Synchrotron X-ray crystallography techniques: time-resolved aspects of data collection

1992 ◽  
Vol 340 (1657) ◽  
pp. 221-232 ◽  
Keyword(s):  
Data Collection ◽  
Three Dimensional ◽  
Protein Crystal ◽  
Measure Data ◽  
Laue Pattern ◽  
Data Sets ◽  
Time Resolved ◽  
Spatial Overlap ◽  
X Ray Crystallography ◽  
Unit Cells

Synchrotron X-radiation (SR) is intense, polychromatic and collimated. These properties are exploited routinely now to measure data at high resolution from proteins or from large unit cells (e.g. viruses), particularly using a monochromatized short wavelength beam. The time needed to measure protein crystal data-sets (in rotation geometry) can be quick (hours or minutes) compared with laboratory sources (weeks or days). Even so, more rapid data collection is of interest for timeresolved macromolecular crystallography. White beam : stationary crystal (Laue) geometry at SR sources offers shorter exposure times (seconds and less with film). Laue data-sets can be sensitive to subtle structural differences. Technical challenges still presented by SR Laue patterns include the energy overlap of low-resolution data and the spatial overlap of spots, both of which affect the completeness of data-sets. Some energy deconvolution is already possible by the use of multiple films. The spatial overlap problem can be alleviated by the use of three-dimensional detectors, such as a ‘toast-rack’ of plates. Monitoring of a crystalline process, via the Laue pattern, requires time slicing detector systems (e.g. based on CCDS) to be developed.

MRPC (Missing Regions in Polypeptide Chains): a knowledgebase

2019 ◽  
Vol 52 (6) ◽  
pp. 1422-1426
Author(s):  
Rajendran Santhosh ◽  
Namrata Bankoti ◽  
Adgonda Malgonnavar Padmashri ◽  
Daliah Michael ◽  
Jeyaraman Jeyakanthan ◽  
...  
Keyword(s):  
Protein Structures ◽  
Three Dimensional ◽  
Protein Molecule ◽  
Data Bank ◽  
Protein Crystal ◽  
Dimensional Structure ◽  
Protein Structure Analysis ◽  
Three Dimensional Structure ◽  
X Ray Crystallography ◽  
Polypeptide Chains

Missing regions in protein crystal structures are those regions that cannot be resolved, mainly owing to poor electron density (if the three-dimensional structure was solved using X-ray crystallography). These missing regions are known to have high B factors and could represent loops with a possibility of being part of an active site of the protein molecule. Thus, they are likely to provide valuable information and play a crucial role in the design of inhibitors and drugs and in protein structure analysis. In view of this, an online database, Missing Regions in Polypeptide Chains (MRPC), has been developed which provides information about the missing regions in protein structures available in the Protein Data Bank. In addition, the new database has an option for users to obtain the above data for non-homologous protein structures (25 and 90%). A user-friendly graphical interface with various options has been incorporated, with a provision to view the three-dimensional structure of the protein along with the missing regions using JSmol. The MRPC database is updated regularly (currently once every three months) and can be accessed freely at the URL http://cluster.physics.iisc.ac.in/mrpc.

scholarly journals A multicrystal diffraction data-collection approach for studying structural dynamics with millisecond temporal resolution

IUCrJ ◽  
2016 ◽  
Vol 3 (6) ◽  
pp. 393-401 ◽  
Author(s):  
Robin Schubert ◽  
Svetlana Kapis ◽  
Yannig Gicquel ◽  
Gleb Bourenkov ◽  
Thomas R. Schneider ◽  
...  
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Temporal Resolution ◽  
Diffraction Data ◽  
Structural Changes ◽  
Structural Information ◽  
Data Sets ◽  
Enzymatic Reactions ◽  
Time Resolved ◽  
Site Specific

Many biochemical processes take place on timescales ranging from femtoseconds to seconds. Accordingly, any time-resolved experiment must be matched to the speed of the structural changes of interest. Therefore, the timescale of interest defines the requirements of the X-ray source, instrumentation and data-collection strategy. In this study, a minimalistic approach forin situcrystallization is presented that requires only a few microlitres of sample solution containing a few hundred crystals. It is demonstrated that complete diffraction data sets, merged from multiple crystals, can be recorded within only a few minutes of beamtime and allow high-resolution structural information of high quality to be obtained with a temporal resolution of 40 ms. Global and site-specific radiation damage can be avoided by limiting the maximal dose per crystal to 400 kGy. Moreover, analysis of the data collected at higher doses allows the time-resolved observation of site-specific radiation damage. Therefore, our approach is well suited to observe structural changes and possibly enzymatic reactions in the low-millisecond regime.

