scholarly journals A suite of software for processing MicroED data of extremely small protein crystals

2014 ◽  
Vol 47 (3) ◽  
pp. 1140-1145 ◽  
Author(s):  
Matthew G. Iadanza ◽  
Tamir Gonen
Keyword(s):  
Structure Determination ◽  
Ad Hoc ◽  
Source Code ◽  
Three Dimensional ◽  
Molecular Replacement ◽  
X Ray ◽  
X Ray Crystallography ◽  
Software Packages ◽  
Transmission Electron ◽  
Standard Software

Electron diffraction of extremely small three-dimensional crystals (MicroED) allows for structure determination from crystals orders of magnitude smaller than those used for X-ray crystallography. MicroED patterns, which are collected in a transmission electron microscope, were initially not amenable to indexing and intensity extraction by standard software, which necessitated the development of a suite of programs for data processing. The MicroED suite was developed to accomplish the tasks of unit-cell determination, indexing, background subtraction, intensity measurement and merging, resulting in data that can be carried forward to molecular replacement and structure determination. Thisad hocsolution has been modified for more general use to provide a means for processing MicroED data until the technique can be fully implemented into existing crystallographic software packages. The suite is written in Python and the source code is available under a GNU General Public License.

scholarly journals Low-Zpolymer sample supports for fixed-target serial femtosecond X-ray crystallography

2015 ◽  
Vol 48 (4) ◽  
pp. 1072-1079 ◽  
Author(s):  
Geoffrey K. Feld ◽  
Michael Heymann ◽  
W. Henry Benner ◽  
Tommaso Pardini ◽  
Ching-Ju Tsai ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Three Dimensional ◽  
Two Dimensional ◽  
X Ray Diffraction ◽  
Fixed Target ◽  
X Ray ◽  
X Ray Crystallography ◽  
Transmission Electron ◽  
Linac Coherent Light Source ◽  
Efficient Alternative

X-ray free-electron lasers (XFELs) offer a new avenue to the structural probing of complex materials, including biomolecules. Delivery of precious sample to the XFEL beam is a key consideration, as the sample of interest must be serially replaced after each destructive pulse. The fixed-target approach to sample delivery involves depositing samples on a thin-film support and subsequent serial introductionviaa translating stage. Some classes of biological materials, including two-dimensional protein crystals, must be introduced on fixed-target supports, as they require a flat surface to prevent sample wrinkling. A series of wafer and transmission electron microscopy (TEM)-style grid supports constructed of low-Zplastic have been custom-designed and produced. Aluminium TEM grid holders were engineered, capable of delivering up to 20 different conventional or plastic TEM grids using fixed-target stages available at the Linac Coherent Light Source (LCLS). As proof-of-principle, X-ray diffraction has been demonstrated from two-dimensional crystals of bacteriorhodopsin and three-dimensional crystals of anthrax toxin protective antigen mounted on these supports at the LCLS. The benefits and limitations of these low-Zfixed-target supports are discussed; it is the authors' belief that they represent a viable and efficient alternative to previously reported fixed-target supports for conducting diffraction studies with XFELs.

scholarly journals Structure of catalase determined by MicroED

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Brent L Nannenga ◽  
Dan Shi ◽  
Johan Hattne ◽  
Francis E Reyes ◽  
Tamir Gonen
Keyword(s):  
Structure Determination ◽  
Three Dimensional ◽  
Bovine Liver ◽  
Electron Crystallography ◽  
X Ray ◽  
X Ray Crystallography ◽  
Continuous Rotation ◽  
Bovine Liver Catalase ◽  
Diffraction Patterns ◽  
Atomic Analysis

