scholarly journals X-ray free-electron laser studies reveal correlated motion during isopenicillin N synthase catalysis

2021 ◽  
Vol 7 (34) ◽  
pp. eabh0250
Author(s):  
Patrick Rabe ◽  
Jos J. A. G. Kamps ◽  
Kyle D. Sutherlin ◽  
James D. S. Linyard ◽  
Pierre Aller ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Free Electron ◽  
Free Electron Laser ◽  
Enzyme Dynamics ◽  
Time Resolved ◽  
X Ray ◽  
Unique Reaction ◽  
Solution Studies ◽  
Correlated Motion ◽  
Serial Femtosecond Crystallography

Isopenicillin N synthase (IPNS) catalyzes the unique reaction of l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) with dioxygen giving isopenicillin N (IPN), the precursor of all natural penicillins and cephalosporins. X-ray free-electron laser studies including time-resolved crystallography and emission spectroscopy reveal how reaction of IPNS:Fe(II):ACV with dioxygen to yield an Fe(III) superoxide causes differences in active site volume and unexpected conformational changes that propagate to structurally remote regions. Combined with solution studies, the results reveal the importance of protein dynamics in regulating intermediate conformations during conversion of ACV to IPN. The results have implications for catalysis by multiple IPNS-related oxygenases, including those involved in the human hypoxic response, and highlight the power of serial femtosecond crystallography to provide insight into long-range enzyme dynamics during reactions presently impossible for nonprotein catalysts.

Time-Resolved Serial Femtosecond Crystallography at the European X-ray Free Electron Laser

2019 ◽  
Author(s):  
Marius Schmidt ◽  
Suraj Pandey ◽  
Adrian Mancuso ◽  
Richard Bean
Keyword(s):  
Free Electron ◽  
Free Electron Laser ◽  
Crystallographic Data ◽  
Time Resolved ◽  
X Ray ◽  
Serial Femtosecond Crystallography

Abstract This protocol introduces step by step into the collection of time resolved crystallographic data and their analysis at the European Free Electron Laser.

scholarly journals Pump-Probe Time-Resolved Serial Femtosecond Crystallography at SACLA: Current Status and Data Collection Strategies

2019 ◽  
Vol 9 (24) ◽  
pp. 5505 ◽  
Author(s):  
Eriko Nango ◽  
Minoru Kubo ◽  
Kensuke Tono ◽  
So Iwata
Keyword(s):  
Data Collection ◽  
Free Electron ◽  
Free Electron Laser ◽  
Current Status ◽  
Optical Pump ◽  
Time Resolved ◽  
Pump Probe ◽  
X Ray ◽  
Collection Strategies ◽  
Serial Femtosecond Crystallography

Structural information on protein dynamics is a critical factor in fully understanding the protein functions. Pump-probe time-resolved serial femtosecond crystallography (TR-SFX) is a recently established technique for visualizing the structural changes or reactions in proteins that are at work with high spatial and temporal resolution. In the pump-probe method, protein microcrystals are continuously delivered from an injector and exposed to an X-ray free-electron laser (XFEL) pulse after a trigger to initiate a reaction, such as light, chemicals, temperature, and electric field, which affords the structural snapshots of intermediates that occur in the protein. We are in the process of developing the device and techniques for pump-probe TR-SFX while using XFEL produced at SPring-8 Angstrom Compact Free-Electron Laser (SACLA). In this paper, we described our current development details and data collection strategies for the optical pump X-ray probe TR-SFX experiment at SACLA and then reported the techniques of in crystallo TR spectroscopy, which is useful in clarifying the nature of reaction that takes place in crystals in advance.

scholarly journals Nanosecond pump–probe device for time-resolved serial femtosecond crystallography developed at SACLA

2017 ◽  
Vol 24 (5) ◽  
pp. 1086-1091 ◽  
Author(s):  
Minoru Kubo ◽  
Eriko Nango ◽  
Kensuke Tono ◽  
Tetsunari Kimura ◽  
Shigeki Owada ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Free Electron ◽  
Good Choice ◽  
Cubic Phase ◽  
Liquid Droplets ◽  
Optical Pump ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Crystallography ◽  
Serial Femtosecond Crystallography

X-ray free-electron lasers (XFELs) have opened new opportunities for time-resolved X-ray crystallography. Here a nanosecond optical-pump XFEL-probe device developed for time-resolved serial femtosecond crystallography (TR-SFX) studies of photo-induced reactions in proteins at the SPring-8 Angstrom Compact free-electron LAser (SACLA) is reported. The optical-fiber-based system is a good choice for a quick setup in a limited beam time and allows pump illumination from two directions to achieve high excitation efficiency of protein microcrystals. Two types of injectors are used: one for extruding highly viscous samples such as lipidic cubic phase (LCP) and the other for pulsed liquid droplets. Under standard sample flow conditions from the viscous-sample injector, delay times from nanoseconds to tens of milliseconds are accessible, typical time scales required to study large protein conformational changes. A first demonstration of a TR-SFX experiment on bacteriorhodopsin in bicelle using a setup with a droplet-type injector is also presented.

