>
>
>
>
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> <https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1=DwMFaQ=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=VkLxNlrBECX7o3wLR5zDSntBqBQ_-KWyyTD1q8eaV4c=ePn1ZYT0MZoInhXNiDogieUtXfLlzrI-eFDQPfHajVk=>
>
>
>
>
> --
>
> --
>
> Arne Raasakka
>
> PhD Biochemistry
>
> Department of Biomedicine, University of Bergen
>
> Norway
>
> --
>
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Re: [ccp4bb] A question of density

2020-03-04 Thread Roger Rowlett
Chloride coordination spheres are typically tetrahedral (CN=4) or square
pyramidal (CN=5), and occasionally octahedral (CN=6). Arg and His and well
as amide nitrogens are common protein ligands, and it is possible but not
common to see carboxylates in the coordination sphere.

Roger Rowlett

On Wed, Mar 4, 2020, 1:05 PM Jessica Besaw  wrote:

>
> Hey Roger,
>
> I believe the Chloride spot is a negatively changed anion because it has
> more density that water and it is close to an arginine. Does this chloride
> binding coordination look reasonable?
>
> All of the other water are coordinated to two other side-chains, and do
> not depend on the partially occupied water
>
> Cheers!
>
> Jessica
>
>
>
> On Wed, 4 Mar 2020 at 12:27, Roger Rowlett  wrote:
>
>> In addition, one should carefully consider the chemistry and coordination
>> geometry around the putative chloride ion. Not only do candidates have to
>> be consistent with the observed omit density, but they must also make
>> chemical interaction and geometry sense. I suspect a partial occupancy
>> water may make the most sense from the limited data provided, but only if
>> all the other placed solvent molecules have clearly defined interactions
>> that do not depend solely on the partially occupied water. What does the
>> chloride coordination geometry look like? Is it clearly a chloride ion and
>> not a cation?
>>
>> Roger Rowlett
>> Gordon & Dorothy Kline Professor, Emeritus
>> Colgate University
>>
>> On Wed, Mar 4, 2020, 12:20 PM Nukri Sanishvili 
>> wrote:
>>
>>> Hi Jessica,
>>> You do not say how well is the rest of the structure refined.
>>> First, you should refine the structure best you can, without placing
>>> anything in the unclear blob of your interest so to obtain the best
>>> possible phases and hopefully improve the blob density as well.
>>> Then you should let the BB see what that density looks like. Looking at
>>> only the list of possibilities has very little value without seeing the
>>> density itself.
>>> Best wishes,
>>> Nukri
>>>
>>> On Wed, Mar 4, 2020 at 11:10 AM Jessica Besaw 
>>> wrote:
>>>
>>>> Hello friends,
>>>>
>>>> I have a "blob" of density in an active site of my protein.
>>>>
>>>> I am struggling to determine if I should place a water in this spot, if
>>>> I should model it as a disordered water, if the density may be a ligand
>>>> that I have not considered, or if it should be left as unaccounted for
>>>> density. I would like to publish this structure without compromising the
>>>> science.
>>>>
>>>> I have attached several possibilities that I have considered below.
>>>>
>>>> Any suggestions would be appreciated.
>>>>
>>>> Cheers!
>>>>
>>>> Jessica Besaw
>>>>
>>>>
>>>>
>>>> --
>>>>
>>>> To unsubscribe from the CCP4BB list, click the following link:
>>>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>>>
>>>
>>> --
>>>
>>> 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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>



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Re: [ccp4bb] A question of density

2020-03-04 Thread Roger Rowlett
In addition, one should carefully consider the chemistry and coordination
geometry around the putative chloride ion. Not only do candidates have to
be consistent with the observed omit density, but they must also make
chemical interaction and geometry sense. I suspect a partial occupancy
water may make the most sense from the limited data provided, but only if
all the other placed solvent molecules have clearly defined interactions
that do not depend solely on the partially occupied water. What does the
chloride coordination geometry look like? Is it clearly a chloride ion and
not a cation?

Roger Rowlett
Gordon & Dorothy Kline Professor, Emeritus
Colgate University

On Wed, Mar 4, 2020, 12:20 PM Nukri Sanishvili  wrote:

> Hi Jessica,
> You do not say how well is the rest of the structure refined.
> First, you should refine the structure best you can, without placing
> anything in the unclear blob of your interest so to obtain the best
> possible phases and hopefully improve the blob density as well.
> Then you should let the BB see what that density looks like. Looking at
> only the list of possibilities has very little value without seeing the
> density itself.
> Best wishes,
> Nukri
>
> On Wed, Mar 4, 2020 at 11:10 AM Jessica Besaw 
> wrote:
>
>> Hello friends,
>>
>> I have a "blob" of density in an active site of my protein.
>>
>> I am struggling to determine if I should place a water in this spot, if I
>> should model it as a disordered water, if the density may be a ligand that
>> I have not considered, or if it should be left as unaccounted for density.
>> I would like to publish this structure without compromising the science.
>>
>> I have attached several possibilities that I have considered below.
>>
>> Any suggestions would be appreciated.
>>
>> Cheers!
>>
>> Jessica Besaw
>>
>>
>>
>> --
>>
>> 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] Protein concentration for the initial crystallisation trials

2020-01-08 Thread Roger Rowlett
I usually set a partial screen, maybe 24-48 conditions. If less than half
the wells have precipitate, double protein concentration. If most have
precipitate, maybe reduce protein or halve concentration of screen
reagents. I usually start at 10 mg/mL or so. You can conveniently change
protein conc. by manipulating protein/screen volume ratio.

__
Roger Rowlett

On Wed, Jan 8, 2020, 11:16 AM Armando Albert  wrote:

> Dear all,
> I was wondering how to guess the optimal protein concentration for the
> initial crystallisation trials. Is there any trick or assay other than the
> classic PCT from Hampton?
> Armando
>
> 
>
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Re: [ccp4bb] difficult molecular replacement solution

2019-11-13 Thread Roger Rowlett
 Look at the chain packing in your solution. If the solution is grossly
correct you will notice nice solvent channels and no glaring chain-size
holes and no gross overlaps. The initial maps should look sensible, if
noisy.

If the protein is composed of likely dimers, try a search with dimer units
to simplify the search.

Good luck.

__
Roger Rowlett

On Wed, Nov 13, 2019, 10:33 AM Robert S Phillips  wrote:

> I have been working on a protein structure which has been hard to solve by
> molecular replacement.
>
> Unit cell: (60.6, 172.34, 196.42, 90, 90, 90)
> Space group: P 21 21 21
>
> The problem is that the homologues have only ~20% identity, and there are
> multiple chains in the asymmetric unit.  The question is how many.  It
> could be 4, 5, or 6 chains.
>
> N  solvent   P
>  4  0.602   3.090.225
>  5  0.502   2.470.388
>  6  0.403   2.060.229
>
> I have run PHASER with 4, 5 and 6 chains.  I allowed it to search all
> possible space groups, and P212121 was the best solution.  These are the
> results.
>
> N  LLG   TFZ
> 4  104.97.5
> 5  137.57.7
> 6  166.28.3
>
>  Am I correct to conclude that there are 6 chains in the asymmetric unit?
>
> Rob
>
> Robert S. Phillips
> Professor of Chemistry and of Biochemistry and Molecular Biology
> University of Georgia
> Athens, GA 30602
> Phone: (706) 542-1996
> Fax: (706) 542-9454
> E-mail: rsphill...@chem.uga.edu
> Web:  http://tryptophan.net
> <https://pod51004.outlook.com/owa/redir.aspx?C=ccbf42ffea5f48b1bf8e9bb950454bab=http%3a%2f%2ftryptophan.net>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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>



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Re: [ccp4bb] crysalis pro from rigaku

2019-11-04 Thread Roger Rowlett
The current version of CrysalisPro will directly import several image
formats, or you can convert images to Esperanto format to import. The
latter is a bit clumsy but does work.

__
Roger Rowlett

On Mon, Nov 4, 2019, 9:54 AM Almudena Ponce Salvatierra <
maps.fa...@gmail.com> wrote:

> Dear all,
>
> does any of you have experience with using Crysalis Pro software from
> Rigaku with data that were not collected on a Rigaku instrument?
>
> Any help will be much appreciated.
>
> All the best,
>
> Almudena
>
> --
>
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Re: [ccp4bb] ITC question -dimer vs monomer

2019-10-03 Thread Roger Rowlett
Won't this depend on the relative final concentrations of A and B in the
two experiments? If A going into excess B will have different mass action
considerations that B going into excess A. Even if the final concentrations
of A and B are stoichiometric, the initial stages of the titration will
have very different mass action products for A into B vs. B into A. An
additional wrinkle is the concentrations of A and B relative to the
dissociation constant Kd. The titration curve math gets a little more
complex when the concentration of the species is in the same order of
magnitude as the Kd. There are quite a few examples of bollixed binding
curves in the literature for tight-binding equilibria that ignore the
relationship between Kd and ligand concentrations.  Cooperativity issues
will of course perturb any pure, non-cooperative statistical analysis based
on equilibrium constants.

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor, Emeritus
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
email: rrowl...@colgate.edu

On Thu, Oct 3, 2019 at 11:51 AM Bernhard Rupp 
wrote:

> I am not looking for anything yet – I wonder what – if any – the
> consequences of doing it one way or the other would be.
>
> I am reasonably certain that any difference affects the analysis.
>
>
>
> Thx, BR
>
>
>
> *From:* CCP4 bulletin board  *On Behalf Of *Keller,
> Jacob
> *Sent:* Thursday, October 3, 2019 17:41
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] ITC question -dimer vs monomer
>
>
>
> I don’t understand what you are trying to do—are you trying to show, by
> the difference in ITC response, that the predictions you made about the
> oligomerization are true?
>
>
>
> JPK
>
>
>
> +
>
> Jacob Pearson Keller
>
> Research Scientist / Looger Lab
>
> HHMI Janelia Research Campus
>
> 19700 Helix Dr, Ashburn, VA 20147
>
> Desk: (571)209-4000 x3159
>
> Cell: (301)592-7004
>
> +
>
>
>
> The content of this email is confidential and intended for the recipient
> specified in message only. It is strictly forbidden to share any part of
> this message with any third party, without a written consent of the sender.
> If you received this message by mistake, please reply to this message and
> follow with its deletion, so that we can ensure such a mistake does not
> occur in the future.
>
>
>
> *From:* CCP4 bulletin board  *On Behalf Of *Bernhard
> Rupp
> *Sent:* Thursday, October 3, 2019 11:06 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] ITC question -dimer vs monomer
>
>
>
> Hi Fellows,
>
>
>
> please let me ask the respective experts an ITC question: I have 2
> proteins, stable and dialyzed in identical buffer.
>
> A is a monomer and B an obligate dimer. I suspect that eventually a A2B2
> dimer will form.
>
>
>
> Intuitively, it should make a difference whether I titrate the dimer with
> the monomer or vice versa.
>
> In the first case, a momomer would initially meet a lot of free dimers,
> and I would expect that randomly, a AB2 complex
>
> is more likely to form than a A2B2 (let’s disregard any more complex
> colligative/cooperative effects).
>
>
>
> If I drip the dimer into the monomer pool, it is quite likely that the B
> dimer meets 2 free As, and I get right away a higher population of A2B2s.
>
>
>
> Maybe at dilutions of ITC and with sufficient equilibration that is not an
> issue at all (again, absent any cooperative effects that might alter the
> first Kd vs. the second, despite the sites on the dimer are at least
> initially equivalent).
>
>
>
> Can someone guide me towards literature about this or perhaps share some
> first-hand experience?
>
>
>
> Many thanks, BR
>
>
>
> --
>
> Bernhard Rupp
>
> http://www.hofkristallamt.org/
> 
>
> b...@hofkristallamt.org
>
> +1 925 209 7429
>
> +43 676 571 0536
>
> --
>
> Many plausible ideas vanish
>
> at the presence of thought
>
> --
>
>
>
>
> --
>
> 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] Another difficult MR case

2019-08-29 Thread Roger Rowlett
EPMR is also a good alternative for finding multiple placements per ASU.

If a reasonable packing MR solution can be found and there is significant
NCS, then DM followed by NCS enhanced automated model building from initial
MR phases can do wonders. Improvement goes as the square root of NCS
numbers, though, so 2 is not a great improvement, e.g.

__
Roger Rowlett

On Thu, Aug 29, 2019, 1:42 PM Jonathan Cooper <
0c2488af9525-dmarc-requ...@jiscmail.ac.uk> wrote:

> It would be useful to know the number of molecules per asymmetric unit and
> the sequence similarity of the search model and target. There is always
> Molrep to try which is good at NCS ;-
>
> Sent from Yahoo Mail on Android
> <https://go.onelink.me/107872968?pid=InProduct=Global_Internal_YGrowth_AndroidEmailSig__AndroidUsers_wl=ym_sub1=Internal_sub2=Global_YGrowth_sub3=EmailSignature>
>
> On Thu, 29 Aug 2019 at 18:30, David Briggs
>  wrote:
> Following on from Ivan's suggestion, SIMBAD might he worth a shot.
>
> https://journals.iucr.org/d/issues/2018/07/00/rr5159/
>
> The other thing you might try is handing the MR phases to the density
> modifying and autobuilding program of your choice, increasing the number of
> cycles by $arbitarylargenumber and then leaving it to run for a few
> hours/over night.
>
> This has worked for me in the past when resolution was decent, phaser had
> found an obviously correct MR solution, but the domain placed was only
> ~30-40% of the total scattering mass of the ASU, and more conventional
> refinement was not yielding decent maps outside the aforementioned domain.
>
> Good luck!
>
> Dave
>
> --
> Dr David C. Briggs
> Senior Laboratory Research Scientist
> Signalling and Structural Biology Lab
> The Francis Crick Institute
> London, UK
> ==
> about.me/david_briggs
>
> --
> *From:* CCP4 bulletin board  on behalf of Phil
> Jeffrey 
> *Sent:* Thursday, August 29, 2019 5:24:48 PM
> *To:* CCP4BB@JISCMAIL.AC.UK 
> *Subject:* Re: [ccp4bb] Another difficult MR case
>
> Are you *sure* there's no translational NCS ?
>
> For example your first molecular replacement solution out of Phenix shows
>
> EULER  293.6   27.7  288.7
> FRAC -0.02  0.02  0.02
> (that's "first molecule at origin in P1")
>
> and
>
> EULER  294.0   27.9  288.8
> FRAC -0.37  0.02  0.02
>
> which is essentially the same orientation, and a translation down one
> crystallographic axis (a*)
>
> And this suggests to me that either Xtriage or Phaser is missing
> something here.  Does Phaser find translational NCS in its initial data
> analysis ?  Unmodeled translational NCS could cause significant problems
> with the molecular replacement search.
>
> Phil Jeffrey
> Princeton
>
>
>
>
> On 8/29/19 11:28 AM, Napoleão wrote:
> > Deal all,
> > Sorry for the long post.
> > I have a data set obtained from a crystal produced after incubating a
> > protease with a protein which is mostly composed by an antiparallel beta
> > sheet. I have tried numerous approaches to solve it, and failed.
> > Molecular replacement using Phaser, and the protease or the protein as a
> > template yields no solution. However, molecular replacement using only
> > part of the beta sheet yields LLG=320 TFZ==28.0 (see below).
> >
> > The apparently good data extends to 1.9 A, as processed by XDS, and the
> > space group is P1 (pointless agree). XDS info below:
> >
> > SPACE_GROUP_NUMBER=1
> > UNIT_CELL_CONSTANTS=44.4372.2977.30  97.802  89.939 101.576
> >
> >   ab  ISa
> >   9.647E-01  3.176E-03   18.07
> >
> >   RESOLUTION NUMBER OF REFLECTIONSCOMPLETENESS R-FACTOR
> > R-FACTOR COMPARED I/SIGMA   R-meas  CC(1/2)  Anomal  SigAno   Nano
> > LIMIT OBSERVED  UNIQUE  POSSIBLE OF DATA   observed
> > expected  Corr
> >   1.90   24890   19149 23814   80.4%  58.1%
> > 63.7%114820.77 82.2%63.8* 30.694 492
> >  total  163756  125884146938   85.7%  10.6%
> > 10.8%757443.78 15.0%99.0*-30.7615834
> >
> >
> > Xtriage in Phenix 1.16-3549 gives me all green lights (print below),
> > suggesting the data presents no twinning, no translational NCS, no ice
> > rings and is not anisotropic.
> >
> https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Ffullonline.org%2Fscience%2Fphenix_xtriage_green.pngdata=02%7C01%7C%7Cde40f97d75824ea6519308d72c9d6fe0%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C0%7C637026927009132543sdata=RMV8abo87ybMyxaqD%2FH

Re: [ccp4bb] low resolution map with unmodelled map

2019-08-19 Thread Roger Rowlett
I would back way up in the process and verify that the basics are correct.
Ten molecules in the ASU is unusual unless your molecules are forming
defined oligomers, e.g., 2.5 tetramers, etc. Precise predictions of
monomers in the ASU by Matthews number is increasingly unreliable above 2-3
monomers. You have to look at lattice packing to be more sure of the
correct number. If there are defined oligomers, e.g.dimers, the initial
search may work better with oligomers as the search unit than monomers.

How confident are you of the space group and unit cell assignment? What
does the lattice packing of the MR solution look like? (You can do this in
Coot.) Do protein molecules in the lattice make sensible contacts? Are
there serious overlaps? Can you see solvent channels? Can you see
symmetry-related oligomers? I'd sort this out before proceeding further. If
these check out, then there are ways of using noncrystallographic symmetry
in your ASU to significantly improve your initial maps for rebuilding,
although sequence assignment may still be challenging at this resolution.

Roger Rowlett


On Mon, Aug 19, 2019, 8:15 PM Zhu Qiao  wrote:

> Sorry for the initial message. I tried to attach Matthews coefficient
> calculation figure but failed to do so, which resulted the message as not
> plain text.  Below is my question, thanks.
>
> I collected one dataset and processed it to 3.6 angstrom. my protein is
> quite small with only 14 kDa. It is estimated over ten molecules in one ASU
> based on Matthew coefficient calculation.
>
> However, only ten molecules can be correctly placed with good fitting. I
> can observe extra Fo-Fc electron density maps there needed to be modelled.
> But Fo-Fc electron density maps are discontinuous.
>
> I tried to fix the ten molecules as the partial solution in phaser and
> search more molecules, either resulted in no solution or the newly added
> molecules didn't fit in the map after refinement.
>
> So I manually built the poly alanine chain in order to decrease the R
> factor. I built around 200 amino acids into the final model. but these poly
> alanine model can hardly be interpreted to remodel as my target protein
> because of the low resolution. Currently the ten molecules model has a
> Rwork/free as 0.33/0.36. The model with poly alanine chain has a
> Rwork/free as 0.30/0.34.  I can already extract the useful information
> based on the well fitted ten molecules. And the fitting of the poly alanine
> models could just for better model refinement.
>
> Can I get some suggestions regarding this kind of issue, what's the
> general practise for such situation. Can I deposit the model with poly
> alanine fitted and labelled it unidentified to pdb?
>
> Thanks for any suggestions and replies.
>
> Best Regards
> Qiao Zhu
>
> 
>
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[ccp4bb] FFT bug, CCP4i job file configuration

2019-08-06 Thread Roger Rowlett
FYI,

When FFT is run in the CCP4i interface, the output file destination is
configured incorrectly. Examining the batch file that is created for the
job, it appears that CCP4i ignores the MAPOUT filename and substitutes the
file name of the temporary file, which is (alas) deleted in the last step.
There is a workaround by using the full file path in the MAPOUT line
instead of the project directory and local file name.

Cheers,

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor, Emeritus
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
email: rrowl...@colgate.edu



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[ccp4bb] Position opening - Colgate University

2019-08-03 Thread Roger Rowlett
Please find attached a description of a position opening at Colgate
University that may be of interest to a protein crystallographer interested
in working with undergraduates in a research-rich, research supportive
environment. Colgate is equipped with an Oxford Diffraction home source,
Gryphon dropsetter, and a Scorpion screen maker, as well as other essential
tools for a teacher-scholar interested in protein structure and function.
In addition, Colgate is also only 90 minutes from CHESS.

