Sometimes (sadly, not always) the problem with thin plates is that they get
damaged in mounting. If none of the tricks suggested work, you might try
seeing if you get any better data from the existing crystals using those tennis
racket shaped loops that give more support to the thin plate.
>
Dear Bei,
It can sometimes be difficult to tell a real molecular replacement solution
from noise at low resolution.
In addition to Eleanor's excellent advice, you might try to use your
potential derivative data to test the top molrep solutions, even if their
statistics are crappy. Try mak
If its old and out of warrenty, see if you have a local shop that can do it.
We found the guys in physics here are great with such things (and a lot cheaper
than official company repair guys).
=
Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The Uni
With Flp recombinase - DNA complexes, a C-terminal His tag triggered a
different (but sadly not better) crystal form, and the His side chains packed
against the bases at the end of a neighboring DNA duplex.
=
Phoebe A. Rice
Dept. of Biochemistry & Molecular
Try adding DNA then dialyzing to low salt (in some microdialyer).
=
Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?fa
91 585 4616
>>> http://www.cnb.csic.es/~mjvanraaij
>>>
>>>
>>>
>>> On 25 Apr 2012, at 09:41, Jan Dohnalek wrote:
>>>
>>>> There have been other manipulations with user-input values. We could not
>>>> input solvent content
I just noticed that the PDB has changed the stated resolution for one of my old
structures! It was refined against a very anisotropic data set that extended
to 2.2 in the best direction only. When depositing I called the resolution 2.5
as a rough average of resolution in all 3 directions, but
I'd suggest:
- Find a dinosaur from my generation who can suck one into a capillary and
check diffraction at room T.
- Try using those loops that look like miniature tennis paddles to give the
crystal a little more support
- To minimize strain on the crystal when pulling it out of the drop, tr
puter this nice structure that I
> was looking for!"), there is no need to invent
> anything.
>
> About a wrongly fit compound, the reviewer can ask
> images about the model in a map calculated at a
> specific sigma and in different orientations.
>
> Maria
>
Can we leverage this to push journals to routinely allow reviewers access
coordinates and maps?
Outright fraud is outrageous, but I'm actually more worried about ligands fit
to marginal density and other issues of under-supervised model building.
=
Phoebe
Ah, an old pet peeve resurfaces!
English is complicated and "data" is by now an English word.
To use a somewhat strained analogy, at the quantum level, the word has a
singular and a plural form, and at the classical-mechanics level, the word is a
mass noun.
Most crystallographers use the wor
I probably it depends on whether you've got gunk or a functionally relevant
oligomer in that void volume. Is it active?
RecA and Rad51 both form open-ended head-to-tail oligomers and yet they still
crystallize.
=
Phoebe A. Rice
Dept. of Biochemistry & Molecu
I'm enjoying this discussion.
It also seems like a good spot to inject my standard plea for better treatment
of anisotropy in things like "table 1" of papers and PDB deposition forms.
When you have Ewald's football (American football), like many nucleic
acid-ophiles do, one number simply isn't
As the proud owner of a carefully organized, highly annotated VMS backup tape
(reel-to-reel, of course), my main concern is that paper is the only format
that we'll be able to count on reading a decade (or more) from now.
=
Phoebe A. Rice
Dept. of Biochemistry
We've seen nice in vivo activity (on purpose) from proteins cloned under T7
promoters but transformed into non-DE3 cells. In fact, friends working with
more zesty enzymes who wanted a more "tunable" in vivo assay have had to mutate
the ribosome binding sites for proteins under T7 promotors to k
>
>> Can I be dogmatic about this ?
>
>I wish you could, but I don't think so, because even though those
>sources call it that, others don't. I agree with your thinking, but
>usage is usage.
And 10,000 lemmings can't be wrong?
Hi all,
For those who teach xtallography - we found some plastic turtles that can be
snapped together in an amazing variety of space groups. Worked well in a
workshop for our students, so I thought I'd share the shopping tip. They're
called Reptangles, and we got them from Amazon.
http://ww
What is Kd?
Also, in reply to earlier posts: it is sadly common in crystallizing large
protein-DNA complexes to go through a couple dozen different duplexes and
several dismally-diffracting crystal forms before finding a good one.
Phoebe
>From: CCP4 bulletin board (on behalf of umar faroo
One more consideration:
Since organization is not one of my greatest talents, I would be absolutely
delighted if a databank took over the burden of archiving my raw data for me.
