From: Jacob Keller
Date: 10 February 2012 20:25
Dear Crystallographers,
I am looking for references which discuss the validity of the
assertion that multiple crystal structures of the same or similar
proteins can be considered freeze-frame snapshots of actual
conformations assumed in solution. In a way, the assertion seems
almost definitely true to me, but on the other hand, I could imagine
some objections as well. Seems there should be some classic literature
here...
All the best,
Jacob
----------
From: James Stroud
How could they not be snapshots of conformations adopted in solution?
James
----------
From: Nat Echols
compact lattice and freezing it to 100K isn't necessarily
representative of "solution", especially when your solution contains
non-physiological amounts of salt and various organics (and possibly
non-physiological pH too).
-Nat
----------
From: Jacob Keller
same protein solved under different conditions, or several homologs
solved under similar conditions, or both. Further, let's say some
structural element, perhaps a helix, assumes different mannerisms in
the various structures. Can I reasonably assert that these structures
are snapshots of the protein which would have existed under
physiological conditions, and assemble the structures to a unifying
conception of the helical motion, or must I assume these are artifacts
of bizarre solution conditions, and one has nothing to do with the
other? Or perhaps every case/protein is unique, in which case no
general rule can be followed, amounting approximately to the same
thing?
Jacob
----------
From: Robert Immormino
Hi Jacob,
Lorena Beese has a few systems where snapshots of reaction mechanisms
have been looked at structurally.
Here are two such papers:
Long, SB, Casey, P., Beese, LS (2002) The reaction path of protein
farnesyltransferase at atomic resolution. Nature Oct 10;
419(6907):645-50.
http://www.ncbi.nlm.nih.gov/pubmed?term=The%20reaction%20path%20of%20protein%20farnesyltransferase%20at%20atomic%20resolution
J. R. Kiefer, C. Mao, J. C. Braman and L. S. Beese (1998) "Visualizing
DNA replication in a catalytically active Bacillus DNA polymerase
crystal" Nature 6664:304-7.
http://www.ncbi.nlm.nih.gov/pubmed?term=Visualizing%20DNA%20replication%20in%20a%20catalytically%20active%20Bacillus%20DNA%20polymerase%20crystal
Cheers,
-bob
----------
From: David Schuller
Date: 10 February 2012 20:25
Dear Crystallographers,
I am looking for references which discuss the validity of the
assertion that multiple crystal structures of the same or similar
proteins can be considered freeze-frame snapshots of actual
conformations assumed in solution. In a way, the assertion seems
almost definitely true to me, but on the other hand, I could imagine
some objections as well. Seems there should be some classic literature
here...
All the best,
Jacob
----------
From: James Stroud
How could they not be snapshots of conformations adopted in solution?
James
----------
From: Nat Echols
On Fri, Feb 10, 2012 at 12:29 PM, James Stroud <xtald00d@gmail.com> wrote:
> How could they not be snapshots of conformations adopted in solution?
Packing billions of copies of an irregularly-shaped protein into a> How could they not be snapshots of conformations adopted in solution?
compact lattice and freezing it to 100K isn't necessarily
representative of "solution", especially when your solution contains
non-physiological amounts of salt and various organics (and possibly
non-physiological pH too).
-Nat
----------
From: Jacob Keller
> How could they not be snapshots of conformations adopted in solution?
Let me clarify--sorry about that. Consider several structures of thesame protein solved under different conditions, or several homologs
solved under similar conditions, or both. Further, let's say some
structural element, perhaps a helix, assumes different mannerisms in
the various structures. Can I reasonably assert that these structures
are snapshots of the protein which would have existed under
physiological conditions, and assemble the structures to a unifying
conception of the helical motion, or must I assume these are artifacts
of bizarre solution conditions, and one has nothing to do with the
other? Or perhaps every case/protein is unique, in which case no
general rule can be followed, amounting approximately to the same
thing?
Jacob
----------
From: Robert Immormino
Hi Jacob,
Lorena Beese has a few systems where snapshots of reaction mechanisms
have been looked at structurally.
