Great experience at BioMAX

On June 30, 2017 Professor Jette Sandholm Kastrup, University of Copenhagen were granted two shifts of beamtime at BioMAX by the Program Advisory Committee (PAC) and the MAX IV Laboratory Management for the project “Molecular recognition of agonists, antagonists and positive allosteric modulators at ionotropic glutamate receptors”. The ionotropic glutamate receptors (iGluRs) are highly abundant in the central nervous system (CNS) and mediate fast synaptic neurotransmission. Dysfunction of the glutamatergic system has been associated with various diseases in the CNS, e.g. depression, Parkinson’s and Alzheimer’s diseases and epilepsy. The iGluRs are for example considered an attractive and appropriate target for the discovery of cognitive enhancers.

Three researchers (Associate professor Karla Frydenvang, Professors Michael Gajhede and Jette Sandholm Kastrup) went to MAX IV with one dewar loaded with ~60 protein crystals. At the BioMAX beamline experienced beamline scientists met them. One of the first comments from Uwe Müller was: “If you want to test 40 crystals manually (the robot for crystal mounting not working yet), you will need to work like dogs”. So, we did! Everything worked out very well at the BioMAX beamline and the computer rooms and laboratories were great. “It was a fantastic experience”, says Jette Sandholm Kastrup.

 

Professor Jette Sandholm Kastrup manually mounting a crystal of the ligand-binding domain of an ionotropic glutamate receptor in complex with glutamate and a novel positive allosteric modulator. Associate professor Karla Frydenvang to the right is assisting her.
Professor Jette Sandholm Kastrup manually mounting a crystal of the ligand-binding domain of an ionotropic glutamate receptor in complex with glutamate and a novel positive allosteric modulator. Associate professor Karla Frydenvang to the right is assisting her.

Several data sets were collected at the beamline, which led to three new structures (wildtype and two mutants of the receptor) in complex with glutamate and a novel positive allosteric modulator. Full data sets could be collected in less than 15 sec and the best data set was collected at 1.3 Å resolution. Beamline scientist Uwe Müller wrote at LinkedIn: “First structure phased at the beamline! Good team work!” The initial electron density maps are of excellent quality, allowing unambiguous positioning of glutamate and the novel positive allosteric modulator.

 

Fo-Fc electron density map contoured at 3 sigma, clearly showing the location of glutamate in the structure.
Fo-Fc electron density map contoured at 3 sigma, clearly showing the location of glutamate in the structure.
After struggling with crystallization of the complex, Professor Jette sandholm Kastrup found it difficult to get her arms down when the very well-defined electron density for the ligands appeared at the screen.
After struggling with crystallization of the complex, Professor Jette sandholm Kastrup found it difficult to get her arms down when the very well-defined electron density for the ligands appeared at the screen.