Phoebe
=
Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The Universit
Some things that are a good sign for having DNA as well as protein in your
crystal:
1) You get different (or no) crystals if you add or substract a base or two
from the DNA ends. Of course, we did get fooled by this logic once when one
particular oligo seemed to be contaminated with an easily
Its also a bit too simple to count secondary structure restraints as 2
restraints per residue, because if they're tight enough on, say, an alpha
helix, in combination with other geometry restraints (good bond angles, no
clashes, etc) you could probably turn the backbone of the entire helix into
Gamma delta resolvase catalytic domain stock solutions used to make a nice
clear jelly at 4 degrees, but it was perfectly reversible by warming the sample
to room T. In fact, one mutant crystallized in the stock tube after a few
trips in and out of the fridge. The crystals didn't diffract very
Is there finally, at long last, one convention for nucleic acids? I wonder how
many cumulative person-years of exasperation this @#$% issue has caused?
And please note, even Mother Nature herself, let alone synthetic chemists,
occassionally attaches U to deoxyribose or T to plain ribose.
===
beta sheets in really well-phased low-resolution maps should look sort of like
walls, but in an imperfect map they might by rather spotty.
I'd be very leary of making any conclusions from molecular replacement at such
low resolution. For a good control, try "solving" your data set with a
simil
Some people on this list seem to think that reviewers are on the whole lazy and
irresponsible and in need of some sort of public ridicule.
Please don't forget that reviewing manuscripts, while an important duty for
members of the community, is already in practice a completely thankless
voluntee
Good point. And solubilizing tags can sometimes drag unfolded garbage into
solution with them.
=
Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/0
My favorite test for truth in molecular replacement solutions is whether or not
the map shows density for something that you know about but the computer
doesn't. Take out a base pair or two, or delete some nice fat side chains, and
see if they still show up in the density when you use that mode
This brings up a philosophical caveat that one should be very careful about
labeling one set of potential results "negative" and another "positive" before
even doing the experiment, because its all too easy to see what you hoped to
see even if it was only marginally there.
The best experimental
Based on the dents in my own forehead from banging it on similar brick walls,
it would probably be more efficient to get a heavier atom with fewer binding
sites, and work your way in from there. If your data set really only extends
to 6A, even when you do find all those Se atoms their phasing p
We actually screen with unpurified oligos (up to just over 30nt) and it works
just fine. Saves lots of time & $$.
If we get hopefull crystals, we pay for purification or do it ourselves by
gel. Sometimes it makes the crystals better and sometimes it doesn't.
Phoebe
=
As suggested, you can probably get good purification of heparin.
If your pet protein has a known specific binding site, you can make it a
personalized column by PCR'ing up arrays of directly repeated binding sites.
The repeats will mis-anneal in subsequent rounds, giving rise to longer and
long
Congratulations on your amazing discovery, which immediately suggests many new
lines of inquiry:
Does dark matter affect macromolecular stability? Can it explain the
difficulty some students have in sample preparation? Is it found in higher
concentrations in brains that are thought to be dens
>> - they all know what B is and how to look for regions of high B
>> (with, say, pymol) and they know not to make firm conclusions about H-bonds
>> to flaming red side chains.
>
>But this "knowledge" may be quite wrong. If the flaming red really indicates
>large vibrational motion then yes, on
I've now polled 4 fairly savvy "end users" of crystal structures and there
seems to be a consensus:
- they all know what B is and how to look for regions of high B (with, say,
pymol) and they know not to make firm conclusions about H-bonds to flaming red
side chains.
- None of them would ever t
My 2 cents worth on the stereo-dependent:
1) They have carpal tunnel syndrome that makes it painful to keep the molecule
in motion while rebuilding it (NOTE: enough constant mouse-wiggling and you
will get carpal tunnel problems if you don't have them yet!)
2) They work on big, low-resolution s
I'm allergic to red nitrogens and blue oxygens, and I'm tired of changing the
default way new files are colored every time I restart coot (I know it save the
whole previous session, but sometimes I don't want to see that set of molecules
again). I must be missing a way to save the preferred def
Depending on what the expected activity is, its worth considering the
highly-depressing possibility that the activity seen in the impure sample was
due to impurities: for example, barely-visible-on-a-gel chaperones can give a
nice ATP hydrolysis signal, and DNA ligases float about with an AMP c
You've probably done it already, but solvent flattening/flipping/massaging at
80% solvent should provide a cheap thrill with regard to phase improvement.
=
Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
htt
It sounds a bit silly after that nice theoretical discussion, but I would try
poking the existing oily blobs with a hair. Since you may be close to xtal
conditions, stirring up the equilibrium a bit may help nucleate something.