scholarly journals New methods in time-resolved Laue pump–probe crystallography at synchrotron sources

2015 ◽  
Vol 22 (2) ◽  
pp. 280-287 ◽  
Author(s):  
Philip Coppens ◽  
Bertrand Fournier
Keyword(s):  
Ground State ◽  
Initial Assessment ◽  
Ratio Method ◽  
Ground State Structure ◽  
Data Sets ◽  
State Structure ◽  
Time Resolved ◽  
Structure Factors ◽  
Unit Cells ◽  
Diffraction Patterns

Newly developed methods for time-resolved studies using the polychromatic and in particular the pink-Laue technique, suitable for medium and small-size unit cells typical in chemical crystallography, are reviewed. The order of the sections follows that of a typical study, starting with a description of the pink-Laue technique, followed by the strategy of data collection for analysis with the RATIO method. Novel procedures are described for spot integration, orientation matrix determination for relatively sparse diffraction patterns, scaling of multi-crystal data sets, use of Fourier maps for initial assessment and analysis of results, and least-squares refinement of photo-induced structural and thermal changes. In the calculation of Fourier maps a ground-state structure model, typically based on monochromatic results, is employed as reference, and the laser-ON structure factors for the Fourier summations are obtained by multiplying the reference ground-state structure factors by the square root of the experimental ON/OFF ratios. A schematic of the procedure followed is included in the conclusion section.

scholarly journals BraggNet: integrating Bragg peaks using neural networks

2019 ◽  
Vol 52 (4) ◽  
pp. 854-863 ◽  
Author(s):  
Brendan Sullivan ◽  
Rick Archibald ◽  
Jahaun Azadmanesh ◽  
Venu Gopal Vandavasi ◽  
Patricia S. Langan ◽  
...  
Keyword(s):  
Neural Network ◽  
Neural Networks ◽  
Negative Control ◽  
Protein Crystal ◽  
Data Sets ◽  
Data Set ◽  
X Ray ◽  
X Ray Crystallography ◽  
Integrated Intensities ◽  
Neutron Crystallography

Neutron crystallography offers enormous potential to complement structures from X-ray crystallography by clarifying the positions of low-Z elements, namely hydrogen. Macromolecular neutron crystallography, however, remains limited, in part owing to the challenge of integrating peak shapes from pulsed-source experiments. To advance existing software, this article demonstrates the use of machine learning to refine peak locations, predict peak shapes and yield more accurate integrated intensities when applied to whole data sets from a protein crystal. The artificial neural network, based on the U-Net architecture commonly used for image segmentation, is trained using about 100 000 simulated training peaks derived from strong peaks. After 100 training epochs (a round of training over the whole data set broken into smaller batches), training converges and achieves a Dice coefficient of around 65%, in contrast to just 15% for negative control data sets. Integrating whole peak sets using the neural network yields improved intensity statistics compared with other integration methods, including k-nearest neighbours. These results demonstrate, for the first time, that neural networks can learn peak shapes and be used to integrate Bragg peaks. It is expected that integration using neural networks can be further developed to increase the quality of neutron, electron and X-ray crystallography data.

scholarly journals A Medipix quantum area detector allows rotation electron diffraction data collection from submicrometre three-dimensional protein crystals

2013 ◽  
Vol 69 (7) ◽  
pp. 1223-1230 ◽  
Author(s):  
Igor Nederlof ◽  
Eric van Genderen ◽  
Yao-Wang Li ◽  
Jan Pieter Abrahams
Keyword(s):  
Electron Diffraction ◽  
Data Collection ◽  
Diffraction Data ◽  
Three Dimensional ◽  
Protein Crystal ◽  
Protein Crystals ◽  
Electron Diffraction Data ◽  
Single Shot ◽  
X Ray ◽  
Area Detector

