Poster Session IV

Conference: 2020: 70th ACA Annual Meeting
08/06/2020: 4:00 PM  - 5:00 PM 
PS4 
Poster Session 
Virtual  

Description

The ACA holds three evening poster sessions. Poster sessions are organized by the Poster Chairs and feature presentations covering a range of crystallography topics. Poster presentations may not seem as prominent as oral presentations, but they offer a terrific opportunity to interact with other scientists in your field in a structured way.

Presentations

Recent Developments SAXS using MetalJet X-ray Source

High-end x-ray scattering techniques such as SAXS, BIO-SAXS, non-ambient SAXS and GISAXS rely heavily on the x-ray source brightness for resolution and exposure time. Traditional solid or rotating anode x-ray tubes are typically limited in brightness by when the e-beam power density melts the anode. The liquid-metal-jet technology has overcome this limitation by using an anode that is already in the molten state. With bright compact sources, time resolved studies could be achieved even in the home laboratory. We report brightness of 6.5 x 1010 photons/(s·mm2·mrad2·line) over a spot size of 10 µm FWHM. Over the last years, the liquid-metal-jet technology has developed from prototypes into fully operational and stable X-ray tubes running in more than 8 labs over the world. Multiple users and system manufacturers have been now routinely using the metal-jet anode x-ray source in high-end SAXS set-ups. With the high brightness from the liquid-metal-jet x-ray source, novel techniques that was only possible at synchrotron before can now also be used in the home lab. Examples involving in-situ measurements and time resolution such as SEC-SAXS or growth kinetics with temporal resolution on the order of seconds will be shown. This presentation will review the current status of the metal-jet technology specifically in terms of stability, lifetime, flux and optics. It will furthermore refer to some recent SEC-SAXS and bio-SAXS data from users. 

View Proposal 357

Author

Anasuya Adibhatla, Excillum Inc Grafton, MA 

The SHAPES Program for the Reconstruction of Protein Envelopes from X-ray Solution Scattering Data

SHAPES is a computer program intended for the convenient, efficient and reliable reconstruction of protein molecular envelopes from x-ray solution scattering data (J.Badger, J. Appl. Cryst. (2019) 52, 937-944). The program represents the protein volume using a set of volume-filling elements ('beads') that interact with each other via a modified 6-12 Lennard-Jones potential. A Monte Carlo reconstruction process drives the beads to coalesce into a space equal to the expected partial specific volume of the target protein while maintaining a relatively uniform packing density. The final set of bead positions fits the pair-distribution function derived from x-ray scattering data. Convenience features built into the program include the capability of performing multiple reconstruction trials within a single run and the output of ready-to-use molecular volumes. SHAPES is written in python and is compatible with python 2 and 3 interpreters. It is freely available as an open source software under a GNU GPLv3 software license from http://saxs2shapes.com. In addition to providing transparency in program operation, the availability of source code allows for local adaptation, extension and experimentation with use and application of reconstruction methodologies. Although relatively fast when compared to most other available programs for ab initio envelope reconstruction, considerable acceleration may be achieved by adapting repetitive array-based operations to employ functions available in the numpy module (R. Von Dreele, personal communication). Trials of SHAPES with simulated and real data show that accurate and definitive molecular envelopes may be recovered from a wide range of structure types. However, reconstructions of protein envelopes from x-ray solution scattering data are inherently less reliable when the shape of the target protein or protein assembly is highly elongated or flattened. The difficulty in determining correct solutions from these types of sample manifests itself in the appearance of inconsistent solutions over replicate trials and in nonphysical solutions, where the volume-defining beads are broken into many clusters. Recent work with SHAPES includes an option for providing an additional interaction term between beads that tends to prevent them from dissolving into sub-clusters. Application of this option permits the reconstruction of protein shapes from some of the more problematic examples. 

View Proposal 271

Author

John Badger, Consultant San Diego, CA 

High-pressure Neutron Diffraction on WAND2in a Paris-Edinburgh Press

Pressure is a useful tool to increase the reactivity of materials that can aid in and even enable synthesis of novel materials. However, this higher reactivity can also be a hindrance to experiments as samples interact with the apparatus generating the pressure. For example, the diamonds used to generate pressure in one of the most common high pressure cells – the diamond anvil cell – interact with samples at the extremes of pressure. One such system where this phenomenon is a particular hindrance is in high-pressure studies of lithium. Li is often used as a prototype system for the nearly-free electron model. With the application of pressure this simple metal displays a wealth of complex phase behavior as a result of the increased interplay between the core and valence electrons; However, despite decades of theoretical and experimental research dedicated to understanding the complex nature of lithium at high pressure, many conflicting results exist and large regions of pressure-temperature conditions remain unexplored. This is in part due to a lack of high-quality data as a result of lithium's low atomic number, making x-ray diffraction structural investigations difficult. It is however also due to lithium's propensity to break the diamond anvils used in high pressure experiments. The exact mechanism behind this lithium-induced anvil failure at high pressure is unclear and no experimental evidence on the lithium-diamond has been reported. The work presented here will address this short-coming with neutron diffraction data collected on diamond-lithium mixtures pressurized in a Paris-Edinburgh press on WAND[sup]2[/sup] at the High Flux Isotope Reactor at Oak Ridge National Laboratory. 

