Growing Crystals
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.
View the full movie in MPEG format by clicking one of the two corresponding links below.
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.
view low resolution movie view high resolution movie
download the low resolution movie download the high resolution movie
Notes on lysozyme crystal growth movie
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.
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Gaining time in a losing battle ...
The Crystal Structure of Vancomycin
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.
The structure is particularly illuminating, because it consists of a dimer with two binding pockets for the D-Ala-D-Ala- peptide tail of the nascent cell wall. One binding pocket is filled with an acetate that mimics the peptide, whereas the other is closed like a trap-door by the asparagine side-chain. The antibiotic dimer can bind two peptides coming from opposite directions, interfering with the cross-linking of the cell wall.
Schäfer et al., Structure 4 (1996) 1509-1515; Loll et al., J.Am.Chem.Soc. 119 (1997) 1516-1522.
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The 1001-atom saga
Ab initio methods of solving the phase problem break the 1000-atom barrier
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.).
A large number of previously resistant structures in the 100-1000 atom range have been solved recently by these ab initio methods. The apparently unassailable 1000-atom barrier fell for the first time during a Workshop on ab initio methods in Erice, Sicily, in May 1997 with the solution of the (unfortunately well known) structure of the triclinic form of HEW Lysozyme, which just happens to have 1001 unique protein atoms! When some heavier atoms are also present even larger structures can be solved; the current record stands at 30kD for a cytochrome protein that includes 8 iron atoms.
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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"
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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
"Kristallstrukturuntersuchungen zum Katalyse- und Regulationsmechanismus der Tyrosin-regulierten 3-Deoxy-D-arabino-Heptulosonat-7-Phosphat-Synthase aus Saccharomyces cerevisiae"
by
Verena König
"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
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