Cheers,

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor, Emeritus
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
email: rrowl...@colgate.edu



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1


colgate-u-position.pdf
Description: Adobe PDF document


Re: [ccp4bb] Questionable Ligand Density: 6MO0, 6MO1, 6MO2

2019-07-19 Thread Roger Rowlett
If it is as it appears, it is disappointing to see this in JACS. I would 
expect better. Unfortunately, reviewers don't always get a lot of 
information to judge quality of structures (which has been discussed 
extensively  on prior occasions on this board), so some trust is 
involved that what data is presented by the authors is representative. I 
see the authors duly presented a 2Fo-Fc map and a Fo-Fc omit map in one 
of the figures, but it appears from a personal examination that the 
Fo-Fc omit map presented may not resemble what one gets when personally 
examining the deposited data, and some protein chain refinement problems 
are immediately evident. That is a bit concerning. High-pressure 
research, coupled with (perhaps) a lack of understanding, experience, 
and supervision is a potentially dangerous ethical stew.


Correcting erroneous published data is challenging. I share the 
frustration of others when attempting to challenge scientifically 
questionable published results. My trip down the rabbit hole (not 
related directly to crystallography) nearly identical to that related 
previously here. Spend your own time writing a full paper rebuttal (ugh) 
or move on and concentrate resources on your own work.


_
Roger S. Rowlett
Gordon & Dorothy Kline Professor, Emeritus
Department of Chemistry
Colgate University
email: rrowl...@colgate.edu

On 7/19/2019 2:24 PM, Robbie Joosten wrote:


Even though PDB-REDO cannot salvage this model without extensive 
rebuilding which is what Tristan showed, it is fun to look at the maps 
and B-factors near the ligand. The B-factors go way up and the 
negative difference density disappears, as does most of the 2mFo-DFc 
density. It’s obviously not news to most people on the CCP4BB, 
properly refining B-factors is really important!


Cheers,

Robbie

*From:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf 
Of *Bärbel Blaum

*Sent:* Friday, July 19, 2019 17:24
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* Re: [ccp4bb] Questionable Ligand Density: 6MO0, 6MO1, 6MO2

Hi Rhys,

the reported B-factors for the “ligands” are all way below the 
reported B-factors for the protein chains, with the worst of the three 
complexes reporting unitless numbers 23.2 and 64.8, respectively, just 
to highlight *one* striking feature of the data collection and 
refinement table. So even with the limited info normally available to 
reviewers this table should have raised a red flag. After the 
re-refinement suggested by others, i.e. your own proper assessment of 
the crystallographic data, if you do not find noteworthy density you 
may want to contact the article’s editor with your results. If you 
work in this field, i.e. really care about this paper scientifically 
and you are not afraid to confront the authors you could suggest 
writing a comment/direct response article but in my opinion that would 
only make sense if you can be sure beforehand that it will be linked 
visibly to the actual paper, else it will be a waste of time. And 
don’t forget that just because one or some of the authors did a bad 
job at the crystallographic end other findings of the paper might 
still be solid – in collaborations often one author is unable to 
critically evaluate another author’s contribution and this would not 
be the first case were good synthetic or biological work is presented 
along with a bad crystal structure.


By the way and a bit ironically this protein may have suffered bad 
crystallography/scientific practice before - I think it was one of the 
fake Krishna Murthy structures, right? The associated (now retracted) 
article I mean is here


https://www.sciencedirect.com/science/article/pii/S002228360093924X?via%3Dihub 



Kind regards, Bärbel

---

Bärbel Blaum, PhD

Inthera Bioscience AG

Einsiedlerstrasse 34

CH-8820 Waedenswil

Switzerland

E-Mail: baerbel.bl...@intherabio.com

Phone: +41 43 477 94 72--

*Von: *CCP4 bulletin board  im Auftrag von 
"Manfred S. Weiss" 

*Antworten an: *"Manfred S. Weiss" 
*Datum: *Freitag, 19. Juli 2019 um 16:03
*An: *
*Betreff: *Re: [ccp4bb] Questionable Ligand Density: 6MO0, 6MO1, 6MO2

Hi Rhys,

all three structures are at modest resolution and they don't seem to
be properly refined. At least they are all below average. I wonder
how this paper made it past the referees.

I haven't checked the paper, but there are ways and means how to
deal with weakly bound ligands in the best possible way. One aspect
is to improve the phases as much as possible without having the ligand
present. This was obviously NOT done. Another way is to use the
PANDDA approach, which relies on having many data sets available.
I suppose that this was also not done.

The best way to check is to delete the ligand and so some extensive
refinement in order to remove the phase bias introduced by the
ligand. Only then you can reliably assess whether something is there
or not.

Cheers, Manfred

Am 19.07.2019 um 15:21 schrieb Rhys Grinter:


Re: [ccp4bb] analytical gel filtration columns

2019-04-17 Thread Roger Rowlett
How about BioGel-A50m with an exclusion limit of 50 million Da? I've used
A15m for complexes as large as 500 kDa and it seemed to work well.

__
Roger Rowlett

On Wed, Apr 17, 2019, 11:11 AM Mohinder Pal 
wrote:

> Dear all,
>
> I would like to gel filter a multi protein complex (1.4MDa) as the final
> purification step. I have tried tandem Superose 6 columns and this complex
> elutes close to void volume as it is a very elongated molecule.
>
>
> I was wondering if someone could suggest different analytical columns for
> better resolution as I plan to add more components to this existing
> complex.
>
> Best wishes,
>
> Mohinder Pal
>
> --
> "Whatever you’re meant to do, do it now. The conditions are
> always impossible.”
> Doris Lessing
> --
>
>
>
>
>
> --
>
> 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] SO4 or PO4

2019-02-16 Thread Roger Rowlett
Two things to look at that could provide a clue:

Examine the anomalous map for some density over the central atom. Sulfur
will often, but not always have significant anomalous density depending on
the wavelength and quality of data set.

Phosphate is normally HPO4= or H2PO4-. Look for phosphate donor to acceptor
hydrogen bonding contacts. Sulfate rarely has donor to acceptor hydrogen
bonding contacts, as it is SO4= at any reasonable pH.

Roger Rowlett

On Sat, Feb 16, 2019, 4:06 AM 张士军 <21620150150...@stu.xmu.edu.cn wrote:

> Dear all
>
> I have got a crystal grown at the condition both have ion of SO4 and PO4,
> and the diffraction resolution is very well, but the problem is coming: how
> to tell which is which just from electron density? I think they are exactly
> same. Thanks a lot !!!
>
> Beat Regards
>
> Shijun
>
> --
>
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Re: [ccp4bb] Sulphate or phosphate?

2018-07-31 Thread Roger Rowlett
You might be able to distinguish sulfate from phosphate by examining
hydrogen bonding partners. Phosphate can donate one or two hydrogen bonds
at neutral pH values, whereas sulfate is usually only a hydrogen bond
acceptor. (Having said that, we have published a structure where a sulfate
clearly interacts with a glutamate, the later of which which may or may not
be protonated at near neutral pH.)

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

tel: (315)-723-7245
email: rrowl...@colgate.edu


On Tue, Jul 31, 2018 at 9:38 AM, David Schuller 
wrote:

> How can one distinguish between a sulphate or phosphate in an electron
> density map? Both are present in the mother liquor, and resolution is in
> the range of 1.75 - 2.25 A
>
>
> --
> ===
> All Things Serve the Beam
> ===
>David J. Schuller
>modern man in a post-modern world
>MacCHESS, Cornell University
>schul...@cornell.edu
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] Sulphur SAD at home source

2018-04-03 Thread Roger Rowlett
Iodine is ideally suited for Cu K-alpha SAD phasing, and iodide ions can 
normally be easily added by soaking crystals in potassium iodide 
containing solutions, which can be done at the time of cryopreservation. 
A quick lit search will turn up the appropriate protocols. For 
structural genomics work where MR was unsuccessful or unusable, iodide 
soaks were found to work as much as 80% of the time.


I've used iodide-soaked lysozyme for an XRD teaching lab and 
undergraduate research student training, and SAD phasing works really 
well on an overnight data collection on our Oxford Diffraction PX-ultra 
system. It's worth a shot, and very easy to do. Many proteins will 
tolerate soaking, especially if crystallized from salts.

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor Emeritus
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 4/3/2018 10:46 AM, Eleanor Dodson wrote:
Well - the S f" is only ~ 0.5 at Cu Kalpha so the signal will be very 
weak..
Very accurate data may get a solution but you first have to position 
the S atoms...


Much easier to try to make a heavy atom derivative!

Eleanor

On 3 April 2018 at 15:26, Manoj Saxena 
<1d16aa30e8a1-dmarc-requ...@jiscmail.ac.uk 
> wrote:


Hi All,

I am writing to seek advice on doing  sulphur SAD data collection
at Cu based home source for a protein that is 12 KDa and has 6 S
atoms.
I have seen some links online and some references but would be
grateful if
you can share your know-how for success with this.
Like what multiplicity of data would be good to aim for and
data processing tips.
Inputs from people who have tried and failed would also be highly
appreciated.

Thank you
Manoj Saxena
University of Puerto Rico







Re: [ccp4bb] Yet another "what's my blob" thread

2017-10-02 Thread Roger Rowlett
Blob one is something possibly distorted by alternate partial occupancy
binding sites near a symmetry axis. Blob 2 could be a chloride ion, or if
that is not enough density, maybe bicarbonate. Blob 1 seems to be attracted
two His residues, of the things in your mixture, Mg(II) seems possible or
Cl-. Blob 2 is less likely to be  metal around those Arg residues, but Cl-
or bicarbonate are possible and well-known in this kind of coordination
environment. Cl- is in your mixture, and bicarbonate can accumulate,
especially at alkaline pH from atmospheric CO2.

Cheers,

Roger Rowlett

On Oct 2, 2017 6:06 PM, "Lucas" <lucasbleic...@gmail.com> wrote:

I'm in the later stages of solving a structure which contains two
tetramers in the asymetric unit. I found these two blobs (in
equivalent positions on each tetramer) with positively charged
residues around it. Crystallization condition is Magnesium chloride,
Bis-tris and PEG3350. While the second blob looks like a metal, the
first one has a weird shape even though they are expected to have the
same thing. Any ideas?

Lucas


Re: [ccp4bb] Stable Refinement as Low(ish) resolution

2017-07-12 Thread Roger Rowlett
Besides empirically adjusting the weighting factor for X-ray data to
increase the geometric constraints, have you tried jelly-body refinement or
refinement with external constraints? The latter two methods can be helpful
when refining low resolution data.

Roger Rowlett

On Jul 12, 2017 7:33 PM, "Christine Gee" <chr...@gmail.com> wrote:

> Hi Rhys,
>
> In Refmac, you can change the weighting of the Xray terms vs the geometry
> restraints rather than use what is automatically assigned. Have you tried
> this? In Phenix you can use secondary structure restraints (probably you
> can do this in Refmac too, but I haven't tried) and try checking the box
> optimize stereochemistry/Xray weight. This will probably help if your over
> fitting is moving things into density but making the geometry sub optimal.
>
> Regards
> Christine
>
> On Wed, Jul 12, 2017 at 4:17 PM, Rhys Grinter <rhys.grin...@monash.edu>
> wrote:
>
>> Dear All,
>>
>> I'm currently in the process of refining a low(ish) resolution structure
>> at 3.2 Ang, with a fair level of anisotropy. I processed the data through
>> the anisotropy server (https://services.mbi.ucla.edu/anisoscale/), which
>> elliptically truncated the data to 4.0, 3.8 and 3.2 Ang. This really
>> improved the maps and allowed me to trace the majority of the chain and
>> build most side chains.
>>
>> The R-factors are reasonable (0.29 work and 0.35 free respectively). but
>> I'm having trouble with over fitting in refinement as I continue to refine.
>> What parameters/restraints would the community generally use when refining
>> this kind of structure? Additionally Refmac doesn't seem to read the
>> structure factors from the anisotropy server output file properly, giving
>> vastly inflated R values and strange looking maps.
>>
>> Cheers,
>>
>> Rhys
>>
>> --
>> Dr Rhys Grinter
>> Sir Henry Wellcome Fellow
>> Monash University
>> +61 (0)3 9902 9213 <+61%203%209902%209213>
>> +61 (0)403 896 767 <+61%20403%20896%20767>
>>
>
>


Re: [ccp4bb] crystallization optimization

2017-07-12 Thread Roger Rowlett
Yes, we have our screenmaking robot programmed to set 50% diluted screens
and frequently employ this when a large fraction of the undiluted screens
result in precipitation. We have found quite a few productive hits this
way, as some previously grungy wells often present crystals when diluted.

Cheers,

Roger Rowlett


On Jul 12, 2017 12:01 PM, "Alun R Coker" <alun.co...@ucl.ac.uk> wrote:

> So, if we have a commercial 96 well screen where more than around 60% of
> the drops precipitate. It may be worth diluting the whole screen say (30ul
> screen and 20ul water in each well) and repeating . rather than
> diluting the protein.
>
> Has anyone ever tried this?
>
> All the best,
>
> Alun
>
> On 12/07/17 16:54, Frank von Delft wrote:
>
> Yes, exactly.  Thanks for doing the Right Thing and posting the actual
> diagram.
>
>
> On 12/07/2017 16:26, Patrick Shaw Stewart wrote:
>
>
> Alun
>
> I agree Frank's point is very interesting - and he intriguingly refers us
> to the phase diagram.
>
> Is the point that Line A is longer than Line B ?
>
> Best wishes
>
> Patrick
>
>
>
>
>
>
>
>
>
>
>
> On 12 July 2017 at 14:40, Alun R Coker <alun.co...@ucl.ac.uk> wrote:
>
> Hi Everyone,
>
> Franks point is really interesting. We routinely reduce the protein
> concentration when we see too many precipitated wells, but we never dilute
> the screen. Has anyone tried this?
>
> All the best,
>
> Alun
>
> On 12/07/17 08:48, Frank von Delft wrote:
>
> The point I was failing to make:  reducing either protein or precipitant
> concentration will indeed reduce nucleation, but often won't get you bigger
> or more single crystals:  it will just make the appearance of crystals less
> reliable.
>
> The way to get big single reliable crystals is to *increase* protein and
> *greatly* reduce precipitant.
>
> (Even better:  do seeding.  Like Vicky said.  Incredible how often people
> don't bother to do seeding, yet it solves so many problems.)
>
> phx
>
>
> On 12/07/2017 07:50, Vicky Tsirkone wrote:
>
> Dear Frank,
>
> I may see in the attached pic several nucleation points and a considerable
> amount of microcrystals. Based to my knowledge decreasing the concentration
> of the precipitant and/or the protein concentration would be a reasonable
> approach to refine the initial hits.
> By checking the diagram as you correctly mentioned you may see that the
> fine tuning of protein and precipitant concetration may lead to the
> desirable result without reaching the precipitation zone.
>
> Patrick just check your screens. Just a rule of thumb, if you see
> precipitation in the ~60% of your drops then you should definitely reduce
> the protein concentration.
>
> ps dont forget to try the *streak seeding*, as well.
>
> Have a nice day and again good luck.
>
> Vicky
>
> On Wed, Jul 12, 2017 at 8:50 AM, Frank von Delft <
> frank.vonde...@sgc.ox.ac.uk> wrote:
>
> Actually, you should try *increasing* the protein concentration - a lot.
> But be prepared to drop the precipitant concentration to almost nothing (1
> or 2% isn't "low").
>
> To understand why, look at the phase diagram and what we assume about
> vapour diffusion.  (Which I'm assuming is what you're doing.)
>
>
> On 12/07/2017 06:28, Vicky Tsirkone wrote:
>
> Dear Patrick,
>
> You may reduce the protein concentation, as well.
> Another option could be the *streak seeding* by exploiting the drop of
> your initial condition.
>
> Good luck,
>
> V.T.
>
> On Mon, Jul 10, 2017 at 7:17 PM, Patrick Shaw Stewart <
> patr...@douglas.co.uk> wrote:
>
>
> Microseed them into two or three random screens.
>
> Search for MMS and rMMS online.
>
> Good luck
>
> Patrick
>
>
>
>
> On 10 July 2017 at 15:47, Liuqing Chen <519198...@163.com> wrote:
>
> hello everyone!
> I get a condition (10% w/v PEG 6000, 100mm HEPES PH7.0) in which my
> protein grow small  needle like crystals, how can i optimize it to get
> bigger crystals?  the attach is the crystals  figure.
> thanks in advance
> sincerely
> Liuqing Chen
>
>
>
>
> --
>  patr...@douglas.co.ukDouglas Instruments Ltd.
>  Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
>  Directors: Peter Baldock, Patrick Shaw Stewart
>
>  http://www.douglas.co.uk
>  Tel: 44 (0) 148-864-9090 <01488%20649090>US toll-free 1-877-225-2034
> <%28877%29%20225-2034>
>  Regd. England 2177994, VAT Reg. GB 480 7371 36
>
>
>
>
>
>
> --
> Dr Alun R. Coker
> Senior Lecturer
> Wolfson Institute for Biomedical Research
> Un

Re: [ccp4bb] Dynamic light scattering instrument

2017-05-02 Thread Roger Rowlett

We've had a Malvern Zetasizer for many years.

___
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 5/2/2017 12:13 PM, Jie Liu wrote:

Dear all,

We are interested in using dynamic light scattering to evaluate 
crystallizability of proteins. What instrument would you recommend?


Many thanks and with best regards.

Jie





Re: [ccp4bb] protein precipitation reg

2017-03-29 Thread Roger Rowlett
Exactly. Tris is very cheap. HEPES not so much. On the other hand, 
zwitterionic buffers have significant advantages in terms of controlling 
inorganic anion or cation concentrations.


___
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 11:53 AM, David Briggs wrote:

It doesn't cost as much as HEPES, iirc.

On Wed, 29 Mar 2017, 16:36 Keller, Jacob, <kell...@janelia.hhmi.org 
<mailto:kell...@janelia.hhmi.org>> 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
<mailto:CCP4BB@JISCMAIL.AC.UK>] *On Behalf Of *Roger Rowlett
*Sent:* Wednesday, March 29, 2017 11:10 AM
*To:* CCP4BB@JISCMAIL.AC.UK <mailto: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 <mailto: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

--
--

David Briggs PhD
https://about.me/david_briggs

<https://about.me/david_briggs?promo=email_sig_source=email_sig_medium=email_sig_campaign=external_links>





Re: [ccp4bb] protein precipitation reg

2017-03-29 Thread Roger Rowlett
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





Re: [ccp4bb] Nitrate versus Carbonate

2016-11-11 Thread Roger Rowlett
Once again, chemical intuition may help. At neutral pH values, sulfate 
is going to be present at SO4(2-), whereas phosphate will be present as 
H2PO4(-) or HPO4(2-). The hydrogen bond network supporting binding may 
be able to offer clues. Sulfate is not likely to have any H-bond 
acceptors in its ligand sphere (e.g. no interactions with carbonyl 
oxygens or aspartate/glutamate).


Of course, having said that, we did publish a structure in which 
bicarbonate has an apparent Glu-O acceptor-acceptor interaction that 
makes no sense if you believe the Glu is deprotonated. We concluded that 
the Glu residue must be protonated in this structure even as the 
solution pH was near neutrality else there would be significant 
electrostatic repulsion. (Plus, there was a nice precedent for this 
ligand sphere around bicarbonate.)


Cheers,

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

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

On 11/11/2016 11:49 AM, Gulcin Gulten wrote:
Similarly, how do you differentiate a phosphate ion than sulfate just 
based on electron density if data is not at atomic resolution?


Thanks!


On Fri, Nov 11, 2016 at 3:52 AM, Harry Powell > wrote:


Hi all

Sticking to the first question, if you don't restrict yourself to
_X-ray_ crystallography but use your local neutron source instead,
it should be straightforward (subject to all the normal caveats).

On 10 Nov 2016, at 23:02, Tim Gruene wrote:


Dear JPK,

to answer your first question, at atomic resolution you would
notice a density
difference between N and C. At a little less resolution you might
still
measure difference in bond length.

Regrds,
Tim

On Thursday, November 10, 2016 8:41:43 PM CET Keller, Jacob wrote:

Dear Crystallographers,

I don't think there is any feasible way crystallographically to
distinguish
between nitrate and carbonate or bicarbonate-correct? But that
is not my
main question.

My main question is: given that nitrate and carbonate are both very
important and also very different physiologically, and therefore
they must
be distinguished/recognized by cells, how is this done, since
the ions are
so similar in structure? Is there some aspect of these ions that
differs
dramatically of which I am not aware? What kind of "handles" could a
protein grab onto to distinguish between nitrate and
carbonate/bicarbonate?