View Proposal 407

Author

Mary-Ellen Donnelly, Oak Ridge National Laboratory Oak Ridge, TN 

Additional Author(s)

Bianca Haberl, Oak Ridge National Laboratory Oak Ridge, TN 
Yan Wu, Oak Ridge National Laboratory Oak Ridge, TN 
Emily Kroll, Oak Ridge National Laboratory Oak Ridge, TN 
Matthias Frontzek, Oak Ridge National Laboratory Oak Ridge, TN 
Jamie Molaison, Oak Ridge National Laboratory Oak Ridge, TN 

How Low Can You Go? Exploring the Lower Limit of Crystal on a Home X-ray Source

There is a misconception in the literature (Gruene, [i]et. al.[/i], 2018) that one needs crystals as large as 50 µm on a side to perform single X-ray diffraction studies. It has also been suggested that MicroED measurements are needed to probe smaller samples. In this presentation, we will demonstrate that this is simply not true. Furthermore, we will show the lower limit of what is possible with a home X-ray source is somewhere just above the upper range of what is accessible to MicroED, that is, about 1-3 µm. We will also compare some X-ray and ED structural quality and make some suggestions regarding classification of different types of structures based on quality of the results. Gruene, T.; Wennmacher, J. T. C.; Zaubitzer, C.; Holstein, J. J.; Heidler, J.; Fecteau-Lefebvre, A.; De Carlo, S.; Müller, E.; Goldie, K. N.; Regeni, I.; Li, T.; Santiso-Quinones, G.; Steinfeld, G.; Handschin, S.; van Genderen, E.; van Bokhoven, J. A.; Clever, G. H.; Pantelic, R. Rapid Structure Determination of Microcrystalline Molecular Compounds Using Electron Diffraction. [i]Angew. Chem. Int. Ed.[/i] (2018). [b]57[/b], 16313-16317;. 

Proposals


Author

Joseph Ferrara, Rigaku Americas Corp The Woodlands, TX 

Additional Author(s)

Mathias Meyer, Rigaku Polska Sp. z o.o. Wroclaw, Poland 
Takashi Satow, Rigaku Corporation Tokyo, Japan 
Fraser White, Rigaku Europe SE Neu-Isenburg
Akihito Yamano, Application Laboratories, Rigaku Corporation
Takashi Matsumoto, Rigaku Corp. Akishima

Synthesis and characterization of metastable, crystalline st12 germanium

This work uses neutron scattering to characterize a metastable crystalline phase of germanium recovered from high pressure. Such metastable phases of silicon and germanium exhibit interesting functionality. They could potentially yield a Si or Ge structure with ideal band gap characteristics for solar power conversion, improved thin-film characteristics or – in the form of a hydride - even become a useful material for very high temperature superconductivity. Several of such metastable, crystalline phases can be recovered from the metallic high-pressure polymorph of Si and Ge, the so-called β-Sn phase (I4[sub]1[/sub]/amd). This metallic polymorph forms upon room temperature compression to ~11 GPa from the standard diamond cubic Si or Ge (Fd-3m). Upon decompression, this transition is not reversible and instead these metastable phase form. The exact crystal structure that is nucleated is thereby dependent on the exact decompression parameters such as temperature, rate or hydrostaticity. However, the need for synthesis pressures above ~10 GPa has typically limited the recoverable sample volumes. Hence, the majority of studies have been conducted computationally and fewer experimental characterizations have been performed. Thus, there are many open questions on the behavior and characteristics of these metastable phases. Here, we focus on the simple tetragonal st12 structure of Ge (P4[sub]3[/sub]2[sub]1[/sub]2) by combining synthesis capabilities of the SNAP diffractometer of the Spallation Neutron Source with in situ high pressure diffraction on the WAND[sup]2[/sup] beamline of the High Flux Isotope Reactor and inelastic neutron scattering on recovered samples on the ARCS beamline of the Spallation Neutron Source. The st12 structure is synthesized using double-toroidal diamond anvils in a Paris-Edinburgh press from small pieces of a Ge wafer. The sample is pressurized to above ~15 GPa and kept at maximum pressure for several hours to ensure full conversion to the β-Sn phase. The transition pathways is confirmed by in situ diffraction on WAND[sup]2[/sup]. Rietveld refinement of the Ge material under pressure confirms that all diamond-cubic material was indeed converted to the metallic β-Sn phase. It is noteworthy that this experiment represents the first use of the double-toroidal anvils on the WAND2 beamline and that pressures above 10 GPa were achieved for the first time at the HFIR facility. Several such pellets were then measured on ARCS using incident energies of 30, 50, and 70 meV, which were combined to provide a fine resolution phonon density of states. The result closely matches the DFT predictions, although subtle differences may be detected. Thus, in summary, these findings yield new insights into the potential use of the st12 phase as future semiconductor material and also open avenues for further characterization of such metastable phases of Si and Ge. [b]Acknowledgments[/b]: This work was conducted at the SNAP, ARCS and WAND2 beamlines of the Spallation Neutron Source and the High Flux Isotope Reactor, respectively, both DoE Office of Science User Facilities operated by Oak Ridge National Laboratory. 

View Proposal 329

Author

Bianca Haberl, Oak Ridge National Laboratory Oak Ridge, TN 

Additional Author(s)

Mary-Ellen Donnelly, Oak Ridge National Laboratory Oak Ridge, TN 
Yan Wu, Oak Ridge National Laboratory Oak Ridge, TN 
Emily Kroll, Oak Ridge National Laboratory Oak Ridge, TN 
Matthias Frontzek, Oak Ridge National Laboratory Oak Ridge, TN 
Jamie Molaison, Oak Ridge National Laboratory Oak Ridge, TN 
Garrett Granroth, Oak Ridge National Laboratory Oak Ridge, TN 

BioXTAS RAW 2.0: The latest in SAXS data analysis

BioXTAS RAW is a graphical-user-interface-based free open-source Python program for reduction and analysis of small-angle X-ray solution scattering (SAXS) data. RAW 2.0 brings python 3 compatibility, faster integration using pyFAI, and numerous other improvements both small and large. The software is designed for biological SAXS data and enables creation and plotting of one-dimensional scattering profiles from two-dimensional detector images, standard data operations such as averaging and subtraction and analysis of radius of gyration and molecular weight, and advanced analysis such as calculation of inverse Fourier transforms and 3D reconstructions. It also allows easy processing of inline size-exclusion chromatography coupled SAXS data and data deconvolution using the evolving factor analysis method. It provides an alternative to closed-source programs such as Primus and ScÅtter for primary data analysis. Because it can calibrate, mask and integrate images it also provides an alternative to synchrotron beamline pipelines that scientists can install on their own computers and use both at home and at the beamline. RAW is currently used by at least four SAXS beamlines worldwide, including two in North America (the MacCHESS BioSAXS beamline at CHESS and BioCAT at the APS). It is also used at numerous home source SAXS instruments worldwide and is distributed by SAXSLAB/Xenocs with some of their SAXS instruments. 

