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.

scholarly journals Evaluation of microflow configurations for scale inhibition and serial X-ray diffraction analysis of crystallization processes

Lab on a Chip ◽  
2020 ◽  
Vol 20 (16) ◽  
pp. 2954-2964
Author(s):  
Mark A. Levenstein ◽  
Yi-Yeoun Kim ◽  
Liam Hunter ◽  
Clara Anduix-Canto ◽  
Carlos González Niño ◽  
...  
Keyword(s):  
Crystal Growth ◽  
Diffraction Analysis ◽  
X Ray Diffraction ◽  
Time Resolved ◽  
X Ray ◽  
Scale Inhibition ◽  
Serial Crystallography

Exploration of crystal growth in microchannels led to effective scale inhibition, enabling time-resolved injector-based serial crystallography-on-a-chip.

scholarly journals Mapping the Conformational Landscape of a Dynamic Enzyme by XFEL and Multitemperature Crystallography

2015 ◽  
Author(s):  
Daniel A Keedy ◽  
Lillian R Kenner ◽  
Matthew Warkentin ◽  
Rahel Woldeyes ◽  
Michael C Thompson ◽  
...  
Keyword(s):  
Protein Function ◽  
Fixed Target ◽  
Time Resolved ◽  
Conformational Ensembles ◽  
Catalytic Function ◽  
Abrupt Changes ◽  
Temperature Dependences ◽  
Conformational Landscape ◽  
Conformational Coupling ◽  
New Generation

Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function. Here we compare the conformational ensembles of CypA by fixed-target X-ray free electron laser (XFEL) crystallography and multitemperature synchrotron crystallography. The “diffraction-before-destruction” nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA. We monitored the temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated above, but not below, the glass transition temperature (~200 K) and reveal abrupt changes in protein flexibility that provide all-atom insight into conformational coupling. Together, our XFEL data and multitemperature analyses motivate a new generation of time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.  

scholarly journals Well-based crystallization of lipidic cubic phase microcrystals for serial X-ray crystallography experiments

2019 ◽  
Vol 75 (10) ◽  
pp. 937-946 ◽  
Author(s):  
Rebecka Andersson ◽  
Cecilia Safari ◽  
Petra Båth ◽  
Robert Bosman ◽  
Anastasya Shilova ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Protein Structures ◽  
Cubic Phase ◽  
Crystal Formation ◽  
X Ray Diffraction ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Crystallography ◽  
Lipidic Cubic Phase ◽  
Serial Crystallography

Serial crystallography is having an increasing impact on structural biology. This emerging technique opens up new possibilities for studying protein structures at room temperature and investigating structural dynamics using time-resolved X-ray diffraction. A limitation of the method is the intrinsic need for large quantities of well ordered micrometre-sized crystals. Here, a method is presented to screen for conditions that produce microcrystals of membrane proteins in the lipidic cubic phase using a well-based crystallization approach. A key advantage over earlier approaches is that the progress of crystal formation can be easily monitored without interrupting the crystallization process. In addition, the protocol can be scaled up to efficiently produce large quantities of crystals for serial crystallography experiments. Using the well-based crystallization methodology, novel conditions for the growth of showers of microcrystals of three different membrane proteins have been developed. Diffraction data are also presented from the first user serial crystallography experiment performed at MAX IV Laboratory.

Conventional X-ray diffraction approaches to the study of enzyme mechanism: serine proteinases, aminoacyl-tRNA synthetases and xylose isomerase

1992 ◽  
Vol 340 (1657) ◽  
pp. 311-321 ◽  
Keyword(s):  
Catalytic Mechanism ◽  
Xylose Isomerase ◽  
Enzyme Mechanism ◽  
X Ray Diffraction ◽  
Molecular Mechanics Calculations ◽  
Time Resolved ◽  
X Ray ◽  
Trna Synthetases ◽  
Substrate Analogues ◽  
Aminoacyl Trna Synthetases

