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1.  Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1 
IUCrJ  2014;1(Pt 5):305-317.
Femtosecond X-ray crystallography allows structural analysis of a difficult-to-crystallize fusion protein that is a potential component of a candidate HIV-1 subunit vaccine.
CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.
doi:10.1107/S2052252514014900
PMCID: PMC4174873  PMID: 25295172
X-ray crystallography; femtosecond nanocrystallography; HIV-1; gp41; membrane-proximal region; cholera toxin B subunit; crystallization; free-electron lasers
2.  Femtosecond protein nanocrystallography—data analysis methods 
Optics express  2010;18(6):5713-5723.
X-ray diffraction patterns may be obtained from individual submicron protein nanocrystals using a femtosecond pulse from a free-electron X-ray laser. Many “single-shot” patterns are read out every second from a stream of nanocrystals lying in random orientations. The short pulse terminates before significant atomic (or electronic) motion commences, minimizing radiation damage. Simulated patterns for Photosystem I nanocrystals are used to develop a method for recovering structure factors from tens of thousands of snapshot patterns from nanocrystals varying in size, shape and orientation. We determine the number of shots needed for a required accuracy in structure factor measurement and resolution, and investigate the convergence of our Monte-Carlo integration method.
PMCID: PMC4038330  PMID: 20389587
3.  Time-resolved protein nanocrystallography using an X-ray free-electron laser 
Aquila, Andrew | Hunter, Mark S. | Doak, R. Bruce | Kirian, Richard A. | Fromme, Petra | White, Thomas A. | Andreasson, Jakob | Arnlund, David | Bajt, Saša | Barends, Thomas R. M. | Barthelmess, Miriam | Bogan, Michael J. | Bostedt, Christoph | Bottin, Hervé | Bozek, John D. | Caleman, Carl | Coppola, Nicola | Davidsson, Jan | DePonte, Daniel P. | Elser, Veit | Epp, Sascha W. | Erk, Benjamin | Fleckenstein, Holger | Foucar, Lutz | Frank, Matthias | Fromme, Raimund | Graafsma, Heinz | Grotjohann, Ingo | Gumprecht, Lars | Hajdu, Janos | Hampton, Christina Y. | Hartmann, Andreas | Hartmann, Robert | Hau-Riege, Stefan | Hauser, Günter | Hirsemann, Helmut | Holl, Peter | Holton, James M. | Hömke, André | Johansson, Linda | Kimmel, Nils | Kassemeyer, Stephan | Krasniqi, Faton | Kühnel, Kai-Uwe | Liang, Mengning | Lomb, Lukas | Malmerberg, Erik | Marchesini, Stefano | Martin, Andrew V. | Maia, Filipe R.N.C. | Messerschmidt, Marc | Nass, Karol | Reich, Christian | Neutze, Richard | Rolles, Daniel | Rudek, Benedikt | Rudenko, Artem | Schlichting, Ilme | Schmidt, Carlo | Schmidt, Kevin E. | Schulz, Joachim | Seibert, M. Marvin | Shoeman, Robert L. | Sierra, Raymond | Soltau, Heike | Starodub, Dmitri | Stellato, Francesco | Stern, Stephan | Strüder, Lothar | Timneanu, Nicusor | Ullrich, Joachim | Wang, Xiaoyu | Williams, Garth J. | Weidenspointner, Georg | Weierstall, Uwe | Wunderer, Cornelia | Barty, Anton | Spence, John C. H. | Chapman, Henry N.
Optics Express  2012;20(3):2706-2716.
We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.
doi:10.1364/OE.20.002706
PMCID: PMC3413412  PMID: 22330507
(170.7160) Ultrafast technology; (170.7440) X-ray imaging; (140.3450) Laser-induced chemistry; (140.7090) Ultrafast lasers; (170.0170) Medical optics and biotechnology
4.  Femtosecond X-ray protein nanocrystallography 
Chapman, Henry N. | Fromme, Petra | Barty, Anton | White, Thomas A. | Kirian, Richard A. | Aquila, Andrew | Hunter, Mark S. | Schulz, Joachim | DePonte, Daniel P. | Weierstall, Uwe | Doak, R. Bruce | Maia, Filipe R. N. C. | Martin, Andrew V. | Schlichting, Ilme | Lomb, Lukas | Coppola, Nicola | Shoeman, Robert L. | Epp, Sascha W. | Hartmann, Robert | Rolles, Daniel | Rudenko, Artem | Foucar, Lutz | Kimmel, Nils | Weidenspointner, Georg | Holl, Peter | Liang, Mengning | Barthelmess, Miriam | Caleman, Carl | Boutet, Sébastien | Bogan, Michael J. | Krzywinski, Jacek | Bostedt, Christoph | Bajt, Saša | Gumprecht, Lars | Rudek, Benedikt | Erk, Benjamin | Schmidt, Carlo | Hömke, André | Reich, Christian | Pietschner, Daniel | Strüder, Lothar | Hauser, Günter | Gorke, Hubert | Ullrich, Joachim | Herrmann, Sven | Schaller, Gerhard | Schopper, Florian | Soltau, Heike | Kühnel, Kai-Uwe | Messerschmidt, Marc | Bozek, John D. | Hau-Riege, Stefan P. | Frank, Matthias | Hampton, Christina Y. | Sierra, Raymond G. | Starodub, Dmitri | Williams, Garth J. | Hajdu, Janos | Timneanu, Nicusor | Seibert, M. Marvin | Andreasson, Jakob | Rocker, Andrea | Jönsson, Olof | Svenda, Martin | Stern, Stephan | Nass, Karol | Andritschke, Robert | Schröter, Claus-Dieter | Krasniqi, Faton | Bott, Mario | Schmidt, Kevin E. | Wang, Xiaoyu | Grotjohann, Ingo | Holton, James M. | Barends, Thomas R. M. | Neutze, Richard | Marchesini, Stefano | Fromme, Raimund | Schorb, Sebastian | Rupp, Daniela | Adolph, Marcus | Gorkhover, Tais | Andersson, Inger | Hirsemann, Helmut | Potdevin, Guillaume | Graafsma, Heinz | Nilsson, Björn | Spence, John C. H.