JPK


***
Jacob Pearson Keller, PhD
Research Scientist
HHMI Janelia Research Campus / Looger lab
Phone: (571)209-4000 x3159 
Email: kell...@janelia.hhmi.org
>
***



-- 
--

Paul Scherrer Institut
Tim Gruene
- persoenlich -
OFLC/102
CH-5232 Villigen PSI
phone: +41 (0)56 310 5297 

GPG Key ID = A46BEE1A


Harry
--
Dr Harry Powell
Chairman of International Union of Crystallography Commission on
Crystallographic Computing
Chairman of European Crystallographic Association SIG9
(Crystallographic Computing)














--
Post-Doctoral Research Associate
Texas A University
Dept. of Biochemistry and Biophysics
Interdisciplinary Life Sciences Building
301 Old Main Drive, Lab 2138
College Station, Texas 77843




Re: [ccp4bb] Nitrate versus Carbonate

2016-11-10 Thread Roger Rowlett
Bicarbonate ion is a weak base and is normally protonated near neutral pH.
(Nitrate is a pathetically weak base and is not protonated at any
reasonable biological pH.) Therefore, bicarbonate will have one hydrogen
bond donor group (the -OH group) and two hydrogen bond acceptor groups,
whereas nitrate will have only acceptor groups. This distinguishing feature
is clearly seen, for example, in the allosteric site of beta-carbonic
anhydrase (2A8D) where the bicarbonate binding site has one critically
placed hydrogen bond acceptor group (carbonyl oxygen of Val47) among a sea
of donor groups. Bicarbonate ions will populate this site in crystals after
a few minutes soak at 100 mM concentration. The same crystals soaked in as
much as 1M (!) nitrate overnight do not bind nitrate ion in this site.

Roger Rowlett

On Nov 10, 2016 3:42 PM, "Keller, Jacob" <kell...@janelia.hhmi.org> wrote:

> Dear Crystallographers,
>
>
>
> I don’t think there is any feasible way crystallographically to
> distinguish between nitrate and carbonate or bicarbonate—correct? But that
> is not my main question.
>
>
>
> My main question is: given that nitrate and carbonate are both very
> important and also very different physiologically, and therefore they must
> be distinguished/recognized by cells, how is this done, since the ions are
> so similar in structure? Is there some aspect of these ions that differs
> dramatically of which I am not aware? What kind of “handles” could a
> protein grab onto to distinguish between nitrate and carbonate/bicarbonate?
>
>
>
> JPK
>
>
>
>
>
> ***
>
> Jacob Pearson Keller, PhD
>
> Research Scientist
>
> HHMI Janelia Research Campus / Looger lab
>
> Phone: (571)209-4000 x3159
>
> Email: kell...@janelia.hhmi.org
>
> ***
>
>
>


Re: [ccp4bb] Two SGs in one droplet?

2016-10-28 Thread Roger Rowlett

I've seen this kind of thing before.

Case 1: two crystal forms in the same droplet, C2 and C222. If you 
looked closely, you could tell them apart and I was pretty good at 
getting a high percentage of the desired space group by looking at the 
crystal forms. The C2 form diffracted better, so I fished for that one.


Case 2: A mixture of crystals, C2 and C2 with the long axis doubled in 
length, caused by asymmetric ligand binding. In the "double-size" C2 
crystal form, ligands bound to only 10 of the 12 chains in the 
double-size ASU, which no longer conformed to two adjacent "normal-size" 
C2 ASUs. In the "normal" size C2 form, all 6 protein chains in the ASU 
bound ligand.


Cheers,

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

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

On 10/28/2016 9:13 AM, Sam Tang wrote:

Dear all

Sorry for going a bit off-topic in this thread.
May I seek your advice as on whether you have experienced that 
crystals being obtained from the same droplet, looking alike under 
microscope (rod shape) and in fact growing possibly from a same 
nuclei, give two space groups after indexing?


I recently obtain crystals for a protein (co-crystallized with a 
nucleic acid ligand) and collected two datasets from synchrotron. 
Although these two crystals are from the same drop, the SG and unit 
cell dimensions are very different:


Xtal1: C121 (156 60 105 90 111 90) (L-test, Pointless shows that there 
is no twinning), ~2.5 Angstrom

Xtal2: P1 (53 60 79 106 105 98), ~3 Angstorm

Would it be possible that the ligand changes the SG of the crystal so 
that only one of the forms contains the ligand?


Any advice is appreciated and thanks a lot in advance for your input.

Regards

Sam Tang
Biochemistry Programme, School of Life Sciences, CUHK





Re: [ccp4bb] suggestion for structure solution of a protein with low sequence identity

2016-10-25 Thread Roger Rowlett
I'll also recommend Buccaneer. You might try using a combination of 
PARROT for density modification and NCS averaging followed by 
autobuilding with BUCCANEER using initial phases from your MR solution. 
You only have two copies of the protein in the ASU, so you only get a 
modest boost in electron density averaging, but maybe every little bit 
helps. This approach was very successful for me with 8-fold NCS and a 
29% identity search model that gave poor but usable initial MR phases 
for 2.4A data.


___
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 10/25/2016 9:19 AM, Dmytro Guzenko wrote:


Hi Vikram,

Try Buccaneer, it works much better at such resolution than Arp/Warp.

Launch it several times with different parameters (e.g. use original 
structure as seed/initial/nothing), then open the results together and 
merge the pieces that look best and have lowest b-factors. Use Buster 
for refinement of the merged model and iterate with Buccaneer again. 
That's what worked for me once to bootstrap a poor MR solution (~20% 
of the structure originally) to a near-complete model at 2.7A.


You are in a sense replicating what phenix.autobuild attempts to do, 
but you'll be able to do it better (and probably faster) manually.


Kind regards,

Dmytro.
On 25/10/16 14:16, Vikram Dalal wrote:

Hi everyone,

We are trying to solve a protein structure of 2.6 A. We have 
processed it with HKL2000. We have even tried processing with mosflm 
and xia2. It is in C2221 space group (checked by pointless) and data 
is not twinned. It has 31% identical with a search model and has 57% 
sequence coverage. There are 2 subunits in asu.


I did not get proper phases with MOLREP and phaser. I have tried 
Balbes (R free 50) and Mr BUMP (R free 54).But, density from balbes 
look some reasonable.
So i have refined it in ccp4i and phenix differently and then model 
build it by coot. My protein has 377 Amino acid. But, I stucked at R 
free 41 and now i have amino acid 50 to 369 in both chains, still 49 
amino acid at N terminal and 8 amino acid at C terminal are missing 
and some loops are missing in between too.


*I have tried the ARP/wARP, but it does not work for it. *
*
*
*I have even tried phase and build of phenix but condition remain 
same. i got stuck at R free at 42 and same around 49 amino acids at N 
terminal and 8 amino acids at C terminal and some internal loops 
still absent.*

*
*
*Thank you in advance.*



*
*













Re: [ccp4bb] Structural biology software that does not run on Windows or gives important Windows-specific problems

2016-10-14 Thread Roger Rowlett
Except for graphics-intensive programs (e.g., Coot, Pymol), it is 
possible to run Linux within a VM in windows, and you can even share 
files with the other OS. I actually do it the other way 'round, running 
Windows in Linux via wine to have a one-box solution for processing data 
from our Oxford Diffraction instrument for which the software, 
CrysalisPro (unfortunately) runs only on Windows at the present.


I would personally find doing my entire workflow in Windows somewhat 
excruciating, especially for maintaining multiple student workstations 
and data servers. Linux is much easier to maintain for protein 
crystallography work, I think, for a variety of reasons.


___
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 10/14/2016 11:14 AM, Mark J van Raaij wrote:

Dear All,

our institution requires me to provide a reasoning not to buy a Windows 
computer (I want to buy a new MacOSX system), so I am looking for software that 
does not run or is limited on Windows.

Not available:
(Auto)SHARP
ARPWARP

Available on Windows but with significant limitations
Phenix (no MR-Rosetta, no parallelization)
CCP4 (limitations on file-names)

Please correct me if pertinent and provide additional examples if possible.

Gratefully yours,

Mark

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
http://wwwuser.cnb.csic.es/~mjvanraaij


Re: [ccp4bb] Ambiguous metal ion at active site.

2015-07-09 Thread Roger Rowlett
If the metal center is a stable complex then ICP-OES or XRF (e.g. TXRF)
methods can easily identify and quantify metals present in a small sample
of protein.

Roger Rowlett
Dear All

I have solved a structure of a metal-ion dependent exonuclease enzyme. In
homologous structures, two or three Manganese ions are present at catalytic
center. However, I have used 2 mM MgCl2 in protein purification buffer. I
tried to fit both of these metal ions at catalytic center but in both cases
it still shows green density (Sigma level ~ 7) in difference map and low
b-factor (10) for these metal ions. For better understanding I have
attached the screenshot of metal ions with difference map on. Please
suggest me the possible reasons or methods to validate the presence of any
other metal ions at catalytic center.

Thanks in advance.

Regards
Dilip Kumar
Research Associate
Chemical and Systems Biology Unit
CSIR-Institute of Genomics  Integrative Biology
Delhi-110025


Re: [ccp4bb] Phaser solution and solvent content for arp warp

2015-06-29 Thread Roger Rowlett
I think I would be tempted to chainsaw one of the ensemble chains of 
2IT8 (they look very similar except for side-chain disposition) and use 
1 or 2 of these as search models in a Phaser run. If this works, you 
should see good Z-values and the final result inspected in Coot should 
show good molecular packing with clear solvent channels and no isolated 
molecules. Electron density should look reasonable, and if there are any 
known non-protein features (like metal ions) they should clearly show up 
in difference density.


If the initial Phaser solution looks OK in terms of packing, I would be 
tempted to take the phased generated and, using the sequence of your 
protein, subject it to auto-building with Parrot and Buccanner, using 
2-fold NCS (if you do in fact have 2 molecules in the ASU). With any 
luck, you should be able to auto-build 90% of your model. This approach 
worked for us for a medium resolution problem with 8-fold symmetry for a 
really marginal search model.


___
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 6/29/2015 3:10 PM, xaravich ivan wrote:

Hi everyone,
I think I have finally got a solution in Phaser (screen shot attached) 
as the TFZ  is 10.
However the solution PDB has 30 molecules in it as the search model 
was an NMR solution.
As I have 0.944 Angs resolution data pretty complete, I thought of 
building the initial model in ArpWarp.
I prepared a new PDB file form the Phaser solution output with only 
one molecule instead of 30. Prepared the sequence file in pir format 
for the target. Now the wilson plot says B-factor of 3.66 and solvent 
content 0.98 even if I cut off the low resolution data to 8.0 from 
20.0 and increase the high resolution to 1.0.
Initially when I calculated the mathews coefficient it showed the best 
estimate to be 2 molecules in the assymmetric unit.

Am I missing something again?

Thanks in advance,
Ivan



Re: [ccp4bb] distorted phosphate molecule geometry after refinement

2015-06-22 Thread Roger Rowlett
The metal ion is looking reasonable, and it is also chemically sensible 
based on the crystallization conditions. Add another water around the 
remaining positive difference density and see if the resulting geometry 
looks remotely octahedral. From the one, view provided, it does appear 
to be an approximately octahedral coordination sphere. If so, this 
interpretation might be a winner. If this is a surface site, it is very 
possible that the nickel ion (and associated waters) are at less than 
100% occupancy.


Depending on the wavelength selected and the quality of data collected, 
you might have some anomalous scattering that would help confirm the 
presence of nickel.


Cheers,

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

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

On 6/22/2015 7:27 PM, Keller, Jacob wrote:


Definitely Ni, and maybe add another two waters to fill in the 
density. Regarding B-factors, it depends on those of the surrounding 
side chains, and should be a bit higher than theirs. Also, since it’s 
probably not a biologically-relevant Ni site, it would have low 
affinity and therefore you could plausibly lower the occupancy to make 
it work (b-factor would decrease.)


JPK

*From:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf 
Of *ansuman biswas

*Sent:* Monday, June 22, 2015 7:04 PM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* Re: [ccp4bb] distorted phosphate molecule geometry after 
refinement


I tried refining with a phosphorylated His (NEP, attached figures 1 
and 2 ). After refinement the geometry looks fine, there are no short 
contacts and the B-factors on the attached phosphate are ~30A2, except 
for one O-atom which is at 50 A2. However, some positive density is 
showing up in the Fo-Fc map.




I also tried refining with unmodified His, but with a Ni-ion and 2 
water molecules. Ni was present in the crystallization condition. 
After refinement (3rd Fig attached), there is hardly much positive 
density in Fo- Fc map. However, the B-factors of the added Ni and 
water molecules are ~50A2. The Ni coordination site can have both His 
(predominant) and Lys.




The data was collected at wavelength 0.9A and has resolution 2.3A

Please suggest.



Regards,

Ansuman

On Tuesday, 23 June 2015 1:13 AM, Shane Caldwell 
shane.caldwel...@gmail.com mailto:shane.caldwel...@gmail.com wrote:


It's probably much less likely than metal coordination and it's hard 
to judge from only one angle, but phospho-histidine might be something 
else to consider.


http://www.jbc.org/content/276/5/3247.full

Shane

On Mon, Jun 22, 2015 at 2:15 PM, Roger Rowlett rrowl...@colgate.edu 
mailto:rrowl...@colgate.edu wrote:


I agree...one possibility is a M-His(2)-Lys-(OH2) site. Possible metal 
ions would include Zn(II), although Lys is a relatively rare ligand in 
zinc-metalloenzyme sites.


Cheers,

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

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



On 6/22/2015 11:20 AM, Keller, Jacob wrote:

Looks to me like a metal binding site with those histidines, 
perhaps--any chance of that? That might also explain the weird 
geometry issues.


JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK 
mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dale Tronrud

Sent: Monday, June 22, 2015 11:17 AM
To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] distorted phosphate molecule geometry after 
refinement


It is possible that your PO4 has its atoms labeled with the wrong 
chirality.  Yes, I know that PO4 is not chiral when you ignore 
hydrogen atoms and single/double bonds but adding labels creates an 
unnatural chriality.  Try your refinement again after switching the 
labels on two oxygen atoms.


Dale Tronrud

On 6/22/2015 7:48 AM, ansuman biswas wrote:

Dear CCP4 users,

I am working on a protein from a hyperthermophilic archaeon.

I have collected mutliple X-Ray datasets, both from home source and
synchrotron and always found a clear density for tetrahedral geometry,
co-ordinated by two histidines and one lysine.

I tried fitting phosphate there, but its geometry always gets
distorted after each refinement cycle (Refmac 5.8.0073). Also I found
some short contacts between the coordinated residues and phosphate
which were very difficult to remove.

I am attaching a figure with the density and phosphate.

Kindly suggest -
1. if this may be a possible modification of any of the associated
residues, and the code of the modified residue to be used.

2. If the ligand requires separate restraints during refinement, I am
using the restrained refinement option available

Re: [ccp4bb] distorted phosphate molecule geometry after refinement

2015-06-22 Thread Roger Rowlett
The metal ion is looking reasonable, and it is also chemically sensible 
based on the crystallization conditions. Add another water around the 
remaining positive difference density and see if the resulting geometry 
looks remotely octahedral. From the one, view provided, it does appear 
to be an approximately octahedral coordination sphere. If so, this 
interpretation might be a winner. If this is a surface site, it is very 
possible that the nickel ion (and associated waters) are at less than 
100% occupancy.


Depending on the wavelength selected and the quality of data collected, 
you might have some anomalous scattering that would help confirm the 
presence of nickel.


Cheers,

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

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

On 6/22/2015 7:27 PM, Keller, Jacob wrote:


Definitely Ni, and maybe add another two waters to fill in the 
density. Regarding B-factors, it depends on those of the surrounding 
side chains, and should be a bit higher than theirs. Also, since it’s 
probably not a biologically-relevant Ni site, it would have low 
affinity and therefore you could plausibly lower the occupancy to make 
it work (b-factor would decrease.)


JPK

*From:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf 
Of *ansuman biswas

*Sent:* Monday, June 22, 2015 7:04 PM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* Re: [ccp4bb] distorted phosphate molecule geometry after 
refinement


I tried refining with a phosphorylated His (NEP, attached figures 1 
and 2 ). After refinement the geometry looks fine, there are no short 
contacts and the B-factors on the attached phosphate are ~30A2, except 
for one O-atom which is at 50 A2. However, some positive density is 
showing up in the Fo-Fc map.




I also tried refining with unmodified His, but with a Ni-ion and 2 
water molecules. Ni was present in the crystallization condition. 
After refinement (3rd Fig attached), there is hardly much positive 
density in Fo- Fc map. However, the B-factors of the added Ni and 
water molecules are ~50A2. The Ni coordination site can have both His 
(predominant) and Lys.




The data was collected at wavelength 0.9A and has resolution 2.3A

Please suggest.



Regards,

Ansuman

On Tuesday, 23 June 2015 1:13 AM, Shane Caldwell 
shane.caldwel...@gmail.com mailto:shane.caldwel...@gmail.com wrote:


It's probably much less likely than metal coordination and it's hard 
to judge from only one angle, but phospho-histidine might be something 
else to consider.


http://www.jbc.org/content/276/5/3247.full

Shane

On Mon, Jun 22, 2015 at 2:15 PM, Roger Rowlett rrowl...@colgate.edu 
mailto:rrowl...@colgate.edu wrote:


I agree...one possibility is a M-His(2)-Lys-(OH2) site. Possible metal 
ions would include Zn(II), although Lys is a relatively rare ligand in 
zinc-metalloenzyme sites.


Cheers,

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

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



On 6/22/2015 11:20 AM, Keller, Jacob wrote:

Looks to me like a metal binding site with those histidines, 
perhaps--any chance of that? That might also explain the weird 
geometry issues.


JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK 
mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dale Tronrud

Sent: Monday, June 22, 2015 11:17 AM
To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] distorted phosphate molecule geometry after 
refinement


It is possible that your PO4 has its atoms labeled with the wrong 
chirality.  Yes, I know that PO4 is not chiral when you ignore 
hydrogen atoms and single/double bonds but adding labels creates an 
unnatural chriality.  Try your refinement again after switching the 
labels on two oxygen atoms.


Dale Tronrud

On 6/22/2015 7:48 AM, ansuman biswas wrote:

Dear CCP4 users,

I am working on a protein from a hyperthermophilic archaeon.

I have collected mutliple X-Ray datasets, both from home source and
synchrotron and always found a clear density for tetrahedral geometry,
co-ordinated by two histidines and one lysine.

I tried fitting phosphate there, but its geometry always gets
distorted after each refinement cycle (Refmac 5.8.0073). Also I found
some short contacts between the coordinated residues and phosphate
which were very difficult to remove.

I am attaching a figure with the density and phosphate.

Kindly suggest -
1. if this may be a possible modification of any of the associated
residues, and the code of the modified residue to be used.

2. If the ligand requires separate restraints during refinement, I am
using the restrained refinement option available

Re: [ccp4bb] distorted phosphate molecule geometry after refinement

2015-06-22 Thread Roger Rowlett
I agree...one possibility is a M-His(2)-Lys-(OH2) site. Possible metal 
ions would include Zn(II), although Lys is a relatively rare ligand in 
zinc-metalloenzyme sites.


Cheers,

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

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

On 6/22/2015 11:20 AM, Keller, Jacob wrote:

Looks to me like a metal binding site with those histidines, perhaps--any 
chance of that? That might also explain the weird geometry issues.

JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dale 
Tronrud
Sent: Monday, June 22, 2015 11:17 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] distorted phosphate molecule geometry after refinement

It is possible that your PO4 has its atoms labeled with the wrong 
chirality.  Yes, I know that PO4 is not chiral when you ignore hydrogen atoms 
and single/double bonds but adding labels creates an unnatural chriality.  Try 
your refinement again after switching the labels on two oxygen atoms.

Dale Tronrud

On 6/22/2015 7:48 AM, ansuman biswas wrote:

Dear CCP4 users,

I am working on a protein from a hyperthermophilic archaeon.

I have collected mutliple X-Ray datasets, both from home source and
synchrotron and always found a clear density for tetrahedral geometry,
co-ordinated by two histidines and one lysine.

I tried fitting phosphate there, but its geometry always gets
distorted after each refinement cycle (Refmac 5.8.0073). Also I found
some short contacts between the coordinated residues and phosphate
which were very difficult to remove.

I am attaching a figure with the density and phosphate.

Kindly suggest -
1. if this may be a possible modification of any of the associated
residues, and the code of the modified residue to be used.

2. If the ligand requires separate restraints during refinement, I am
using the restrained refinement option available at the top of the
GUI for refmac.

Thanking you,
yours sincerely,
Ansuman Biswas,
PhD student,
Dept. of Physics,
IISc



Re: [ccp4bb] CCP4 installation problem

2015-06-16 Thread Roger Rowlett

Appu,

You will have to edit your .tcshrc (startup) file to point to the 
correct setup script with a line like this:


source /usr/local/xtal/ccp4-6.5/ccp4.setup-csh

If you are using a different shell, e.g. bash, you will have to edit the 
appropriate startup file, e.g. .bashrc.


