Dear CCP Bulletin Board readers,
The issue of hydrogen atoms is an interesting one that my lab has been
pursuing for
a number of years on the flavoenzyme, cholesterol oxidase. Hydrogen
atoms typically
become visible in the density maps higher than 1.2Å (this is the
accepted boundary
for defining atomic resolution). That being said the density is clear
for these atoms only when
the "heavy atoms" to which they are bonded are not overly
mobile...otherwise the
single electron of the hydrogen becomes smeared out and not visible
anymore. In
our experience the first hydrogens that become visible in the maps are
those attached
to the main chain (alpha carbon and amide hydrogens that are part of the
peptide bond).
In many ways however the more interesting ones are those on ionizable
side chains. In
the case of enzymes, these are the ones that are likely to be involved
in catalysis. One
typically needs resolutions higher than 1.0 Å to see these atoms...plus
you need alot of
order in your structure.
Anthony's comments about offset density for predicted hydrogen
positions is particularly relevant in protein structures. The internal
core region of a protein
molecule has a unique microenvironement and interesting effects can be
seen as a
result. We have recently reported such interesting effects as a
function of
pH in our structure (see Nature Chemical Biology, 2006, 2, 259-264).
When you reach resolutions higher than 0.8-0.9Å you
should consider doing multipole refinements as has been done with aldose
reductase
at 0.66Å (see Cell Mol Life Sci., 2004, 61, 774-82)
At that resolution the density around the atoms can be treated as non
spherical due to
polarization effects. Such refinements allow a much more detailed view
of the density
distribution and give important information about electrostatics.
This has been pursued for many years now on small molecules. Work by
Lecomte and colleagues with the program MoPro are making these types of
refinements
a reality for macromolecules which diffract to very high resolutions.
These studies are
important as they tell us alot about the unique environment within a
large macromolecule
and how this environement confers stability and chemistry to the molecule.
Of course if you really want to see hydrogens most convincingly neutron
diffraction is the way
to move forward. Large crystals and perdeuterated protein make such
studies
most feasible.
We can learn alot about the chemistry of a protein....one electron at a
time!
Alice
Anthony Addlagatta wrote:
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Hi Pietro,
A while ago, I had a chance to work with an ultra-high resolution
(0.86 Ang) of a small protein, BPTI (Acta Crystallogr., 2001, D57,
649-663). We have done few experiments on that data. One such was
dealing with H-atoms. We could locate several H-atoms in the
difference density map in the core of the protein, particularly on
the C-alpha and the amide N atoms (on some water molecules too!). We
added them as riding atoms in the SHELXL. To our surprise, there was
still some density near the H-atoms. We realized that the position of
some of the H-atoms, particularly on C-alpha's in a beta strand is in
fact is not at the predicted position but is offset by 0.1 to 0.2 Ang
in the direction towards a carbonyl oxygen in the neighboring strand.
We concluded that this offset is because of the possible C-H...O
hydrogen which is less accepted in biocrystallographer community.
I like your idea of adding H-atoms (probably not refining them but
only as riding, may be at resolutions better than 1.0 Ang.). But keep
in mind that not all the H-atoms are NOT in the ideal positions.
Happy hydrogenation!
Anthony
________________________________
Anthony Addlagatta, PhD
HHMI Research Associate
Institute of Molecular Biology
University of Oregon
Eugene, OR-97403
Phone: (541) 346-5867
Fax: (541)346-5270
Web: http://darkwing.uoregon.edu/~anthony
On Jan 13, 2007, at 6:56 AM, Pietro Roversi wrote:
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Dear all,
let's see if I can make a smooth transition of the
bulletin board to a different topic.
Marc Schiltz mentioned that H atoms are not routinely
refined because more often than not the resolution of the data does
not warrant their refinement - except I think there are a few H
atoms in proteins whose positions _are_ uniquely defined by the
position of the atom thy are bonded to, and the atoms bonded to that
e.g. the amide Hs, and the Hs on the Calpha, Asn and Gln amide Hs,
Proline Hs, and so on. In fact, most Hs are a pretty sure bet except
the Ser, Tyr and Thr -OH moieties, the -SH of Cys, the terminal -CH3
of Ala, Met, Ile, Val, Leu, Thr, and the ones bound to N on Arg,
Lys, His side chains. Some stereochemical guesswork could be done on
those but I do not want to open a can of worms there.
Anyway, those H atoms could be added at positions that are
essentially correct within the typical resolution of the data, at
zero parameter cost. They scatter, there are quite a few of them,
and they would actually provide extra non-bonded restraints given
that the H-X bond has a direction and a length (for example
MolProbity uses H atom addition to detect clashes pointing to
suboptimal parts of the model). This is a case of a very reliable
prior which we should incorporate all the more in our model, given
that the data are uninformative as to that part of the model.
This is already done for example in Shelx with riding H atoms
- so an option that would "add safely locatable Hydrogens" would
help all refinement programs.
As the Morris cars fans magazine's title goes: "Minor Matters"
Ciao
Pietro
--
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Alice Vrielink
Department of Chemistry & Biochemistry
University of California, Santa Cruz Office Phone: (831) 459 5126
1156 High Street Lab Phone: (831) 459 3929
Santa Cruz, CA Fax: (831) 459-3139
95064 Email: [EMAIL PROTECTED]
USA [EMAIL PROTECTED]
Home page: http://bio.research.ucsc.edu/people/vrielink
"Proper education is not inculcation. Apart from developing skills and imparting a
core of indispensable information, education is a process of widening experience, of
fostering a spirit of inquiry, and thereby of creating the basis for disciplined
judgement" M. I. Finley
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