scholarly journals Current status of protein micro-crystallography at SPring-8

2014 ◽  
Vol 70 (a1) ◽  
pp. C333-C333 ◽  
Author(s):  
Kunio Hirata ◽  
Yoshiaki Kawano ◽  
Keitaro Yamashita ◽  
Go Ueno ◽  
Takaaki Hikima ◽  
...  
Keyword(s):  
Data Collection ◽  
Current Status ◽  
Photon Flux ◽  
Protein Structure Analysis ◽  
Experimental Conditions ◽  
Automatic Data ◽  
Sports Science ◽  
Experimental Phasing ◽  
Raster Scan ◽  
Data Collections

Protein micro-crystallography is one of the most advanced technologies for protein structure analysis. In order to realize this, an undulator beamline, named BL32XU, was constructed at SPring-8. The beamline can provide beam with size of 0.9 x 0.9 µm and photon flux of 6E10 photons/s. The beam size can be easily changed by users from 1 to 10 µm square with the same flux density. Through three years user operation, we have established several key systems for efficient protein micro-crystallography. One of them is the software for precise positioning of micro-crystals in `raster scan'. SHIKA is a program with GUI which searches diffraction spots in a plenty of low dose diffraction images obtained in raster scan. Finally, it generates 2D map of crystal positions based on the number of spots or spot intensities. Parameters and thresholds in peak search have been empirically optimized for LCP crystals and it provides robust results. Another system is for the data collection strategy. Almost all successful data collections were conducted via `helical data collection' on BL32XU using the line-focused beam. The GUI software, named KUMA, enables estimation of an accumulated dose and suggests suitable experimental conditions for helical data collection. The system is proven to be useful for experimental phasing using tiny LCP crystals of membrane proteins[1-3]. Based on them, the rapid and automatic data collection system using protein micro-crystals is under development. The new CCD detector, Rayonix MX225HS, was installed for faster data acquisition in 10 Hz with the pixel size of 78 µm square. The new SHIKA using GPUs is under development for faster and more accurate crystal alignment. Following this step, KUMA system can suggest experimental conditions for each crystal found on the loop. We also report about the effects of higher dose rate in protein crystallography up to the order of 100 MGy/s. This work was supported by Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

scholarly journals Guidelines for de novo phasing using multiple small-wedge data collection

2021 ◽  
Vol 28 (5) ◽  
pp. 1284-1295 ◽  
Author(s):  
Seiki Baba ◽  
Hiroaki Matsuura ◽  
Takashi Kawamura ◽  
Naoki Sakai ◽  
Yuki Nakamura ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Data Collection ◽  
De Novo ◽  
Optimal Dose ◽  
Dose Dependence ◽  
Systematic Investigation ◽  
Quality Data ◽  
Anomalous Scattering ◽  
Automatic Data ◽  
Experimental Phasing

Intense micro-focus X-ray beamlines available at synchrotron facilities have achieved high-quality data collection even from the microcrystals of membrane proteins. The automatic data collection system developed at SPring-8, named ZOO, has contributed to many structure determinations of membrane proteins using small-wedge synchrotron crystallography (SWSX) datasets. The `small-wedge' (5–20°) datasets are collected from multiple crystals and then merged to obtain the final structure factors. To our knowledge, no systematic investigation on the dose dependence of data accuracy has so far been reported for SWSX, which is between `serial crystallography' and `rotation crystallography'. Thus, herein, we investigated the optimal dose conditions for experimental phasing with SWSX. Phase determination using anomalous scattering signals was found to be more difficult at higher doses. Furthermore, merging more homogeneous datasets grouped by hierarchical clustering with controlled doses mildly reduced the negative factors in data collection, such as `lack of signal' and `radiation damage'. In turn, as more datasets were merged, more probable phases could be obtained across a wider range of doses. Therefore, our findings show that it is essential to choose a lower dose than 10 MGy for de novo structure determination by SWSX. In particular, data collection using a dose of 5 MGy proved to be optimal in balancing the amount of signal available while reducing the amount of damage as much as possible.

scholarly journals Data collection tools for challenging samples at the MX beamlines at Diamond

2014 ◽  
Vol 70 (a1) ◽  
pp. C329-C329
Author(s):  
Ralf Flaig
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Special Focus ◽  
Data Set ◽  
Automatic Data ◽  
Standard Data ◽  
Experimental Phasing ◽  
The Difference ◽  
The Uk ◽  
Collection Strategies