You will likely find the offending line pointing to the old installation 
in your run command (startup) file and can edit it.


Cheers,

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

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu
On 6/16/2015 11:13 AM, Appu kumar wrote:

Hello,
I have downloaded 'ccp4-6.5 and deleted the older version of ccp4 
installed. After installing the new version, it is giving the 
following problem on executing the ccp4i command.


Error in startup script: couldn't read file 
/usr/local/xtal/ccp4-6.2.0/share/dbccp4i/ClientAPI/dbClientAPI.tcl: 
no such file or directory

while executing
source [FileJoin [GetEnvPath DBCCP4I_TOP] ClientAPI dbClientAPI.tcl]
(file 
/home/appu/software/destination/ccp4-6.5/share/ccp4i/src/projectdirs.tcl 
line 23)

invoked from within
source [SearchPath TOP src projectdirs.tcl]
(file 
/home/appu/software/destination/ccp4-6.5/share/ccp4i/src/system.tcl 
line 3379)

invoked from within
source [file join $env(CCP4I_TOP) src system.tcl]
(file 
/home/appu/software/destination/ccp4-6.5/share/ccp4i/bin/ccp4i.tcl 
line 79)

invoked from within
source [file join  $env(CCP4I_TOP) bin ccp4i.tcl]
(file /home/appu/software/destination/ccp4-6.5/bin/ccp4i line 12)





I have deleted ccp4-6.2.0 version. Somehow ccp4-6.5 is looking for the 
older version upon execution.

I will be thankful your your advice.
Appu





Re: [ccp4bb] Easy way to generate symmetry-related protein chains?

2015-05-22 Thread Roger Rowlett
If you mean generation of pdb coordinates of specific symmetry chains (not
just viewing) then you can do this with the symexp command in pymol. Select
the desired symmetry partners and save as pdb. You may want to edit
duplicate chain id labels in coot or a text editor.

Roger Rowlett
On May 22, 2015 8:25 AM, Mark J van Raaij mjvanra...@cnb.csic.es wrote:

 Just wondering if there is an easy way to generate symmetry-related
 chains, necessary for instance to join protein chains into the biologically
 relevant multimers.
 What I do now is look up the correct symmetry and translation operator in
 COOT or PYMOL and input that in PDBSET, but there may be easier ways.

 in the CCP4bb archive I found the following tip for COOT:

 Extensions - Modelling - Symm Shift Reference Chain Here.

 but that does not appear to be available in COOT, or not anymore.

 Mark J van Raaij
 Dpto de Estructura de Macromoleculas
 Centro Nacional de Biotecnologia - CSIC
 c/Darwin 3
 E-28049 Madrid, Spain
 tel. (+34) 91 585 4616
 http://www.cnb.csic.es/~mjvanraaij



Re: [ccp4bb] CYS mod

2015-05-13 Thread Roger Rowlett
Are the Cys residues in question on the surface of the protein? DMSO 
(which is in the crystallization mix) is a weak oxidant and could 
conceivably contain impurities like dimethylsulfide and methanethiol 
which could form difulfides with surface Cys residues. Or there could 
have been sulfides carried over from the protein extraction and 
purification steps. Does -S-S-CH3 fit the density with correct geometry?

___
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 5/13/2015 9:49 AM, Bio Physics wrote:
Is there any reason to be S-S-CH3 modification of a specific CYS! 
Others CYSs are fine.


On Tue, May 12, 2015 at 5:27 PM, Finke Aaron (PSI) aaron.fi...@psi.ch 
mailto:aaron.fi...@psi.ch wrote:


What are the bond lengths- or, rather, the centers from blob to
blob? Looks a bit short to be S-O, could be S-S-CH3?

Cheers,
Aaron
--
Dr. Aaron Finke
Postdoctoral Fellow
Swiss Light Source
WSLA/217
CH-5232 Villigen-PSI
phone: +41 56 310 5652 tel:%2B41%2056%20310%205652
e-mail: aaron.fi...@psi.ch mailto:aaron.fi...@psi.ch

On May 13, 2015, at 0:15, Bio Physics biophysics.w...@gmail.com
mailto:biophysics.w...@gmail.com wrote:


No, it is not sulfenic acid or so, I had that in differend
structure. It is L shaped from S, and linear if I put SCX mutaion
(another 1.4A way a nice blob). My resolution is 1.3 A.



On Tue, May 12, 2015 at 4:18 PM, Artem Evdokimov
artem.evdoki...@gmail.com mailto:artem.evdoki...@gmail.com wrote:

Looks like a wrong shape for tetrahedral acid
...

On May 12, 2015 4:07 PM, Boaz Shaanan bshaa...@bgu.ac.il
mailto:bshaa...@bgu.ac.il wrote:

Hi,


As I've just written to David, maybe it's sulfenic acid
(result of radiation damage?). I've had those in some
cases. See attached scheme for the chemistry and other
possibilities for Cys modification by oxidation.

   Boaz

/Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

E-mail: bshaa...@bgu.ac.il mailto:bshaa...@bgu.ac.il
Phone: 972-8-647-2220  Skype: boaz.shaanan
Fax: 972-8-647-2992 or 972-8-646-1710 /
//
//
/

/


*From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK
mailto:CCP4BB@JISCMAIL.AC.UK] on behalf of Bio Physics
[biophysics.w...@gmail.com
mailto:biophysics.w...@gmail.com]
*Sent:* Tuesday, May 12, 2015 11:57 PM
*To:* CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK
*Subject:* Re: [ccp4bb] CYS mod


Hi All,

I have same type of modified CYS !!!
No BME.

On Tue, May 12, 2015 at 2:59 PM, David Schuller
schul...@cornell.edu mailto:schul...@cornell.edu wrote:

So far almost everyone is suggesting BME.

I didn't do the purification  crystallization
myself, but I am told:

Buffer was HEPES
cryo was ethylene glycol, glycerol and DMSO.
Some MgSO4 and KPO4
No BME, but maybe some DTT.

Purified from a proteobacterium.




On 05/12/15 15:20, Dyda wrote:

What is in buffer? Perhaps DTT and cacodylate?

Fred
 
[32m***
Fred Dyda, Ph.D.  Phone:301-402-4496
tel:301-402-4496
Laboratory of Molecular Biology Fax: 301-496-0201
tel:301-496-0201
DHHS/NIH/NIDDK e-mail:fred.d...@nih.gov
mailto:e-mail%3afred.d...@nih.gov
Bldg. 5. Room 303
Bethesda, MD 20892-0560   URGENT message e-mail:
2022476...@mms.att.net
mailto:2022476...@mms.att.net
Google maps coords: 39.000597, -77.102102

http://www2.niddk.nih.gov/NIDDKLabs/IntramuralFaculty/DydaFred

***
[m



-- 
===

All Things Serve the Beam

===
 

Re: [ccp4bb] Enzyme kinetics

2015-05-08 Thread Roger Rowlett
In these coupled enzyme assays, concentrations of [E1], [S], [E2] and 
[NADH] need to be chosen such that the rate of the second reaction is at 
least one to two orders of magnitude faster than the first, otherwise 
the measured rate -d[NADH]/dt will not be rate-limited by -d[S]/dt. 
Normally this is accomplished by using relatively high [E2] and [NADH] 
and relatively low [E1]. When varying the pH of the reaction, care must 
be taken to ensure that this condition is maintained, as the coupled 
reaction (P1 - P2) may also have a pH-rate variation. You can do a 
crude pH-rate profile by choosing a single concentration of [S] and 
assaying at various pH values to establish the approximate pH optimum. 
However, to establish the true pH optima you would need to establish 
values of kcat and kcat/Km at each pH value by measuring rates at 
various [S] values and fitting to the Michaelis-Menten equation (if M-M 
behavior is appropriate for E1).


Cheers,

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

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

On 5/8/2015 9:23 AM, rohit kumar wrote:

Dear all,

Sorry for off topic discussion. i have a doubt for the enzyme kinetic.


Inline image 2
Above is the reaction of my interest. it is a couple reaction.

I want to determine the Km and Vmax for the E1 enzyme by the help of 
E2 enzyme by decreasing the amount of NADH (at 340 nm).


if i don't know the optimum pH for E1.  So is it ok, for publication 
point of view, to  determine  the Km and Vmax value of E1 enzyme at pH 
7.5 ( a physiological pH) .


Suppose if i determine the optimum pH of E1 by the help of E2 enzyme, 
that will be solely depend on the behaviour of E2 at different pH (if 
i am not wrong).



Please suggest.

Thanks in advance.






--
WITH REGARDS
Rohit Kumar Singh
Lab. no. 430,
P.I. Dr. S. Gourinath,
School of Life Sciences,
Jawaharlal Nehru University
New Delhi -110067




Re: [ccp4bb] Off topic - Mercury Lamp for Akta

2015-05-05 Thread Roger Rowlett
I wouldn't mind knowing how to source this lamp as well. But FYI, the 
lamps are usable for years after the FPLC gives you the dire low 
intensity warning. We just ignore it until the lamp goes completely 
dark or it's impossible to measure normal absorbances. We've used the 
current lamp for many, many years and it stays on all the time.


___
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 5/5/2015 11:05 AM, Bonsor, Daniel wrote:


The mercury lamp (28-4042-25) offered by GE Healthcare for Akta 
systems come with the housing and is priced at $1345. We have plenty 
of sparing housing units and are interested in just the lamp. Does 
anyone here have any cheaper alternatives/suggestions than buying the 
lamp and housing?


Thanks in advance.

Dan

Daniel A Bonsor PhD.

Sundberg Lab

Institute of Human Virology

University of Maryland, Baltimore

725 W Lombard Street N370

Baltimore

Maryland

MD 21201

Tel: (410) 706-7457

http://www.sundberglab.org/Home.html





Re: [ccp4bb] cryo condition

2015-05-04 Thread Roger Rowlett
We rarely use glycerol anymore, because it seems to fail so often for 
many of our current proteins. Try glucose, 25-30%. This is most 
conveniently done by weighing 125-150 mg of glucose in a microcentrifuge 
tube, then addding well solution to the 0.5 mL mark and mixing until 
completely dissolved. Then you can try dunking crystals in the cryo 
solution, or, you can try the no-fail method (which does fail on 
occasion) of cryoprotecting directly in the crystallization drop by slow 
addition of the cryoprotectant. See 
http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Mounting+Protein+Crystals. 
We have often found the slow addition of a glucose cryoprotectant works 
for fragile, high solvent content crystals that are prone to cracking 
under osmotic stress.


Other alternatives include high concentrations of sodium malonate 
(1-2M), or high concentrations of sodium formate (I think around 4 M?). 
These could also be introduced gradually if required.


Good luck!

___
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 5/4/2015 2:43 PM, Faisal Tarique wrote:

Hello everyone

Can anybody suggest me a cryo condition for a crystal obtained in
MIDAS screen of Molecular Dimension:

G1 0.1MTris8.0G10.1Mpotassium chloride25% v/vSOKALAN®CP7 0.1MHEPES7.0

G20.3Mammonium formate20% v/vSOKALAN® CP 5 0.1MHEPES7.0

Crystals are in beautiful cuboid shaped but all sorts of PEG
combinations and Glycerol formulation failed to prevent it from
cracking and dissolving.

Has anybody faced a similar situation as mentioned above and what
precaution was taken to prevent it from cracking or dissolving.

Your suggestions will be of immense help

Thanks in advance


Re: [ccp4bb] Cleaved peptide density!

2015-04-22 Thread Roger Rowlett
If your protease depends on an oxyanion hole for stabilizing the 
transition state, fluoride ions are known to be a potent inhibitor of 
these proteases. (It is a quasi-diagnostic test for serine-type 
proteases, and related cysteine proteases.) This might allow you to get 
reactant bound without cleavage. It's worth a shot.


Cheers,

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

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

On 4/22/2015 3:40 PM, Radisky, Evette S., Ph.D. wrote:


We had a similar situation with a catalytically dead serine protease.  
Initially I was excited to think we might be seeing residual catalytic 
activity of the mutant enzyme on a highly specific substrate; however, 
the activity turned out to result from contamination with a very small 
amount of wt enzyme, likely as a result of using the same affinity 
column to purify both.  When we incubated the enzyme preparation with 
PMSF (an inhibitor that covalently modifies the catalytic serine and 
would not have affected the mutant), we eliminated the residual 
activity of the enzyme preparation. However, in our case we never were 
able to get crystals with the intact substrate, which apparently was 
not compatible with our crystal form.


Is there a covalent inhibitor of your cysteine protease that you could 
use to pre-treat your enzyme, to see if this eliminates the activity?  
If so this might help distinguish between residual activity of the 
mutant vs. contamination with wt enzyme.


Good luck!

Evette

Evette S. Radisky, Ph.D.
Associate Professor and Senior Associate Consultant

Department of Cancer Biology
Mayo Clinic Cancer Center
Griffin Cancer Research Building, Rm 310
4500 San Pablo Road
Jacksonville, FL 32224
(904) 953-6372

http://www.mayo.edu/research/labs/proteases-cancer/

*From:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf 
Of *Dipankar Manna

*Sent:* Monday, April 20, 2015 2:42 PM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* [ccp4bb] Cleaved peptide density!

Dear Crystallographers,

I am working with a cysteine protease. I co-crystallized the protease 
with some small chemically synthesised peptides of 7 amino acid 
residues. I mutated the active Cysteine residue with Alanine to avoid 
the peptide cleavage so that I can get the whole peptide bound with my 
protein. But interesting I got the density for a cleaved peptide with 
4 amino acids instead of the whole peptide. The resolution is 1.4 A, I 
can see the clear cleavage and the cleavage occurred exactly at the 
same peptide bond where it should. But I do not know how!


Now my question is why I am getting the cleaved peptide as I already 
mutated the active residue Cysteine with Alanine (this mutant did no 
show any activity when I checked with SDS-PAGE).


If anybody has the same kind of experience please advice me.

Thanks in advance.

Best,

Dipankar

--

Dipankar Manna

Research Scholar

Department of Chemistry

University of Oslo

Oslo, Norway





Re: [ccp4bb] HPLC vs FPLC for protein purification

2015-04-20 Thread Roger Rowlett
On Apr 20, 2015 8:28 PM, Roger Rowlett rrowl...@colgate.edu wrote:

 Depends on what you want to accomplish... If you have a liter of crude
 lysate, capacity should be one of the choices. A step gradient is fast
 but low resolution; a gradient elution has more resolution but will eat
 buffer and take much longer. Lower stationary phase particle size to get
 more resolution and flow rates must go down, etc etc. Choices must be made
 to achieve the desired goal in the desired time with available resources.

 Roger Rowlett
 On Apr 20, 2015 8:14 PM, Christopher Colbert 
 christopher.colb...@ndsu.edu wrote:

   Hi Roger,

  Which 2 do you pick?

  Cheers,

  Chris

   --
 Christopher L. Colbert, Ph.D.
  Assistant Professor
 Department of Chemistry and Biochemistry
 North Dakota State University
 P.O. Box 6050 Dept. 2710
 Fargo, ND 58108-6050
 PH: (701) 231-7946
 FAX: (701) 231-8324

   From: Roger Rowlett rrowl...@colgate.edu
 Reply-To: Roger Rowlett rrowl...@colgate.edu
 Date: Monday, April 20, 2015 7:07 PM
 To: CCP4BB@JISCMAIL.AC.UK CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] HPLC vs FPLC for protein purification

   Many protein purification media have large particle sizes or will not
 mechanically stand excessive pressure without crushing. (Superdex is an
 example of the latter.) In general, smaller stationary phase particle sizes
 give rise to higher selectivity and separation efficiency at the expense of
 flow rate and possibly capacity. Higher pump pressures are required to get
 flow through columns packed with tiny particles sizes. To borrow an
 analytical chemistry maxim: speed, resolution, capacity: pick any two.

 Roger Rowlett
 On Apr 20, 2015 6:45 PM, xaravich ivan xaravich.i...@gmail.com wrote:

 Hi CCP4eans,

  Do you guys have any preference in purifying a protein by SEC in FPLC
 system or using a solvent based HPLC system after the initial affinity
 column purification. Where would you prefer HPLC purification over standard
 FPLC?
 I have routinely seen HPLC based purification of organic molecules and
 small peptides but not so much of proteins.
 What in your experience are the Pros and Cons of each, in the field of
 protein purification?
 Any suggestions/insights welcome.

  Thanks,
 Ivan




Re: [ccp4bb] HPLC vs FPLC for protein purification

2015-04-20 Thread Roger Rowlett
Many protein purification media have large particle sizes or will not
mechanically stand excessive pressure without crushing. (Superdex is an
example of the latter.) In general, smaller stationary phase particle sizes
give rise to higher selectivity and separation efficiency at the expense of
flow rate and possibly capacity. Higher pump pressures are required to get
flow through columns packed with tiny particles sizes. To borrow an
analytical chemistry maxim: speed, resolution, capacity: pick any two.

Roger Rowlett
On Apr 20, 2015 6:45 PM, xaravich ivan xaravich.i...@gmail.com wrote:

 Hi CCP4eans,

 Do you guys have any preference in purifying a protein by SEC in FPLC
 system or using a solvent based HPLC system after the initial affinity
 column purification. Where would you prefer HPLC purification over standard
 FPLC?
 I have routinely seen HPLC based purification of organic molecules and
 small peptides but not so much of proteins.
 What in your experience are the Pros and Cons of each, in the field of
 protein purification?
 Any suggestions/insights welcome.

 Thanks,
 Ivan



Re: [ccp4bb] Crystallisation of a minority fraction monomers

2015-04-08 Thread Roger Rowlett
The problem with crystallization is that is selects for the least 
soluble, most packable species. Sometimes that works against what you 
would like to know. That could include oligomerization state as well as 
conformational state. For example, some of the allosteric carbonic 
anhydrases stubbornly crystallize only in the T-state, despite 
crystallization conditions that are known to preferentially stabilize 
the R-state, and for which the predominant R-state population can be 
confirmed by other methods.


Cheers,

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

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

On 4/8/2015 9:07 AM, Sebastiaan Werten wrote:

Dear all,

we are currently working on a protein that is known to exist in a 
monomer-dimer equilibrium. At the high concentrations used for 
crystallisation assays, the dimer is predominant and the monomer 
practically undetectable.


Nevertheless, one of the crystal forms that we have obtained contains 
the monomeric species, not the dimer.


I was wondering if anyone is aware of similar (published) cases, and 
if the phenomenon as such has been discussed in detail anywhere?


I did literature searches but so far couldn't find anything.

Any pointers would be much appreciated!

Best wishes,

Sebastiaan Werten.





Re: [ccp4bb] Number of Molecules in Asymetric Unit

2015-04-01 Thread Roger Rowlett
In the CCP4i GUI you will find Phaser Cell Content Analysis under the 
Analysis tab. IIRC, this calls up the Matthews Probability Calculator. 
It will give you a good idea of the likely number of search models in 
the ASU. For large numbers, e.g. 3, the most probable number is likely 
to be unreliable and you might try smaller numbers and observing the 
packing of the partial solutions to get a better clue of the correct number.


Cheers,

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

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

On 4/1/2015 11:45 AM, amro selem wrote:

Dear CCP4 Community ,
which program i should use in ccp4 backage to find how many molecules 
in Asymetric Unit .

All the best
Amr




Re: [ccp4bb] No MR solution

2015-04-01 Thread Roger Rowlett
There are 8 possible space groups in the P422 family. Did you search in all
of these? How many search models are likely to fit in the unit cell based
on  Matthews analysis? Then there are questions about model selection and
preparation. How identical is the search model to the target? Does it make
sense to search for the domains separately? Did you truncate the model side
chains?

There are lots of ways to skin the cat depending on the problems
encountered. 3.0 A may be challenging for a low identity search model.

__
Roger Rowlett
Gordon  Dorothy Kline Professor
Department of Chemistry
Colgate University
On Apr 1, 2015 11:06 AM, Sekharreddy M vijayasekk...@gmail.com wrote:

 HI All,
   I am trying to find MR solution for one of the protein crystals
 with 3.0A resolution, but not able find solution using Phaser (PHENIX).The
 protein has two domains.and the probable best space group identified was
 p4212(HKL2000).

  I am novice in using crystallization tools for solving structures.I am
 stuck at this stage  and I appreciate any help in this regard.

 Thank you.



Re: [ccp4bb] Cis-peptide bond checking

2015-02-16 Thread Roger Rowlett
Great reminder. But there are real non proline cis peptide bonds, including
highly conserved motifs in active sites, e.g. 3UAO and its homologs. I
would hope these don't get corrected.

