Membrane protein megahertz crystallography at the European XFEL

The world’s first superconducting megahertz repetition rate hard X-ray free-electron laser (XFEL), the European XFEL, began operation in 2017, featuring a unique pulse train structure with 886 ns between pulses. With its rapid pulse rate, the European XFEL may alleviate some of the increasing demand for XFEL beamtime, particularly for membrane protein serial femtosecond crystallography (SFX), leveraging orders-of-magnitude faster data collection. Here, we report the first membrane protein megahertz SFX experiment, where we determined a 2.9 Å-resolution SFX structure of the large membrane protein complex, Photosystem I, a > 1 MDa complex containing 36 protein subunits and 381 cofactors. We address challenges to megahertz SFX for membrane protein complexes, including growth of large quantities of crystals and the large molecular and unit cell size that influence data collection and analysis. The results imply that megahertz crystallography could have an important impact on structure determination of large protein complexes with XFELs. The European X-ray free-electron laser (EuXFEL) in Hamburg is the first XFEL with a megahertz repetition rate. Here the authors present the 2.9 Å structure of the large membrane protein complex Photosystem I from T. elongatus that was determined at the EuXFEL.

Steffen Hauf | Hans Fangohr | Anton Barty | Saša Bajt | Henry N. Chapman | Marc Messerschmidt | Petra Fromme | Matthias Frank | Marius Schmidt | Peter Schwander | Jolanta Sztuk-Dambietz | Matthew A. Coleman | Thomas Michelat | Monica Turcato | Richard A. Kirian | Abbas Ourmazd | John C. H. Spence | Manuela Kuhn | Sandor Brockhauser | Cyril Danilevski | Alessandro Silenzi | Jose M. Martin-Garcia | Alexandra Ros | Thomas A. White | Chen Xu | Janusz Szuba | Adrian P. Mancuso | Mark S. Hunter | Gianpietro Previtali | Krzysztof Wrona | Johan Bielecki | Romain Letrun | Grant Mills | Britta Weinhausen | Valerio Mariani | Djelloul Boukhelef | Max O. Wiedorn | Katerina Dörner | Shatabdi Roy-Chowdhury | Yaroslav Gevorkov | Ahmad Hosseinizadeh | Nadia A. Zatsepin | Luis Maia | Richard Bean | Tokushi Sato | Nasser Al-Qudami | Maurizio Manetti | Thomas D. Grant | Yoonhee Kim | H. Chapman | A. Mancuso | M. Frank | A. Barty | S. Bajt | M. Messerschmidt | M. Hunter | T. White | J. Spence | P. Fromme | R. Kirian | A. Ourmazd | P. Schwander | V. Mariani | J. Bielecki | S. Aplin | Chen Xu | C. Danilevski | L. Maia | S. Brockhauser | T. Michelat | H. Fangohr | S. Hauf | D. Boukhelef | J. Szuba | M. Turcato | K. Wrona | R. Fromme | T. Grant | J. Knoška | M. Wiedorn | K. Dörner | O. Yefanov | N. Zatsepin | C. Luna-Chavez | A. Ros | I. Sarrou | M. Coleman | S. Botha | J. Yang | R. Bean | J. Mondal | B. Bruce | Tokushi Sato | J. Martín-García | F. Januschek | A. Silenzi | J. Sztuk-Dambietz | M. Kuhn | Shatabdi Roy-Chowdhury | J. Coe | Marius Schmidt | Steve Aplin | Sabine Botha | Christopher Kupitz | Iosifina Sarrou | Barry D. Bruce | M. Shelby | Raimund Fromme | Oleksandr M. Yefanov | Jesse Coe | Gihan K. Ketawala | Chris Gisriel | Cesar Luna-Chavez | Natasha E. Stander | Stella Lisova | Austin Echelmeier | Jorvani Cruz Villarreal | Juraj Knoska | Victoria Mazalova | Jay-How Yang | Alex Jones | Jose D. Meza | Gerrit Brehm | Erin Discianno | Zachary Dobson | Friederike Januschek | Jyotirmoy Mondal | Silvan Schön | Megan L. Shelby | Mohamed H. Abdellatif | James D. Zook | C. Gisriel | Y. Gevorkov | M. Abdellatif | Yoonhee Kim | B. Weinhausen | A. Hosseinizadeh | R. Letrun | Austin Echelmeier | Jorvani Cruz Villarreal | N. Al-Qudami | Christopher Kupitz | M. Manetti | Z. Dobson | Gerrit Brehm | V. Mazalova | E. Discianno | Gianpietro Previtali | Stella Lisova | Jorvani Cruz Villarreal | G. Mills | A. Jones | J. D. Meza | Silvan Schön | J. Martin-Garcia | Gihan Ketawala | M. Hunter | S. Schön | Victoria Mazalova