MicroED is a recently developed method that uses electron diffraction for structure determination from very small three-dimensional crystals of biological material. Previously we used a series of still diffraction patterns to determine the structure of lysozyme at 2.9 Å resolution with MicroED (<xref ref-type="bibr" rid="bib26">Shi et al., 2013</xref>). Here we present the structure of bovine liver catalase determined from a single crystal at 3.2 Å resolution by MicroED. The data were collected by continuous rotation of the sample under constant exposure and were processed and refined using standard programs for X-ray crystallography. The ability of MicroED to determine the structure of bovine liver catalase, a protein that has long resisted atomic analysis by traditional electron crystallography, demonstrates the potential of this method for structure determination.

scholarly journals 4-(4-Chlorophenyl)-4,5-dihydro-1H-1,2,4-triazole-5-thione

Molbank ◽  
10.3390/m1047 ◽  
2019 ◽  
Vol 2019 (1) ◽  
pp. M1047 ◽  
Author(s):  
Chien Yeo ◽  
Ainnul Azizan ◽  
Edward Tiekink
Keyword(s):  
Hydrogen Bonds ◽  
Structure Determination ◽  
Title Compound ◽  
Uv Spectroscopy ◽  
Three Dimensional ◽  
Membered Ring ◽  
X Ray ◽  
X Ray Crystallography ◽  
Orthogonal Relationship ◽  
Ir And Uv Spectroscopy

The title compound, 4-(4-chlorophenyl)-4,5-dihydro-1H-1,2,4-triazole-5-thione (1), was synthesized by a hetero-cyclization reaction of 4-chlorophenyl isothiocyanate and formic hydrazide. Compound 1 was characterized by a single-crystal X-ray structure determination as well as 1H and 13C{1H} NMR, IR, and UV spectroscopy, and microelemental analysis. X-ray crystallography on 1 confirms the molecule exists as the thione tautomer and shows the five-membered ring to be planar and to form a dihedral angle of 82.70(5)° with the appended chlorophenyl ring, indicating an almost orthogonal relationship. In the molecular packing, supramolecular dimers are formed via thioamide-N–H⋯S(thione) hydrogen bonds and these are connected by C=S⋯π(triazolyl) and C-Cl⋯π(triazolyl) interactions, leading to a three-dimensional architecture.

Artificial intelligence to solve the X-ray crystallography phase problem: a case study report.

2021 ◽  
Author(s):  
Irene Barbarin-Bocahu ◽  
Marc GRAILLE
Keyword(s):  
Structure Prediction ◽  
Three Dimensional ◽  
Structural Similarity ◽  
Molecular Replacement ◽  
Learning Approaches ◽  
Phase Problem ◽  
Data Set ◽  
X Ray ◽  
X Ray Crystallography ◽  
Eukaryotic Mrnas

The determination of three dimensional structures of macromolecules is one of the actual challenge in biology with the ultimate objective of understanding their function. So far, X-ray crystallography is the most popular method to solve structure, but this technique relies on the generation of diffracting crystals. Once a correct data set has been obtained, the calculation of electron density maps requires to solve the so-called phase problem using different approaches. The most frequently used technique is molecular replacement, which relies on the availability of the structure of a protein sharing strong structural similarity with the studied protein. Its success rate is directly correlated with the quality of the models used for the molecular replacement trials. The availability of models as accurate as possible is then definitely critical. Very recently, a breakthrough step has been made in the field of protein structure prediction thanks to the use of machine learning approaches as implemented in the AlphaFold or RoseTTAFold structure prediction programs. Here, we describe how these recent improvements helped us to solve the crystal structure of a protein involved in the nonsense-mediated mRNA decay pathway (NMD), an mRNA quality control pathway dedicated to the elimination of eukaryotic mRNAs harboring premature stop codons.