When protein crystals are submicrometre-sized, X-ray radiation damage precludes conventional diffraction data collection. For crystals that are of the order of 100 nm in size, at best only single-shot diffraction patterns can be collected and rotation data collection has not been possible, irrespective of the diffraction technique used. Here, it is shown that at a very low electron dose (at most 0.1 e− Å−2), a Medipix2 quantum area detector is sufficiently sensitive to allow the collection of a 30-frame rotation series of 200 keV electron-diffraction data from a single ∼100 nm thick protein crystal. A highly parallel 200 keV electron beam (λ = 0.025 Å) allowed observation of the curvature of the Ewald sphere at low resolution, indicating a combined mosaic spread/beam divergence of at most 0.4°. This result shows that volumes of crystal with low mosaicity can be pinpointed in electron diffraction. It is also shown that strategies and data-analysis software (MOSFLMandSCALA) from X-ray protein crystallography can be used in principle for analysing electron-diffraction data from three-dimensional nanocrystals of proteins.

scholarly journals Microfocus diffraction from different regions of a protein crystal: structural variations and unit-cell polymorphism

2018 ◽  
Vol 74 (5) ◽  
pp. 411-421 ◽  
Author(s):  
Michael C. Thompson ◽  
Duilio Cascio ◽  
Todd O. Yeates
Keyword(s):  
Principal Component ◽  
Unit Cell ◽  
Protein Crystal ◽  
Data Sets ◽  
Structural Variations ◽  
Data Set ◽  
X Ray ◽  
Unit Cells ◽  
Factor Data ◽  
Dynamics Simulations

Real macromolecular crystals can be non-ideal in a myriad of ways. This often creates challenges for structure determination, while also offering opportunities for greater insight into the crystalline state and the dynamic behavior of macromolecules. To evaluate whether different parts of a single crystal of a dynamic protein, EutL, might be informative about crystal and protein polymorphism, a microfocus X-ray synchrotron beam was used to collect a series of 18 separate data sets from non-overlapping regions of the same crystal specimen. A principal component analysis (PCA) approach was employed to compare the structure factors and unit cells across the data sets, and it was found that the 18 data sets separated into two distinct groups, with largeRvalues (in the 40% range) and significant unit-cell variations between the members of the two groups. This categorization mapped the different data-set types to distinct regions of the crystal specimen. Atomic models of EutL were then refined against two different data sets obtained by separately merging data from the two distinct groups. A comparison of the two resulting models revealed minor but discernable differences in certain segments of the protein structure, and regions of higher deviation were found to correlate with regions where larger dynamic motions were predicted to occur by normal-mode molecular-dynamics simulations. The findings emphasize that large spatially dependent variations may be present across individual macromolecular crystals. This information can be uncovered by simultaneous analysis of multiple partial data sets and can be exploited to reveal new insights about protein dynamics, while also improving the accuracy of the structure-factor data ultimately obtained in X-ray diffraction experiments.

A modified Pinkerton-type helium gas-flow system for high-accuracy data collection at the X3 SUNY synchrotron beamline at NSLS

2001 ◽  
Vol 34 (1) ◽  
pp. 76-79 ◽  
Author(s):  
Lynn Ribaud ◽  
Guang Wu ◽  
Yuegang Zhang ◽  
Philip Coppens
Keyword(s):  
Data Collection ◽  
Gas Flow ◽  
Flow System ◽  
Three Dimensional ◽  
Large Data ◽  
Large Data Sets ◽  
Typical Result ◽  
Data Sets ◽  
Helium Gas ◽  
Synchrotron Light

As the combination of high-intensity synchrotron sources and area detectors allows collection of large data sets in a much shorter time span than previously possible, the use of open helium gas-flow systems is much facilitated. A flow system installed at the SUNY X3 synchrotron beamline at the National Synchrotron Light Source has been used for collection of a number of large data sets at a temperature of ∼16 K. Instability problems encountered when using a helium cryostat for three-dimensional data collection are eliminated. Details of the equipment, its temperature calibration and a typical result are described.

scholarly journals The role of wavelength and source in the search for sulfur-atom positions evaluated in two case studies: lysozyme at room temperature and cryo apocrustacyanin A1

2004 ◽  
Vol 37 (4) ◽  
pp. 555-564 ◽  
Author(s):  
Michele Cianci ◽  
John R. Helliwell ◽  
David Moorcroft ◽  
Andrzej Olczak ◽  
James Raftery ◽  
...  
Keyword(s):  
Synchrotron Radiation ◽  
Data Collection ◽  
Room Temperature ◽  
Protein Crystal ◽  
Medium Size ◽  
Data Sets ◽  
X Rays ◽  
Anomalous Signal ◽  
Selection Of