Nature  2011;470(7332):73-77.
X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded1-3. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction ‘snapshots’ are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source4. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes5. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (~200 nm to 2 μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes6. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.
doi:10.1038/nature09750
PMCID: PMC3429598  PMID: 21293373
5.  Time-resolved protein nanocrystallography using an X-ray free-electron laser 
Aquila, Andrew | Hunter, Mark S | Bruce Doak, R. | Kirian, Richard A. | Fromme, Petra | White, Thomas A. | Andreasson, Jakob | Arnlund, David | Bajt, Saša | Barends, Thomas R. M. | Barthelmess, Miriam | Bogan, Michael J. | Bostedt, Christoph | Bottin, Hervé | Bozek, John D. | Caleman, Carl | Coppola, Nicola | Davidsson, Jan | DePonte, Daniel P. | Elser, Veit | Epp, Sascha W. | Erk, Benjamin | Fleckenstein, Holger | Foucar, Lutz | Frank, Matthias | Fromme, Raimund | Graafsma, Heinz | Grotjohann, Ingo | Gumprecht, Lars | Hajdu, Janos | Hampton, Christina Y. | Hartmann, Andreas | Hartmann, Robert | Hau-Riege, Stefan | Hauser, Günter | Hirsemann, Helmut | Holl, Peter | Holton, James M. | Hömke, André | Johansson, Linda | Kimmel, Nils | Kassemeyer, Stephan | Krasniqi, Faton | Kühnel, Kai-Uwe | Liang, Mengning | Lomb, Lukas | Malmerberg, Erik | Marchesini, Stefano | Martin, Andrew V. | Maia, Filipe R.N.C. | Messerschmidt, Marc | Nass, Karol | Reich, Christian | Neutze, Richard | Rolles, Daniel | Rudek, Benedikt | Rudenko, Artem | Schlichting, Ilme | Schmidt, Carlo | Schmidt, Kevin E. | Schulz, Joachim | Seibert, M. Marvin | Shoeman, Robert L. | Sierra, Raymond | Soltau, Heike | Starodub, Dmitri | Stellato, Francesco | Stern, Stephan | Strüder, Lothar | Timneanu, Nicusor | Ullrich, Joachim | Wang, Xiaoyu | Williams, Garth J. | Weidenspointner, Georg | Weierstall, Uwe | Wunderer, Cornelia | Barty, Anton | Spence, John C. H | Chapman, Henry N.
Optics express  2012;20(3):2706-2716.
We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 μs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.
PMCID: PMC3413412  PMID: 22330507
6.  Structure-factor analysis of femtosecond microdiffraction patterns from protein nanocrystals 
A complete set of structure factors has been extracted from hundreds of thousands of femtosecond X-ray diffraction patterns from randomly oriented Photosystem I membrane protein nanocrystals, using the Monte Carlo method of intensity integration. The data, collected at the Linac Coherent Light Source, are compared with conventional single-crystal data collected at a synchrotron source, and the quality of each data set was found to be similar.
A complete set of structure factors has been extracted from hundreds of thousands of femtosecond single-shot X-ray microdiffraction patterns taken from randomly oriented nanocrystals. The method of Monte Carlo integration over crystallite size and orientation was applied to experimental data from Photosystem I nanocrystals. This arrives at structure factors from many partial reflections without prior knowledge of the particle-size distribution. The data were collected at the Linac Coherent Light Source (the first hard-X-ray laser user facility), to which was fitted a hydrated protein nanocrystal injector jet, according to the method of serial crystallography. The data are single ‘still’ diffraction snapshots, each from a different nanocrystal with sizes ranging between 100 nm and 2 µm, so the angular width of Bragg peaks was dominated by crystal-size effects. These results were compared with single-crystal data recorded from large crystals of Photosystem I at the Advanced Light Source and the quality of the data was found to be similar. The implications for improving the efficiency of data collection by allowing the use of very small crystals, for radiation-damage reduction and for time-resolved diffraction studies at room temperature are discussed.
doi:10.1107/S0108767310050981
PMCID: PMC3066792  PMID: 21325716
nanocrystals; femtosecond diffraction; free-electron lasers; Monte Carlo methods; protein microdiffraction

Results 1-6 (6)