Techniques that have been used to study enzyme mechanism by conventional steady-state crystallographic techniques are reviewed. Substrates and substrate analogues can often be diffused into crystals, but occasionally co-crystallization is necessary. The poor solubility of substrates and inhibitors may pose a problem. Even if a substrate is present at adequate concentration, it may not be observed by X -ray diffraction. To observe a substrate, special measures may be needed to stop enzyme action, but sometimes this is not necessary because an equilibrium is established. Inhibitors may usefully model a particular reaction state, but one must always question whether the inhibitor provides a correct model. Stabilization of a transition state is often discussed, but rarely achieved. Where practicable, protein engineering can provide a powerful tool to test proposals about the catalytic mechanism. Molecular mechanics calculations can also be useful. These themes are developed in relation to enzymes studied in the authors’ laboratory. Many of the same problems are encountered in the application of time-resolved techniques to the study of enzyme mechanism.

Spatial localization of unknown proteins in the endoplasmic reticulum predicts functions

2007 ◽  
Vol 30 (4) ◽  
pp. 84
Author(s):  
Michael D. Jain ◽  
Hisao Nagaya ◽  
Annalyn Gilchrist ◽  
Miroslaw Cygler ◽  
John J.M. Bergeron
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Folding ◽  
Protein Synthesis ◽  
Protein Function ◽  
Protein Translocation ◽  
Spatial Localization ◽  
Predict Protein Function ◽  
Insight Into ◽  
Soluble Fractions ◽  
Er Membrane

Protein synthesis, folding and degradation functions are spatially segregated in the endoplasmic reticulum (ER) with respect to the membrane and the ribosome (rough and smooth ER). Interrogation of a proteomics resource characterizing rough and smooth ER membranes subfractionated into cytosolic, membrane, and soluble fractions gives a spatial map of known proteins involved in ER function. The spatial localization of 224 identified unknown proteins in the ER is predicted to give insight into their function. Here we provide evidence that the proteomics resource accurately predicts the function of new proteins involved in protein synthesis (nudilin), protein translocation across the ER membrane (nicalin), co-translational protein folding (stexin), and distal protein folding in the lumen of the ER (erlin-1, TMX2). Proteomics provides the spatial localization of proteins and can be used to accurately predict protein function.

scholarly journals Direct observations of shock-induced melting in a porous solid using time-resolved x-ray diffraction

2020 ◽  
Vol 4 (6) ◽  
Author(s):  
A. Mandal ◽  
B. J. Jensen ◽  
M. C. Hudspeth ◽  
S. Root ◽  
R. S. Crum ◽  
...  
Keyword(s):  
Porous Solid ◽  
X Ray Diffraction ◽  
Time Resolved ◽  
X Ray ◽  
Direct Observations

scholarly journals Bulk modulus of H2O across the ice VII–ice X transition measured by time-resolved x-ray diffraction in dynamic diamond anvil cell experiments

2021 ◽  
Vol 103 (6) ◽  
Author(s):  
A. S. J. Méndez ◽  
F. Trybel ◽  
R. J. Husband ◽  
G. Steinle-Neumann ◽  
H.-P. Liermann ◽  
...  
Keyword(s):  
Bulk Modulus ◽  
Diamond Anvil Cell ◽  
X Ray Diffraction ◽  
Time Resolved ◽  
X Ray ◽  
Diamond Anvil

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.

Polydomain Structure of Epitaxial PbTiO3 films on MgO

1997 ◽  
Vol 493 ◽  
Author(s):  
S. P. Alpay ◽  
A. S. Prakash ◽  
S. Aggarwal ◽  
R. Ramesh ◽  
A. L. Roytburd ◽  
...  
Keyword(s):  
Domain Structure ◽  
Pulsed Laser ◽  
Laser Deposition ◽  
X Ray Diffraction ◽  
X Ray ◽  
Theoretical Predictions ◽  
The Stability ◽  
Increasing Temperature ◽  
Stable Domains ◽  
Domain Stability

ABSTRACTA PbTiO3(001) film grown on MgO(001) by pulsed laser deposition is examined as an example to demonstrate the applications of the domain stability map for epitaxial perovskite films which shows regions of stable domains and fractions of domains in a polydomain structure. X-ray diffraction studies indicate that the film has a …c/a/c/a… domain structure in a temperature range of °C to 400°C with the fraction of c-domains decreasing with increasing temperature. These experimental results are in excellent agreement with theoretical predictions based on the stability map.

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