Synchrotron radiation beamlines offer automated data collection with faster and larger detectors, a choice of wavelength(s), intense beams and fine collimation. An increasing output of protein crystal structures sustains an interest in streamlining data collection protocols. Thus, more and more investigators are looking into the use of the anomalous signal from sulfur to obtain initial phase information for medium-size proteins. This type of experiment ideally requires the use of synchrotron radiation, softer X-rays and cryocooling of the sample. Here the results are reported of an investigation into locating the weak,i.e.sulfur, anomalous scatterers in lysozyme using rotating anode or synchrotron radiation data recorded at room temperature. It was indeed possible to locate the sulfur atoms from a lysozyme crystal at room temperature. Accurate selection of images during scaling was needed where radiation damage effects were detected. Most interestingly, comparisons are provided of high-redundancy data sets recorded with synchrotron radiation at λ = 2.0 and 1.488 Å, and with CuKα and MoKα radiation. Apocrustacyanin A1 was also investigated; from the results of a very high redundancy data collection using softer synchrotron X-rays and a cryo-cooled crystal, it was possible to find the sulfur atoms.

Faster acquisition of structure-factor amplitudes in surface X-ray diffraction experiments

2004 ◽  
Vol 37 (3) ◽  
pp. 395-398 ◽  
Author(s):  
X. Torrelles ◽  
J. Rius
Keyword(s):  
Three Dimensional ◽  
Direct Methods ◽  
Lorentz Factor ◽  
Test Material ◽  
Data Sets ◽  
X Ray Diffraction ◽  
X Ray ◽  
Lack Of Information ◽  
Unit Cells ◽  
Order Of Magnitude

The traditional way of measuring intensities in surface X-ray diffraction experiments is by performing rocking scans. Owing to experimental time limitations, this rather slow procedure leads to incomplete data sets for large surface unit cells. This lack of information not only may affect adversely the accuracy of the refined atomic positions, but also hinders the application of three-dimensional direct methods of structure solution. Here the viability of the alternative data collection strategy proposed by Specht & Walker [J. Appl. Cryst.(1993),26, 166–171] is investigated by using as test material a 50 Å thick NBCO thin film grown on an SrTiO3(001) substrate. This procedure, which is based on stationaryLscans,i.e.the sample is kept fixed during theLscan, shortens the measuring time by an order of magnitude. In this study, special attention has been paid to the validation of the predicted theoretical Lorentz factor, which is specific for this experimental setup.

S-SAD phasing of monoclinic histidine kinase fromBrucella abortuscombining data from multiple crystals and orientations: an example of data-collection strategy anda posteriorianalysis of different data combinations

2015 ◽  
Vol 71 (7) ◽  
pp. 1433-1443 ◽  
Author(s):  
Sebastián Klinke ◽  
Nicolas Foos ◽  
Jimena J. Rinaldi ◽  
Gastón Paris ◽  
Fernando A. Goldbaum ◽  
...  
Keyword(s):  
Data Collection ◽  
Structure Determination ◽  
Histidine Kinase ◽  
Structural Information ◽  
Data Sets ◽  
X Rays ◽  
X Ray Crystallography ◽  
Data Collection Strategy ◽  
Sad Phasing ◽  
The Impact

The histidine kinase (HK) domain belonging to the light–oxygen–voltage histidine kinase (LOV-HK) fromBrucella abortusis a member of the HWE family, for which no structural information is available, and has low sequence identity (20%) to the closest HK present in the PDB. The `off-edge' S-SAD method in macromolecular X-ray crystallography was used to solve the structure of the HK domain from LOV-HK at low resolution from crystals in a low-symmetry space group (P21) and with four copies in the asymmetric unit (∼108 kDa). Data were collected both from multiple crystals (diffraction limit varying from 2.90 to 3.25 Å) and from multiple orientations of the same crystal, using the κ-geometry goniostat on SOLEIL beamline PROXIMA 1, to obtain `true redundancy'. Data from three different crystals were combined for structure determination. An optimized HK construct bearing a shorter cloning artifact yielded crystals that diffracted X-rays to 2.51 Å resolution and that were used for final refinement of the model. Moreover, a thorougha posteriorianalysis using several different combinations of data sets allowed us to investigate the impact of the data-collection strategy on the success of the structure determination.

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