[1]  Steffen Hauf,et al.  Megahertz data collection from protein microcrystals at an X-ray free-electron laser , 2018, Nature Communications.

[2]  Georg Weidenspointner,et al.  Time-resolved protein nanocrystallography using an X-ray free-electron laser , 2012, Optics express.

[3]  Roberto Dinapoli,et al.  The Adaptive Gain Integrating Pixel Detector at the European XFEL , 2018, Journal of synchrotron radiation.

[4]  Randy J. Read,et al.  Acta Crystallographica Section D Biological , 2003 .

[5]  Petra Fromme,et al.  Three-dimensional structure of cyanobacterial photosystem I at 2.5 Å resolution , 2001, Nature.

[6]  R. Feidenhansl The European X-ray Free-Electron Laser , 2017 .

[7]  Sébastien Boutet,et al.  Direct observation of ultrafast collective motions in CO myoglobin upon ligand dissociation , 2015, Science.

[8]  Anton Barty,et al.  CrystFEL: a software suite for snapshot serial crystallography , 2012 .

[9]  P. Emsley,et al.  Features and development of Coot , 2010, Acta crystallographica. Section D, Biological crystallography.

[10]  Albert J. M. Duisenberg,et al.  Indexing in single‐crystal diffractometry with an obstinate list of reflections , 1992 .

[11]  Daniel Beisel,et al.  An anti-settling sample delivery instrument for serial femtosecond crystallography , 2012 .

[12]  Anton Barty,et al.  Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography , 2017 .

[13]  K. Schmidt,et al.  Gas dynamic virtual nozzle for generation of microscopic droplet streams , 2008, 0803.4181.

[14]  Anton Barty,et al.  Double-flow focused liquid injector for efficient serial femtosecond crystallography , 2017, Scientific Reports.

[15]  M. Hunter,et al.  Toward structure determination using membrane-protein nanocrystals and microcrystals. , 2011, Methods.

[16]  Anton Barty,et al.  OnDA: online data analysis and feedback for serial X-ray imaging1 , 2016, Journal of applied crystallography.

[17]  Steffen Hauf,et al.  Data Analysis Support in Karabo at European XFEL , 2018 .

[18]  Gianluca Geloni,et al.  The European X-ray Free-Electron Laser , 2015 .

[19]  Marcin Sikorski,et al.  The Single Particles, Clusters and Biomolecules and Serial Femtosecond Crystallography instrument of the European XFEL: initial installation1 , 2019, Journal of synchrotron radiation.

[20]  B. Schmitt,et al.  Megapixels @ Megahertz – The AGIPD high-speed cameras for the European XFEL , 2019, Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment.

[21]  Roberto Dinapoli,et al.  The adaptive gain integrating pixel detector AGIPD a detector for the European XFEL , 2011 .

[22]  Marius Schmidt Time-Resolved Macromolecular Crystallography at Pulsed X-ray Sources , 2019, International journal of molecular sciences.

[23]  Randy J. Read,et al.  Phaser crystallographic software , 2007, Journal of applied crystallography.

[24]  Henry N. Chapman,et al.  Correction: Corrigendum: X-ray holography with a customizable reference , 2014, Nature Communications.

[25]  Anton Barty,et al.  Recent developments in CrystFEL , 2016, Journal of applied crystallography.

[26]  Randy J. Read,et al.  Overview of the CCP4 suite and current developments , 2011, Acta crystallographica. Section D, Biological crystallography.