I've seen this work more than once, although usually with things w
Especially if you're dealing with lysate, I suspect the best way to do it is
with magnetic Ni beads that you lift up and out of the gunk, to help avoid
false positives from aggregating stuff that SDS/urea/guan would all "elute".
But why do you want X to remain on the column/beads? Removing Y bu
Its may be an interesting question of pseudosymmetry. The best thing would be
to find a local mentor who could have a good hands-on look, but here are some
thoughts:
>From the space group tables you will see that P212121 has half as many
>asymmetric units as C2221, and C2221 has crystallograph
I'm all in favor of (rational) length limits for the text - almost everybody's
prose is easier to read when they've been forced to cut 10-20%.
However, some journals include references in the character limit, and that just
encourages unscholarly behavoir.
Phoebe
Another unfortunate aspect of this sort of editorial policy is that many of
these papers contain almost no technical information at all, except for the
supplement. I've started to avoid using Nature papers for class discussions
becuase they leave the students so puzzled, and with a
glossiness-
Journal editors need to know when the reviewer they trusted is completely out
to lunch. So please don't just silently knuckle under!
It may make no difference for Nature, but my impression has been that rigorous
journals like JMB do care about review quality.
Phoebe
===
Another issue with these statistics is that the PDB insists on a single value
of "resolution" no matter how anisotropic the data. Especially in the
outermost bins, Rmerge could be ridiculously high simply because the data only
exist in one out of 3 directions.
Phoebe
===
I know people sometimes have good reasons, but people should be very careful
about "carving" a map so close to the atoms - one could end up seriously
misrepresenting the quality of the map.
=
Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The Unive
Dear Yang,
You might try acrylamide instead of agarose, since the caging effect of the
tighter matrix might keep the complex together better.
I'm not sure why MgATP? If your protein only binds in the presence of ATP you
might need a non-hydrolyzable analog?
Your concentrations are also vast
Dear Qing,
Many structures have been solved that way. Make sure you try solvent
modification (flattening / flipping) on your map. This is because SAD will
give two equally-probably estimates for the phase. The initial, unmodified map
will use the average of the two, but solve modification s
Have you tried expression tricks like Rosetta cells? Testing different
colonies and/or starting from fresh transformants? Sometimes that matters.
If your protein is an oligomer and your contaminants are degradation products,
you might try adding some urea. If desparate, you could spike the fr
There's no theoretical reason it can't be that long.
BUT it is possible, especially for problematic diffraction
patterns (e.g. from a badly-diffracting, cracked crystal)
for the software to pick some wacky value in its attempt to
fit spots that don't really all belong in the same pattern.
Could any of those strong spots at 2.1 be ice?
Original message
>Date: Tue, 20 Jul 2010 16:13:31 -0700
>From: Lijun Liu
>Subject: Re: [ccp4bb] Residual densities!
>To: CCP4BB@JISCMAIL.AC.UK
>
> Pavel,
> Thanks for the quick response. I just listed out
> the Fo-Fc column of th
What would be wrong with WORDS? They were such a clever
invention. I can tell the difference between colors, but it
takes a second step to figure out what they mean anyway. Why
not just write "no info" over the gray ones? And a 1-word
caption on all the little icons would help, IMHO.
Phoebe
it it doesn't provide
enough phasing power to crack the problem, you can use the
positions of Se peaks in anomalous diff maps to check possible
molecular replacement solutions.
Good luck!
Phoebe Rice
Original message
>Date: Fri, 21 May 2010 20:23:23 +0530
>From: Vineet Gaur
You could make use of product binding energy to drive the
reaction forward while the substrate/product is bound to the
enzyme. But enzymes that pull that trick are barely
"enzymes" - they stay stuck to the first product they make
until something else uses some energy to release it.
You can't
Move the beam stop back? My lab has grown quite a few
crystals that only diffract to very low resolution.
Phoebe (with sympathy!)
Original message
>Date: Mon, 19 Apr 2010 11:35:11 +0800
>From: tat cheung cheng
>Subject: [ccp4bb] Re： [ccp4bb] Mysterious Crystals?
>To: CCP4BB@JIS
Many thanks to all who suggested references!
