Breakthrough in femtosecond X-ray protein nanocrystallography

4 Feb 2011

Professor Petra Fromme, a Principal Investigator of Subtask 2 at the Center, and members of her lab, Ingo Grotjohan and Raimund Fromme, have recently been involved in international collaboration study on time-resolved structure determination of membrane proteins. A recent breakthrough publication in Nature based on results of this study is entitled “Femtosecond X-ray protein nanocrystallography”.

Conventional X-ray crystallography requires crystals of good quality. So far more than sixty thousand crystal structures of the proteins had been resolved; however, only 0.4% of these proteins are membrane proteins. Obtaining atomic structure of membrane proteins that are significant players in cellular processes is limited by difficulties in crystallization and fragility of crystals. The new breakthrough method of X-ray crystal structure determination allows working with fragile crystals of membrane proteins or large DNA protein complexes that are as small as 200 nm. The novelty of the technique is in using femtosecond X-ray lasers and a stream of nanocrystals flowed with a superjet speed across the beam of the powerful laser at ambient temperature. Growing large crystals is a tedious task. Obtaining a suspension of many nanocrystals is much easier. Free electron lasers at Linac Coherent Light Source (LCLS) at Stanford Linear Accelerator Center (SLAC) provide ultra-fast X-ray (10 fs) pulses of extremely high intensity (1016 Watt per square cm). The laser pulses induce the diffraction of the electrons on atoms of the crystals. The speed of the flow of the nanocrystals is adjusted in such a way that each pulse of X-ray hits single crystal at a time and produces a diffraction pattern, which is recorded. For a good spatial resolution a plenty of femtosecond snapshots of the diffraction patterns should be accumulated and averaged. Quanta of X-ray radiation are pulsed with a frequency of 1 pulse every 30 milliseconds, which converts to 1800 per minute. At such a rate accumulation of one million diffraction patterns would take about nine hours, a little bit longer than a normal working day.

The power of the laser pulse is enough to destroy any solid material placed in the beam. However, time resolution plays a crucial role in the measurements. The duration of the laser pulse is 10 fs (10-14 sec). It turns out that during this time the snapshot of the diffraction pattern could be achieved without crystal destruction. In this work authors report on a crystal structure of the Photosystem I complex from cyanobacteria. Averaging of more than 3,000,000 diffractions from individual photosystem I nanocrystals (~200 nm to 2 μm in size) allowed obtaining a three-dimensional data set with 8.5 Å resolution. The study showed that using 10 - 70 fs pulses did not affect the observed resolution. However, pulses with longer duration (200 fsec) cause spatial disordering.

Researchers plan to improve the resolution by increasing pulse irradiance and using pulses of shorter duration, which will allow in the future further reducing damage of the crystals and performing experiments on smaller nanocrystals or even single molecules. The ultimate goal is to obtain structural information without crystallization or molecule’s ordering, an obstacle in structure study of large membrane proteins and DNA/RNA protein complexes. Petra Fromme plans to adopt the new technique for structure determination of the analogs of the photosynthetic oxygen evolving complexes that will catalyze the oxidation of water to O2 and hydrogen ions.


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