Diamond Light Source [1] is the UK third generation synchrotron facility located south of Oxford and it started with the user programme in early 2007. Currently, there are five operational macromolecular crystallography (MX) beamlines that provide state of the art facilities to the user community and eventually there will be seven beamlines dedicated to MX [2]. All MX beamlines provide tools for standard data collection but given the increasing complexity and associated challenges with bigger macromolecular complexes, membrane proteins, smaller crystals, and radiation damage, different approaches are often required to get the best possible data out of these samples. Tools for sample location and characterization are a first step. Often, because of radiation damage and sample deterioration, multiple crystals are needed in order to obtain a complete data set and a number of tools and different experiment setups that help to address this problem will be described, including use of suitable software tools to get the best data set, fast data collection, crystal humidity control, in situ screening and use of a mini kappa goniometer. These tools enable new data collection strategies which can make the difference towards a successful structure determination. A special focus will be on the use and potential of multi-axis goniometers. Given the limited amount of beam time and ever faster data acquisition rates, quick decision making during the beam time becomes more important. Therefore, data collection strategies and crystal and diffraction image characterization are provided automatically. Very shortly after the data collection has finished the results from our automatic data processing routines are available and we also provide difference electron density map, molecular replacement and experimental phasing pipelines.

scholarly journals Multi-position data collection and dynamic beam sizing: recent improvements to the automatic data-collection algorithms on MASSIF-1

2018 ◽  
Vol 74 (5) ◽  
pp. 433-440 ◽  
Author(s):  
Olof Svensson ◽  
Maciej Gilski ◽  
Didier Nurizzo ◽  
Matthew W. Bowler
Keyword(s):  
Data Collection ◽  
Beam Diameter ◽  
Automatic Data ◽  
Computing Power ◽  
Large Numbers ◽  
Position Data ◽  
Crystal Complex ◽  
Automatic Data Collection ◽  
Data Collections ◽  
Selection Of

Macromolecular crystallography is now a mature and widely used technique that is essential in the understanding of biology and medicine. Increases in computing power combined with robotics have not only enabled large numbers of samples to be screened and characterized but have also enabled better decisions to be taken on data collection itself. This led to the development of MASSIF-1 at the ESRF, the first beamline in the world to run fully automatically while making intelligent decisions taking user requirements into account. Since opening in late 2014, the beamline has processed over 42 000 samples. Improvements have been made to the speed of the sample-handling robotics and error management within the software routines. The workflows initially put into place, while highly innovative at the time, have been expanded to include increased complexity and additional intelligence using the information gathered during characterization; this includes adapting the beam diameter dynamically to match the diffraction volume within the crystal. Complex multi-position and multi-crystal data collections have now also been integrated into the selection of experiments available. This has led to increased data quality and throughput, allowing even the most challenging samples to be treated automatically.

A Novel Method of Data Collection for Automated Electron Tomography Based upon Pre-cal1bration of Image Shifts and Defocus Changes

2001 ◽  
Vol 7 (S2) ◽  
pp. 78-79
Author(s):  
Ulrike Ziese ◽  
Ries Janssen ◽  
Willie Geerts ◽  
Theo van der Krift ◽  
Auke van Balen ◽  
...  
Keyword(s):  
Data Collection ◽  
3D Reconstruction ◽  
Electron Tomography ◽  
Ccd Camera ◽  
Current Status ◽  
Imaging Method ◽  
Major Step ◽  
Experimental Conditions ◽  
Digital Format ◽  
Tilt Series

Electron tomography is a three-dimensional (3D) imaging method with transmission electron microscopy (TEM) that provides high-resolution 3D images of structural arrangements. with electron tomography a series of images is acquired of a sample that is tilted over a large angular range (±70°) with small angular tilt increments. For the 3D-reconstruction, the images of the tilt series are aligned relative to each other and the 3D-reconstruction is computed. Electron tomography is the only technique that can provide 3D information with nm-scale resolution of individual and unique samples. Routine application of electron tomography will comprise a major step forward in the characterization of complex materials and cellular arrangements. When collecting tilt series for electron tomography image shifts and defocus changes have to be corrected for by the human operator. The repetitive correction of these changes is highly time consuming, error prone and very hard to carry out under low-dose imaging conditions.Many practical problems are overcome when electron tomography data collection is performed in an automated fashion. Automation includes the (a) image acquisition on a (digital) CCD camera, which implies that (b) changes in image position and defocus can be detected by on-line image processing and (c) immediately be corrected for by computer control of the microscope, (d) Finally, tilt series are directly available in digital format for subsequent processing. Typically, carrying out such an experiment would take a day, and the actual data collection 2-4 hours. in spite of the enormous progress made in terms of data collection speed during the last few years, the current status of automated tomography still does not meet the requirements that would make it a routinely applicable tool. For a great number of biological assays and research projects, results obtained under different experimental conditions have to be compared, and series of experiments have to be carried out. Therefore, we propose a novel approach for recording a tilt series that significantly increases data collection speed, and widens the applicability of the technique.