View Proposal 170

Author

Jesse Hopkins, BioCAT (Sector 18, APS), Illinois Institute of Technology Downers Grove, IL 

Additional Author(s)

Richard Gillilan, MacCHESS, Cornell Univ Ithaca, NY 
Soren Skou, Xenocs Nordic Horsholm, Denmark 

BioCAT beamline enables high quality equilibrium and time resolved biological solution SAXS

The BioCAT beamline (Sector 18) at the Advanced Photon source is a state of the art facility for biological solution scattering. The beamline features advanced size exclusion chromatography small angle x-ray scattering (SEC-SAXS) coupled to multiangle light scattering (MALS), dynamic light scattering (DLS), and refractive index (RI) detectors. These additional techniques allow accurate determination of molecular weight, even for poorly separated species, and measurement of the radius of hydration (Rh), which can provide complementary information to the standard SAXS determined radius of gyration. The beamline also has unique time resolved capabilities, with chaotic continuous flow mixers enabling time resolved SAXS measurements from ~100 us to 100 ms, as well as more conventional stopped flow mixers (time points >= 1 ms) and laminar continuous flow mixers (time points ~10 ms to 1 s), available for users. These capabilities, alongside expert support and an on-site fully equipped wet lab make BioCAT a premier facility of biological SAXS. In addition to SAXS, BioCAT is the only beamline in the Americas that routinely supports fiber and muscle diffraction experiments. 

View Proposal 172

Author

Jesse Hopkins, BioCAT (Sector 18, APS), Illinois Institute of Technology Downers Grove, IL 

Additional Author(s)

Thomas Irving, Biology, Illinois Inst of Technology Chicago, IL 
Srinivas Chakravarthy, BioCAT (Sector 18, APS), Illinois Institute of Technology Argonne, IL 

CryoDiscovery (TM): A machine learning platform for automated Cryogenic electron microscopy class average selection

Cryogenic electron microscopy (Cryo-EM) produces high-resolution 3D images at angstrom levels used by researchers across a broad range of fields including structural biology, life Science, materials science, nanotechnology, semiconductors, energy, environmental science, and food science. Advancements in microscopy hardware enable production of 2D and 3D micrographs with near Angstrom resolution but require exponentially increasing data processing and storage capability. Images generated by cryo-EM are visually noisy, and each project can produce more than 100,000 images and take weeks to arrive at one viewable 3D structure. Many steps in the cryo-EM workflow require manual intervention and analysis that can take several weeks and result in errors due to user bias, time waiting and user fatigue. Current image processing and data analysis solutions are not well-integrated, requiring extensive manual user involvement and long wait times before assessing image quality. Here we describe our development of machine learning models for automation of single particle classification during cryo-EM image processing with repeatable accuracy levels and integrated into the cryo-EM workflow for easy deployment with a new machine learning platform, called CryoDiscovery (Figure 1). We tested several Convolution Neural Network (CNN) designs for ML training and inference using a private set of over 20,000 images and metadata files. CNN architectural considerations include network depth, activation function and hyperparameters. Our CNN processed image data via a layered approach, iteratively through repeated transformations (in the "hidden" layers) to extract features before classifying them (in the "output" layer) 2-D and 3-D class selection. CNN models were trained using image data found in mrcs files, non-image metadata found in star files, and image annotations (ground truth) found in selection files using a computer with a dual socket 2nd gen Intel Xeon® CPU (8 cores each) with 4 NVIDIA 2070-Ti GPUs and 96GB of physical memory. Data preparation was conducted by trained researchers prior to ML training, and consisted of image retrieval, resolution normalization, image augmentation, and metadata selection. Verification of our models was done by analyzing maximum prediction accuracy with low variance, and false negatives to minimize misclassification of good data, and the impact of using metadata to improve model prediction accuracy. Model boosting was used to generate strong prediction algorithms and more consistent results from multiple simple models [1]. Three models were trained sequentially and used for inferencing, as shown in Figure 2. The third model was used when the first two models disagreed for the production data. Fourier Shell Correlation vs. Resolution (1/Aº) [2] was used to verify that the resolution (at threshold) meets published results. Secondly, we calculated the Mean Square Error (MSE) of 2D predicted images vs. ground truth images to provide a leading indicator of 3D model differences. Lastly, we examine Structural Similarity Index (SSIM) for structure level comparison [3 & 4]. Our prediction results reached over 90% accuracy with only a 3% false negative rate (Figure 3). These image processing steps (2D classification, 3D Init model and classification, & 3D Refinement) took only hours to complete with our system. In order to verify the model with larger datasets, we verified our ML inference results using publicly available datasets, such as EMPIAR, and other private and public datasets. The objective of this research is to produce a software tool that consistently classifies particles with a high-level of accuracy and is easily integrated into the cryo-EM workflow. The approach will be to increase the training and validation datasets from a wide range of users and particle types (research labs, proteins, etc.), utilize existing convolutional neural network frameworks and develop new techniques running experiments to optimize the models, integrate the prototype into established cryo-EM workflows for end-to-end processing, and produce a delivery method for easy deployment. The expected results will improve accuracy and productivity reducing the time to produce cryo-EM 3D structures from weeks to hours. 1. Schapire, R., A Brief Introduction to Boosting. Proceedings of the Sixteenth International Joint Conference on Artificial Intelligence, 1999. 2. Liao, H.Y. and J. Frank, Definition and estimation of resolution in single-particle reconstructions. Structure, 2010. 18(7): p. 768-775. 3. Ndajah, P., et al. SSIM image quality metric for denoised images. in Proc. 3rd WSEAS Int. Conf. on Visualization, Imaging and Simulation. 2010. 4. Brunet, D., A study of the structural similarity image quality measure with applications to image processing. 2012. 

