Our main reason for thinking that the nanocrystals were not twinned is
because the "regular" crystals of the same form, grown under very
similar conditions were not twinned (1jb0). The space group, unit cell,
and indeed the structure in the ASU were all the same. In fact, I don't
think anyone has grown twinned crystals of this protein?
But, to address Colin's question:
In Figure 2 of the Nature paper we showed some "Shrinkwrap"
reconstructions of the crystal shape from the spot shape. For those who
don't know Shrinnkwrap, it works a lot like "dm" or "solomon", except
you have 99.9% solvent content (the area outside the crystal), so it is
fairly robust. In all the cases I have seen, there is only one blob in
the shape reconstruction, indicating a single (non-twinned) crystal. In
fact, many of them look like a hexagonal prismatic solid, which is what
samples of these crystals look like under EM. This was done for several
images, but certainly not all of them. I don't think any single human
being has looked at all of the images.
However, maybe Colin would be interested in the "double hits"? The
nanocrystals were diluted to avoid them, but every so often two crystals
are caught in a single shot, and you see two lattices on the detector.
Autoindexing generally fails in these cases, but the distance between
the two crystallites should be apparent as a modulation of the fringes.
Colin, maybe you could ask Henry Chapman or John Spence about that?
-James Holton
MAD Scientist
On 2/10/2011 3:14 AM, Colin Nave wrote:
James
All great stuff.
ØThe individual crystals were not twinned (or at least I would be VERY
surprised if they were)
Can this be tested? The spots should show fringes around them
corresponding to the shape transform of the nano-crystal. If twinned,
the intensities of the fringes should be modified due to the contrast
between the two types of domain for the particular reflection. If it
worked, one would be equivalent to grinding up the crystal using the
coherent beam.
Probably difficult as would have to be done independently for each
crystal.
Colin
*From:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf
Of *James Holton
*Sent:* 10 February 2011 01:20
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* Re: [ccp4bb] First images of proteins and viruses caught
with an X-ray laser
The twin fraction for REFMAC was exactly 0.5. The individual crystals
were not twinned (or at least I would be VERY surprised if they were),
but they do belong to the space group P63, and autoindexing will give
you one of the two possible axis conventions at random. So, of the
15445 crystals that were merged, there were about ~7700 indexed one
way and 7700 indexed the other way. It is hard to tell exactly.
Mergeing the data "blindly" (which we did) will result in a "twinned
data set" where each merged h,k,l has an equal contribution from
k,h,-l. This was all discussed in the data processing paper PMID:
20389587.
Obviously, there are a number of things you can think of for resolving
this indexing ambiguity and removing the "twinning effect", and in
fact, this is what I thought was really cool about this particular
kind of data collection and why I encouraged the people doing all the
work (the other 87 authors) to go ahead with a "twinnable" space
group. On the first pass, you can just take advantage of TWIN
refinement in REFMAC, but in the future, when the indexing improves
(probably using post-refinement) it may be possible to de-twin a
crystal system that actually suffers from "real" twinning. That is, a
"twin domain" cannot be smaller than a mosaic block (otherwise the
h,k,l and -k,h,-l structure factors would add as phased Fs, not
|F|^2). So, since nanocrystals are essentially single mosaic blocks
(smaller than the coherence length of the beam), you could take your
twinned crystals and either grind them up or re-optimize for smaller
crystals (counterintuitive!), and then resolve the twin domains
"manually". Sort of like Louis Pasteur and his tartaric acid crystals.
Anyway, I thought that was a cool idea, but like so many other cool
things, it had to be cut from the Nature paper. Admittedly, the
problem has not actually been solved yet. This is why we used REFMAC
in TWIN mode.
-James Holton
MAD Scientist
On Wed, Feb 9, 2011 at 3:29 PM, Jon Schuermann <schue...@anl.gov
<mailto:schue...@anl.gov>> wrote:
According to the paper, the data was refined in REFMAC in 'twin mode'
which, I believe, calculates the R-factor using a non-conventional
R-factor equation which usually lower than the conventional R-factor.
I believe this is dependent on the twin fraction which wasn't
mentioned in the paper (or supplementary info) unless I missed it.
Jon
--
Jonathan P. Schuermann, Ph. D.
Beamline Scientist
NE-CAT, Building 436E
Advanced Photon Source (APS)
Argonne National Laboratory
9700 South Cass Avenue
Argonne, IL 60439
email:schue...@anl.gov <mailto:schue...@anl.gov>
Tel: (630) 252-0682
Fax: (630) 252-0687
On 02/09/2011 05:11 PM, James Holton wrote:
This was "molecular replacement" from 1jb0, so the phases came from
the model. Probably more properly called "direct refinement" since
all we did was a few cycles of rigid body. Personally, I was quite
impressed by how good the R factors were, all things considered.
-James Holton
MAD Scientist
On Wed, Feb 9, 2011 at 2:56 PM, Bernhard Rupp (Hofkristallrat a.D.)
<hofkristall...@gmail.com <mailto:hofkristall...@gmail.com>> wrote:
Any idea where then phases came from?
BR
-----Original Message-----
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK
<mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of Thomas
Juettemann
Sent: Wednesday, February 09, 2011 12:16 PM
To: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] First images of proteins and viruses caught with an
X-ray laser
Thank you for clarifying this James. Those details are indeed often
lost/misinterpreted when the paper is discussed in journal club, so your
comment was especially helpful.
Best wishes,
Thomas
On Wed, Feb 9, 2011 at 20:38, James Holton <jmhol...@lbl.gov
<mailto:jmhol...@lbl.gov>> wrote:
>
> As one of the people involved (I'm author #74 out of 88 on PMID
> 21293373), I can tell you that about half of the three million
> snapshots were blank, but we wanted to be honest about the number that
> were collected, as well as the "minimum" number that were needed to
> get a useful data set. The blank images were on purpose, since the
> nanocrystals were diluted so that there would be relatively few
> double-hits. As many of you know, multiple lattices crash autoindexing
algorithms!
>
> Whether or not a blank image or a failed autoindexing run qualifies as
> "conforming to our existing model" or not I suppose is a matter of
> semantics. But yes, I suppose some details do get lost between the
> actual work and the press release!
>
> In case anyone wants to look at the data, it has been deposited in the
> PDB under 3PCQ, and the detailed processing methods published under
PMID:
> 20389587.
>
> -James Holton
> MAD Scientist
>
> On 2/9/2011 10:38 AM, Thomas Juettemann wrote:
>>
>> http://www.nanowerk.com/news/newsid=20045.php
>>
>> http://home.slac.stanford.edu/pressreleases/2011/20110202.htm
>>
>> I think it is pretty exciting, although they only take the few
>> datasets that conform to their existing model:
>>
>> "The team combined 10,000 of the three million snapshots they took to
>> come up with a good match for the known molecular structure of
>> Photosystem I."
>
>