Roger Rowlett
On Feb 16, 2015 5:09 AM, Tristan Croll tristan.cr...@qut.edu.au wrote:

  Dear all,


  My apologies for the spam-like nature of my post, but I would like to
 draw your attention to an important issue (outlined in an upcoming short
 communication to *Acta D*, which will appear at doi:10.1107/S1399004715000826
 once it's online). At present, neither the structural quality checks in
 commonly-used crystallography packages nor those run on deposition of a
 structure to the PDB are flagging the presence of non-proline *cis *peptide
 bonds. This has led to the presence of many erroneous *cis *bonds
 creeping into the PDB - primarily in low-resolution structures as one would
 expect, but I have identified clearly erroneous examples in structures with
 resolutions as high as 1.3 Angstroms. From my analysis, I estimate that a
 few thousand structures have been affected to some extent, with the worst
 cases having as high as 3% of their peptide bonds in *cis*. Particularly
 if you have published anything 2.5 Angstroms in the past few years, may I
 gently suggest that you make a quick double-check of your deposited
 structures? This can be done quickly and simply in Coot
 (Extensions-Modelling-Residues with Cis peptide bonds).


  Best regards,


  Tristan





Re: [ccp4bb] how to reduce protein solubility

2015-02-16 Thread Roger Rowlett
Bringing the pH closer to the measured pI would definitely help. The pI can
be measured on an IEF gel. Glycosylation, if applicable, could dramatically
increase solubility.

Roger Rowlett
On Feb 16, 2015 11:33 PM, Mattiroli,Francesca 
francesca.mattir...@colostate.edu wrote:

  Hi all,

 I am struggling with a protein complex that is too soluble. I have reached
 about 20 mg/ml but I still observe very little precipitation (clear drops
 in 90-95% of the tested conditions). The proteins are expressed in insect
 cells and going to higher concentration is not easily achievable.
 I have tried different buffer conditions (salt concentration and pH) and I
 am testing temperatures. I am at a loss with what to try next.
 Do you think PTMs (phosphorylation, acetylation) might be causing this?
 Any input on how to decrease solubility?

 Thank you very much in advance,

 Francesca





Re: [ccp4bb] adding a Cobalt atom within Coot

2015-02-02 Thread Roger Rowlett
This is a known bug in Coot 0.7.x. You can add the metal ion correctly 
using the Get Monomer dialog and selecting CO as the monomer. This is 
fixed in Coot 0.8.1.


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

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

On 2/2/2015 9:40 AM, Almudena Ponce Salvatierra wrote:

Hi,

this sounds weird but I am adding a Cobalt atom within Coot, with the 
option place atom at pointer, then I select other and type in the 
textbox CO. It adds an Atom that it calls A, why does this happen?


Any ideas?

Thanks in advance.

Best wishes,

Almudena

--
Almudena Ponce-Salvatierra
Macromolecular crystallography and Nucleic acid chemistry
Max Planck Institute for Biophysical Chemistry
Am Fassberg 11 37077 Göttingen
Germany



Re: [ccp4bb] PHASER MR solution

2015-01-28 Thread Roger Rowlett
The Z-values may be marginal, but of course you should inspect the 
solution in Coot to see if the electron density makes sense, and the 
packing of the protein molecules in the unit cell (look at symmetry 
mates) is sensible. If this passes the sniff test, then you should clean 
up your model (make it consistent with the target protein) and try to 
refine it. If your target and model are really different in terms of 
gaps, insertions, etc., and your electron density is good, this would be 
a good opportunity to do some auto-building with Buccaneer based on your 
initial phase solution, for example, to get to a better starting point 
for refinement. If you can't refine the MR solution and drive R and 
Rfree down significantly, you may still have some problems. With 70% 
sequence identity, I would expect your model to be pretty close to your 
target, and autobuilding may not be necessary to get to a good starting 
point, although this can often save you some time.


___
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 1/28/2015 12:25 PM, jeorgemarley thomas wrote:

Hi, all

As per the suggestions, I hv done with the phaser MR and the solution 
has come with screw axes P 21 21 21. here I am attaching the output 
text from Mr and sol file. So Now should I go ahead with this? Please 
suggest.


Thank you very much in advance !

On Tue, Jan 27, 2015 at 9:33 PM, jeorgemarley thomas 
kirtswab...@gmail.com mailto:kirtswab...@gmail.com wrote:


Thank you so much to all for your kind concern.



Jeorge

On Mon, Jan 26, 2015 at 5:55 PM, Kay Diederichs
kay.diederi...@uni-konstanz.de
mailto:kay.diederi...@uni-konstanz.de wrote:

Dear Jeorge,

you'll find some information about this in

http://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/Space_group_determination
. A practical and easy way is described in
http://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/Pointless

HTH,

Kay

On Mon, 26 Jan 2015 11:24:27 +0100, Tim Gruene
t...@shelx.uni-ac.gwdg.de mailto:t...@shelx.uni-ac.gwdg.de wrote:

Dear Jeorge,

XDS make no claim to determine the SPACE GROUP but rather the
LAUE
GROUP, as only the latter is taken into account during data
integration.

This is definitely so during the indexing step (IDXREF.LP),
but even in
CORRECT, when systematic absences are sometimes indicated,
XDS does not
really choose the space group.

Best,
Tim

On 01/26/2015 05:46 AM, jeorgemarley thomas wrote:
 Hello Dr. Randy
 Here is the IDXREF.LP I got in which, on the basis of
quality of fit, I
 went for this space group well I would also try for the
other screw axes.
 So should I Integrate the data from beginning with all
possible screw axes
 of orthogonal space group?  I am attaching the IDXREF.LP
screen shot here.

 Jeorge

 On Sun, Jan 25, 2015 at 5:00 PM, Roger Rowlett
rrowl...@colgate.edu mailto:rrowl...@colgate.edu wrote:

 Did you search all 8 possibilities of screw axes, e.g.
P2221, P21212,
 P212121, etc?

 Roger Rowlett
 On Jan 25, 2015 4:50 AM, jeorgemarley thomas
kirtswab...@gmail.com mailto:kirtswab...@gmail.com
 wrote:

 Hi, I have processed the data using XDS and space group
found to be P 2 2
 2 (16) and I used the phaser MR for first phase
determination. The model I
 have used has has more than 70 % sequence identity, when
I run the phaser I
 got the message which I have attached here. And only sum.
file I got as an
 output. Does any one have suggestion what should I do ? I
would highly
 appreciate your kind suggestions. Thank you in advance.





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

GPG Key ID = A46BEE1A









Re: [ccp4bb] Queries regarding bead beater and french press.

2015-01-12 Thread Roger Rowlett
The Bead Beater has a 15, 40, and 350 mL chambers. I haven't used mine 
to homogenize yeast, but I suspect it is similar in performance to E. 
coli disruption. (Different bead sizes are used for yeast than 
bacteria.) We get excellent, gentle disruption of E. coli in 8 minutes 
total. A French Press takes a loong time for more than 40-50 mL 
of lysate, and often requires multiple passes for complete disruption. 
Beads and cell wall debris can be easily removed by centrifugation for 
direct application on your FPLC.


We moved to Bead Beaters from a French Press more than a decade ago and 
haven't looked back. We can do one liter overexpression preps (20-30 g 
wet packed cells) in the 40 mL chamber.


Cheers,

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

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

On 1/12/2015 10:44 AM, Johnson Luwang wrote:

Dear All,
I am looking for an equipment that can do /Saccharomyces 
cerevisiae/ cells lysis (say about 100-200ml lysate volume). I have 
used the Constant Systems cell disruptor TS 0.75kW model (at another 
facility)  that can go up to 40kpsi.  It works perfect for my yeast 
cell lysis experiment.   We were planning to set up similar facility 
at our place, but some of my colleagues suggested me about bead 
beater  for the same purpose.  Quite frankly I don't have much idea 
about bead beater. And I need the suggestions from the experts who 
have used these two systems (french press and bead beater). Is the 
bead beater better than the french press for yeast cell lysis? If so 
can you suggest me a bead beater model which is the best for our purpose?


Many thanks.

Best Regards,
Johnson L.W.







Re: [ccp4bb] Some advices on model modification

2015-01-06 Thread Roger Rowlett
How you approach this will depend substantially on the sequence identity 
between your target and your potential MR models. Definitely remove all 
non-protein atoms from your search model, as these are not likely at all 
to be present, or present at these positions, in your target. In 
addition to using a program like Chainsaw to truncate your model, you 
might also consider using models truncated at the N- or C-terminus (if 
these are relatively mobile or ill-defined), or consider searching with 
multimers (e.g., dimers) for targets that are homo-oligomers with 
expected quaternary structure symmetry. For difficult targets in which 
there were significant gaps and insertions in the target compared to the 
available search models, we used homology-based protein folding 
prediction (e.g., Phyre) to prepare search models. For marginal models, 
it is possible to get a reasonable crude solution using MR, followed by 
density modification and autobuilding to trace a better solution (e.g. 
PARROT  BUCCANEER), especially if you have NCS in your target. This 
worked really nicely for us for 3UAO, where search models had 30% or 
less identity.


Cheers,

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

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

On 1/6/2015 8:41 AM, vellieux wrote:

Hello,

Keeping the waters in a model used for molecular replacement search is 
not a very good idea.


I'd suggest first that you use a program like Chainsaw and possibly 
additional programs to fine-tune your search model to the problem that 
you are trying to solve.


Also, reading material such as the ccp4 study weekend proceedings 
devoted to molecular replacement and similar material (that are 
available on the internet) would be a good idea as well. The problems 
you are encountering have been discussed in the literature.


Fred.


On 06/01/15 13:06, allen price wrote:


Dear all:

I got a dataset at 2.8 angstron. I have tried several ways such as 
phaser, MRBUMP,BALBES,but still can't solve the


data,which means I have to edit my model. Maybe I'd better cut it off 
or delete the water or loop. I really have no


idea,as it is my first time to do such things, I alway used the whole 
model to mr. Could anyone give me some advice?


what kind  of software do you guys use? really need you help!

Best regards,

Allen



--
Fred. Vellieux (B.Sc., Ph.D., hdr)

IBS / ELMA
Campus EPN
71 avenue des Martyrs
CS 10090
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Tel: +33 457428605
Fax: +33 476501890




Re: [ccp4bb] CrystalClear tape rolls

2014-12-01 Thread Roger Rowlett
I use 3 clear duck tape. Amazon has it in packages of 6 rolls. Seems to
work OK, with maybe one well in a 2x96 well screen interacting with the
adhesive. I think it's an isopropanol or dioxane condition.

Roger Rowlett
On Dec 1, 2014 5:32 AM, Mark J van Raaij mjvanra...@cnb.csic.es wrote:

 Dear All,

 Just wondering what the current situation on CrystalClear tray sealing
 tape is.
 Molecular Dimensions, Hampton, Jena seem to be selling mainly sheets or
 strips precut for plates - I guess related with the fact that the consumer
 box sealing tape changed specifications and now clouds over with certain
 reservoir solutions. See:
 https://www.mail-archive.com/ccp4bb@dl.ac.uk/msg00622.html
 We are stilll using rolls which came with plates we bought years back, but
 now only have two rolls left - so I am wondering if there is a more
 economic solution than buying sheets or strips.

 Greetings,

 Mark

 Mark J van Raaij
 Lab 20B
 Dpto de Estructura de Macromoleculas
 Centro Nacional de Biotecnologia - CSIC
 c/Darwin 3
 E-28049 Madrid, Spain
 tel. (+34) 91 585 4616
 http://www.cnb.csic.es/~mjvanraaij



Re: [ccp4bb] workstation crystallography

2014-11-11 Thread Roger Rowlett
I'm running home-built Ubuntu boxes with old, plain-vanilla CPUs (e.g., 
Q9300 or core i3/i5/i7) and 6-8Gbyte of RAM, and a cheap Nvidia video 
card (e.g. GT 9xxx or GT 620).This is more than sufficient to do routine 
structure solution. Any contemporary desktop or laptop computer should 
be sufficient, although if running Linux I have not had good luck with 
integrated Intel graphics. (If you want stereo display, you have to 
choose a compatible graphics card and video monitor.) It's not like the 
old days, where you needed special, dedicated hardware (remember Silicon 
Graphics?) to get the graphics and computing performance required. 
Current technology is more than sufficient for routine work.


___
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 11/11/2014 8:27 AM, abhishek jamwal wrote:

Dear ccp4 bb members,

I need to buy a desktop workstation for the purpose of running 
crystallography related applications. I have short-listed HP's Z420 
and Dell's T7600, I chose this because their configuration description 
looks impressive (8 cores, 16 threads, 3.6 GHz processor etc.). 
However, I have no practical idea about their performance.


Can anyone , who has experience with these workstations comment on 
 performance ? And whether these workstations are optimal/suboptimal 
for the desired purpose ?


what other options do I have apart from dell and hp ? Please suggest.

*Is desktop iMac a good option for this purpose ?*


many thanx in advance


abhishek









Re: [ccp4bb] Adding water molecule and metal atom

2014-10-24 Thread Roger Rowlett
Adding metal ions will work properly through the Get Monomer dialog in 
0.7.2. Until Coot is updated (either manually or through a new CCP4 
release) this is a reasonable workaround.

___
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 10/24/2014 10:02 AM, Pamela J Focia wrote:


Until the Coot bug is worked out, why not simply change the water 
molecule you want to be an ion into the atom you want it to be in the 
pdb file with a text editor?


-=pam


On Oct 24, 2014, at 5:09 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de 
mailto:t...@shelx.uni-ac.gwdg.de wrote:



Dear Jeorge,

from the second last thread, I guess you are using Coot 0.7.2?
Apparently this is a bug that you get around by updating Coot.

Regards,
Tim

On 10/24/2014 10:13 AM, jeorgemarley thomas wrote:

Hi All,

First of all sorry to ask such simple question over here. I have added
water molecule in my protein molecule in coot, also some Ba atom. I have
added the water molecule manually as when it was added automatically the
water added everywhere it find lobes of electron density. And also 
when I

add Ba atom it gets converted as water molecule.

Your kind help will be greatly appreciated
Regards

Jeorge



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

GPG Key ID = A46BEE1A



---


Pamela J. Focia, Ph.D.
Research Assistant Professor

Structural Biology Facility Manager
Robert H. Lurie Comprehensive Cancer Center
in the Departments of:
Molecular Pharmacology and Biological Chemistry, Feinberg  School of 
Medicine,

and Molecular Biosciences,  Northwestern University
303 E. Chicago Ave., S-215,  Chicago 60611
o (312)503-0848
c (312)286-3274
f (312)503-5349
fo...@northwestern.edu mailto:fo...@northwestern.edu












Re: [ccp4bb] Adding zink and coper by COOT

2014-10-23 Thread Roger Rowlett
There is a bug in version 0.7.2 Coot that causes metal ions added via 
the place atom at pointer to be a water. However, if you add the metal 
ions through the Get Monomer dialog I think it will work OK.


___
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 10/23/2014 1:32 PM, amro selem wrote:

Dear ccp4bb Community ,
i hope this email finds you well, i am trying to do add Zink and 
Copper to my Superoxid dismuase Enzyme by Coot , i encountered two 
problems.
1- after adding Zink and copper by place atom at pointer   other  
 writing ZN OR CU then running Refmac5 , both atom is turned to water
2- i traied to  added  atom and merge the atoms with coordinate file 
out side the coot. the atoms are added but i see red patches arround 
them. i also added calcium instead then i replaced them bz editing the 
PDB file.

3- the Rfactor 16 and R free is 22.1
 so my question
how can i add zink and copper  in right way. second what is the red 
patches mean?

thank you in advance
Amr






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

2014-10-17 Thread Roger Rowlett
You've tried the obvious things. Lowering the IPTG concentration doesn't 
really work all that well, at least in my hands. We find we can still 
get maximal expression from many promoters like trc with only 0.2 mM 
IPTG, and the response doesn't modulate well: it's either on or off. 
Growing at lower temperature, like 18-22 deg C sometimes works, but 
sometimes not. Here are a few more approaches:


1. Use leaky expression in a pET vector system. Just don't induce it
   at all and grow for 4-18 hours. The slow expression may allow for
   protein folding before kinetic trapping in protein tangles. You may
   want to combine this with lower temperatures and less rich media
   (e.g., LB instead of TB) to slow growth and protein synthesis.
2. Put multiple, tandem copies of your gene behind the promoter. I'm
   not sure how this works, but it may tie up enzymes transcribing the
   message from the plasmid to slow mRNA transcription and thus
   translation. I never really understood the chemistry behind this,
   and no one could explain it to me in a cohereht way. However it
   works, it can slow protein expression enough to allow for soluble,
   instead of precipitated protein. This works like a charm to express
   the alpha subunit of trp synthase. The vector has 4 tandem copies of
   the gene after a trc promoter. You can make 1000 mg of soluble
   protein per 100 mL of culture (!) (I didn't mess up my zeros.)
3. Express your protein with a solubilizing tag on the N-terminus. I
   think there are some commercial vectors that put NusA in front of
   your gene. This may allow for soluble expression. NusA is supposedly
   better than 6X His tagging for making soluble protein. (Hasn't
   worked for me yet, but the literature suggests some successes.)
4. Try a different promoter. We use a modified version of pTrc99 that
   has at trc promoter instead of the T7 promoter. It is tightly
   controlled by IPTG, with no leaky expression. It can be a fierce
   promoter, but it may not be as fierce as T7, and can give you
   different results.

Have fun! And good luck!

___
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 10/17/2014 8:51 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] comparisons - views

2014-10-17 Thread Roger Rowlett
I've had a Gryphon for 2+ years and use it in an undergraduate 
environment. It's been trouble-free, and there are no instrument 
consumables, just blocks and trays. OK, I do have to feed it deionized 
water and a PCR tube of diluted protein for each set. It can set a 
96-well tray in under 2 minutes. The basic protocol I use is 200+200 nL 
drops. The software is easy enough to use that my undergrads know how to 
program it to do 1 or 2 drop screens or partial plates. I don't have the 
LCP module but you can get that installed or retro-fitted.


I'm pretty sure the acquisition cost of the Gryphon is much less than 
the Mosquito and NT8. I squeezed mine on a NSF-RUI grant as project 
research equipment.


Cheers,

___
Roger S. Rowlett
Gordon  Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
On 10/17/2014 1:56 AM, Dean Derbyshire wrote:


Apologies for the slightly off topic… thought this was the best way to 
get views from a wide (relevant) audience.


Any views on differences – pros and cons – and experiences with:

Mosquito; Gryphon and NT8?

And similarly with Minstrel and Rock imager.  Related to that last 
‘comparison’ what’s the prevailing thoughts on SONICC vs standard UV 
laser technology… any experiences with coping phase separation or 
condensation ?


Thanks

Dean

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   Box 1086

   SE-141 22 Huddinge

   SWEDEN

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Direct: +46 8 54683219

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Re: [ccp4bb] Off topic: reformatting screens from Falcons into deepwell blocks

2014-10-01 Thread Roger Rowlett
The Art Robbins Scorpion has this function baked in the the operating 
software. When I was shopping for liquid handling robots for 
screen-making, only the Emerald Biosystems Opti-Matrix and the Scorpion 
were affordable for a small lab. The Scorpion can handle more solutions 
at one time.


___
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 10/1/2014 10:38 AM, Blankenfeldt, Wulf wrote:


Dear colleagues,

we are trying to find a cost-effective solution to reformat commercial 
crystallisation screens from 15ml-Falcons into 1- or 2ml-deepwell 
blocks using a liquid handling robot.


We have spoken to various vendors but are a bit shocked by the prices 
they ask for their solutions. However, since the machines we have seen 
seem to be aiming at flexibility while we are looking for something 
simple to do just a single task, we hope that there are simpler 
machines out there that we are simply unaware of.


I’d therefore like to ask this community for advice – is there any 
robot that you can recommend for this task?


Thanks in advance,

Wulf




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Re: [ccp4bb] frozen pellet insoluble protein

2014-09-29 Thread Roger Rowlett
I have experience with some proteins that don't tolerate freeze-thawing 
very well. It's hard to say exactly what the physical chemistry of this 
is, but it probably relates to (1) aggregation due to high concentration 
or protein or salts during the freezing process as water is removed, 
and/or (2) pH shifts due to changes in pKa of buffers/proteins as the 
temperature is lowered. Usually freezing in whole cells is less 
problematic than freezing purified protein solutions, but there are no 
absolutes. One protein we worked on could only be stabilized from cradle 
to grave in 20% glycerol, 100 mM DTT, and 4 deg C. Would not tolerate 
freezing, ever. Not even in cell pellets. Died at 25 deg C in a couple 
of hours--had to work quickly to do kinetics. Worst...protein...to work 
on...ever.