View Proposal 299

Author

Narassimha Kumar, Health Technology Innovations Portland, OR 

Additional Author

Gershon Wolfe, Health Technology Innovations Portland, OR 

The Rigaku HyPix-Arc 150: The First Curved Photon Counting Detector

The Rigaku HyPix-6000HE, a hybrid photon counting (HPC) detector is a very popular detector resulting from the properties: high sensitivity, almost no electronic noise, large dynamic range and single pixel top-hat point spread function. Available on all the Synergy-S diffractometers, it yields accurate and precise data in a reduced amount of time, while working in ambient conditions with no need of cooling or vacuum. The latest addition to the HyPix family, the HyPix-Arc 150 possesses 50% more pixels than the HyPix-6000HE, in addition to being curved. The curvature of the detector surface provide several benefits such as: - Greater data coverage while keeping the size and weight of the detector moderate. The HyPix-Arc 150 covers up to 150º of data in a single image. - Reduced data collection time and in turn reduced radiation decay for sensitive crystals. - Reduced reflection distortion, leading to more compact reflections. This, in turn, leads to better data quality and less overlaps between close-together reflections. In this work, we present results from both small molecule and protein data collected on the microfocus sealed tube equipped Rigaku XtaLAB Synergy-S: - Complete data to 0.837 Å on the Friedel mates on a crystal of chlorothiazide crystal in space group P1. - A charge density study. - Structure solution of lysozyme by S-SAD phasing. 

View Proposal 179

Author

Pierre Le Magueres, Rigaku Americas Corporation The Woodlands, TX 

Additional Author(s)

Joseph Ferrara, Rigaku Americas Corp The Woodlands, TX 
Mark Del Campo, Life Sciences, Rigaku Americas Corporation The Woodlands, TX 
Mathias Meyer, Rigaku Polska Sp. z o.o. Wroclaw, Poland 
Hiroyuki Kanda, Rigaku Corporation Tokyo, Japan 
Takashi Satow, Rigaku Corporation Tokyo, Japan 
Przemyslaw Stec, Rigaku Polska Wroclaw, Poland 

Enhancing Protein Crystallization Screening Results Using Engineered Nucleation Features

Protein structure determination remains a critical facet in understanding cellular functions and developing treatments for disease, and macromolecular X-ray crystallography remains the benchmark method for determining these structures at atomic resolution. Despite advances in high throughput crystallization workflows, the time- and sample-consuming challenges of crystallization still result in 80% of crystallization screens failing to produce positive hit information, let alone crystals of diffraction quality. DeNovX is developing crystallization platforms that use engineered nucleation features (ENFs) to improve crystallization outcomes while retaining the requisite diffraction quality. Overall, DeNovX has demonstrated 1.1-8.7-fold increases in crystallization hit percentages while reducing time to crystallization by an average of 40% for a group of proteins known to crystallize (e.g., lysozyme, BPT, etc.) and for proteins with less well characterized crystallization behavior. In order to assess crystal quality, synchrotron X-ray diffraction data were collected and structure determinations were conducted for control samples and for crystals formed more rapidly using ENFs, and the resolution and quality metrics are comparable to their relevant benchmark structures in the PDB. In addition, crystallization using ENFs generates an average of 2.5-fold more crystalline material, which is beneficial in emerging techniques like fixed-target serial crystallography that are acutely sample-destructive and can require hundreds of crystals per study. 

View Proposal 342

Author

Kyle Nordquist, DeNovX Brookfield, IL 

Additional Author(s)

Andrew Howard, Biological Sciences, Illinois Inst of Technology
Okba Hammadi, Illinois Institute of Technology Chicago, IL 
Kevin Schaab, DeNovX Chicago, IL 
Youngchang Kim, Argonne National Lab Lemont, IL 
Gyorgy Babnigg, Argonne National Lab Lemont, IL 
Andrew Bond, DeNovX Chicago, IL 

Crystallization of Heme Dioxygenases IDO and TDO Facilitate Structure-Based Design of Cancer Immunology Therapeutics

The heme dioxygenases IDO and TDO catabolize tryptophan into kynurenine using the heme iron as a central binding component of the substrate. Multiple tumor cells have been shown to over express IDO and TDO, and the elevated kynurenine metabolite is thought to suppress the host immune response, promoting tumor cell survival and proliferation. These metalloproteins are thus targets for cancer immunology, as inhibition of IDO and TDO restores tryptophan levels, relieving the immune suppression. This presentation describes our work on the purification and crystallization of heme-bound IDO and TDO to facilitate selective structure-based drug design and the discovery of tool compounds. 