scholarly journals Conformational flexibility of adenine riboswitch aptamer in apo and bound states using NMR and an X-ray free electron laser

2019 ◽  
Vol 73 (8-9) ◽  
pp. 509-518
Author(s):  
Jienv Ding ◽  
Monalisa Swain ◽  
Ping Yu ◽  
Jason R. Stagno ◽  
Yun-Xing Wang
Keyword(s):  
Conformational Changes ◽  
Free Electron ◽  
Free Electron Laser ◽  
Messenger Rna ◽  
Bound States ◽  
Conformational Dynamics ◽  
Thermal Fluctuation ◽  
Binding Pocket ◽  
X Ray ◽  
Aptamer Domain

Abstract Riboswitches are structured cis-regulators mainly found in the untranslated regions of messenger RNA. The aptamer domain of a riboswitch serves as a sensor for its ligand, the binding of which triggers conformational changes that regulate the behavior of its expression platform. As a model system for understanding riboswitch structures and functions, the add adenine riboswitch has been studied extensively. However, there is a need for further investigation of the conformational dynamics of the aptamer in light of the recent real-time crystallographic study at room temperature (RT) using an X-ray free electron laser (XFEL) and femtosecond X-ray crystallography (SFX). Herein, we investigate the conformational motions of the add adenine riboswitch aptamer domain, in the presence or absence of adenine, using nuclear magnetic resonance relaxation measurements and analysis of RT atomic displacement factors (B-factors). In the absence of ligand, the P1 duplex undergoes a fast exchange where the overall molecule exhibits a motion at kex ~ 319 s−1, based on imino signals. In the presence of ligand, the P1 duplex adopts a highly ordered conformation, with kex~ 83 s−1, similar to the global motion of the molecule, excluding the loops and binding pocket, at 84 s−1. The µs–ms motions in both the apo and bound states are consistent with RT B-factors. Reduced spatial atomic fluctuation, ~ 50%, in P1 upon ligand binding coincides with significantly attenuated temporal dynamic exchanges. The binding pocket is structured in the absence or presence of ligand, as evidenced by relatively low and similar RT B-factors. Therefore, despite the dramatic rearrangement of the binding pocket, those residues exhibit similar spatial thermal fluctuation before and after binding.

scholarly journals Liquid sample delivery techniques for serial femtosecond crystallography

2014 ◽  
Vol 369 (1647) ◽  
pp. 20130337 ◽  
Author(s):  
Uwe Weierstall
Keyword(s):  
Flow Rate ◽  
Free Electron ◽  
Repetition Rate ◽  
Free Electron Laser ◽  
Room Temperature ◽  
Liquid Flow Rate ◽  
Free Electron Lasers ◽  
Liquid Sample ◽  
X Ray ◽  
Serial Femtosecond Crystallography

X-ray free-electron lasers overcome the problem of radiation damage in protein crystallography and allow structure determination from micro- and nanocrystals at room temperature. To ensure that consecutive X-ray pulses do not probe previously exposed crystals, the sample needs to be replaced with the X-ray repetition rate, which ranges from 120 Hz at warm linac-based free-electron lasers to 1 MHz at superconducting linacs. Liquid injectors are therefore an essential part of a serial femtosecond crystallography experiment at an X-ray free-electron laser. Here, we compare different techniques of injecting microcrystals in solution into the pulsed X-ray beam in vacuum. Sample waste due to mismatch of the liquid flow rate to the X-ray repetition rate can be addressed through various techniques.

scholarly journals Two mirror X-ray pulse split and delay instrument for femtosecond time resolved investigations at the LCLS free electron laser facility

2016 ◽  
Vol 24 (11) ◽  
pp. 11768 ◽  
Author(s):  
Nora Berrah ◽  
Li Fang ◽  
Brendan F Murphy ◽  
Edwin Kukk ◽  
Timur Y. Osipov ◽  
...  
Keyword(s):  
Free Electron ◽  
Free Electron Laser ◽  
Time Resolved ◽  
X Ray ◽  
Laser Facility

scholarly journals Direct autocorrelation of soft-x-ray free-electron-laser pulses by time-resolved two-photon double ionization of He

2009 ◽  
Vol 80 (2) ◽  
Author(s):  
R. Mitzner ◽  
A. A. Sorokin ◽  
B. Siemer ◽  
S. Roling ◽  
M. Rutkowski ◽  
...  
Keyword(s):  
Free Electron ◽  
Free Electron Laser ◽  
Laser Pulses ◽  
Double Ionization ◽  
Time Resolved ◽  
X Ray ◽  
Two Photon
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