Here are two such papers:
Long, SB, Casey, P., Beese, LS (2002) The reaction path of protein
farnesyltransferase at atomic resolution. Nature Oct 10;
419(6907):645-50.
http://www.ncbi.nlm.nih.gov/pubmed?term=The%20reaction%20path%20of%20protein%20farnesyltransferase%20at%20atomic%20resolution
J. R. Kiefer, C. Mao, J. C. Braman and L. S. Beese (1998) "Visualizing
DNA replication in a catalytically active Bacillus DNA polymerase
crystal" Nature 6664:304-7.
http://www.ncbi.nlm.nih.gov/pubmed?term=Visualizing%20DNA%20replication%20in%20a%20catalytically%20active%20Bacillus%20DNA%20polymerase%20crystal
Cheers,
-bob
----------
From: David Schuller
On 02/10/2012 03:25 PM, Jacob Keller wrote:
How could that possibly be the case when any structure is an average of all the unit cells of the crystal over the timespan of the diffraction experiment?Dear Crystallographers,
I am looking for references which discuss the validity of the
assertion that multiple crystal structures of the same or similar
proteins can be considered freeze-frame snapshots of actual
conformations assumed in solution. In a way, the assertion seems
almost definitely true to me, but on the other hand, I could imagine
some objections as well. Seems there should be some classic literature
here...
----------
From: Roger Rowlett
I believe the most justifiable assumption one can make is that crystal structures are likely to represent the least soluble conformations of a protein under the conditions of crystallization (which might be a broad range of conditions, including physiological). This can be quite vexing if you are studying an allosteric protein and one of the two conformations is typically much less soluble than the other. BTDT. I'm sure others have had the same experience.
Having said that, the solvent content of protein crystals (which is close to that of cellular conditions), the observation of enzymatic activity in many protein crystals, and the *general* concordance of XRD and NMR structures of proteins (when both have been determined) leads one to believe that XRD structures are likely representative of physiologically relevant conformations.
Cheers,
Having said that, the solvent content of protein crystals (which is close to that of cellular conditions), the observation of enzymatic activity in many protein crystals, and the *general* concordance of XRD and NMR structures of proteins (when both have been determined) leads one to believe that XRD structures are likely representative of physiologically relevant conformations.
Cheers,
From: Jacob Keller
Interesting to juxtapose these two responses:
James Stroud:
>How could they not be snapshots of conformations adopted in solution?
David Schuller:> How could that possibly be the case when any structure is an average of all
> the unit cells of the crystal over the timespan of the diffraction
> experiment?
JPK> the unit cells of the crystal over the timespan of the diffraction
> experiment?
----------
From: James Stroud
So the implication is that some of these treatments might allow the protein to overcome energetic barriers that are prohibitive in solution--after the protein is already in the solid state and not in solution any more?
Another view is that crystallization is a result of stabilizing conformations that are accessible in solution.
On the point of physiological relevance, it wasn't mentioned in the original question.
James
----------
From: George
>Packing billions of copies into a compact lattice
Not so compact there is 40-80% water
>freezing it to 100K
We have frozen many times protein solutions in liquid nitrogen and then thaw
and were working OK
> non-physiological amounts of salt and various organics
What is the amount of salt and osmotic pressure in the cell??>non-physiological pH too
What is the non-physiological pH too? I am sure that some enzymes they are
not working in pH 7. Also most of the proteins they have crystallized in pH
close to 7 so I would not say non-physiological.
George
PS There are lots of solution NMR structures as well supporting the
physiological crystal structures
----------
From: James Stroud
The contrast seems to boil down to the semantics of the word "snapshot".
In my definition, I assume that the uncertainty of a structure is an intrinsic quality of the structure and thus included in the meaning of "snapshot". Part of that uncertainty comes from averaging.
James
----------
From: Ethan Merritt
On Friday, February 10, 2012 12:51:03 pm Jacob Keller wrote:
> Interesting to juxtapose these two responses:
>
> James Stroud:
> >How could they not be snapshots of conformations adopted in solution?
>
> David Schuller:
> > How could that possibly be the case when any structure is an average of all
> > the unit cells of the crystal over the timespan of the diffraction
> > experiment?
This pair of perspectives is the starting point for the introductory> Interesting to juxtapose these two responses:
>
> James Stroud:
> >How could they not be snapshots of conformations adopted in solution?
>
> David Schuller:
> > How could that possibly be the case when any structure is an average of all
> > the unit cells of the crystal over the timespan of the diffraction
> > experiment?
rationale I usually present for TLSMD analysis.
The crystal structure is a snapshot, but just like a photographic snapshot
it contains blurry parts where the camera has captured a superposition
of microconformations. When you photograph an object in motion, those
microconformations correspond to a trajectory purely along time.
In a crystallographic experiment, the microconformations correspond
to samples from a trajectory in solution. Separation in time has
been transformed into separation in space (from one unit cell to
another). A TLSMD model tries to reproduce the observed blurring by
modeling it a samples from a trajectory described by TLS displacement.
The issue of averaging over the timespan of the diffraction experiment
is relevant primarily to individual atomic vibrations, not so much to
what we normally mean by "conformations" of overall protein structure.