View Proposal 205

Author

Angela Oh, Genentech South San Francisco, CA 

Comprehensive strategy for quick determination of protein structures

A special advantage of protein crystallography is quick determination of target proteins for biological science and high-throughput structure determination of a large number of protein-compound complexes for pharmaceutical science. To achieve the quick structure determination, utilization of well-experienced protein crystallization technique and the latest approach of structure determination are crucial. We developed a standard protocol to obtain well-diffracted crystals through a lot of projects. At the first step, our integrated crystallization robot is useful for the initial crystallization screening. If crystals appeared at several conditions, we evaluate their crystal quality and choose relatively good crystals based on the snapshot images using X-ray. When obtained crystals have insufficient quality, cryoprotectant screening is effective to improve the crystal quality in many cases. Qualities of various crystals have been improved using our strategy when original crystals were of poor quality (1, 2, 3, 4). MR-native SAD method is quite useful for quick structure determination with semi-automatic model building program. Diffraction data collection using lower energy X-ray like 1.9 Å or 2.7 Å wavelength is essential for measurement of an anomalous signal from sulfur atoms (5). Here we show several examples of crystal structure determination using MR-native SAD method. In some cases, MR-native SAD method easily gave high quality model without manual model building despite MR method gave poor model which was too difficult to fix problems by manually. References 1. Hayashi et al., (2017) Cell Reports 20, 2876. 2. Senda et al., (2016) Crystal growth & Design 16, 1565. 3. Sumita et al., (2016) Molecular Cell 61, 1. 4. Hayashi et al., (2012) Cell Host Microbe 12, 20. 5. Liebschner et al., (2016) Acta Crystallogr. D 72, 728. 

View Proposal 274

Author

Miki Senda, High Energy Accelerator Research Organization (KEK) Tsukuba

Additional Author

Toshiya Senda, Structural Bio Research Ctr Inst of Materials Structure , High Energy Accelerator Research Org Tsukuba

Zero Ramachandran outliers does not guarantee a “good” model

Ramachandran plots report the distribution of the (φ, ψ) torsion angles of the protein backbone and are one of the best quality metrics of experimental structure models. Typically, validation software reports the number of residues belonging to "outlier", "allowed" and "favored" regions. While "zero unexplained outliers" can be considered the current "gold standard", this can be misleading if deviations from expected distributions, even within the favored region, are not considered. We therefore revisited the Ramachandran Z-score (Rama-Z), a quality metric introduced more than two decades ago, but underutilized. We describe a re-implementation of the Rama-Z score in the Computational Crystallography Toolbox along with a new algorithm to estimate its uncertainty for individual models; final implementations are available both in Phenix and in PDB-REDO. We discuss the interpretation of the Rama-Z score and advocate including it in the validation reports provided by the Protein Data Bank. We also advocate reporting it alongside the outlier/allowed/favored counts in structural publications. Figure shows examples of Ramachandran plots: Left: a good-looking Ramachandran plot for (1ix9, 0.9 Å), Middle: an obviously bad Ramachandran plot (5a9z, 4.7 Å) and Right: a suspicious Ramachandran plot (6dzv, 4.2 Å). PDB ID code of the models in top right corner. Triplets of numbers on the bottom right on each plot indicate, from top to bottom: percentage of residues in favored and outlier regions, Rama-Z. This research was supported by the NIH (grant GM063210), the Phenix Industrial Consortium, and by the Netherlands Organization for Scientific Research (NWO; Vidi grant 723.013.003). This work was partially supported by the US Department of Energy under Contract DE-AC02-05CH11231. 

View Proposal 165

Author

Oleg Sobolev, Lawrence Berkeley National Laboratory Berkeley, CA 

Additional Author(s)

Pavel Afonine
Nigel Moriarty, Lawrence Berkeley National Laboratory Berkeley, CA 
Maarten Hekkelman, Division of Biochemistry, The Netherlands Cancer Institute Amsterdam, The Netherlands 
Robbie Joosten, Division of Biochemistry, The Netherlands Cancer Institute Amsterdam, The Netherlands 
Anastassis Perrakis, Division of Biochemistry, The Netherlands Cancer Institute Amsterdam, The Netherlands 
Paul Adams, Lawrence Berkeley Laboratory

Mail-in and remote access for solution scattering at the LiX beamline

At the Life Science X-ray Scattering (LiX) beamline at NSLS-II, we have developed capabilities for fully automated solution scattering data collection and data processing [1]. While it has been our plan all along to support mail-in and remote user access, the COVID-19 pandemic has made it a necessity. We report our current status in preparation for supporting remote user experiments, which we will make available to users for the 2020-2 cycle starting in mid-July. Central to our implementation is an Opentron liquid handling robot that we use to transfer samples from standard 96-well plates to our 18-position sample holder. Sample plates and sample holders are tracked using bar codes and QR codes, respectively, to ensure accurate identification of the samples. Remote access is realized through NX. Once the users' samples are ready for measurements, the user is allowed to run a simple GUI to start measurements and monitor the process of data collection. Alternatively, overnight static measurements can be started by beamline staff on all samples that have been accumulated. Additional software tools have been developed to ensure data quality and assist data analysis. As soon as the processed data in hdf5 file and analysis results are available, they can be accessed either from the users' home institution via Globus, or using the computing resources at NSLS-II. [1] Solution scattering at the Life Science X-ray Scattering (LiX) beamline L. Yang, S. Antonelli, S. Chodankar, J. Byrnes, E. Lazo and K. Qian J. Synchrotron Rad. (2020). 27, 804-812, https://doi.org/10.1107/S1600577520002362 

View Proposal 370

Author

Lin Yang, Brookhaven National Laboratory Upton, NY 

Additional Author(s)

Shirish Chodankar, Brookhaven National Laboratory Upton, NY 
James Byrnes, Brookhaven National Laboratory Upton, NY 