Cheers,

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

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

On 9/29/2014 11:02 AM, Andreas Förster wrote:

Dear all,

I've encountered people who refuse to freeze cells and always lyse 
fresh pellets.  Better protein, they say.  I've never had reason to do 
so myself, or even to believe in their voodoo.  Up until now, maybe.


My protein expresses well and is almost all in the soluble fraction in 
an expression test from a fresh pellet.  The large-scale expression 
from the same pellet, now frozen and thawed, yielded 90% insoluble 
protein.


If it's the freezing that dooms the protein, I'm happy to redo the 
fermentor run.  Are there other examples out there of this?


Thanks.


Andreas






Re: [ccp4bb] refine structure with mtz file in P212121 or P222?

2014-09-05 Thread Roger Rowlett
As always, look at the unit cell packing of your alternative solutions. 
In all likelihood one of these two solutions from Phaser should pack 
sensibly in the unit cell, and the other will not. You may get some sort 
of quasi-reasonable-looking electron density out of the wrong solution 
initially, but usually not.


It is possible that you indexed, integrated, and scaled in P222 and 
P212121, but if you do a full automated search in Phaser it will look 
for solutions in both space groups, and may have written out a solution 
in the same spacegroup both times.  (Check the log file or PDB header.)


Cheers,

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

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

On 9/5/2014 11:54 AM, Wei Shi wrote:

Hi all,
I am working with a protein-ligand complex structure. The data was 
indexed with XDS and mtz file is generated using Phenix---Reflection 
tools---Reflection file editor. The space group used was P222. Then, I 
used the Phenix---Molecular replacement to find a solution (try all 
possible in same pointgroup), and the MR solution was in space group 
P212121. And then, I reindexed the data in XDS using space group 
P212121 and then generate the mtz file in space group P212121. So, I 
have two mtz file, in space group P212121 and P222.
When I refine the MR solution with the different mtz files, the map 
generated is not same for the ligand. The statisitcs is better with 
P222 mtz file than P212121 mtz file. I am wondering why the density 
for the ligand and the statistics is different using mtz file in 
different space groups and which mtz file should I use to get the 
final structure model.


Thank you so much!

Best,
Wei


Re: [ccp4bb] Off topic: Precast gels

2014-08-29 Thread Roger Rowlett
Define cheap. Several vendors offer SDS PAGE minigels for about $10 US a
pop. I get mine from Novex.

Roger Rowlett
On Aug 29, 2014 2:22 AM, Theresa Hsu theresah...@live.com wrote:

 Dear all

 Would anyone knows of source of cheap precast SDS-page gels?

 Thank you.



Re: [ccp4bb] Exporting Omit Maps for use in Pymol using CCP4i FFT

2014-08-20 Thread Roger Rowlett
Did you enter a valid pdb filename for a coordinate file when selecting the
option 'cover all atoms in pdb'?

__
Roger Rowlett
Gordon  Dorothy Kline Professor
Department of Chemistry
Colgate University
On Aug 20, 2014 3:54 PM, Wei Shi wei.shi...@gmail.com wrote:

 Hi all,
 I am trying to display F0-Fc omit map in PyMol. I got the mtz file from
 Phenix, and now use CCP4i FFT to generate the map for PyMol. I followed the
 instructions from the below link:

 http://www.p212121.com/2010/04/26/display-a-mtz-file-in-pymol/

 And, I constantly got the following error message:
 There is no file name for parameter EXTEND_XYZIN. Do you want to abort
 this run to enter the file name before running?

 I don't know how to enter the file name for parameter EXTEND_XYZIN. Does
 any of you happen to know how to do this? Thank you so much!

 Best,
 Wei



Re: [ccp4bb] Removing PEG3350

2014-08-20 Thread Roger Rowlett
Excellent references. PEG 3350 appears to be hydrodynamically equivalent 
to a 20 kD globular protein. So for efficient separation, your protein 
needs to be significantly larger than 20 kDa on a GEC column. In a 
centrifugal filter (which is very inefficient--you need many exchanges 
and dilutions with buffer to get nearly quantitative removal) it is 
possible that snaking of linear polymer molecules through the pores 
might contribute to slightly more efficient removal than expected based 
solely on hydrodynamic radius.


GEC or a desalting column is definitely the quickest way to do this, if 
possible. Flow rates may have to be slow (hence a typical flow rate 
column separation) to allow for efficient distribution of solutes in the 
sample solution if it has increased viscosity.


Cheers,

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

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

On 8/20/2014 4:18 PM, Reza Khayat wrote:

Hi,

I managed to significantly reduce the viscosity of the PEG solution via buffer 
exchange using a 100kDa MWCO ultrafiltration device. The following papers have 
fantastic tables of solutes with their hydrodynamic radii. Definitely worth a 
read, followed by printing and posting of the tables on walls next to the FPLC 
:)

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3055910/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1304934/


Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070
www.khayatlab.org


 Original message 

Date: Wed, 20 Aug 2014 18:57:07 +
From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Alexander Aleshin 
aales...@sanfordburnham.org)
Subject: Re: [ccp4bb] Removing PEG3350
To: CCP4BB@JISCMAIL.AC.UK

 I meant application of GF as an ion exchange
 column.

   Oh, my goodness! Ion exchange is something else!
   It should read buffer-exchange = desalting column.
   On Aug 20, 2014, at 11:48 AM, Alexander Aleshin
   wrote:

 Dear Remie,
 I meant application of GF as an ion exchange
 column. You can use special ion exchange columns,
 but our lab often uses preparative GF columns for
 this task.  We just load the column, keeping
 sample volume   the void volume. Thus, we do not
  concentrate a protein before an ion exchange,
 only after it. But that is inevitable. When I am
 afraid to loose a protein during its
 concentrating, I concentrate shoulders of the
 eluted peak first, then add a central part.
 My point was that it might be okay to exchange
 buffers by concentrating a protein, but other
 molecules like Peg3K would not penetrate the
 membrane as well as water or salts do, as a result
 their reduction in concentration will be
 unreliable. Like, you do a 10 fold
 concentrating/delusion of a solution, but the
 final concentration of PEG3K will drop only by 3
 fold...
 Alex
 On Aug 19, 2014, at 9:42 AM, Remie wrote:

   Hi Alex,
   I disagree with you even though GF is always the
   last step in my purifications.
   Because it involves concentration before and
   after the GF so during the concentration you can
   already be doing the buffer exchange.
   You use GF when you want to purify other protein
   impurities if they are different sizes. Of
   course it has other uses too. But not quite
   practical for just changing buffer also
   considering the amount of protein you could be
   loosing along the process. If one is careful,
   centripreps are best for concentrating and
   changing the buffer. I tell you this from
   experience with large hard to express proteins.
   Best of luck,
   Remie
   On Aug 19, 2014, at 10:45 AM, Alexander Aleshin
   aales...@sanfordburnham.org wrote:

 Remie,
 Actually, concentrating of a protein solution
 is not the best approach to removing low MW
 impurities, gel filtration chromatography is
  more reliable and ... faster.
 Regards,
 Alex
 On Aug 19, 2014, at 7:03 AM, Remie Fawaz-Touma
 wrote:

   Hi Reza, I had to do this before.
   This protocol works for any PEG and any
   chemical to be removed from a solution:
   buffer exchange into the new buffer you want
   your protein to be in. There are ways to do
   that by 15 mL Amicon concentrators from
   millipore for large volumes, or if your
   protein is already concentrated, there are
   some small 0.5 mL concentrators from
   millipore as well.
   The key is to keep your spinning at low
   speeds (concentrators manuals will tell you)
  

Re: [ccp4bb] Enigmatic electron density attached to Cys residue

2014-08-19 Thread Roger Rowlett
The planarity and symmetry rules out a mixed disulfide with DTT. Wrong
size/shape for glycerol. Without looking at your crystallization reagents,
I would have suggested something like an S-hydroxycysteine adduct of
something like 2,4-pentanedione or a dehydrated MPD alkene. Could
pentanedione be a degradation product of PEG200? Did any MPD get into the
mix? We have seen spurious oxidation of Cys to Cys-OH in thiol proteins,
e,g, PDB 3UAO.

Good luck. With that high quality ED, you'd think it would be easy to ID...

Roger Rowlett

Dear all,



We are currently working on a small GTPase. The structure has been solved
to 1.4 A with two molecules in the ASU. In the difference electron density
we can clearly see difference density (in one monomer) attached to a Cys
residue.



The protein has been expressed in E. coli. For crystallization experiments
the GTPase was incubated with GMPPNP, that had been dissolved in 20 mM
HEPES pH 7.5 and 50 mM NaCl. Prior to crystallization

the protein was stored in a buffer composed of 20 mM HEPES pH 7.5, 200 mM
NaCl, 5 mM, Mg acetate, 2 mM DTT and 2% (v/v) glycerol.



The protein crystallized under the following conditions:

28% (v/v) PEG 200, 5% (w/v) PEG 3000 and 100 mM MES buffer at pH 6.0



The anomalous signal is too weak to judge the positions of the sulphur
atoms. We have performed MS analysis on the protein before crystallization
and on dissolved protein crystals. MS revealed a mass difference of about
135 Da, indicating that some chemistry must have went on in the
crystallization drop.



The extra electron density has a planar shape and is quite symmetric. We
have placed some dummy water molecules in the density. Distances are given
in A in the PNG file.



Attached files



coot1.png: 2FoFc electron density map (blue) @ sigma=1 and FoFc electron
density map (blue) @ sigma=+3 after phenix.refine

coot2.png: same electron densities as in coot1.png, with dummy atoms placed

coot3.png: same electron densities as in coot1.png, side view



Thanks for your time and efforts.



Cheers,



Bernhard

-- 
Dr. Bernhard Loll
Freie Universitaet Berlin
Fachbereich Biologie, Chemie, Pharmazie
Institut fuer Chemie und Biochemie
AG Strukturbiochemie
Takustr. 6
D-14195 Berlin
Germany

Phone: +49 (0) 30 838-57348
Fax:   +49 (0) 30 838-457348
Email: l...@chemie.fu-berlin.de
Homepage: http://www.bcp.fu-berlin.de/chemie/bc/ag/agwahl/


[ccp4bb]

2014-08-19 Thread Roger Rowlett
Some things to try to increase solubility:

1. Move the buffer pH away from the expected pI. Proteins have minimum
solubility near their pI values.
2. Add solubilizing agents to the solution, e.g., 20-50% glycerol. (This
may alter you crystallization screening strategy)
3. Include some inert salt in the solution to minimize electrostatic
interactions, e.g. 100-500 mM NaCl.

Ultimately, your protein just may not be very soluble. That is potentially
OK...it will ppt and maybe xtallize well at low concentration.

Roger Rowlett
On Aug 19, 2014 1:52 PM, Prashant Deshmukh prashantbiophys...@gmail.com
wrote:

 Hi,
 i am concentrating my protein using centricon filter, but it is
 precipitated soon. Please help me solving this problem.
 Thanks.
 Prashant Deshmukh
 Dept. of Biophysics,
 NIMHANS,
 Bangalore 560 029,
 E-mail:prashantbiophys...@gmail.com
 Mob.No.: +919620986525



Re: [ccp4bb] Zinc binding protein expressed from insect cells

2014-08-15 Thread Roger Rowlett

Harvey,

Depending on the zinc-binding site, it may not bind Fe(II) at all. 
Zn(II) and Fe(II) have very different preferred ligand binding 
environments. For many zinc-metalloenzymes, substitution with Fe(II) 
would be difficult to impossible. In general, you will find it very 
difficult to make your non-defined expression medium zinc-deficient. 
Zinc is a very common component of complex media, and is also a very 
common adventitious contaminant. Ideally, you will want to include the 
metal ion in the expression medium so that it can be incorporated at the 
time of protein synthesis. In many cases, this will enhance the 
stability of the synthesized protein. For bacterial overexpression at 
very high protein levels, 10-100 uM metal ion is more than plenty. More 
than that is actually toxic in bacterial systems, as it may impede 
critical iron transport into cells. But we have found that complex media 
already contains more than enough zinc to populate overexpressed 
proteins. We only supplement when we are trying to make 
metallosubstituted protein, in which case we use defined zinc-deficient 
media and supplement with a compatible metal ion (e.g., Co(II)) at 
10-100 uM maximum in bacterial systems. Even that is tricky, as we need 
some trace metals to populate other metalloenzymes without introducing 
too much in the way of zinc-containing impurities.


Most zinc-metalloenzymes will be immune to metal chelation by DTT or 
BME, as the protein-metal binding constants will be orders of magnitude 
higher. (Values  10^(12) are typical.) Even EDTA is not enough for many 
(most?) Cys(2)His(2) or Cys(2)His(OH2) sites. It is very unlikely that 
1-5 mM DTT will be able to extract Zn from a metalloenzyme binding site. 
(We stored a particularly unstable Cys-rich zinc-metalloprotein in 100 
mM DTT(!) and 2 mM EDTA (!!) and 50% glycerol and it is stable 
indefinitely at -20 deg C without detectable Zn loss.)


You may have to evaluate the metal-binding strength of your protein 
experimentally or by comparison to homologous proteins. If the binding 
constan is expected to be 10^(10), I don't think you need to worry too 
much about DTT or BME.


Cheers,

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

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

On 8/15/2014 11:03 AM, Harvey Rodriguez wrote:

Dear all,

Sorry for the non-crystallographic question. Currently I am working on 
a zinc binding protein which is expressed in insect cells and may 
contain 4-6 zinc ions. As we know, so many zinc binding proteins can 
absorb the iron ions from the culture medium and the protein looks 
from yellow to dark red when concentrated. But when I concentrate the 
protein, I didn’t see the red color even in the very high 
concentration. I am just wondering if a zinc binding protein is 
expressed from insect or mammalian cells, can the zinc binding sites 
grab the irons instead of zinc or the zinc binding site can be empty 
loaded if there is not enough zinc in the culture medium? If so, do I 
need to include some zinc salt into the culture medium when doing 
expression or I can add some zinc ions when purifying? Usually, how 
much zinc and at which step of purification can we add the zinc into 
the solution when doing purification?


Another question is that we know DTT can react with the heavy atoms to 
form the insoluble sulfide precipitates and if the zinc binding 
protein is purified with DTT at a final concentration of 1-5 mM, can 
it strip the zinc ions from the protein?


I am appreciated if someone has this kind of experimental experiences 
and thanks in advance!


Heng




Re: [ccp4bb] CC-half value ??

2014-08-14 Thread Roger Rowlett
Exactly. Aimless will give you suggested resolution cutoffs based on CC 1/2
in the log file.

Roger Rowlett
On Aug 14, 2014 5:04 PM, conan仙人指路 conan_...@hotmail.com wrote:

 Hi Faisal,

   CC-half standard is valuable in evaluating the cut-off of highest
 resolution. Sometimes even if I/sigI is close to 1 and completeness is not
 as high, if CC-half is still significant, it may be worth incorporate the
 extra high-res shell data and extend the resolution. Again, if only the
 reliability and unbias are carefully confirmed, and the apparent
 significant CC-half is not due to an artifact of some other factors like
 ice ring etc.
 (Ref: Karplus PA and Diederichs K. 2012 Science 336, 1030-1033
 https://www.pubmed.com/pubmed/22628654)

   It has yet to be appreciated by most population of the crystallography
 society, unlike the I/sigI, completeness, Rsym. In particular, Rsym has
 gradually less a direct measurement of the data quality and or determinant
 of resolution cut-off.

 Best,
 Conan

 Hongnan Cao, Ph.D.
 Department of Biochemistry
 Rice University

 --
 Date: Fri, 15 Aug 2014 01:39:48 +0530
 From: faisaltari...@gmail.com
 Subject: [ccp4bb] CC-half value ??
 To: CCP4BB@JISCMAIL.AC.UK

 Dear all

 How CC-half value of a data set determines the maximum resolution limit
 during data processing ?? Although much we know about the Rsym and I/Isig
 values of the highest resolution shell while processing the data, what are
 the parameters we need to check related to CC-half values ??

 --
 Regards

 Faisal
 School of Life Sciences
 JNU




Re: [ccp4bb] Have everyone had a Scorpion Screen Builder or a Dragonfly screen optimizer?

2014-08-05 Thread Roger Rowlett
We acquired a very early Scorpion and have been using it for a year now.
The software is very easy to use, and you can store reagents in standard 15
or 50 ml plastic tubes in metal racks. We've had as many as 42 solutions in
use at once for a complex screen. It is almost infinitely customizable in
terms of dispensing options. Auto sensing of reagent volumes is a big plus
in the current software. Software and firmware support is good, and the
software is improved and new features added periodically. We usually use it
to dispense custom optimization screens in Greiner blocks for our Gryphon
but it will accommodate a wide variety of trays. It's pretty economical of
tips and dispenses viscous solutions adequately. No complaints. It works as
advertised and is quite affordable. We've yet to use all the features. Ours
is operated by undergraduates. A typical 96 well screen takes a cup of
coffee, about 10-15 minutes to dispense. No way we're going back to hand
dispensing.

Roger Rowlett
Gordon  Dorothy Kline Professor
Colgate University
On Aug 5, 2014 5:05 PM, Joseph Ho sbddintai...@gmail.com wrote:

 Dear all:


 We are interested in purchasing either a Scorpion Screen Builder from
 ARI or a Dragonfly from TTP labtech for setting up the grid screen. I
 am wondering if anyone can share their personal experience or opinions
 with me.

 Your help/comments are highly appreciated.

 Joseph



Re: [ccp4bb] packing test PHASER

2014-06-17 Thread Roger Rowlett
Increase the number of allowed clashes in Phaser, re-run it then look at 
the packing of the solution found and identify the source of the 
clashes. Possibilities for the clash issue include:


1. Wrong space group
2. Flexible loops or termini in search model not present  or
   differently arranged in your crystal target

Once you look at the packing in Coot or Pymol, you will have a good idea 
of what to do next. If the problem is flexible loops or misplaced N- or 
C-termini, you can delete these regions from your search model (they are 
not helping you phase anyway) and re-run Phaser with an appropriately 
truncated search model.


Cheers,

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

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

On 6/17/2014 10:44 AM, Almudena Ponce Salvatierra wrote:

Dear ccp4 users,

I get the following message from Phaser when I do a molecular 
replacement with two ensembles. One of the ensembles is placed but the 
second one is not placed, and then it says this:


Solutions with Z-scores greater than 13.0 (the threshold indicating a 
definite


solution) were rejected for failing packing test

#1 TFZ=16.8 PAK=487

#2 TFZ=17.4 PAK=440

#3 TFZ=17.0 PAK=312

#4 TFZ=16.7 PAK=294

#5 TFZ=16.8 PAK=227

#6 TFZ=16.6 PAK=284

#7 TFZ=16.2 PAK=219

#8 TFZ=16.3 PAK=287

#9 TFZ=16.1 PAK=186

#10 TFZ=16.6 PAK=277

#11 TFZ=16.7 PAK=204

#12 TFZ=16.8 PAK=404

#13 TFZ=15.9 PAK=271

#14 TFZ=15.8 PAK=194

#15 TFZ=14.9 PAK=229

#16 TFZ=16.4 PAK=368

#17 TFZ=17.3 PAK=194

#18 TFZ=15.2 PAK=240

#19 TFZ=16.1 PAK=325

#20 TFZ=15.3 PAK=455

#21 TFZ=16.5 PAK=298

#22 TFZ=16.6 PAK=290

#23 TFZ=15.2 PAK=259

#24 TFZ=16.2 PAK=194

#25 TFZ=16.1 PAK=314

#26 TFZ=16.3 PAK=194

#27 TFZ=16.3 PAK=387

#28 TFZ=15.6 PAK=193

#29 TFZ=15.7 PAK=219

#30 TFZ=15.6 PAK=474

#31 TFZ=15.1 PAK=194

#32 TFZ=15.4 PAK=339

#33 TFZ=15.5 PAK=210

#34 TFZ=15.2 PAK=300

#35 TFZ=15.6 PAK=186

#36 TFZ=16.2 PAK=216

#37 TFZ=14.9 PAK=182

#38 TFZ=15.7 PAK=279

#39 TFZ=15.5 PAK=285

#40 TFZ=15.0 PAK=374

#41 TFZ=15.4 PAK=404

#42 TFZ=15.3 PAK=185

#43 TFZ=15.6 PAK=227

#44 TFZ=14.8 PAK=364

#45 TFZ=15.7 PAK=193

#46 TFZ=14.5 PAK=448

#47 TFZ=14.6 PAK=219

#48 TFZ=15.5 PAK=210

#49 TFZ=15.7 PAK=189

#50 TFZ=15.0 PAK=193

#51 TFZ=15.0 PAK=281

#52 TFZ=15.2 PAK=202


And the list still continues for a bit. How should I think about this? 
I would assume that the clashes are too many only by seeing those 
numbers, but maybe there is something else to take into account?