Three-Dimensional Structure Of C3D From Low Temperature Scanning Transmission Electron Microscopy and X-Ray Crystallography

2000 ◽  
Vol 6 (S2) ◽  
pp. 294-295
Author(s):  
D.J. Martin ◽  
F.P. Ottensmeyer
Keyword(s):  
Electron Microscopy ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
X Rays ◽  
Complement Protein ◽  
X Ray ◽  
X Ray Crystallography ◽  
Scanning Transmission Electron ◽  
Transmission Electron ◽  
Scanning Transmission

Macromolecular structure can be solved by x-ray crystallography to atomic resolution provided that the molecule can be crystallized, that the crystals diffract x-rays to high resolution, and that the phases of the diffracted x-rays can be determined. Though the resolution of single particle imaging by electron microscopy is lower than that of x-ray diffraction by crystals, electron microscopy can directly image a large molecular weight range of macromolecules in a non-crystalline environment, and provide the basis for the three-dimensional reconstruction of these structures. To investigate combining structural information from x-ray crystallography and electron microscopy for unknown structures, we have imaged a small protein of known structure (1), the 35 kDa human complement protein fragment C3d, in a scanning transmission electron microscope (STEM). The intention is to eventually combine the knowledge of electron densities and molecular boundaries from electron microscopy to assist in phase determination in x-ray crystallography.

scholarly journals PHENIX: a comprehensive Python-based system for macromolecular structure solution

2010 ◽  
Vol 66 (2) ◽  
pp. 213-221 ◽  
Author(s):  
Paul D. Adams ◽  
Pavel V. Afonine ◽  
Gábor Bunkóczi ◽  
Vincent B. Chen ◽  
Ian W. Davis ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Numerical Data ◽  
Crystallographic Structure ◽  
Biological Processes ◽  
X Ray ◽  
X Ray Crystallography ◽  
Software Packages ◽  
Structure Solution ◽  
Repeated Use ◽  
Subjective Input

Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics.PHENIXhas been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.

scholarly journals PROBABILISTIC ENSEMBLES FOR IMPROVED INFERENCE IN PROTEIN-STRUCTURE DETERMINATION

2012 ◽  
Vol 10 (01) ◽  
pp. 1240009 ◽  
Author(s):  
AMEET SONI ◽  
JUDE SHAVLIK
Keyword(s):  
Protein Structure ◽  
Structure Determination ◽  
Protein Structures ◽  
Three Dimensional ◽  
Protein Structure Determination ◽  
Complex Problem ◽  
Approximate Inference ◽  
X Ray ◽  
X Ray Crystallography ◽  
Inference Methods

Protein X-ray crystallography — the most popular method for determining protein structures — remains a laborious process requiring a great deal of manual crystallographer effort to interpret low-quality protein images. Automating this process is critical in creating a high-throughput protein-structure determination pipeline. Previously, our group developed ACMI, a probabilistic framework for producing protein-structure models from electron-density maps produced via X-ray crystallography. ACMI uses a Markov Random Field to model the three-dimensional (3D) location of each non-hydrogen atom in a protein. Calculating the best structure in this model is intractable, so ACMI uses approximate inference methods to estimate the optimal structure. While previous results have shown ACMI to be the state-of-the-art method on this task, its approximate inference algorithm remains computationally expensive and susceptible to errors. In this work, we develop Probabilistic Ensembles in ACMI (PEA), a framework for leveraging multiple, independent runs of approximate inference to produce estimates of protein structures. Our results show statistically significant improvements in the accuracy of inference resulting in more complete and accurate protein structures. In addition, PEA provides a general framework for advanced approximate inference methods in complex problem domains.

scholarly journals Covering complete proteomes with X-ray structures: a current snapshot

2014 ◽  
Vol 70 (11) ◽  
pp. 2781-2793 ◽  
Author(s):  
Marcin J. Mizianty ◽  
Xiao Fan ◽  
Jing Yan ◽  
Eric Chalmers ◽  
Christopher Woloschuk ◽  
...  
Keyword(s):  
Homology Modeling ◽  
Structure Determination ◽  
Large Scale ◽  
Structural Model ◽  
Target Selection ◽  
Three Dimensional ◽  
X Ray ◽  
X Ray Crystallography ◽  
Knowledge Based ◽  
Wide Range