RCSB PDB Next-generation Data Delivery and Search Services

RCSB Protein Data Bank (PDB) provides tools for analysis and visualization of 3D structures of biological macromolecules stored in the PDB archive. Recently-introduced Search and Data Delivery APIs offer comprehensive functionality and high performance at RCSB.org. The new services represent a complete overhaul of the software/data management architecture, transforming a monolithic application into a micro-service-oriented and cloud-ready resource. The data model is based on the PDBx/mmCIF dictionary (http://mmcif.wwpdb.org/) with extensions that facilitate usage and delivery for the RCSB PDB website and web services. For Data delivery (https://data.rcsb.org), a GraphQL interface allows arbitrary retrieval of data across the entire data model. To the best of our knowledge, this represents a first in Structural Bioinformatics. Search services (https://search.rcsb.org) are supported by a powerful Search API with a JSON-based Domain Specific Language (DSL). Arbitrary boolean logic search is now possible across all fields available in our data model. Importantly, a search aggregator layer seamlessly combines text searches from the Elasticsearch engine with specialized bioinformatics algorithms that perform searches against macromolecular sequence and/or atomic coordinate data. Examples of the searches integrated by the aggregator are mmseqs2 sequence search (1), BioZernike structure shape search (2), and sequence motif search. Users of existing services are strongly encouraged to migrate to the new APIs before November 2020, when legacy RCSB PDB APIs (REST search and fetch) will be discontinued. RCSB PDB is funded by the National Science Foundation (DBI-1832184), the US Department of Energy (DE-SC0019749), and the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and National Institute of General Medical Sciences of the National Institutes of Health under grant R01GM133198. 1) Mirdita M, Steinegger M and Soeding J. MMseqs2 desktop and local web server app for fast, interactive sequence searches. Bioinformatics, doi: 10.1093/bioinformatics/bty1057 (2019). Dmytro Guzenko, Stephen K. Burley, Jose M. Duarte. Real time structural search of the Protein Data Bank (2020) bioRxiv doi: https://doi.org/10.1101/845123 2) Dmytro Guzenko, Stephen K. Burley, Jose M. Duarte. Real time structural search of the Protein Data Bank (2020) bioRxiv doi: https://doi.org/10.1101/845123 

View Proposal 251

Author

Christine Zardecki, Rutgers Proteomics, RCSB Protein Data Bank Piscataway, NJ 

Additional Author(s)

Jose M. Duarte, RCSB Protein Data Bank CA 
Charmi Bhikadiya, RCSB Protein Data Bank NJ 
Chunxiao Bi, RCSB Protein Data Bank CA 
Sebastian Bittrich, RCSB Protein Data Bank CA 
Li Chen, RCSB Protein Data Bank NJ 
Dmytro Guzenko, RCSB Protein Data Bank CA 
Robert Lowe, RCSB Protein Data Bank NJ 
Joan Segura, RCSB Protein Data Bank CA 
Yana Valasatava, RCSB Protein Data Bank CA 
John Westbrook
Stephen Burley, RCSB Protein Data Bank, Rutgers University Piscataway, NJ 

Evaluating crystallographic likelihood functions using numerical quadratures.

Intensity-based likelihood functions in crystallographic applications have the potential to enhance the quality of structures derived from marginal diffraction data. Their usage however is complicated by the ability to efficiently compute these targets functions. Here a numerical quadrature is developed that allows for the rapid evaluation of intensity-based likelihood functions in crystallographic applications. By using a sequence of change of variable transformations, including a non-linear domain compression operation, an accurate, robust, and efficient quadrature is constructed. The approach is flexible and can incorporate different noise models with relative ease. 

View Proposal 237

Author

Petrus Zwart Berkeley, CA 

Additional Author

D. Elliot Perryman, U. Tennessee Knoxville, TN 

Crystal Structures of Large-Volume Commercial Pharmaceuticals

As part of a continuing project, the challenging room-temperature crystal structures of four commercial pharmaceutical APIs have been solved by Monte Carlo simulated annealing techniques using synchrotron X-ray powder diffraction data (11-BM at APS), and optimized using density functional techniques. Atorvastatin calcium trihydrate (Lipitor®), (C33H34FN2O5)2Ca(H2O)3 crystallizes in space group P1 (#1) with a = 5.44731(4), b = 9.88858(16), c = 29.5925(10) Å, a = 95.859(3), β = 94.211(1), g = 105.2790(1)°, V = 1521.277(10) Å3, and Z = 1. The structure was solved by removing the O atoms from the carboxylate groups of the anion, and using a CaO6 fragment. Pimecrolimus (Elidel), C43H68ClNO11, crystallizes in space group P21 (#4) with a = 15.28864(7), b = 13.31111(4), c = 10.95529(5) Å, β = 96.1542(3)̊, V = 2216.649(9) Å3, and Z = 2. By default, simulated annealing programs did not give enough torsional degrees of freedom, so the macrocycle was broken, and re-formed at a low success rate. Ivermectin hemihydrate ethanolate, (C48H74O14)(H2O)0.5(C2H5OH)0.68, crystallizes in space group I2 (#5) with a = 14.94878(15), b = 9.26938(4), c = 39.27263(30) Å, β = 94.4017(7)̊, V = 5425.80(5) Å3, and Z = 4. A reduced cell search yielded another solvate, and the guest species were identified using difference Fourier and spectroscopic techniques. Ceftriaxone sodium hemiheptahydrate (Rocefin), C18H16N8O7S3Na2(H2O)3.5, crystallizes in space group C2 (#5) with a = 30.56495(19), b = 4.75245(3), c = 18.55021(18) Å, β = 90.3551(7)̊, V = 2694.521(24) Å3, and Z = 4. Some of the water molecules were difficult to locate conventionally, and were placed by progressively searching for smaller voids. Other new structures may be discussed as they become available. 