[27]  N. Nelson,et al.  Structure and function of wild-type and subunit-depleted photosystem I in Synechocystis. , 2018, Biochimica et biophysica acta. Bioenergetics.

[28]  Ji-Xin Cheng,et al.  Selective detection of protein crystals by second harmonic microscopy. , 2008, Journal of the American Chemical Society.

[29]  Mitchell D. Miller,et al.  Structural enzymology using X-ray free electron lasers , 2016, Structural dynamics.

[30]  Huixia Yang,et al.  Molecular mechanism of photosystem I assembly in oxygenic organisms. , 2015, Biochimica et biophysica acta.

[31]  W. Delano The PyMOL Molecular Graphics System , 2002 .

[32]  Anton Barty,et al.  Accurate determination of segmented X-ray detector geometry. , 2015, Optics express.

[33]  Garth J. Williams,et al.  Time-resolved serial crystallography captures high-resolution intermediates of photoactive yellow protein , 2014, Science.

[34]  H. N. Chapman,et al.  Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography , 2016, Nature.

[35]  Georg Weidenspointner,et al.  Femtosecond X-ray protein nanocrystallography , 2011, Nature.

[36]  P. Zwart,et al.  Towards automated crystallographic structure refinement with phenix.refine , 2012, Acta crystallographica. Section D, Biological crystallography.

[37]  C. Betzel,et al.  Three-dimensional structure of system I of photosynthesis at 6 Å resolution , 1993, Nature.

[38]  N. Pannu,et al.  REFMAC5 for the refinement of macromolecular crystal structures , 2011, Acta crystallographica. Section D, Biological crystallography.

[39]  P. Fromme,et al.  Photosystem I, an Improved Model of the Stromal Subunits PsaC, PsaD, and PsaE* , 1999, The Journal of Biological Chemistry.

[40]  P. Fromme,et al.  Localization of Two Phylloquinones, QK and QK′, in an Improved Electron Density Map of Photosystem I at 4-Å Resolution* , 1999, The Journal of Biological Chemistry.

[41]  Owen Johnson,et al.  iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM , 2011, Acta crystallographica. Section D, Biological crystallography.

[42]  Anton Barty,et al.  Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography , 2014, Nature Communications.

[43]  S. Boutet,et al.  3D printed droplet generation devices for serial femtosecond crystallography enabled by surface coating. , 2019, Journal of applied crystallography.

[44]  Steffen Hauf,et al.  Megahertz serial crystallography , 2018, Nature Communications.

[45]  Garth J. Williams,et al.  High-Resolution Protein Structure Determination by Serial Femtosecond Crystallography , 2012, Science.

[46]  G. Nass Kovács,et al.  Sample delivery for serial crystallography at free-electron lasers and synchrotrons , 2019, Acta crystallographica. Section D, Structural biology.

[47]  J.N.Galayda The New LCLS-II Project : Status and Challenges , 2014 .

[48]  M. J. Adams Preparation and Analysis of Protein Crystals , 1983 .

[49]  Petra Fromme,et al.  Improved isolation and crystallization of photosystem I for structural analysis , 1998 .

[50]  Anton Barty,et al.  Enzyme intermediates captured “on the fly” by mix-and-inject serial crystallography , 2017, bioRxiv.

[51]  A. Ros,et al.  Electric Triggering for Enhanced Control of Droplet Generation. , 2019, Analytical chemistry.

[52]  Anton Barty,et al.  Cheetah: software for high-throughput reduction and analysis of serial femtosecond X-ray diffraction data , 2014, Journal of applied crystallography.

[53]  H. Chapman,et al.  Rapid sample delivery for megahertz serial crystallography at X-ray FELs , 2018, IUCrJ.

[54]  Anton Barty,et al.  XGANDALF – extended gradient descent algorithm for lattice finding , 2019, Acta crystallographica. Section A, Foundations and advances.

[55]  P. Fromme,et al.  Photosystem I at 4 Å resolution represents the first structural model of a joint photosynthetic reaction centre and core antenna system , 1996, Nature Structural Biology.

[56]  Uwe Weierstall,et al.  Liquid sample delivery techniques for serial femtosecond crystallography , 2014, Philosophical Transactions of the Royal Society B: Biological Sciences.