scholarly journals EMBL beamlines for macromolecular crystallography at PETRA III

2014 ◽  
Vol 70 (a1) ◽  
pp. C345-C345 ◽  
Author(s):  
Thomas Schneider ◽  
Gleb Bourenkov ◽  
Michele Cianci ◽  
Johanna Kallio ◽  
Guillaume Pompidor ◽  
...  
Keyword(s):  
Data Collection ◽  
Heavy Atom ◽  
Photon Flux ◽  
Wide Energy Range ◽  
Macromolecular Crystallography ◽  
Experimental Conditions ◽  
High Photon Flux ◽  
Small Crystals ◽  
Wide Range ◽  
Integrated Facility

Since 2012, EMBL Hamburg operates two new beamlines for macromolecular crystallography - P13 and P14 - at PETRA III at DESY (Hamburg, Germany). We exploit the high brilliance and the wide energy range offered by PETRA III to offer a wide range of conditions to fit the experimental conditions to the challenges posed by the samples. P13 provides high photon flux down to 4 keV. With a helium cone and a kappa goniostat, this allows optimized data collection for SAD phasing. Using adaptive mirrors, the focus size (H x V) can be adjusted between 30 x 20 μm^2 and 150 x 100 μm^2 to match the size of the sample. A MARVIN sample changer is in operation for rapid loading and unloading of samples. P14 offers a high photon flux (>10^12 ph/sec at 12 keV into 5 x 5 µm^2). The beamsize can be varied between 1 x 1.5 mm^2 (unfocused) and 5 x 5 µm^2 (fully focused) in less than a minute by moving the KB mirrors in and out of the beam. For small crystals, an MD3 vertical diffractometer with a sphere of confusion smaller than 100 nm offers excellent conditions. Both beamlines are equipped with PILATUS 6M-F detectors for shutter-less data collection and dedicated data processing computers. The beamlines are embedded into the 'Integrated Facility for Structural Biology' offering facilities for sample preparation and characterization, a laboratory specifically equipped for the preparation of heavy atom derivatives, and downstream facilities for data evaluation We will report about the status of the beamlines and describe typical experimental situations (small crystals, large unit cells, serial crystallography, low-energy phasing, small molecules and others).

scholarly journals VMXi: a fully automated, fully remote, high-flux in situ macromolecular crystallography beamline

2019 ◽  
Vol 26 (1) ◽  
pp. 291-301 ◽  
Author(s):  
Juan Sanchez-Weatherby ◽  
James Sandy ◽  
Halina Mikolajek ◽  
Carina M. C. Lobley ◽  
Marco Mazzorana ◽  
...  
Keyword(s):  
Data Collection ◽  
Rapid Screening ◽  
Current Status ◽  
Photon Flux ◽  
Cubic Phase ◽  
High Flux ◽  
Macromolecular Crystallography ◽  
Multiple User ◽  
Band Pass

VMXi is a new high-flux microfocus macromolecular crystallography beamline at Diamond Light Source. The beamline, dedicated to fully automated and fully remote data collection of macromolecular crystals in situ, allows rapid screening of hundreds of crystallization plates from multiple user groups. Its main purpose is to give fast feedback at the complex stages of crystallization and crystal optimization, but it also enables data collection of small and delicate samples that are particularly difficult to harvest using conventional cryo-methods, crystals grown in the lipidic cubic phase, and allows for multi-crystal data collections in drug discovery programs. The beamline is equipped with two monochromators: one with a narrow band-pass and fine energy resolution (optimal for regular oscillation experiments), and one with a wide band-pass and a high photon flux (optimal for fast screening). The beamline has a state-of-the-art detector and custom goniometry that allows fast data collection. This paper describes the beamline design, current status and future plans.

scholarly journals LED Illumination Spectrum Manipulation for Increasing the Yield of Sweet Basil (Ocimum basilicum L.)

Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 344
Author(s):  
Md Momtazur Rahman ◽  
Mikhail Vasiliev ◽  
Kamal Alameh
Keyword(s):  
Growth Rate ◽  
Fold Increase ◽  
Photosynthetic Photon Flux Density ◽  
Ocimum Basilicum ◽  
Photon Flux ◽  
Photon Flux Density ◽  
White Led ◽  
Experimental Conditions ◽  
Sweet Basil ◽  
Optimal Illumination

Manipulation of the LED illumination spectrum can enhance plant growth rate and development in grow tents. We report on the identification of the illumination spectrum required to significantly enhance the growth rate of sweet basil (Ocimum basilicum L.) plants in grow tent environments by controlling the LED wavebands illuminating the plants. Since the optimal illumination spectrum depends on the plant type, this work focuses on identifying the illumination spectrum that achieves significant basil biomass improvement compared to improvements reported in prior studies. To be able to optimize the illumination spectrum, several steps must be achieved, namely, understanding plant biology, conducting several trial-and-error experiments, iteratively refining experimental conditions, and undertaking accurate statistical analyses. In this study, basil plants are grown in three grow tents with three LED illumination treatments, namely, only white LED illumination (denoted W*), the combination of red (R) and blue (B) LED illumination (denoted BR*) (relative red (R) and blue (B) intensities are 84% and 16%, respectively) and a combination of red (R), blue (B) and far-red (F) LED illumination (denoted BRF*) (relative red (R), blue (B) and far-red (F) intensities are 79%, 11%, and 10%, respectively). The photosynthetic photon flux density (PPFD) was set at 155 µmol m−2 s−1 for all illumination treatments, and the photoperiod was 20 h per day. Experimental results show that a combination of blue (B), red (R), and far-red (F) LED illumination leads to a one-fold increase in the yield of a sweet basil plant in comparison with only white LED illumination (W*). On the other hand, the use of blue (B) and red (R) LED illumination results in a half-fold increase in plant yield. Understanding the effects of LED illumination spectrum on the growth of plant sweet basil plants through basic horticulture research enables farmers to significantly improve their production yield, thus food security and profitability.

scholarly journals Twenty Years of Data Linkage in The Australian Longitudinal Study on Women’s Health

2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Colleen Loos ◽  
Gita Mishra ◽  
Annette Dobson ◽  
Leigh Tooth
Keyword(s):  
Longitudinal Study ◽  
Data Collection ◽  
Women’S Health ◽  
Women's Health ◽  
Linked Data ◽  
Data Linkage ◽  
Data Access ◽  
National Study ◽  
Australian Longitudinal Study ◽  
Data Collections

IntroductionLinked health record collections, when combined with large longitudinal surveys, are a rich research resource to inform policy development and clinical practice across multiple sectors. Objectives and ApproachThe Australian Longitudinal Study on Women’s Health (ALSWH) is a national study of over 57,000 women in four cohorts. Survey data collection commenced in 1996. Over the past 20 years, ALSWH has also established an extensive data linkage program. The aim of this poster is to provide an overview of ALSWH’s program of regularly up-dated linked data collections for use in parallel with on-going surveys, and to demonstrate how data are made widely available to research collaborators. ResultsALSWH surveys collect information on health conditions, ageing, reproductive characteristics, access to health services, lifestyle, and socio-demographic factors. Regularly updated linked national and state administrative data collections add information on health events, health outcomes, diagnoses, treatments, and patterns of service use. ALSWH’s national linked data collections, include Medicare Benefits Schedule, Pharmaceutical Benefits Scheme, the National Death Index, the Australian Cancer Database, and the National Aged Care Data Collection. State and Territory hospital collections include Admitted Patients, Emergency Department and Perinatal Data. There are also substudies, such as the Mothers and their Children’s Health Study (MatCH), which involves linkage to children’s educational records. ALSWH has an internal Data Access Committee along with systems and protocols to facilitate collaborative multi-sectoral research using de-identified linked data. Conclusion / ImplicationsAs a large scale Australian longitudinal multi-jurisdictional data linkage and sharing program, ALSWH is a useful model for anyone planning similar research.

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