View Proposal 109

Author

James Kaduk, Chemistry, Illinois Inst of Technology Naperville, IL 

Additional Author(s)

Ryan Hodge, North Central College Naperville, IL 
Nicholas Boaz, North Central College Naperville, IL 
Shivang Bhaskar, Illinois Mathematics and Science Academy Aurora, IL 
Diana Gonzalez, Illinois Mathematics and Science Academy Aurora, IL 
Joseph Golab, Illinois Mathematics and Science Academy Aurora, IL 
Amy Gindhart, ICDD Newtown Square, PA 
Thomas Blanton, ICDD

Pushing the Limits of Microfocus X-Ray Sealed Tube Sources for Biological and Chemical Crystallography

The structure determination on ever smaller and weakly diffracting crystals is one of the biggest challenges in the development of in-house X-ray analytical equipment for chemical and biological crystallography, which continuously raises the requirements for modern X-ray sources and detectors. Nowadays, modern low power microfocus X-ray sealed tube sources, such as the Incoatec Microfocus Soure IμS, define the state-of-the-art for most in-house X-ray diffraction equipment, as they deliver intensities in the range of rotating anodes, yet maintain all the comfort of a sealed tube system. Throughout the past years, we have continuously improved the performance of the IµS by optimizing critical parameters in the X-ray tube and adapting the X-ray optics, making the IµS the market-leading microfocus sealed tube X-ray source with more than 1000 sources sold to date world-wide. The latest improvement for the IµS 3.0, the first and only microfocus sealed tube source fully optimized for X-ray diffraction applications, is a dedicated multilayer mirror which delivers an intensity in the range of 8·10[sup]...[/sup]10 phts/s/mm[sup]...[/sup]2 with a divergence that matches the typical mosaicity of weakly diffracting chemical samples. Applications that demand an even higher brightness, such as protein crystallography, benefit from our recently introduced unique new class of microfocus sealed tube sources which uses diamond as a heat sink to cool the anode. This IμS DIAMOND combines the performance of a modern 1 kW microfocus rotating anode with all the comfort of a conventional microfocus sealed tube source, and is now available for Cu-Kα, Mo-Kα and Ag-Kα radiation. We will be presenting selected results showing the impact of these recent developments on the data quality. 

View Proposal 294

Author

Juergen Graf Geesthacht

Additional Author(s)

Tobias Stuerzer, Bruker AXS GmbH Karlsruhe
Matthew Benning, Bruker AXS LLC Madison, WI 
Holger Ott, Bruker AXS GmbH Karlsruhe
Paul Radcliffe, incoatec GmbH Geesthacht
Jenss Schmidt-May, incoatec GmbH Geesthacht
Carsten Michaelsen, incoatec GmbH Geesthacht

Towards Synergy of Macromolecular and Small Molecule Crystallography

Small molecule and macromolecular crystallographers use the same technique and work in close proximity, yet they are mostly separate in practice, many times even using separate diffractometers in the same room. Our newest diffractometers were designed around the concept of synergy of techniques. Here, we use our newest hybrid photon counting detector the HyPix-Arc150 to demonstrate this synergy. We use the same instrument to collect atomic resolution data sets of lysozyme with sucrose bound and of sucrose by itself. Both crystal structures will be presented. 

View Proposal 292

Author

Mark Del Campo, Life Sciences, Rigaku Americas Corporation The Woodlands, TX 

Additional Author(s)

Pierre Le Magueres, Rigaku Americas Corporation The Woodlands, TX 
Joseph Ferrara, Rigaku Americas Corp The Woodlands, TX 

DECOR: The Database of Educational Crystallographic Online Resources, Updates and Prospectus

The Database of Educational Crystallographic Online Resources (DECOR) is the worlds first repository for educational resources for the teaching of crystallography and diffraction. The site, available at decor.cst.temple.edu, permits the sharing and downloading of educational resources such as practice problems, visual aids, animations, and more. The site is currently organized into three basic resource layouts: 1) Resources by Type, where visitors can browse for homework problems, presentations slides, or animations across topics. 2) Resources by Topic, where visitors can look for resources relating to particular subject matter such as the reciprocal lattice, scattering, or symmetry. 3) Links, which sends visitors to other sites that provide convenient resources for crystallographic education. The purpose of the poster is to make attendees aware of this teaching resource, and to present the current state of the site, and plans to upgrade and enhance it in the near future. 

View Proposal 413

Author

Michael Zdilla, Chemistry, Temple Univ Philadelphia, PA 

Implementing Best Practices At The National Center For CryoEM Access And Training

The mission of NCCAT (National Center for CryoEM Access and Training) is twofold: to provide nationwide access to advanced cryoEM technical capabilities, and to assist users in the development of cryoEM skills needed for independent research. NCCAT provides access to state-of-the-art equipment required to solve structures to the highest possible resolution using cryoEM methods. At the center, we bring the most current best practices to assist researchers access new technologies and accelerate their research. In particular, a significant bottleneck to the generation of high-resolution structures is sample preparation. The method of preparing vitrified samples by blotting followed by plunging into liquid ethane or propane has changed little in the past couple of decades apart from the development of automated blotting devices. This technique takes expertise to master and can produce a range of ice thicknesses. The ability to determine ice thickness routinely during screening or data collection provides a helpful guide to deciding which areas to image or indeed whether further imaging on a grid should be abandoned. Towards that goal, we have implemented a way to measure ice thickness directly into our data acquisition and processing workflow. In addition, to aid with the reproducibility of this technique NCCAT has access to a new type of grid that is essentially "self-blotting" and can be used in conjunction with a robotic device that dispenses small volumes (tens of pL) of a sample using a piezoelectric nozzle. When small volumes of sample are applied to these grids it is rapidly wicked away, leaving behind a thin film that is then rapidly vitrified, which may assist with samples intractable by traditional methods. Taken together we aim to lower the barriers of access and cross-train biomedical researchers to broadly utilize cryoEM techniques. 