Thanks a lot in advance.

Best wishes,

Almudena



--
Almudena Ponce-Salvatierra
Macromolecular crystallography and Nucleic acid chemistry
Max Planck Institute for Biophysical Chemistry
Am Fassberg 11 37077 Göttingen
Germany





Re: [ccp4bb] metal chelation

2014-05-19 Thread Roger Rowlett

The answer depends on a number of questions:

 * What metal ion are you trying to eliminate?
 * What kind of metal-binding site is involved?
 o A peripheral or loose binding site? (e.g. surface calcium
   ions)--these may respond to chelators
 o An active site coordinated metal? (e.g., metalloenzyme)--these
   can be refractory

Many metalloenzymes are not going to give up their metal to chelators, 
or just any chelator, or at all. Denaturation, dialysis, and refolding 
is an extreme way of removing metal ions to make apoprotein. Won't work 
for every protein. Chelation can be highly specific, that is one 
chelator may work, while another, similar one, will not.


Some metal ions are notoriously difficult to eliminate, because they are 
adventitious trace contaminants in nearly everything, e.g. zinc and 
maybe even iron. (Plastic-ware seems to be often loaded with trace iron, 
and also is capable of adsorbing metal ions form solution.) To make 
apo-enzymes from zinc proteins, you have to go to heroic efforts to 
ensure that glassware, water, buffers, and reagents are zinc-free, 
especially if you don't have high (mM) concentrations of protein to work 
with.


A His-tag is very likely to snag adventitious metals from solution, and 
can often mess up metal analysis for metalloproteins by providing 
extra metal. If this is a problem for your application, you may want 
to consider removing the His-tag.


If you are making apoenzyme to get a different metal installed 
(metallosubstitution), there are slightly easier ways to do that than 
going through the apoenzyme route.


Cheers,

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

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

On 5/19/2014 1:20 PM, SUBSCRIBE CCP4BB Adam Brummett wrote:

Hello All,

   I apologize for the non-crystal related question. I am trying to get a fully 
metal-free apo enzyme. The 6x His construct is consistently purified with some 
metal (20-30%). I have attempted chelating away the metal with up to 30 mM EDTA 
and DFO and then dialyzing it away, but this has shown little to no effect. Any 
thoughts or recommendations would be greatly appreciated. Thanks.

Adam




Re: [ccp4bb] metal chelation

2014-05-19 Thread Roger Rowlett

Adam,

We developed a protocol (loosely based on a few previous literature 
reports) for metallo-substitution of beta-carbonic anhydrase (a 
zinc-metalloenzyme) with cobalt(II). The metal ion in this enzyme is 
extremely refractory toward extraction with chelators, and the protein 
will not denature/refold at all.


Briefly, our protocol involves overexpressing protein in minimal media 
in the presence of 10-100 uM Co(II) ion. This allows us nearly 100% 
metallosubstitution of Co(II) for Zn(II) during the overexpression 
phase. We have verified that our commercially prepared minimal media is 
quite metal-free. The protocol is described here:


 * Katherine M. Hoffmann,† Dejan Samardzic,* Katherine van den Heever,*
   and Roger S. Rowlett§, “Co(II)-substituted Haemophilus influenzae
   β-Carbonic Anhydrase: Spectral Evidence for Allosteric Regulation by
   pH and Bicarbonate Ion,” Arch. Biochem. Biophys. 2011, 511, 80-87.

A couple of tricks we discovered:

 * Thiamin supplementation is required for good growth in minimal media
 * We have to use conditioned plastic expression flasks for this to
   work well. Acid-washed flasks result in no cell growth. Flasks that
   have been used to do regular overexpression runs, but have been
   simply well rinsed out with deionized water work reproducibly well.
   I'm pretty sure that the walls of the conditioned flasks are
   providing sufficient trace metal ions for growth, without swamping
   out our supplemental Co(II) ion with contaminant ions.

FWIW, every time we do ICP analysis of metalloenzymes that are 
His-tagged, we nearly always get non-stoichiometric extra metal ion. 
It's maddening when you are trying to establish protein:metal 
stoichiometry, or determine accurate protein concentrations this way.


Cheers,

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

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

On 5/19/2014 3:04 PM, Nadir T. Mrabet wrote:

Hi Adam,
I have not read all the thread as it came all at once and late (9:00pm 
here).
I believe the best way to strip a protein of metals is to first adsorb 
it onto a solid support (e.g. IEX) and then use a sufficiently low-pH 
(say equal or below 6) buffer that contains also EDTA.

You will probably need several washes but it works!
Also be aware that EDTA binds well to several proteins.
HTH,
Nadir Mrabet
Pr. Nadir T. Mrabet
Structural  Molecular Biochemistry
N-gere - INSERM U-954
University of Lorraine, Nancy
School of Sciences and Technologies
 School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet at univ-lorraine.fr
Cell.: +33 (0)6.11.35.69.09

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On 19/05/2014 20:21, Adam Brummett wrote:
Thank you everyone for the comments and suggestions. To answer a few 
questions:


-I do not use a treated buffer system. I have just used the nano-pure 
water. I have looked into Chelex, but before I bought it I wanted to 
see if you all recommended it. I was trying to avoid this, but it may 
not be possible now.


-the active site does bind metals and is promiscuous in binding, so 
it is not know if the His tag or active is the source of 
contamination, but cleavage is not an option for us. The biding of 
metal is going to be needed for phasing, so good point Tim, hopefully 
just not in the His site.


-thank you Vivoli for the protocol, seems very thorough. Have you had 
success with it? I anticipate I'll need to go down this road.


-Roger, the metals you mentioned (Zn and Fe) are the problem and I 
expect to have to go to heroic measures to get an apo enzyme . But 
you did mention easier ways of getting metal substituted. I have some 
evidence that I can do this. Do you have any other thoughts on this 
matter? Maybe a reference to something similar (non-apo but could 
substitute?


Thank you all so much for the help and advice.

-Adam


On May 19, 2014, at 12:55 PM, Roger Rowlett rrowl...@colgate.edu 
mailto:rrowl...@colgate.edu wrote:



The answer depends on a number of questions:

  * What metal ion are you trying to eliminate?
  * What kind of metal-binding site is involved?
  o A peripheral or loose binding site? (e.g. surface calcium
ions)--these may respond to chelators
  o An active site coordinated metal? (e.g.,
metalloenzyme)--these can be refractory

Many metalloenzymes

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

2014-05-15 Thread Roger Rowlett
A logarithmic plot of cumulative entries to the PDB is approximately linear
and shows a growth rate of about 15% per year. That means it doubles in
size about every 5 years at current growth rate.

Roger Rowlett
On May 15, 2014 4:23 AM, Colin Nave colin.n...@diamond.ac.uk wrote:

 From James's figure, assuming perfect lossless compression, the
 information content of the PDB is 20GB or about 2X10**11 bits
 The information content of the universe has been estimated to be 2**305
 bits or 10**92 bits (this might or might not be changing).
 The PDB is said to be growing exponentially. If we know the coefficients,
 we can work out when the PDB takes over the present universe. This would be
 time to retire.
 Can anyone do this?
 Thanks
   Colin


 From: James Holton [mailto:jmhol...@lbl.gov]
 Sent: 14 May 2014 16:19
 To: ccp4bb
 Subject: Re: [ccp4bb] PDB passes 100,000 structure milestone


 I think 249 GB is uncompressed.  My local copy of the PDB only takes up 20
 GB, or one Blu-Ray.

 I can remember a time when the whole of the PDB fit onto a single CD-ROM.
  The PDB booth at the ACA meeting would hand them out for free!  That was
 impressive to me because CD-R disks were really expensive (to an
 undergraduate like me anyway), and I had to figure out how to do
 multi-session writes so I could back up my whole hard drive 2 or 3 times
 before I filled one up.  And, of course, I had to take out my hard drive
 and go over to that really wealthy lab that had a CD writer to do that.
  Each write took about an hour, and didn't always work.  Ah, those were the
 days.

 But yes, it is impressive how so much effort by so many people over so
 many years can be compressed into such a tiny space.  Is it not a strange
 fate that we should suffer so much fear and doubt for so small a thing?

 -James Holton
 MAD Scientist



 On 5/14/2014 7:15 AM, MARTYN SYMMONS wrote:
 I reckon it's two box sets of 25 discs each  - am I calculating that
 wrong? Maybe room for a 'making of' feature

 ;)

 
 From: Jon Agirre jon.agi...@york.ac.ukmailto:jon.agi...@york.ac.uk
 To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
 Sent: Wednesday, 14 May 2014, 14:28
 Subject: Re: [ccp4bb] PDB passes 100,000 structure milestone

 249GB? That's a whole lot of DVDs!

 On 14 May 2014 14:08, MARTYN SYMMONS martainn_oshioma...@btinternet.com
 mailto:martainn_oshioma...@btinternet.com wrote:
 Although the line boasting that the PDB adds up to 'more than 249 GBbytes
 (sic) of storage' was obviously written by someone from a pre i-tunes
 generation
 http://www.wwpdb.org/news/news_2014.html#13-May-2014
 ;)

 -M.

 
 From: mesters mest...@biochem.uni-luebeck.demailto:
 mest...@biochem.uni-luebeck.de
 To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
 Sent: Wednesday, 14 May 2014, 13:41
 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.dehttp://www.biochem.uni-luebeck.de/
 http://www.iobcr.orghttp://www.iobcr.org/
 [cid:image001.png@01CF701D.0C132EE0]
  [cid:image002.jpg@01CF701D.0C132EE0]
 --
 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
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 which arise as a result of e-mail transmission. If verification is required
 please request a hard-copy version. Please send us by fax any message
 containing deadlines as incoming e-mails are not screened for response

Re: [ccp4bb] Issue with Molecules per Asymmetric Unit for Molecular Replacement

2014-05-15 Thread Roger Rowlett
For P212121, I would put money on something divisible by 2 for the total 
molecules per asu. Anything from 6-12 might be likely. One of the early 
structures I worked on had 6 molecules per asu, which was darn near 
impossible to find using momomers (at the time). The way it was 
eventually solved was by finding a reasonable tetramer solution, then 
looking at the packing of that model to see what was missing and 
determine how much more would fit into the asu. Turned out to be another 
(obvious) dimer, which did the trick. The Matthews coefficient predicted 
something like 9 molecules per asu, which was not close to the actual 
answer. When n=large number, the Matthews coefficient does not easily 
identify a unique, most likely solution, but a range of reasonable 
solutions.


One possible strategy would be to look for some partial solutions to 
give you a better clue of the actual packing in the asu. If you find 
there is a reasonable looking dimer pair, try searching with multiple 
dimers, etc.

___
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 5/15/2014 8:29 PM, Toth, Eric wrote:

Have you tried fixing the molecule that looks correct and searching for others? 
You might have greater than one but less than 9 molecules per ASU.

When you do this, try imposing severe restraints on the packing function. This 
worked for me in Phaser with a difficult case. My anecdotal experience is that, 
when you have lots of molecules per asu, the correct solution gets swamped by 
poorly-packed solutions if the default packing penalties are used.

Good luck.

Sent from my iPhone


On May 15, 2014, at 6:50 PM, Matthew Bratkowski mab...@cornell.edu wrote:

Hello all,


I am working on the structure of a small protein in space group P212121.  The 
protein is monomeric in solution based on gel filtration analysis.  The 
Matthews Coefficeint program indicates that 9-10 molecules per asymmetric unit 
results in ~50% solvent content, while 1 molecule per asymmetric unit results 
in ~95% solvent.

  I tried molecular replacement with a search model which is essentially 
identical in sequence to my protein, and searched for 9 or 10 molecules/asu.  
Using MolRep with 9 or 10 molecules/asu, I get poor contrast scores around 
1-1.5.  However, when using Phaser, I get a solution with one molecules/asu.  
Likewise, when I went back and tried MolRep with 1 molecule/asu, I got a 
contrast score of 3.12.  This model still has some issues, but looks more 
correct compaired to models created with 9 or 10  molecules/asu.

It seems highly unlikely that a crystal would contain 95% solvent, but is there 
any possiblility that this could be the case?  Assuming that the Matthews 
coefficient is correct, does anyone have an idea why MR seems to work better 
for 1 molecule/asu with 95% solvent content compared to 9-10 molecules with 50% 
solvent content? Alternatively, is there any reason why the Matthews 
coefficient could be calculating incorrectly?  Any suggestions would be helpful.

Thanks,
Matt


Re: [ccp4bb] metal ion coordination

2014-04-17 Thread Roger Rowlett

See below.

Ceeers,

___
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 4/17/2014 4:13 PM, Faisal Tarique wrote:

Dear all

Can anybody please explain what is the classical metal ion 
coordination for Mg2+, Ca+ and Na+ with Oxygen atom and the average 
distance with these metal ions


You can find answers at the MESPEUS database 
(http://mespeus.bch.ed.ac.uk/MESPEUS/)
..does the distance vary with the type of metal ion and its 
coordination with oxygen atom
Yes. There are typical metal-ligand bond distances that are sensitive 
to the metal ion, coordination number, and ligand atom.
..what is the best way to identify the correct metal ion in the 
electron density in the vicinity of negatively charged molecule mostly 
oxygen containing molecule..
A combination of both the bond distances and the coordination geometry 
may give you a clue. Bear in mind that X-ray bond-distances in proteins 
may not be that accurate--not like small molecule crystallography. But 
the distances can still be useful nonetheless.
In one of my paper the reviewer has asked me to check whether the 
octahedrally coordinated Mg+ is  Ca+ ion..and similarly raised doubt 
about the identity of the Na+ ion as well..his argument was based on 
metal ion to oxygen distance
This may be challenging, but the MESPEUS database may give you some 
clues as to what the typical ranges of bond distances should be. The 
best way to prove the existence of a bound metal is to do ICP-OES or 
TXRF of your protein sample. Of course, Na, Mg, and Ca are common 
contaminants/adventitious metals, so this can be challenging, too. 
Especially if you need these in your buffer to populate the protein.
..I am attaching the figure with this mail..i request you to please 
shed some light on this area and help me in clearing some doubts 
regarding this.


--
Regards

Faisal
School of Life Sciences
JNU



Re: [ccp4bb] Coot error

2014-04-15 Thread Roger Rowlett
Try yum install glib2-devel (as root). I haven't used CentOS/Fedora in 
a while, but I think this is the right package.


Cheers,

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

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

On 4/15/2014 8:24 AM, Monica Mittal wrote:

Hi all
I am facing a problem while installing coot in CentOS. After 
configuring its showing No package'glib-2.0' found. Either it asks me 
to adjust PKG_CONFIG_PATH env variable if i installed in non standard 
prefix or i can set set env variables GLIB_CFLAGS and GLIB_LIBS to 
avoid need to call pkg-config.


But how to do any of the above. I dont  have idea. So guidance on this 
will be highly appreciated.


Thanx
Monica


Re: [ccp4bb] About crystallization diffraction problem

2014-04-10 Thread Roger Rowlett

A couple of thoughts:

 * We have actually managed to grow quite a few crystals like this.
   Sometimes they are not single crystals, but stacks of mis-aligned
   plates that come apart easily when handled or subjected to osmotic
   stress. Sometimes these stacks give great-looking diffraction
   patterns, but they turn out to be multiple lattices :(  (You find
   this out when you try to index your beautiful diffraction images and
   it runs off the rails.) We got around this issue by taking our
   crystallization condition and doing a full additive screen
   (everything we had in the reagent drawer that looked like an
   additive, salts, solvents, etc.) at 50-200 mM or 5-10%. We found a
   condition that gave slightly thicker plates that were single
   crystals and not stacks of thin plates, and those crystals
   cryoprotected and diffracted well.
 * If your plates are actually single crystals, but are just fragile or
   have high solvent content and are being torn apart by osmotic
   stresses introduced by your cryoprotectant, I highly recommend
   trying the cryomixes described in Vera  Stura, Cryst. Growth Des.
   (2014) 14, 427-435. For crystals grown in PEGs, one or more of these
   mixes are like magic with fragile crystals. For salt conditions, 2.5
   M lithium sulfate is also very dependable. Saved our bacon for a
   couple of high solvent content crystals recently.

Cheers,

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

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

On 4/10/2014 1:59 PM, Anamika Singh wrote:

Dear All,

I want to get some help regarding crystallization for one of the 
protein I am working. This is a recombinant protein of molecular 
weight of 17.5 kDa.I am getting crystals shape like thin plates in 5 
days, in different conditions like bis tris pH 6.5-8, HEPES pH 6-8 
with .1M nacl , .01M DTT having PEG 3350, PEG 6000 as precipitant. But 
whenever we used to put crystal in cryoprotectant like 20 % ethylene 
glycol, glycerol, MPD it used to split like layers of some tree barks.
And the crystal which were diffracting not getting diffracted above 
3.0 Angstrom.


Please help me out.

Thanks in advance
--
Anamika




Re: [ccp4bb] High Salt Cryo

2014-02-18 Thread Roger Rowlett
How about a short swish in well solution + 25-30% glucose? Doesn't take 
long to cryoprotect, just a quick sufrace coat. Sodium malonate? We just 
froze some really fragile crystals from 1.8 M sodium formate in 3 M 
sodium malonate and they held up really well. (Still didn't improve 
their diffraction, though--but at least they did not crack or disintegrate.)


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

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

On 2/18/2014 1:08 PM, Katherine Sippel wrote:

Hi all,

I'm looking for a cryo condition for high NaCl (3+ M) crystallization 
condition. I would do it the proper way, but our beam/cryostream is down.


I've tried a bunch of things at the moment. Ethylene glycol and PEG 
400 nuke the crystals immediately even at low concentrations. 
Prolonged exposure to glycerol and sucrose starts to break them down 
so I'm thinking that the diffraction will probably suffer. I can't 
find any reports of NaCl's viability as a cryosalt. I've got 
Paratone/Paraffin oil/Mitegen's LV cryo oil on tap but I was hoping to 
not put all my eggs in one basket.


I tried the ISRDB database through archive.com http://archive.com 
without any luck (no search function). I've gone to the PDB searching 
for similar crystallization conditions and looked up the papers for 
their cryos, but they are all glycerol. Google gives me the same.


I thought I'd see if anyone on the bb has an anecdotal this worked 
for us story. I would love to hear it.


Thank you for your time,
Katherine

--
Nil illegitimo carborundum/- /Didactylos




Re: [ccp4bb] suggestions for cryoprotectant

2014-02-06 Thread Roger Rowlett
Lots of choices. I usually try the crystallization solution + 30% glucose
first. Glycerol or ethylene glycol are other possibilities here. Another
possibility is 2.5-3.0 M sodium malonate at a similar pH.

Roger Rowlett
On Feb 6, 2014 11:40 PM, Deepak Thankappan Nair deepaktn...@gmail.com
wrote:

 Hello,
 Does anybody know what would be a good cryoprotectant for the following
 condition:
 800 mM Sodium phosphate monobasic/1200 mM Potassium phosphate dibasic 100
 mM Sodium acetate/Aceticacid pH4.5

 Thanks
 Deepak




Re: [ccp4bb] off-topic: bug busting

2014-02-04 Thread Roger Rowlett
BeadBeater. http://biospec.com/. Gentle, aerosol-free way to break 
15-350 mL of cell paste (2-150 g wet packed cells).


Cheers,

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

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

On 2/4/2014 11:49 AM, Phoebe A. Rice wrote:
Some time ago, there was a nice discussion of cost-effective, wimpy 
protein-friendly ways to break open E. coli.  We're thinking about 
replacing an aging sonicator. If people have a favorite gizmo, could 
they repeat that advice?

thank you,
  Phoebe Rice

++

Phoebe A. Rice
Dept. of Biochemistry  Molecular Biology
The University of Chicago

773 834 1723; pr...@uchicago.edu mailto:pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/

http://www.rsc.org/shop/books/2008/9780854042722.asp





Re: [ccp4bb] preparation of citrate buffer pH3-6.5

2014-01-30 Thread Roger Rowlett
The easiest way to produce repeatable conditions is to titrate a stock
solution (say 1M) of citric acid with NaOH to the desired pH and use that
to mix your screen. That's what Hampton does anyway.

