scholarly journals 3D-printed holders for in meso in situ fixed-target serial X-ray crystallography

2020 ◽  
Vol 53 (3) ◽  
pp. 854-859
Author(s):  
Chia-Ying Huang ◽  
Nathalie Meier ◽  
Martin Caffrey ◽  
Meitian Wang ◽  
Vincent Olieric
Keyword(s):  
Data Collection ◽  
Heavy Atom ◽  
Fixed Target ◽  
X Ray ◽  
X Ray Crystallography ◽  
Sample Stability ◽  
3D Printed ◽  
Sponge Phase ◽  
Serial Crystallography

The in meso in situ serial X-ray crystallography method was developed to ease the handling of small fragile crystals of membrane proteins and for rapid data collection on hundreds of microcrystals directly in the growth medium without the need for crystal harvesting. To facilitate mounting of these in situ samples on a goniometer at cryogenic or at room temperatures, two new 3D-printed holders have been developed. They provide for cubic and sponge phase sample stability in the X-ray beam and are compatible with sample-changing robots. The holders can accommodate a variety of window material types, as well as bespoke samples for diffraction screening and data collection at conventional macromolecular crystallography beamlines. They can be used for convenient post-crystallization treatments such as ligand and heavy-atom soaking. The design, assembly and application of the holders for in situ serial crystallography are described. Files for making the holders using a 3D printer are included as supporting information.

Plug-and-play polymer microfluidic chips for hydrated, room temperature, fixed-target serial crystallography

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Deepshika Gilbile ◽  
Megan L. Shelby ◽  
Artem Y. Lyubimov ◽  
Jennifer L. Wierman ◽  
Diana C. F. Monteiro ◽  
...  
Keyword(s):  
Room Temperature ◽  
Microfluidic Chips ◽  
Fixed Target ◽  
Plug And Play ◽  
X Ray ◽  
X Ray Crystallography ◽  
Serial Crystallography

This work presents our development of versatile, inexpensive, and robust polymer microfluidic chips for routine and reliable room temperature serial X-ray crystallography measurements.

scholarly journals 3D-MiXD: 3D-printed X-ray-compatible microfluidic devices for rapid, low-consumption serial synchrotron crystallography data collection in flow

IUCrJ ◽  
2020 ◽  
Vol 7 (2) ◽  
pp. 207-219 ◽  
Author(s):  
Diana C. F. Monteiro ◽  
David von Stetten ◽  
Claudia Stohrer ◽  
Marta Sans ◽  
Arwen R. Pearson ◽  
...  
Keyword(s):  
Data Collection ◽  
Room Temperature ◽  
Time Resolved ◽  
X Ray ◽  
Biologically Relevant ◽  
3D Printed ◽  
Serial Crystallography ◽  
Low X ◽  
Very High

Serial crystallography has enabled the study of complex biological questions through the determination of biomolecular structures at room temperature using low X-ray doses. Furthermore, it has enabled the study of protein dynamics by the capture of atomically resolved and time-resolved molecular movies. However, the study of many biologically relevant targets is still severely hindered by high sample consumption and lengthy data-collection times. By combining serial synchrotron crystallography (SSX) with 3D printing, a new experimental platform has been created that tackles these challenges. An affordable 3D-printed, X-ray-compatible microfluidic device (3D-MiXD) is reported that allows data to be collected from protein microcrystals in a 3D flow with very high hit and indexing rates, while keeping the sample consumption low. The miniaturized 3D-MiXD can be rapidly installed into virtually any synchrotron beamline with only minimal adjustments. This efficient collection scheme in combination with its mixing geometry paves the way for recording molecular movies at synchrotrons by mixing-triggered millisecond time-resolved SSX.

scholarly journals An automated platform for in situ serial crystallography at room temperature

IUCrJ ◽  
2020 ◽  
Vol 7 (6) ◽  
pp. 1009-1018
Author(s):  
Zhong Ren ◽  
Cong Wang ◽  
Heewhan Shin ◽  
Sepalika Bandara ◽  
Indika Kumarapperuma ◽  
...  
Keyword(s):  
Data Collection ◽  
Room Temperature ◽  
Protein Structures ◽  
Routine Data ◽  
Protein Crystals ◽  
X Ray Diffraction ◽  
X Ray ◽  
Serial Crystallography ◽  
Routine Data Collection