Ethan
--
Ethan A Merritt
----------
From: Nat Echols
Just to clarify - I actually think the original assumption that Jacob
posted is generally reasonable. But it needn't necessarily follow
that the conformation we see in crystal structures is always
representative of the solution state; given the extreme range of
conditions in which crystals grow, I would be surprised if there
weren't counter-examples. I'm not familiar enough with the literature
on domain swapping (e.g. diptheria toxin) to know if any of those
structures are crystal packing artifacts.
----------
From: Damian Ekiert
Along the lines of Roger's second point, there was a very nice paper a few years back that found very good agreement between the conformational ensemble sampled by ubiquitin in solution (by NMR) with the ensemble of conformations observed in a large number of crystal structures:
Lange OF, Lakomek NA, Farès C, Schröder GF, Walter KF, Becker S, Meiler J, Grubmüller H, Griesinger C, de Groot BL.
Recognition dynamics up to microseconds revealed from an RDC-derived ubiquitin ensemble in solution.
Science. 2008 Jun 13;320(5882):1471-5. PubMed PMID: 18556554.
Best,
Damian Ekiert
----------
From: Jacob Keller
Isn't calcium-calmodulin one of the archetypical examples of the
crystal structure probably not representing the solution structure
(perhaps because the crystallization pH = 4.5)? Look at that linker
helix--how stable can that be in solution? I don't think a single one
of the NMR ca-calmodulin structures/conformers has the central helix
like that.
Jacob
----------
From: Jon Agirre
Hi Nat,
there are a number of viruses in which a domain swap occurs inside the capsid, with the hinge sequence being highly conserved among their respective families. Perhaps I'm missing your point, but I won't attribute that kind of domain swap to any sort of crystal packing artifact.
Jon--
----------Dr. Jon Agirre
From: Mark Wilson
Hi Jacob,
For Ca2+-CaM, and flexible proteins in general, the average conformation in solution may differ from the most crystallizable conformation. However, any crystallized conformation had to be sampled in solution at some point in order to form a crystal, and thus the crystal structure tells us something about the range of conformations accessible to the protein under the crystallization conditions. In Ca2+-CaM, the presence of MPD is probably more responsible for the continuous central helix than the pH, but early analysis of the thermal factors in that region of the crystal structure predicted flexibility in the center of this helix that was subsequently observed by NMR to be a flexible linker region. More generally, I'd argue that crystal disorder is a subset of solution motion: i.e. disorder observed in crystalline protein almost certainly corresponds to motions that occur in solution (perhaps with altered amplitude), but not all solution motions are observed as disorder in the crystal.
Best regards,
Mark
Mark A. Wilson
----------
From: Zhijie Li
Hi,
There is a interesting paper/tool that might shed a little light on the
debate here:
The paper: http://www.ncbi.nlm.nih.gov/pubmed/19956261
The tool:
http://ucxray.berkeley.edu/ringer/Documentation/ringerManual.htm#Utility
As I remember, this tool claimed to be able to extract information about the
subtle or "hidden" movements of side chains of an enzyme from high-res
crystallographic data.
One thing to note: the authors collected the dataset used in the Nature
paper at RT. However their online manual said they also analyzed 402 hi-res
structure in PDB (all kinds of growth conditions apparently, and most, if
not all, were probably collected under cryo stream) and found an abundance
of alternative side chain conformations. Are all these alternative
conformations relevant to the proteins' native states under physiological
conditions? I guess it must be case-by-case.
JPK: you might find something you are looking for in the nature paper's
reference. The part mentioning the myoglobin and RNase work seems promising.
Good luck.
Zhijie
----------
From: Joel Sussman
2012_02_11
Dear All,
Two really striking examples of "Intrinsically Flexible Proteins" are:
(1) Adenylate kinase: Vonrhein, Schlauderer & Schulz (1995) Structure 3, 483
"Movie of the structural changes during a catalytic cycle of nucleoside monophosphate kinases"
in particular look at:
"video as MPEG white background, closing & opening enzyme (707kb)"
Each "black dot" [upper left, in the morph] indicates an observed crystal structure.
(2) Lac repressor: see Proteopedia page on lac repressor,
morphing from the structure bound to its cognate DNA, to that of the structure bound to its the non-cognate DNA,
best regards,
Joel
----------
From: Poul Nissen
Another good lesson here:
2.
Vestergaard B, Sanyal S, Roessle M, Mora L, Buckingham RH, Kastrup JS, Gajhede M, Svergun DI, Ehrenberg M.
Mol Cell. 2005 Dec 22;20(6):929-38.
----------
From: Nian Huang
Is it possible the solution structure of SAXS, NMR and EM neglect the existence of a very small percentage conformation of the molecule due to the overwhelming signals from the majority conformations? But this state of the molecule is trapped and enriched by the crystallization condition.
Nian
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