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

2021 ◽  
Vol 77 (a1) ◽  
pp. a203-a203
Author(s):  
Deepshika Gilbile ◽  
Megan Shelby ◽  
Artem Lyubimov ◽  
Jennifer Wierman ◽  
Diana Monteiro ◽  
...  
Keyword(s):  
Room Temperature ◽  
Microfluidic Chips ◽  
Fixed Target ◽  
Plug And Play ◽  
Serial Crystallography

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 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.

scholarly journals 2′-O methylation of RNA cap in SARS-CoV-2 captured by serial crystallography

2021 ◽  
Vol 118 (21) ◽  
pp. e2100170118
Author(s):  
Mateusz Wilamowski ◽  
Darren A. Sherrell ◽  
George Minasov ◽  
Youngchang Kim ◽  
Ludmilla Shuvalova ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Untranslated Region ◽  
Drug Target ◽  
Room Temperature ◽  
Fixed Target ◽  
Methyltransferase Activity ◽  
Before And After ◽  
Viral Proliferation ◽  
Serial Crystallography

The genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus has a capping modification at the 5′-untranslated region (UTR) to prevent its degradation by host nucleases. These modifications are performed by the Nsp10/14 and Nsp10/16 heterodimers using S-adenosylmethionine as the methyl donor. Nsp10/16 heterodimer is responsible for the methylation at the ribose 2′-O position of the first nucleotide. To investigate the conformational changes of the complex during 2′-O methyltransferase activity, we used a fixed-target serial synchrotron crystallography method at room temperature. We determined crystal structures of Nsp10/16 with substrates and products that revealed the states before and after methylation, occurring within the crystals during the experiments. Here we report the crystal structure of Nsp10/16 in complex with Cap-1 analog (m7GpppAm2′-O). Inhibition of Nsp16 activity may reduce viral proliferation, making this protein an attractive drug target.

scholarly journals Approach of Serial Crystallography

Crystals ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 854
Author(s):  
Ki Hyun Nam
Keyword(s):  
Radiation Damage ◽  
Room Temperature ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Past ◽  
Wide Range ◽  
Experimental Limitation ◽  
Serial Crystallography ◽  
Collection Strategies

Radiation damage and cryogenic sample environment are an experimental limitation observed in the traditional X-ray crystallography technique. However, the serial crystallography (SX) technique not only helps to determine structures at room temperature with minimal radiation damage, but it is also a useful tool for profound understanding of macromolecules. Moreover, it is a new tool for time-resolved studies. Over the past 10 years, various sample delivery techniques and data collection strategies have been developed in the SX field. It also has a wide range of applications in instruments ranging from the X-ray free electron laser (XFEL) facility to synchrotrons. The importance of the various approaches in terms of the experimental techniques and a brief review of the research carried out in the field of SX has been highlighted in this editorial.

Stable sample delivery in viscous media via a capillary for serial crystallography

2020 ◽  
Vol 53 (1) ◽  
pp. 45-50 ◽  
Author(s):  
Ki Hyun Nam
Keyword(s):  
Room Temperature ◽  
Capillary Tube ◽  
Glucose Isomerase ◽  
Low Flow ◽  
Innovative Technology ◽  
Delivery Method ◽  
Crystal Sample ◽  
X Ray ◽  
Serial Crystallography ◽  
Delivery Medium

Serial crystallography (SX) is an innovative technology in structural biology that enables the visualization of the molecular dynamics of macromolecules at room temperature. SX experiments always require a considerable amount of effort to deliver a crystal sample to the X-ray interaction point continuously and reliably. Here, a sample-delivery method using a capillary and a delivery medium is introduced. The crystals embedded in the delivery medium can pass through the capillary tube, which is aligned with the X-ray beam, at very low flow rates without requiring elaborate delivery techniques, drastically reducing sample consumption. In serial millisecond crystallography using a viscous medium via a capillary, crystals of lysozyme embedded in agarose, which produce an unstable injection stream at atmospheric pressure, and crystals of glucose isomerase embedded in gelatin, which is known to be problematic for open-extruder operation, were stably delivered at a flow rate of 100 nl min−1. The room-temperature crystal structures of lysozyme and glucose isomerase were successfully determined at 1.85 and 1.70 Å resolutions, respectively. This simple but highly efficient sample-delivery method can allow researchers to deliver crystals precisely to an X-ray beam in SX experiments.