Direct observation of functional motions in protein structures is highly desirable for understanding how these nanomachineries of life operate at the molecular level. Because cryogenic temperatures are non-physiological and may prohibit or even alter protein structural dynamics, it is necessary to develop robust X-ray diffraction methods that enable routine data collection at room temperature. We recently reported a crystal-on-crystal device to facilitate in situ diffraction of protein crystals at room temperature devoid of any sample manipulation. Here an automated serial crystallography platform based on this crystal-on-crystal technology is presented. A hardware and software prototype has been implemented, and protocols have been established that allow users to image, recognize and rank hundreds to thousands of protein crystals grown on a chip in optical scanning mode prior to serial introduction of these crystals to an X-ray beam in a programmable and high-throughput manner. This platform has been tested extensively using fragile protein crystals. We demonstrate that with affordable sample consumption, this in situ serial crystallography technology could give rise to room-temperature protein structures of higher resolution and superior map quality for those protein crystals that encounter difficulties during freezing. This serial data collection platform is compatible with both monochromatic oscillation and Laue methods for X-ray diffraction and presents a widely applicable approach for static and dynamic crystallographic studies at room temperature.

scholarly journals Fixed target combined with spectral mapping: approaching 100% hit rates for serial crystallography

2016 ◽  
Vol 72 (8) ◽  
pp. 944-955 ◽  
Author(s):  
Saeed Oghbaey ◽  
Antoine Sarracini ◽  
Helen M. Ginn ◽  
Olivier Pare-Labrosse ◽  
Anling Kuo ◽  
...  
Keyword(s):  
Atmospheric Pressure ◽  
Spectral Mapping ◽  
Fixed Target ◽  
X Ray ◽  
Experimental Scheme ◽  
Novel Structure ◽  
Laser Sources ◽  
Serial Crystallography ◽  
Serial Femtosecond Crystallography

The advent of ultrafast highly brilliant coherent X-ray free-electron laser sources has driven the development of novel structure-determination approaches for proteins, and promises visualization of protein dynamics on sub-picosecond timescales with full atomic resolution. Significant efforts are being applied to the development of sample-delivery systems that allow these unique sources to be most efficiently exploited for high-throughput serial femtosecond crystallography. Here, the next iteration of a fixed-target crystallography chip designed for rapid and reliable delivery of up to 11 259 protein crystals with high spatial precision is presented. An experimental scheme for predetermining the positions of crystals in the chip by means ofin situspectroscopy using a fiducial system for rapid, precise alignment and registration of the crystal positions is presented. This delivers unprecedented performance in serial crystallography experiments at room temperature under atmospheric pressure, giving a raw hit rate approaching 100% with an effective indexing rate of approximately 50%, increasing the efficiency of beam usage and allowing the method to be applied to systems where the number of crystals is limited.

scholarly journals Structural Characterization of Heterodinuclear ZnII-LnIII Complexes (Ln = Pr, Nd) with a Ring-Contracted H2valdien-Derived Schiff Base Ligand

2021 ◽  
Author(s):  
Shabana Noor ◽  
Richard Goddard ◽  
Fehmeeda Khatoon ◽  
Sarvendra Kumar ◽  
Rüdiger W. Seidel
Keyword(s):  
Molecular Structure ◽  
Schiff Base ◽  
Structural Characterization ◽  
Schiff Base Ligand ◽  
Crystal And Molecular Structure ◽  
X Ray ◽  
X Ray Crystallography ◽  
Imidazoline Ring

