Experimental phasing with sticky triangles
Obtaining a suitable heavy-atom derivative is still a challenge in macromolecular crystallography. We have developed a new class of compounds that combines heavy atoms for experimental phasing with functional groups for protein interaction.
The first representative of this novel class is 5-amino-2,4,6-triiodoisophthalic acid (I3C), also
known as the 'the magic triangle'.
I3C contains three iodine atoms that give rise to a strong anomalous signal, also using in-house sources.
The iodine atoms are arranged in an equilateral triangle (with a side of 6.0 Å) which is easily recognised
in the heavy-atom substructure when this compound is used for heavy-atom derivatisation.
The three functional groups present in I3C - two carboxyl and one amino group - make I3C sticky.
Hydrogen bonds may be formed to the protein and involve side chains as well as the protein backbone.
We have demonstrated that I3C is suitable for structure solution using single-wavelength anomalous dispersion (SAD). It has already been used for de novo phasing of macromolecular structures.
The bromine derivative B3C with its three bromine atoms is suitable for multi-wavelength anomalous dispersion (MAD) experiments.
We are currently developing new phasing tools that show enhanced protein binding.
Beck, T., Krasauskas, A., Gruene, T. & Sheldrick, G.M.: "A magic triangle for experimental phasing of macromolecules." Acta Crystallogr. Section D 2008, 64, 1179-1182. [pdf]
For more information on experimental phasing with sticky triangles, please visit Tobias' personal page.
The three pictures below have been taken from a movie documenting the growth of lysozyme crystals using vapour diffusion in a hanging drop over a period of 36 hours.
To avoid thermal influence a cold light source with glass fibre optics
was chosen instead of the one integrated in the microscope. A photograph
was taken every 5 minutes using a digital camera attached to the microscope
and connected to a standard PC. A total of 432 single shots was taken and
after conversion to .gif format the pictures were combined to a huge animated
GIF (~200 MByte!). This was then transformed to an .avi-video file and
finally compressed to an MPEG movie.
We have provided a 'small' version (still 1.8 MByte, sorry!) for slower connections and one with the same size but higher resolution (~ 5.7 MByte) for people with a direct connection to the internet. Both movies take about 30 sec to run. A few notes on some interesting observations are given on the movie site, too (not yet). For off-line viewers these notes are attached at the end of this chapter.
If your browser is not equipped with an mpeg-plugin,
try downloading the zipped movies and view them offline.
- Crystals stop growing before half of the movie is over. This means in 'real life' the growth of these lysozyme crystals terminates about 18 hours after nucleation.
- If looking carefully, you can observe a slight pulsation ('breathing') of the drop. We are not sure but this might be due to a periodic fluctuation in temperature caused by our air condition (shame).
- The continous evaporation of the drop becomes particularly visible at its lower left corner. Appearing light reflections point to a change in surface curvature of the droplet caused by the vapor diffusion. By the time the crystals reach their final size, this reflection does not change anymore.
Gaining time in a losing battle ...
The antibiotic vancomycin is often the last line of defence against deadly streptococcal and straphylcoccal strains such as the superbug Staphylococcus aureus that have developed resistance to penicillin, methicillin and other antibiotics. A precise structure of vancomycin was essential to enable the design of new antibiotics with the hope of gaining a little time in the losing battle against antibiotic resistance.
Despite extensive attempts by crystallographers to determine the structure of vancomycin, it proved almost as resistant as the bacteria. It eventually yielded to a combination of low temperature synchrotron data and a new ab initio method of solving the crystallographic phase problem. Structures of this size (about 400 unique non-hydrogen atoms) were until recently beyond the range of small-molecule direct methods and also unsuitable for standard macromolecular methods.
Schäfer et al., Structure 4 (1996) 1509-1515; Loll et al., J.Am.Chem.Soc. 119 (1997) 1516-1522.
Conventional direct methods of solving the crystallographic phase problem (as used in programs such as MULTAN, SHELXS and SIR) are very effective for structures of up to about 100 unique non-hydrogen atoms, but have had little success with structures larger than about 200 atoms.
This situation has changed dramatically with the introduction of the
Shake & Bake program of the Buffalo group (Weeks, Miller, Hauptman
et al.) and its half-baked competition from Göttingen (Sheldrick,
Usón, Schäfer et al.).
Web-published PhD theses from our group
"Strukturanalyse zum Katalysemechanismus und zur Stabilität der Arylsulfatase A"
by
Rixa von Bülow
"Mersacidin-analoge Typ-B-Lantibiotika - Krsitallsiation, Datensammlung und Strukturaufklärung"
by
Jörg Kärcher
"Röntgenstrukturuntersuchungen an Glycopeptid-Antibiotika und ihren Komplexen mit Zellwandpeptiden Gram-Positiver Bakterien"
by
Christopher Lehmann
"Probleme der modernen hochauflösenden Einkristall-Röntgenstrukturanalyse"
by
Peter Müller
"Strukturelle Untersuchungen an Varianten des Ecballium elaterium Trypsin Inhibitors-II (EETI-II)"
by
Ralph Krätzner
"Die Kristallstruktur der α-Amylase A aus dem hyperthermophilen Bakterium
Thermotoga maritima MSB8"
by
Thomas Pape
"Crystal Structure of Wind, a PDI-Related Protein Required for Drosophila melanogaster Dorsal-Ventral Development"
by
Qingjun Ma
"Studies on the Crystallographic Phasing of Proteins: Substructure Validation and MAD-phased Electron Density Maps at Atomic Resolution"
by
Fabio Dall'Antonia
"Kristallstrukturen der C2B-Domäne von Rabphilin-3A und der PP2C-ähnlichen Phosphatase tPphA von Thermosynechococcus elongatus BP-1"
by
Christine Schlicker
"Crystallographic Analysis of Pathological Crystals, Periplasmic Domain of Ligand-free CitA Sensor Kinase and PDI-related Chaperones"
by
Madhumati Sevvana
"Crystal structures of Aldose Reductase, C2A domain of Rabphilin3A and tests of new restraints"
by
Marianna Biadene