If fine sampling pH, you can mix various ratios of pH 3 and 6.5 buffers.
The pH won't be linear with mixing ratio, but will be easily repeatable.
The actual pH of the final, magic solution can be directly measured if
desired. Calculations will never be exactly right; pKa values are ionic
strength dependent. Better to measure.

Roger Rowlett
On Jan 30, 2014 2:37 AM, sreetama das somon_...@yahoo.co.in wrote:

 Dear All,
We have obtained many tiny protein crystals in a condition
 containing 0.1M citric acid pH 3.5, 2M ammonium sulfate. The crystals are
 too small for mounting in loops.

We intend to vary the salt concentration  pH to obtain larger
 crystals.

Could anyone direct us to some links, or provide us with a
 method (with calculations) to calculate the amounts of citric acid 
 trisodium citrate required to obtain buffers in a range of pH 3 - 6.5?
I have come across online buffer calculators and links where
 the amounts of the components required are mentioned in grams, but none
 explaining how those values were arrived at.

 Thanks  regards,
 sreetama



Re: [ccp4bb] Protein Purification Problem

2014-01-30 Thread Roger Rowlett
Do you really need to remove the NaCl? Some ionic strength is often 
necessary to stabilize proteins. Our routine purification buffers all 
contain at least 100 mM NaCl. This will not usually interfere with 
crystallization screening.


To minimize the probability of aggregation, you need to (1) ensure that 
the buffer pH is not close to the pI--pH 8.00 is a safe choice for many 
proteins, (2) probably maintain 100-150 mM ionic strength, and (3) 
consider solubility-increasing additives, like glycerol. However, 
solubility additives are going to seriously interfere with 
crystallization, but sometimes you have no choice.


Bear in mind that centrifugal concentrators may bind protein. This may 
become noticeable when using small absolute amounts of protein. Usually 
PES (polyethylenesulfone) concentrator membranes have the least protein 
adsorption.


Good luck!

___
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 1/30/2014 8:17 AM, Anindito Sen wrote:

Dear All,

This may be slightly off-the-track question but your feedback will be 
very much appreciated. The situation is-


I obtain a very low amount of the protein of my interest (a 
hetro-dimer) from the construct I am using (only 8% of the total 
amount of protein obtained is the protein of my interest).  After 2 
column purifications (Ni-NTA and St) the concentration of the protein 
is around 0.24 mg/ml (volume- ~1.0 ml)  from a litre of bacterial 
culture and in ~300 mM NaCl present in the elution  buffer.


To reduce the high amount of salt I have I use a desalting column 
which, further lowers the protein concentration significantly.


I need atleast 1.0mg/ml of protein concentration and to the amount of 
~200 microlts for further experiments.


As the last resort I try to use high amount of bacterial culture 
(~6lts) to scale up the yield and use centricon to concentrate the 
protein at various stages.  I am partially successful to obtain 
0.56mg/ml of protein concentration and up to  50 microlts of it.



Another problem is that the protein is  notoriously  prone to 
 aggregation ( 1.5 mg/ml), so dialysis for ~ 2 hrs or so to reduce 
the high salt concentration has failed miserably.


Please do send your feedback.

Thanks and Best Wishes


Andy




*Dr. Anindito Sen (Ph.D)*
*Department of Cell Biology  Anatomy*
*Graduate School of Medicine*
*University of Tokyo*
*Tel  fax: +81-3-5841-3339*





Re: [ccp4bb] preparation of citrate buffer pH3-6.5

2014-01-30 Thread Roger Rowlett
You are correct about certain buffers as interferents. Certain buffer 
species will coordinate with or precipitate silver or mercurous ions 
that are present in the reference electrode compartment of the 
combination pH electrodes. Tris is notorious for clogging the little 
porous frit on the reference electrode, and this, along with reference 
solution cation ion depletion, will drive the electrode crazy until the 
frit is cleared and/or the reference electrode filling solution is 
replenished. Making 1M stock Tris solutions is enough to knock out a pH 
electrode for several hours if you overexpose it to the solution.


Cheers,

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

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu
On 1/30/2014 12:23 PM, Shane Caldwell wrote:

Hi Sreetama,

For most buffers, I use Katherine's method, but in the case of citrate 
I'd recommend just titrating citric acid with NaOH. I've made a pH 
series from citric acid and Na3Cit before, and it's a huge pain. It's 
very difficult to calculate how much of each you'll need, because 
citrate is triprotic and the pKas overlap. When I made a pH series 
this way, I ended up using much more stock than I anticipated, and 
just had an overall unpleasant afternoon.


One other point - high citrate concentrations can cause your pH meter 
to drift, so don't leave the probe in the citrate solution any longer 
than needed, and calibrate it frequently. I'm told this is because 
citrate chelates metals and that throws off the electrode, though 
admittedly I don't know the mechanism and can't find any references to 
back me up - it's just been lab folklore for me. Either way it might 
be worth testing the stability of your electrode over time to get an 
idea.


Shane Caldwell
McGill University


On Thu, Jan 30, 2014 at 10:40 AM, Katherine Sippel 
katherine.sip...@gmail.com mailto:katherine.sip...@gmail.com wrote:


Alternatively you could make a stock solution of citric acid (say
1 M for example) and stock solution of sodium citrate (also 1 M).
Mix them in the appropriate ratio to ballpark the right pH and
just adjust up or down with the stock solution. The concentration
of citrate will be the same no matter the final volume. You can
then dilute that down to whatever your final concentration of
citrate needs to be.

If you are looking for the actual method to do the calculations I
would suggest finding a chemistry textbook and looking at the
chapter on buffering and the Henderson-Hasselbalch equation.

Cheers,
Katherine


On Thu, Jan 30, 2014 at 9:31 AM, Daniel Picot
daniel.pi...@ibpc.fr mailto:daniel.pi...@ibpc.fr wrote:

But you have to be aware that pH depends on the concentration 
of the buffer. This is especially the case for phosphate and

citrate buffer.
Daniel

Le 30/01/2014 15:51, Schnicker, Nicholas J a écrit :

It's a pain but I usually just make each pH of whatever
buffer I'm using (if you make it concentrated then you'll
only have to do it once).  Also, if you haven't already found
it, Hampton has a nice link to calculate volume of components
while designing a tray as long as you tell it the concentrations.

http://hamptonresearch.com/make_tray.aspx

Nick

From: Roger Rowlett rrowl...@colgate.edu
mailto:rrowl...@colgate.edu
Reply-To: Roger Rowlett rrowl...@colgate.edu
mailto:rrowl...@colgate.edu
Date: Thursday, January 30, 2014 at 7:23 AM
To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK
CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] preparation of citrate buffer pH3-6.5

The easiest way to produce repeatable conditions is to
titrate a stock solution (say 1M) of citric acid with NaOH to
the desired pH and use that to mix your screen. That's what
Hampton does anyway.

If fine sampling pH, you can mix various ratios of pH 3 and
6.5 buffers. The pH won't be linear with mixing ratio, but
will be easily repeatable. The actual pH of the final, magic
solution can be directly measured if desired. Calculations
will never be exactly right; pKa values are ionic strength
dependent. Better to measure.

Roger Rowlett

On Jan 30, 2014 2:37 AM, sreetama das
somon_...@yahoo.co.in mailto:somon_...@yahoo.co.in wrote:

Dear All,
   We have obtained many tiny protein crystals in
a condition containing 0.1M citric acid pH 3.5, 2M
ammonium sulfate. The crystals are too small for mounting
in loops.

   We intend to vary the salt concentration  pH
to obtain

Re: [ccp4bb] Lysis of E coli

2014-01-28 Thread Roger Rowlett
We do not have experience with this product. We use a BeadBeater. Can 
handle up to 25-30 g of wet packed cells in the medium beater jar. The 
large jar will handle maybe 3-5x that, but I've never had to go to that 
scale.


___
Roger 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 01/28/2014 08:21 AM, Mark J van Raaij wrote:

Dear All,

Does anyone have experience with the FreezerMill for lysing E coli?
see:
http://www.spexsampleprep.com/products_by_category.aspx?cat=2
It seems to be more for tissues, but perhaps it could also be used for lysing 
reasnoble quantities of E coli - the reason for asking is that I have to decide whether 
it would be useful for us and if so, to support its purchase.

Greetings,

Mark

Mark J van Raaij
Lab 20B
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/~mjvanraaij




Re: [ccp4bb] cryoprotection ideas for salt based condition

2014-01-22 Thread Roger Rowlett

Mahesh,

Try 25-30% glucose. You can gradually add well solution + 37.5-40% 
glucose directly to the drop if your crystals are sensitive to changes 
in osmotic pressure. If your drop is evaporating too quickly, try 
working in the cold room or under oil to slow down evaporation. You can 
find our standard cryoprotection protocol on our wiki here 
http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Mounting+Protein+Crystals 
under No-fail cryoprotection.


Cheers,

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

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

On 1/22/2014 12:28 PM, Mahesh Lingaraju wrote:

Hello folks,

I have crystals for a protein which form in 0.1 M MES pH 6-6.75, 10% 
dioxane and 1.6-2 M Ammonium sulphate. Based on little experience I 
have finding the right cryoprotection for salt based conditions; I 
have tried glycerol (15-25%), Ethylene glycol (20-25%), DMSO (15-20%) 
and increasing ammonium sulphate concentration in presence of 5-10% 
glycerol. The crystals disintegrate in any kind of PEG based cryo. I 
made all these solutions in the mother liquor and tested if they 
freeze clearly before using them. However when I loop the crystals and 
try to soak them in these cryo- mother liquor, a lot of salt crystals 
suddenly form around the protein crystal and I see diffraction only 
from these salt crystals. The best I have been able to get so far is ~ 
9Å diffraction (Home-source) with DMSO as the cryoprotectant.


I have also tried using 1 M sodium malonate as the cryoprotectant but 
my crystals are not too stable in this mother liquor probably because 
I had to lower the ammonium sulphate by ~ 7-10% to make the drop not 
form salt crystals instantly when exposed to air.


Other than trying to make the crystals more cryo-ready by finding 
other hits or may be growing the same crystals with some 
cryoprotectant, I was wondering if any of you have ideas based on your 
experience or suggestions in case I am doing everything wrong in the 
first place.


Thanks for all the help,

Mahesh




Re: [ccp4bb] Phasing with Many Monomers/AU

2014-01-20 Thread Roger Rowlett
I agree. Searching with a larger unit is likely to be successful if you 
have a good idea of the structure of that larger unit. We had an example 
of a low homology (29% identity) MR situation with 8 subunits per ASU 
with twinned data. Not solvable with monomers. Solvable with a dimer 
search model, then feeding that solution to Parrot and Buccaneer for 
density modification and automated chain-building using 8-fold NCS. 
Buccaneer built about 95%+ of the structure correctly.


Cheers,

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

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

On 1/20/2014 8:41 AM, David Schuller wrote:
Is the monomer the biggest unit you have to search with? If there is a 
dimer, tetramer, etc. that is conserved, you could try searching with 
that.




On 01/19/14 14:30, Chris Fage wrote:
Thank you all for your responses. I already have a few ideas about 
how to approach the problem.


One of my concerns with so monomers per asymmetric unit at lower 
resolution was the failure of MR software. Neither PHENIX nor Phaser 
MR have made progress. I am fairly new to anomalous methods, having 
solved only two structures by SeMet-based SAD. I've certainly picked 
up on a number of tricks from the recent messages on heavy atoms, but 
I thought my case might be a little unusual. I am confident the space 
group is P1, as it was the only viable option when I indexed four 
clean albeit low-res datasets.


The monomers are ~38 kDa, and the crystals diffracted to 3.4-3.0 at a 
synchrotron.


The conditions for both native and SeMet crystals are:
8-12% PEG 2000 MME, 0.2 M ammonium sulfate, 0.1 M sodium acetate pH 5.5.

Macromolecular seeding of native crystals into SeMet drops yields the 
needle-like crystals.


Any further input is greatly appreciated!

Regards,
Chris


On Sat, Jan 18, 2014 at 11:14 AM, Chris Fage cdf...@gmail.com 
mailto:cdf...@gmail.com wrote:


Hello Everyone,

I am currently trying to phase a structure with an asymmetric
unit predicted to contain 20-24 monomers (space group P1). The
native crystals, while beautiful in appearance (see attached),
only diffract to ~3.4-3.0 angstroms at best, and SeMet-derived
crystals grow with poor morphology (small needles). Also, based a
fluorescence scan, I know that mercury does not bind appreciably.
Other than screening for a new space group, what options might I
have for phasing this many monomers at lower resolution? Is there
any real chance of solving the structure in this space group?

Thank you in advance for any suggestions!

Regards,
Chris





--
===
All Things Serve the Beam
===
David J. Schuller
modern man in a post-modern world
MacCHESS, Cornell University
schul...@cornell.edu




[ccp4bb] ACA 2014 meeting--Session on engaging students in protein crystallography

2014-01-14 Thread Roger Rowlett
I am co-organizing (with Kraig Wheeler) a session at the 2014 American 
Crystallographic Association Meeting in Albuquerque, NM concerned with 
engaging undergraduate students in protein crystallography. I would like 
to encourage anyone who has involved undergraduate students in protein 
crystallography in a research or instructional laboratory setting to 
submit an abstract. We are probably looking at 30 minute presentations. 
The session description is at the end of this message.


Our past sessions have been well-populated with folks doing nice protein 
crystallography work with undergraduates, and we would love to see some 
new faces again this year. Past talks have described (1) integrating 
protein crystallography research into the teaching lab, (2) some really 
innovative and easy-to-adapt instructional laboratories, (3) how to 
write competitive proposals to acquire instrumentation (ALWAYS popular), 
(4) how to train and involve undergraduate students efficiently, etc.


If you have successfully involved undergraduate students in protein 
crystallography, acquired crystallography instrumentation, or have 
invented a better instructional mousetrap, we want to hear from you! 
National lab, research institution, undergraduate institution, liberal 
arts college--whatever. This session is dedicated to showcasing some 
role models for successful implementation of protein crystallography at 
the undergraduate level at all types of institutions.


*Abstract deadline is January 31. *If you have any questions, or are 
interested/planning on submitting an abstract, please contact me.


_
2.2.1 Engaging Undergraduate Students with X-ray Crystallography

The next generation of crystallography users is rapidly expanding from 
post docs and graduate students to an even younger crowd - 
undergraduates. Success with capturing this younger demographic is now 
well recognized in both formal training and research experiences. This 
session is especially appropriate for faculty wishing to involve 
undergraduates in protein and/or small molecule crystallography, 
including new faculty or those considering academic positions at 
undergraduate institutions. Presentations will address issues of 
integration of crystallography into the curriculum, engaging 
undergraduates in crystallography research, and strategies for faculty 
professional development and instrument acquisition.

__

Cheers,

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

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


Re: [ccp4bb] Cryo solution for crystals grown in magnesium formate

2013-12-16 Thread Roger Rowlett

Junyu,

I haven't tried it personally with this particular solution, but I have 
found that 30% glucose can pretty much cryoprotect any condition I have 
tried it with. If necessary, add cryoprotectant solution (mother liquor 
+ 30% glucose) gradually to minimize osmotic shock and potential 
cracking of crystals.


You can try this without crystals to see if the solution vitrifies as a 
clear solid in liquid nitrogen. If it freezes clear, it is very likely 
to work fine with your crystals.


Cheers,

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

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

On 12/16/2013 4:36 PM, Xiao, Junyu wrote:
Dear all, sorry if this topic does not interest you. I wonder whether 
anyone has experience with freezing crystals grown in ~0.2 M Magnesium 
Formate. Garman and Mitchell suggested that A major anomaly is 
solution 44, 0.2 M magnesium formate, which requires 50% glycerol for 
cryoprotection in their 1996 paper (J Appl. Cryst.  29, 584-587). 
 Since 50% glycerol is kind of harsh, I wonder whether anyone has 
tried alternative cryo protectant. Your kind help will be highly 
appreciated.


Best regards,
Junyu

---
Junyu Xiao, Ph.D.
University of California, San Diego
Leichtag Room 283
9500 Gilman Drive, 0721
La Jolla, CA 92093-0721
Lab phone: 858-822-0684






Re: [ccp4bb] Can Mathew's coefficient tell about a complex

2013-12-04 Thread Roger Rowlett
Looks like 1 or 2 protein units per ASU. Two seems most likely, but I 
have had a bunch of crystals with 67% solvent, too, so 1 is not out of 
the question if less probable. So try both with your favorite MR method 
and see what happens. Crystal packing in the MR solution will tell all.


___
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 12/4/2013 5:06 PM, abbas maqbool wrote:

Dear All,
I crystallized a complex of two proteins and got x-ray data. However I 
dont know if I have got complex or just one of the protein has 
crystallized. Can I check it by mathew's coefficient? If yes how? One 
of my protein is 30 kDa nad other one is 12 kDa.
Actually I calculated Mathew's coeffient (based on Mol. wt of complex 
42000 Da), and results were like that



Cell Volume = 993567
Nmol/asymMathews coeff%solvent
13.94   68
21.9737
31.36


Can any one please explain what does it suggest?

Thanks
Abbas





Re: [ccp4bb] out of topic: liquid handling robot for optimisation

2013-11-29 Thread Roger Rowlett
We're using an Art Robbins Scorpion. In a typical configuration it will
hold three racks of either 8 50 ml Falcon tubes or 24 15 ml Falcon tubes.
We use ours mostly to dispense 96 well optimization blocks with 250-500 uL
per well  to feed our Gryphon. We routinely dispense 50% PEG solutions, but
of course this will be somewhat slow for accuracy. Software is easy to
use--my undergraduates program and use it routinely. PEG blocks take 20-30
min, non-viscous blocks maybe 10 min.

Roger Rowlett
On Nov 29, 2013 5:12 AM, Valerie Biou valerie.b...@ibpc.fr wrote:

 Dear all,

 We are looking for a robot  to mix solutions and prepare optimisation
 plates for crystallisation.
 Ideally, the machine should accurately handle viscous liquids such as 50%
 PEG 4000 (or at least 40%) and start with 10-20 reagents in tubes or plates
 rather than large bottles.
 The programming software should be easy to use but flexible so that rather
 complex mixtures could be made...
 I would be grateful to hear of your experience if you use or have tried
 machines that can do this.

 Best,

 Valerie



 Valerie Biou

 Laboratoire de Biologie Physico-Chimique des Protéines Membranaires
 UMR 7099 CNRS/Univ. Paris Diderot P7
 Institut de Biologie Physico-Chimique
 13 rue Pierre et Marie Curie
 75005 Paris - France
 Tel : +33 (0)1 5841 5099 valerie.b...@ibpc.fr
 Fax : +33 (0)1 5841 5024




Re: [ccp4bb] About protein precipitation problem during dialysis

2013-11-20 Thread Roger Rowlett
Right on target. Many proteins require some ionic strength in the 
solution to maintain solubility and prevent protein aggregation. Usually 
NaCl is used for this purpose. You can also use glycerol to enhance 
solubility, but this may interfere with crystallization if that's the 
next step. NaCl usually interferes less with crystallization. The 
minimum concentration of salt required will have to be determined 
empirically.


For gel exclusion chromatography, it is usually necessary to include at 
least 50 mM NaCl in the buffer to prevent non-specific adsorption to the 
gel medium. I use 100 mM in my GEC columns. If your forget to add the 
salt, your elution times will look very wonky, especially if you are 
trying to use elution data to estimate molecular weight.


Cheers,

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

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

On 11/20/2013 4:37 PM, Tim Gruene wrote:

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Hash: SHA1

Hello Dhana,

use a buffer at least, and some salt - if I remember correctly, gel
filtration resins require 150mM salt concentration for proper
separation (might have been 50mM). Even 150mM may be too little to
keep your protein soluble.

Best,
Tim

On 11/20/2013 09:56 PM, Dhanasekaran Varudharasu wrote:

Dear Crystallographers,

I dialysed a 30 kDa protein (Recombinant protein which was eluted
by 20 mM Tris, 500 mM NaCl, 120 mM imidazole) against water for
overnight. But it gets precipitated after 12 hours. Can anybody
give some suggestion to avoid precipitation. Thanks Dhana

- -- 
Dr Tim Gruene

Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

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[ccp4bb] lib32gfortran3 needed for 64-bit ccp4?

2013-11-11 Thread Roger Rowlett
Just ran into an error running SORTMTZ in CCP4-6.4.0. Looks like SORTMTZ 
requires libgfortran.so.3 32-bit version. I get an error message of the 
missing library if I do not install lib32gfortran3 (32-bit fortran 
libraries). Should this still be necessary for the 64-bit CCP4? I'll 
keep this package on my client workstation install list for now.


Thanks,
___
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


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