Room-Temperature Bonding for Plastic High-Pressure Microfluidic Chips

2007 ◽  
Vol 79 (13) ◽  
pp. 5097-5102 ◽  
Author(s):  
Dieudonne A. Mair ◽  
Marco Rolandi ◽  
Marian Snauko ◽  
Richard Noroski ◽  
Frantisek Svec ◽  
...  
Keyword(s):  
High Pressure ◽  
Room Temperature ◽  
Microfluidic Chips

scholarly journals Methylation of RNA Cap in SARS-CoV-2 captured by serial crystallography

2020 ◽  
Author(s):  
M. Wilamowski ◽  
D.A. Sherrell ◽  
G. Minasov ◽  
Y. Kim ◽  
L. Shuvalova ◽  
...  
Keyword(s):  
Fixed Target ◽  
Innate Response ◽  
Methyltransferase Activity ◽  
X Ray ◽  
Biologically Relevant ◽  
Methylation Reaction ◽  
Before And After ◽  
Serial Crystallography ◽  
Rna Maturation ◽  
First Time

ABSTRACTThe genome of the SARS-CoV-2 coronavirus contains 29 proteins, of which 15 are nonstructural. Nsp10 and Nsp16 form a complex responsible for the capping of mRNA at the 5′ terminus. In the methylation reaction the S-adenosyl-L-methionine serves as the donor of the methyl group that is transferred to Cap-0 at the first transcribed nucleotide to create Cap-1. The presence of Cap-1 makes viral RNAs mimic the host transcripts and prevents their degradation. To investigate the 2′-O methyltransferase activity of SARS-CoV-2 Nsp10/16, we applied fixed-target serial synchrotron crystallography (SSX) which allows for physiological temperature data collection from thousands of crystals, significantly reducing the x-ray dose while maintaining a biologically relevant temperature. We determined crystal structures of Nsp10/16 that revealed the states before and after the methylation reaction, for the first time illustrating coronavirus Nsp10/16 complexes with the m7GpppAm2′-O Cap-1, where 2′OH of ribose is methylated. We compare these structures with structures of Nsp10/16 at 297 K and 100 K collected from a single crystal. This data provide important mechanistic insight and can be used to design small molecules that inhibit viral RNA maturation making SARS-CoV-2 sensitive to host innate response.

scholarly journals An environmental control box for serial crystallography enables multi-dimensional experiments

2021 ◽  
Author(s):  
Pedram Mehrabi ◽  
Davif von Stetten ◽  
Jan-Philipp Leimkohl ◽  
Friedjof Tellkamp ◽  
Eike C Schulz
Keyword(s):  
Protein Function ◽  
Environmental Control ◽  
Xylose Isomerase ◽  
X Ray Diffraction ◽  
Fixed Target ◽  
Time Resolved ◽  
Full Control ◽  
Serial Crystallography ◽  
Increasing Temperature ◽  
Insight Into

We present a new environmental enclosure for fixed-target, serial crystallography enabling full control of both the temperature and humidity. While maintaining the relative humidity to within a percent, this enclosure provides access to X-ray diffraction experiments in a wide temperature range from below 10 C to above 80 C. Coupled with the LAMA method, time-resolved serial crystallography experiments can now be carried out at truly physiological temperatures, providing fundamentally new insight into protein function. Using the hyperthermophile enzyme xylose isomerase, we demonstrate changes in the electron density as a function of increasing temperature and time. This method provides the necessary tools to successfully carry out multi- dimensional serial crystallography.

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