Structural genomics programs have developed and applied structure-determination pipelines to a wide range of protein targets, facilitating the visualization of macromolecular interactions and the understanding of their molecular and biochemical functions. The fundamental question of whether three-dimensional structures of all proteins and all functional annotations can be determined using X-ray crystallography is investigated. A first-of-its-kind large-scale analysis of crystallization propensity for all proteins encoded in 1953 fully sequenced genomes was performed. It is shown that current X-ray crystallographic knowhow combined with homology modeling can provide structures for 25% of modeling families (protein clusters for which structural models can be obtained through homology modeling), with at least one structural model produced for each Gene Ontology functional annotation. The coverage varies between superkingdoms, with 19% for eukaryotes, 35% for bacteria and 49% for archaea, and with those of viruses following the coverage values of their hosts. It is shown that the crystallization propensities of proteomes from the taxonomic superkingdoms are distinct. The use of knowledge-based target selection is shown to substantially increase the ability to produce X-ray structures. It is demonstrated that the human proteome has one of the highest attainable coverage values among eukaryotes, and GPCR membrane proteins suitable for X-ray structure determination were determined.

Beyond X-rays: an overview of emerging structural biology methods

2021 ◽  
Author(s):  
Jason E. Schaffer ◽  
Vandna Kukshal ◽  
Justin J. Miller ◽  
Vivian Kitainda ◽  
Joseph M. Jez
Keyword(s):  
Structure Determination ◽  
De Novo ◽  
Three Dimensional ◽  
Atomic Scale ◽  
X Rays ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Crystallography ◽  
New Methods ◽  
Main Technique

Structural biologists rely on X-ray crystallography as the main technique for determining the three-dimensional structures of macromolecules; however, in recent years, new methods that go beyond X-ray-based technologies are broadening the selection of tools to understand molecular structure and function. Simultaneously, national facilities are developing programming tools and maintaining personnel to aid novice structural biologists in de novo structure determination. The combination of X-ray free electron lasers (XFELs) and serial femtosecond crystallography (SFX) now enable time-resolved structure determination that allows for capture of dynamic processes, such as reaction mechanism and conformational flexibility. XFEL and SFX, along with microcrystal electron diffraction (MicroED), help side-step the need for large crystals for structural studies. Moreover, advances in cryogenic electron microscopy (cryo-EM) as a tool for structure determination is revolutionizing how difficult to crystallize macromolecules and/or complexes can be visualized at the atomic scale. This review aims to provide a broad overview of these new methods and to guide readers to more in-depth literature of these methods.

scholarly journals Improving diffraction resolution using a new dehydration method

2016 ◽  
Vol 72 (2) ◽  
pp. 152-159 ◽  
Author(s):  
Qingqiu Huang ◽  
Doletha M. E. Szebenyi
Keyword(s):  
Escherichia Coli ◽  
Structure Determination ◽  
Three Dimensional ◽  
Archaeoglobus Fulgidus ◽  
Dimensional Structure ◽  
High Quality ◽  
X Ray ◽  
Three Dimensional Structure ◽  
X Ray Crystallography ◽  
Dry Out

The production of high-quality crystals is one of the major obstacles in determining the three-dimensional structure of macromolecules by X-ray crystallography. It is fairly common that a visually well formed crystal diffracts poorly to a resolution that is too low to be suitable for structure determination. Dehydration has proven to be an effective post-crystallization treatment for improving crystal diffraction quality. Several dehydration methods have been developed, but no single one of them is suitable for all crystals. Here, a new convenient and effective dehydration method is reported that makes use of a dehydrating solution that will not dry out in air for several hours. Using this dehydration method, the resolution ofArchaeoglobus fulgidusCas5a crystals has been increased from 3.2 to 1.95 Å and the resolution ofEscherichia coliLptA crystals has been increased from <5 to 3.4 Å.

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