Femtosecond X-ray proteinnanocrystallography

Authors Henry N. Chapman, Petra Fromme, Anton Barty, Thomas A. White, Richard A. Kirian,

Andrew Aquila, Mark S. Hunter, Joachim Schulz, Daniel P. DePonte, Uwe Weierstall, R. Bruce Doak, Filipe R. N. C. Maia, Andrew V. Martin, Ilme Schlichting, Lukas Lomb, Nicola Coppola, Robert L. Shoeman, Sascha W. Epp, Robert Hartmann, Daniel Rolles, Artem Rudenko, Lutz Foucar, Nils Kimmel, Georg Weidenspointner, Peter Holl, Mengning Liang, Miriam Barthelmess, Carl Caleman, Sébastien Boutet, Michael J. Bogan, Jacek Krzywinski, Christoph Bostedt, Saša Bajt, Lars Gumprecht, BenediktRudek, Benjamin Erk, Carlo Schmidt, André Hömke, Christian Reich, Daniel Pietschner, Lothar Strüder, Günter Hauser, Hubert Gorke, Joachim Ullrich, Sven Herrmann, Gerhard Schaller, Florian Schopper, Heike Soltau, Kai-Uwe Kühnel, Marc Messerschmidt, John D. Bozek, Stefan P. Hau-Riege, Matthias Frank, Christina Y. Hampton, Raymond G. Sierra, Dmitri Starodub, Garth J. Williams, Janos Hajdu, Nicusor Timneanu, M. Marvin Seibert, Jakob Andreasson, Andrea Rocker, OlofJönsson, Martin Svenda, Stephan Stern, Karol Nass, Robert Andritschke, Claus-Dieter Schröter, Faton Krasniqi, Mario Bott, Kevin E. Schmidt, Xiaoyu Wang, Ingo Grotjohann, James M. Holton, Thomas R. M. Barends, Richard Neutze, Stefano Marchesini, Raimund Fromme, Sebastian Schorb, Daniela Rupp, Marcus Adolph, Tais Gorkhover, Inger Andersson, Helmut Hirsemann, Guillaume Potdevin, Heinz Graafsma, BjörnNilsson & John C. H. Spence

Problems with conventional X-ray crystallography

Growing the Crystal-Between 0.2mm-1mm shortest dimension-Time Consuming -Membrane proteins problematic

Cryogenic Temperature

Improves Diffraction

Reduces X-ray Damage

X-ray Damage to Light Sensitive Proteins

Femtosecond CrystallographyOverview

How Femtosecond X-Ray over comes these issues

Use of Nanocrystals means easier more frequent crystal growth.

Diffraction data can be taken at room temperature.

Data collection Faster than destruction.(Diffraction before Destruction)

How long is a Femtosecond

Femtosecond is the SI unit of time equal to 10−15 of a second.

That is one quadrillionth, or one millionth of one billionth, of a second.

For context, a femtosecond is to a second as a second is to about 31.7 million years.

Components

Beam

Stream

Screens

BEAM

Free Electron Laser(FEL)

Electrons are not bound to atomic nuclei but travel freely through magnetic structure

Linear Particle Accelerator Coherent Light Source

Linac Coherent Light Source (LCLS) Most Powerful X-ray source in the world A Billions times more powerful than Synchotrons

Stanford UniversityMenlo Park, CA

Beam used in experiment

Photon energy X-ray pulse: 1.8 KeV(6.9A)

Dose: 700Mgy compare to typical 30MGy

Power Density: 1016 W cm-2

Duration: 10, 70, 200 fs @ 30Hz or 1,800/min

STREAM

Photosystem 1

Flowrate: 10µl min-1

Crystal Concentration: 109/ml Protein Concentration 1mg/ml Crystal hit rate 20% 4-μm-diameter column 10mg of protein

Data Collected

1,850,000 X-Ray Pulses

1 / 25,000 Nanocrystals Hit

112,725 Hits(>10 diffraction points)

2,424,394 observed reflections

3,379 unique reflections

a. Upper and lower front detector diffraction pattern

b. Pattern from merged data of 15,000 nanocrystals

Low angle diffraction patterns

Merged 3D data

Data Quality

Can you tell which one is from conventional synchrotron 8.5 A and which is femtosecond method?

We tested the reliability of this approach by comparing the LCLS merged data with data collected at 100 K with 12.4-keV synchrotron radiation from a single crystal of photosystem I cryopreserved in 2 M sucrose. These data sets show good agreement, with a difference metric, Riso, of 22.1% computed over the entire resolution range

To complete our proof of principle, we conducted a rigid-body refinement of the published photosystem I structure (Protein Data Bank ID, 1JB0) against the nanocrystalstructure factors, yielding R/Rfree = 0.25/0.23.

Typical values range from 0.6-0,2

From Text

Problems

Currently at low resolution.

Needs worlds most powerful laser.

Needs to collect a lot of data most of it unusable.

Lots of wasted sample.

Improvements

Using inkjet, timed to match pulse would use 25,000x less sample(0.4µl)

Lower wavelength, higher resolution X-Ray

Tighter beam, using less time 20fs can give good resolution and further limit X-ray damage .

High-Resolution Protein Structure Determination by Serial Femtosecond Crystallography(1.9A)

Science. 2012 Jul 20;337(6092):362-4. doi: 10.1126/science.1217737. Epub 2012 May 31.

Sébastien Boutet,1,* Lukas Lomb,2,3 Garth J. Williams,1 Thomas R. M. Barends,2,3 Andrew Aquila,4 R. Bruce Doak,5 Uwe Weierstall,5 Daniel P. DePonte,4 Jan Steinbrener,2,3 Robert L. Shoeman,2,3 Marc Messerschmidt,1 Anton Barty,4 Thomas A. White,4 Stephan Kassemeyer,2,3 Richard A. Kirian,5 M. Marvin Seibert,1 Paul A. Montanez,1 Chris Kenney,6 Ryan Herbst,6 Philip Hart,6 Jack Pines,6 Gunther Haller,6 Sol M. Gruner,7,8 Hugh T. Philipp,7 Mark W. Tate,7 Marianne Hromalik,9 Lucas J. Koerner,10 Niels van Bakel,11 John Morse,12 Wilfred Ghonsalves,1 David Arnlund,13 Michael J. Bogan,14 Carl Caleman,4 Raimund Fromme,15 Christina Y. Hampton,14 Mark S. Hunter,15 Linda C. Johansson,13 GergelyKatona,13 Christopher Kupitz,15 Mengning Liang,4 Andrew V. Martin,4 Karol Nass,16 Lars Redecke,17,18 Francesco Stellato,4 Nicusor Timneanu,19 Dingjie Wang,5 Nadia A. Zatsepin,5 Donald Schafer,1 James Defever,1 Richard Neutze,13 Petra Fromme,15 John C. H. Spence,5 Henry N. Chapman,4,16 and Ilme Schlichting2,3

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