AbstractSynthesis and structural characterization of two heterodinuclear ZnII-LnIII complexes with the formula [ZnLn(HL)(µ-OAc)(NO3)2(H2O)x(MeOH)1-x]NO3 · n H2O · n MeOH [Ln = Pr (1), Nd (2)] and the crystal and molecular structure of [ZnNd(HL)(µ-OAc)(NO3)2(H2O)] [ZnNd(HL)(OAc)(NO3)2(H2O)](NO3)2 · n H2O · n MeOH (3) are reported. The asymmetrical compartmental ligand (E)-2-(1-(2-((2-hydroxy-3-methoxybenzylidene)amino)-ethyl)imidazolidin-2-yl)-6-methoxyphenol (H2L) is formed from N1,N3-bis(3-methoxysalicylidene)diethylenetriamine (H2valdien) through intramolecular aminal formation, resulting in a peripheral imidazoline ring. The structures of 1–3 were revealed by X-ray crystallography. The smaller ZnII ion occupies the inner N2O2 compartment of the ligand, whereas the larger and more oxophilic LnIII ions are found in the outer O2O2’ site. Graphic Abstract Synthesis and structural characterization of two heterodinuclear ZnII-LnIII complexes (Ln = Pr, Nd) bearing an asymmetrical compartmental ligand formed in situ from N1,N3-bis(3-methoxysalicylidene)diethylenetriamine (H2valdien) through intramolecular aminal formation are reported.

scholarly journals Polyimide mesh-based sample holder with irregular crystal mounting holes for fixed-target serial crystallography

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ki Hyun Nam ◽  
Jihan Kim ◽  
Yunje Cho
Keyword(s):  
Data Collection ◽  
Room Temperature ◽  
Picosecond Laser ◽  
Temperature Structure ◽  
Sample Holder ◽  
Fixed Target ◽  
Random Orientation ◽  
Physical Damage ◽  
Crystal Sample ◽  
Serial Crystallography

AbstractThe serial crystallography (SX) technique enables the determination of the room-temperature structure of a macromolecule while causing minimal radiation damage, as well as the visualization of the molecular dynamics by time-resolved studies. The fixed-target (FT) scanning approach is one method for SX sample delivery that minimizes sample consumption and minimizes physical damage to crystals during data collection. Settling of the crystals on the sample holder in random orientation is important for complete three dimensional data collection. To increase the random orientation of crystals on the sample holder, we developed a polyimide mesh-based sample holder with irregular crystal mounting holes for FT-SX. The polyimide mesh was fabricated using a picosecond laser. Each hole in the polyimide mesh has irregularly shaped holes because of laser thermal damage, which may cause more crystals to settle at random orientations compared to regular shaped sample holders. A crystal sample was spread onto a polyimide-mesh, and a polyimide film was added to both sides to prevent dehydration. Using this sample holder, FT-SX was performed at synchrotron and determined the room-temperature lysozyme structure at 1.65 Å. The polyimide mesh with irregularly shaped holes will allow for expanded applications in sample delivery for FT-SX experiments.

Design of a novel Peltier-based cooling device and its use in neutron diffraction data collection of perdeuterated yeast pyrophosphatase

2010 ◽  
Vol 43 (5) ◽  
pp. 1113-1120 ◽  
Author(s):  
Esko Oksanen ◽  
François Dauvergne ◽  
Adrian Goldman ◽  
Monika Budayova-Spano
Keyword(s):  
Data Collection ◽  
Neutron Diffraction ◽  
Diffraction Data ◽  
Catalytic Mechanism ◽  
Inorganic Pyrophosphatase ◽  
Neutron Diffraction Data ◽  
Cooling Device ◽  
Data Set ◽  
X Ray ◽  
X Ray Crystallography

H atoms play a central role in enzymatic mechanisms, but H-atom positions cannot generally be determined by X-ray crystallography. Neutron crystallography, on the other hand, can be used to determine H-atom positions but it is experimentally very challenging. Yeast inorganic pyrophosphatase (PPase) is an essential enzyme that has been studied extensively by X-ray crystallography, yet the details of the catalytic mechanism remain incompletely understood. The temperature instability of PPase crystals has in the past prevented the collection of a neutron diffraction data set. This paper reports how the crystal growth has been optimized in temperature-controlled conditions. To stabilize the crystals during neutron data collection a Peltier cooling device that minimizes the temperature gradient along the capillary has been developed. This device allowed the collection of a full neutron diffraction data set.

