Thanks to all who responded to my query about cheap, easy, and
foolproof systems for teaching students how to do MIR/SAD
experiments. Below is a summary of responses.
Pat
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Jim Pflugrath: Lysozyme co-crystallizes with KAu(CN)2
You know its there because you get orthorhombic crystals if gold is
incorporated.
Jochen Kuper: We have recently tried Dauters Iodine soak with
lysozyme, though it is not a covalent modification it worked
foolproof :-)!
Dauter Z et al. Novel approach to phasing pro...[PMID: 10666615]
Fred Vellieux: For HEW lysozyme: precipitant is 2 to 6 % (w/v) NaCl
in 0.2 M Na acetate pH 4.7 (the recommended procedure is to prepare a
stock solution of 10% NaCl). For the derivative, use para-chloro
mercuri benzene sulphonic acid. In 15 ml of the above stock solution,
dissolve 0.123g of PCMBS. Then use at 2 to 6% NaCl. Normally the
crystals should grow within 24 to 48 hours.
They are not completely isomorphous with the native crystals though
but this might be sufficient for a course.
Poul Nissen: We use proteinase K that crystallises very well in AmS.
It is also easy to cryoprotect and mount. Excellent derivatives with
e.g. lanthanides, and you routinely get data to the edge on the home
source and stellar experimental maps. On top of that you can also
work on e.g. PMSF and benzamidine soaks for diff. maps etc. IN my
experience far better than HEW. Have no ref at hand.
Bernie Santarsiero: Depending on your time, I'd recommend adding the
use of a native gel band
shift to try a one that doesn't work and one that does.
http://www.doe-mbi.ucla.edu/~sawaya/m230d/Crystallization/
crystallization.html
I know you can use NaBr or NaI to grow lysozyme crystals as well,
and they stick on hydrophobic patches of the enzyme's surface. I do
recall, however, that the crystal system changes from tetragonal to
the triclinic or monoclinic forms.
Jose Antonio Cuesta-Seijo: A 0.5M NaI soak for about 20 seconds
rarely fails for robust, well diffracting crystals.
It is also perfect for SIRAS, but SIR might also work. The only
question is how many iodines to look for, but the method is
straightforward with lysozime, elastase, trypsin... (I personally did
the elastase with I and the trypsin with Br-). And iodines are easy
to see and detect even in a home source as they have significant
anomalous signal and soooo many electrons.
David Roberts: I know you are looking for a recipe for class, but one
thing that was always my favorite magic bullet was osmium salts (I
think hexachloride). Basically, you add some osmium to the dip
(solid), and watch it diffuse into the crystal. The crystal will
turn colors over time, indicating the osmium is in. Osmium is good
as it gives a good anomalous signal (if you are collecting data and
showing that aspect). It's always a great derivative. I'm sure with
lysozyme it would be a good thing. Osmium crystals diffract well (it
doesn't seem to kill crystals as mercury can), and so you usually get
great derivatives from it.
As for a class, I have students solve a MIR structure (one that I
did during my post-doc). I give them a MIR map (that has been
phased) and have them trace it from scratch. I give them a few
starting residues to get them going, and they use coot to build the
molecule (using batons). They then convert it to a polyala
mainchain, add side chains in round 2, and add waters and fix all in
round 3. It's amazing how well it all works. It's a good lesson on
model building and protein structure.
James Holton: Many of us here at ALS use a hydrophobic gadolinium
derivative of tetragonal lysozyme as a "test crystal". The neat
thing about Gd is that it has just as much anomalous signal as Se at
12660 eV. The PDB id is 1H87 and all procedures are described in the
primary reference therein. You can either co-crystallize or soak.
The only tricky part is getting the chelated Gd-DO3A compound. It
was developed as a medical imaging contrast agent, so they want to
sell it to you by the pound. Corrie Ralston here asked the company
for a free sample and they sent ~1 mL. That was 5 years ago, and we
are still using that stock.
Michael Merckel: You might consider Gd-HPDO3A, Acta Cryst. (2002).
D58, 1-9.
Should also give an anomalous signal with in house source.
Neil Paterson: you can grow lysozyme quite easily in 250-400mM NaI
and this phases very nicely from an in-house source (8-9 iodines per
molecule). I can't remember the exact conditions I was using off the
top of my head (think it was 100mg/ml lysozyme and 20-30% peg4k, 0.1M
Na acetate pH 4.6 and NaI)...
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---------------
Patrick J. Loll, Ph. D.
Professor of Biochemistry & Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA 19102-1192 USA
(215) 762-7706
[EMAIL PROTECTED]