Phoebe
From: Matthew Merski
Matthew Merski
Postdoc, Shoichet Group
UCSF
The Molecular Origin of Like-Charge Arginine-Arginine Pairing
in Water
Jií Vondrek, Philip E. Mason, Jan Heyda, Kim D. Collins and
Pavel Jungwirth
J. Phys. Chem. B, 2009, 113
We recently solved a multi-SeMet data set with nasty
pseudotranslational symmetry by telling lies to the software:
"we" (meaning my long-suffering student) indexed it in the
smaller unit cell by picking only the dark spots, found the Se
atoms in that cell, then reconstructed the larger cell
accordi
I'm looking for references where people have tried to at least
vaguely quantify the energetics of stacking the guanidinium
groups of two R's. I'm looking at 3 cases in protein-protein
interfaces, and in each one there's a D or E adjacent, which
presumably helps with the charge-charge repulsion.
Hi
Are these things really cost-effective? That is, compared to
the cost of simply posting a list of evil salts like Mg
ammonium phosphate on the wall and testing a few duds in the
x-ray beam?
Phoebe
Original message
>Date: Wed, 20 Jan 2010 13:59:23 -0500
>From: Gabriel Birrane
>Subje
hat the next one can stack
against its symmetry mate. The flipped base may be too poorly
ordered to see, especially in initial maps. At 3A you
probably can't be sure which base is which, so I'd try
regrowing the crystals with DNAs where some Ts have been
substituted with 5-Br-d
Yes, as much as I like to pick on the tabloids and their
somewhat iffy quality control, it looks like this guy got away
with faking structures for 10 years UNTIL he published in
Nature. Even Acta Cryst has egg on its face.
So the bottom line is that sunlight is indeed the best
disinfectant, and N
We've had good luck (sometimes) searching for nucleic acids
with multiple small pieces - say ask it to find several 5bp
chunks of DNA rather than one 20bp duplex. I think Bill
Scott will agree?
I have a hunch that searching for the protein 1st, at least
at "modest" resolutions, may confuse the
I'd use MAMA to make an extremely simplified mask (either
based on a couple of nicely-positioned spheres, or excessively
smoothed) and they display it with its symmetry mates.
Original message
>Date: Wed, 26 Aug 2009 11:48:57 +0200
>From: Anita Lewit-Bentley
>Subject: [ccp4bb] packing
And I apologize for being crabby about it. I realize its hard
for people from different traditions and different native
tongues to figure out the best way to start an English letter
to people you've never met.
Original message
>Date: Fri, 21 Aug 2009 16:50:28 -
>From: Debajyoti Du
>1. Do people routinely try different lengths of DNA?
Yes, its usually the most important variable because the ends
like to stack against each other to form a pseudo-continuous
helix.
>2. Do you start with blunt or sticky ends?
Both. Plan on a dozen or so different duplexes to start (
Sorry, still need help with this well-trodden territory:
We're badly in need of a new linux PC with stereo and I'm
confused about graphics cards.
We want to recycle the CRT monitor, NuVision emitter and
glasses from an old increasingly unstable machine.
This chart: http://www.nvidia.com/object/
Sorry this has probably come up already, but we're about to
become desperate for a new stereo-capable machine, to be
attached to an existing linux server.
Is there a currently proven-to-work LCD stereo system for
linux that works with Coot and/or O as well as pymol? Or
should we recycle the aging
For something that large, would it be easier and cheaper to
make E. coli do it? Say, fuse your sequence to ubiquitin?
Phoebe
Original message
>Date: Mon, 1 Jun 2009 12:09:00 -0400
>From: Pius Padayatti
>Subject: Re: [ccp4bb] Request recommendation for the peptide
synthesis company
You're not the only one to have had exactly that problem with
exactly that system!
A good ad for the potential utility of JDF (Journal of Dismal
Failure).
Phoebe
Original message
>Date: Thu, 14 May 2009 19:20:58 -0400
>From: matthew.frank...@imclone.com
>Subject: Re: [ccp4bb] Cr
My post-doc recently produced a splendid (for its resolution)
~5A map of a "medium" sized protein-DNA complex using Ta6Br12
clusters. And he's got a good toehold on a ~340kDa complex
using the same clusters. So I'm recently converted to these
little nuggets.
Phoebe
Original message
ames Stroud
>Subject: Re: [ccp4bb] Manipulating electron density
>To: CCP4BB@JISCMAIL.AC.UK
>
>On May 10, 2009, at 12:21 PM, Phoebe Rice wrote:
>> Of COURSE the map will look lovely if you carve it off at 1.5A
>> from your atoms. And your gels will look lovely too if you
&
Of COURSE the map will look lovely if you carve it off at 1.5A
from your atoms. And your gels will look lovely too if you
just touch them up with with some white-out and a sharpie.
Do the honest thing and show the whole truth, using the
z-clipping to get a comprehensible slab.