Zirconium complexes of a cyclopentadienyl-amido ligand with a pendant amine donor via amine and alkane elimination

1996 ◽  
Vol 74 (9) ◽  
pp. 1696-1703 ◽  
Author(s):  
Ying Mu ◽  
Warren E. Piers ◽  
Donald C. MacQuarrie ◽  
Michael J. Zaworotko
Keyword(s):  
Lewis Acids ◽  
Methyl Chloride ◽  
Monoclinic Space Group ◽  
One Pot ◽  
Amido Complexes ◽  
X Ray ◽  
X Ray Crystallography ◽  
Zirconium Complexes ◽  
Alkane Elimination

Zirconium complexes of the multidentate ligand CpHNMeSiN(H)R (SiNR = -SiMe2N-t-butyl; NMe = -CH2CH2NMe2, 1) were prepared and characterized via amine and alkane elimination procedures. Reaction of 1 with Zr(NMe2)4 gave a mixture of bis-amido complexes 2 in which the ligand was 1,2 and 1,3 substituted. This mixture was converted to the analogous dichlorides 3 using Me2NH•HCl and 1,3-3 was purified at this stage; alternatively, 1,3-3 was obtained in one pot from 1 and Zr(NMe2)4 in ≈70% yield. Conversion of 1,3-3 to dimethyl compound (CpNMeSiNR)Zr(CH3)2, 1,3-4, was accomplished via reaction of the dichloride with methyllithium; methide abstraction with the Lewis acids B(C6F5)3 and [Ph3C]+[B(C6F5)4]− generated the cationic alkyls [(CpNMeSiNR)Zr(CH3)]+[R′B(C6F5)3]− (R′ = CH3, 6a; C6F6, 6b), which were characterized by NMR spectroscopy. Zirconium complexes containing 1 ligated as its 1,2 isomer were obtained from alkane elimination reactions between 1 and in situ prepared RnZrCl4−n (R = CH3, n = 3; R = CH2SiMe3, n = 2). 1,2-3 and the methyl chloride complex 1,2-(CpNMeSiNR)Zr(CH3)Cl, 5, were obtained in 18 and 30% yield, respectively. Complex 5 was characterized by X-ray crystallography (monoclinic, space group P21/a, a = 9.6951(10) Å, b = 14.3794(16) Å, c = 14.364(3) Å, V = 1990.3(5) Å3, Z = 4, R = 0.046, Rw = 0.041.) Key words: amine elimination, Cp-amido, zirconium complexes.

scholarly journals Low-Zpolymer sample supports for fixed-target serial femtosecond X-ray crystallography

2015 ◽  
Vol 48 (4) ◽  
pp. 1072-1079 ◽  
Author(s):  
Geoffrey K. Feld ◽  
Michael Heymann ◽  
W. Henry Benner ◽  
Tommaso Pardini ◽  
Ching-Ju Tsai ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Three Dimensional ◽  
Two Dimensional ◽  
X Ray Diffraction ◽  
Fixed Target ◽  
X Ray ◽  
X Ray Crystallography ◽  
Transmission Electron ◽  
Linac Coherent Light Source ◽  
Efficient Alternative

X-ray free-electron lasers (XFELs) offer a new avenue to the structural probing of complex materials, including biomolecules. Delivery of precious sample to the XFEL beam is a key consideration, as the sample of interest must be serially replaced after each destructive pulse. The fixed-target approach to sample delivery involves depositing samples on a thin-film support and subsequent serial introductionviaa translating stage. Some classes of biological materials, including two-dimensional protein crystals, must be introduced on fixed-target supports, as they require a flat surface to prevent sample wrinkling. A series of wafer and transmission electron microscopy (TEM)-style grid supports constructed of low-Zplastic have been custom-designed and produced. Aluminium TEM grid holders were engineered, capable of delivering up to 20 different conventional or plastic TEM grids using fixed-target stages available at the Linac Coherent Light Source (LCLS). As proof-of-principle, X-ray diffraction has been demonstrated from two-dimensional crystals of bacteriorhodopsin and three-dimensional crystals of anthrax toxin protective antigen mounted on these supports at the LCLS. The benefits and limitations of these low-Zfixed-target supports are discussed; it is the authors' belief that they represent a viable and efficient alternative to previously reported fixed-target supports for conducting diffraction studies with XFELs.

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