View Proposal 284

Author

Edward Eng, New York Structural Biology Center New York, NY 

Additional Author

Elina Kopylov, National Center for CryoEM Access and Training New York, NY 

Access modes to the highly automated BioSAXS beamline P12 of EMBL Hamburg

The last decades saw a sharp increase in the use of small angle X-ray scattering (SAXS) for the characterisation of biological macromolecules in solution [1]. SAXS became an important part of the structural biologist's toolbox and dedicated instruments are essential to provide high quality beams and to support the rapidly growing requests to access SAXS beamlines. Here, we present the development of EMBL's BioSAXS beamline (PETRA III ring, Hamburg, Germany) [2]. The reported advances allow for a reliable collection of the weak SAXS signal from biological macromolecules in solution, and for the proper handling and online characterisation/purification of the sample. The high brilliance and low background beamline is equipped with a robotic sample changer [3] and an on-line size exclusion chromatography setup [4]. Data collection and analysis are highly automated, such that the first results can be obtained within a minute after data take. More than 100 user projects (for more than 300 user visits) are measured each year on this instrument. Beyond "standard" bioSAXS measurements, P12 exploits the high flux of the X-ray beam delivered by the PETRA III undulator for fast time resolved measurements. A recently commissioned multilayer monochromator, a EIGER 4M detector as well as a stopped flow device allow time resolved data collection with a dead time of a few ms. A beam chopper and laser triggering devices are now developed to further reduce the dead time of the reaction triggering to perform sub-ms time resolved SAXS experiments at the beamline. Various modes of user access to the beamline will be discussed. The automation at P12 allows mail-in/remote measurements and the user operation is further supported through European funded translational activities such as iNEXT-Discovery. Rapid access is available for urgent (e.g. Covid-19 related) proposals, the BioSAXS group supports service groups and is always open for new collaborations. Recently, a spin-off company, BIOSAXS GmbH (www.biosaxs.com), was founded utilizing the achievements made at EMBL Hamburg to streamline industrial access to synchrotron SAXS. BIOSAXS GmbH presently provides services for numerous pharmaceutical and biotechnological companies ranging from advanced SAXS measurements at the P12 beamline to complete projects involving sample handling, measurements, data analysis and reporting to answer the relevant medical, biological and structural questions. References [1] – Tuukkanen et al., IUCrJ 4(Pt 5):518-528 (2017). [1] – Graewert et al., Current opinion in structural biology 23(5) (2013). [2] – Blanchet et al., Journal of applied crystallography, 48(2) (2015). [3] – Round et al., Acta Crystallographica Section D. 71(1) (2015). [4] – Graewert et al., Scientific reports 5 (2015). 

View Proposal 399

Author

Melissa Graewert, EMBL Hamburg Hamburg

Additional Author(s)

Clement E. Blanchet, EMBL Hamburg
Martin A. Schroer, EMBL Hamburg
Andrey Gruzinov, EMBL Hamburg
Alexey Kikhney, BIOSAXS GmBH
Tobias W. Graewert, BIOSAXS GmBH
Daniel Franke, EMBL Hamburg
Cy M. Jeffries, EMBL Hamburg

Interactive Python Programs for Crystallography

My textbook, Foundations of Crystallography[1] uses MATLAB. Because this is expensive, many of my readers have asked me to use an open source language. The third edition, under preparation, includes many interactive Python crystallography programs. Eight of these prorams will be made available to attendees at this conference along with oral explanations on how to run the programs. Program 1 introduces Python and creates a two-dimensional oblique unit cell and a three-dimensional triclinic cell. Program 2 populates an oblique unit cell of hexamethylbenzene from the fractional coordinates. Program 3 calculates a point group Cayley (or multiplication) table. Program 4 produces a three-dimensional populated unit cell and its projections calculated from crystallographic parameters found in an external database. Program 5 produces the reciprocal cell superimposed on the direct cell using the G and G* matrices. The input is the unit cell parameters a, b, c, α, β, γ. The output is a*, b*, c*, α*, β*, γ*, G, G*, V and V*. Program 6 calculates a powder diffraction pattern where d-spacings use G*. Program 7 calculates the atomic scattering curves for multiple atoms using the Cromer-Mann coefficients. Finally, Program 8 calculates the electron density map for hexamethylbenzene. The first four programs emphasize a holistic approach to the symmetries of point groups and space groups. The last four programs explore experimental crystallography exemplified by the reciprocal lattice, powder diffraction patterns, atomic scattering curves, and finally an electron density map. The calculation of the three-dimensional unit cell is an example of mathematical modelling. The information put into the model is the crystal parameters and space group symmetries. The verification of the model is the identification of the two-dimensional space group symmetries of three independent projections. In all cases, the programs are simple, transparent, and explicit. References [1] Julian, Maureen M. Foundations of Crystallography with Computer Applications, 2nd edition, CRC Press: New York (2015), ISBN: 978-1466552913. 

View Proposal 245

Author

Maureen Julian, Virginia Tech Blacksburg, VA 

Additional Author(s)

Francis T. Julian, Oliver Wyman Digital Princeton, NJ 
Harrison F. Jones, Department of Materials Science and Engineering Blacksburg, VA 

Optimal Box Size and Mask Diameter based on Defocus Distribution for Single Particle Cryo-EM

Recently, it has been demonstrated that single particle analysis (SPA) using 200 keV CryoEM paired with direct electron detector (DED) is capable to reconstruct < 200 kDa protein structures at resolution higher than 3.0 Å. However, the majority of near-atomic resolution cryoEM structures has been determined using 300 keV cryoEMs equipped with DEDs. As a consequence, many of typical parameter settings for cryoEM session and image processing steps are based on the accumulated experience of 300 keV cryoEMs, such as defocus range for EM sessions and amplitude contrast for CTF estimation. Therefore, we revised the parameters for 200 keV acceleration voltage, and found out merely optimizing mask diameter and box size based on defocus distribution of dataset can improve the resolution. 

View Proposal 457

Author

Toshio Moriya