Original messa
My favorite trick was to define domain-wise ncs restraints,
extensively minimize with and without them, then plot the
difference in real-space R factor per residue. Ones that jump
up when restrained are usually involved in crystal packing,
etc, and should be removed from the restraints.
In my clums
Try running the Ni column as fast as possible and putting
concentrated EDTA in the fraction collector tubes before you
start, to minimize opportunities for metal-dependent proteases.
It may not be a magic bullet but it can't hurt.
Phoebe
Original message
>Date: Thu, 19 Mar 2009 23:53:1
We haven't tried SUMO, but had some frustrating results with
GST fusions. They did improve expression and solubility - BUT
in one case the target protein precipitated immediately when
the tag was cleaved off, and resisted all attempts to bring it
back to life. In another case, the fusion protein
Mass action is on the crystal's side.
Two recent examples of proteins that are dimers by standard
solution assays, but form weak/transient/co-factor-dependent
tetramers to function, and those tetramers are seen in the
crystal. (There is good solution data to back up the
relevance of the tetramer i
There are certainly reasons why it shouldn't work:
- the protein may alter the structure - even if its not a
"big bender", it may tweak the groove widths or introduce a
shallow overall bend in a long binding site
- the phosphates move the most between DNA structures, and
unfortunately they also
Just to be a pedantic pain - Km is not necessarily Kd. I
think that assumption only holds if the chemical step
following substrate binding is rate-limiting.
Phoebe
Original message
>Date: Mon, 1 Dec 2008 15:34:59 +0100
>From: mesters <[EMAIL PROTECTED]>
>Subject: Re: [ccp4bb] co-c
I think we still have better luck with longer, slower, more
gentle soaks - but its crystal-dependent.
Try raising the [PEG] at the same time as you raise the
[glycerol].
Phoebe
Original message
>Date: Fri, 31 Oct 2008 18:57:53 -0400
>From: Artem Evdokimov <[EMAIL PROTECTED]>
>Subjec
I was always a big fan of "lego loop" in O for building loops
into dotted lines.
Phoebe
Original message
>Date: Wed, 15 Oct 2008 16:20:26 +0100
>From: Eleanor Dodson <[EMAIL PROTECTED]>
>Subject: Re: [ccp4bb] Poor electron density - polyAla or
PolyGly?
>To: CCP4BB@JISCMAIL.AC.UK
Hi,
Sadly, that happens sometimes.
1) make sure you have some salt in the DNA stock too
2) try (NH4)2SO4 instead of NaCl (just don't add Ca++, and
remember the ionic strength of a given molarity will be higher)
3) if you have to, try more salt
3) try different ends on your oligos - sometimes th
F's.
>>>
>>>
>>>
>>> Marc Schiltz
>>>
>>>
>>>
>>>
>>>
>>> Quoting Jacob Keller <[EMAIL PROTECTED]>:
>>>
>>>
>>>> Does somebody have a .pdf of that French and Wilson
paper?
>>>>
>>>> Thanks in adv
Dear Experts,
At the risk of exposing excess ignorance, truncate makes me
very nervous because I don't quite get exactly what it is
doing with my data and what its assumptions are.
>From the documentation:
... the "truncate" procedure (ke
in
the rather lousy, low-resolution, anisotropic electron
density maps.
Phoebe
Rice PA, Steitz TA.
Model for a DNA-mediated synaptic complex suggested by
crystal packing of gamma delta resolvase subunits.
EMBO J. 1994 Apr 1;13(7):1514-24.
Abdel-Meguid SS, Murthy HM, Steitz TA.
Preliminary X
A layer of dry PEG on the outside of the dialysis tubing
works too, without changing the ionic strength. But you'll
probably get small molecular weight contaminants from the
PEG entering the bag, so you'll have to dialyze against real
buffer afterwards. The whole procedure in the end might be
You should add some salt when you anneal!!!
The duplex is highly negatively charged, so adding even a
small amount (like 10mM NaCl) will help with charge
screening, thus making the two strands less repellant to
each other. Buffer is also always a good idea. At low pH
and high temp you'll hy
If you have high [DTT] in your buffer, you might be
catalyzing the addition of dimethyl arsenic (from the
cacodylate) to some of your cysteines?
Also, 10% glyercol sounds quite low for reproducibly good
freezing (at least in my experience).
Phoebe
Original message
>Date: Mon, 2 Jun 2
I suspect if you compare Ramachandran plots you'll find that
despite the slightly higher Rfree, the ncs-restrained
coordinates already have better geometry.
You probably need to optimize your ncs restraints. For
example:
- remove the restraints all side chains that are in
different crystal pa
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