scholarly journals AlphaFold Protein Structure Database for Sequence-Independent Molecular Replacement

2021 ◽  
Author(s):  
Lawrence Chai ◽  
Ping Zhu ◽  
Jin Chai ◽  
Changxu Pang ◽  
Babak Andi ◽  
...  
Keyword(s):  
Protein Structures ◽  
Data Bank ◽  
Sequence Information ◽  
Molecular Replacement ◽  
E Coli ◽  
Binding Partner ◽  
General Utility ◽  
Structure Database ◽  
Partner Proteins

AbstractCrystallographic phasing recovers the phase information that is lost during a diffraction experiment. Molecular replacement is a dominant phasing method for the crystal structures in the protein data bank. In one form it uses a protein sequence to search a structure database for finding suitable templates for phasing. However, such sequence information is not always available such as when proteins are crystallized with unknown binding partner proteins or when the crystal is that of a contaminant. The recent development of AlphaFold has resulted in the availability of predicted protein structures for all proteins from twenty species. In this work, we tested whether AlphaFold-predicted E. coli protein structures were accurate enough for sequence-independent phasing of diffraction data from two crystallization contaminants for which we had not identified the protein. Using each of more than 4000 predicted structures as a search model, robust molecular replacement solutions were obtained which allowed the identification and structure determination of both structures, YncE and YadF. Our results advocate a general utility of AlphaFold-predicted structure database with respect to crystallographic phasing.

scholarly journals AlphaFold Protein Structure Database for Sequence-Independent Molecular Replacement

Crystals ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1227
Author(s):  
Lawrence Chai ◽  
Ping Zhu ◽  
Jin Chai ◽  
Changxu Pang ◽  
Babak Andi ◽  
...  
Keyword(s):  
Protein Structures ◽  
Data Bank ◽  
Distinct Species ◽  
Sequence Information ◽  
Molecular Replacement ◽  
E Coli ◽  
General Utility ◽  
Structure Database ◽  
Partner Proteins

Crystallographic phasing recovers the phase information that is lost during a diffraction experiment. Molecular replacement is a commonly used phasing method for crystal structures in the protein data bank. In one form it uses a protein sequence to search a structure database to find suitable templates for phasing. However, sequence information is not always available, such as when proteins are crystallized with unknown binding partner proteins or when the crystal is of a contaminant. The recent development of AlphaFold published the predicted protein structures for every protein from twenty distinct species. In this work, we tested whether AlphaFold-predicted E. coli protein structures were accurate enough to enable sequence-independent phasing of diffraction data from two crystallization contaminants of unknown sequence. Using each of more than 4000 predicted structures as a search model, robust molecular replacement solutions were obtained, which allowed the identification and structure determination of YncE and YadF. Our results demonstrate the general utility of the AlphaFold-predicted structure database with respect to sequence-independent crystallographic phasing.

scholarly journals Crystallographic models of SARS-CoV-2 3CLpro: in-depth assessment of structure quality and validation

IUCrJ ◽  
2021 ◽  
Vol 8 (2) ◽  
pp. 238-256
Author(s):  
Mariusz Jaskolski ◽  
Zbigniew Dauter ◽  
Ivan G. Shabalin ◽  
Miroslaw Gilski ◽  
Dariusz Brzezinski ◽  
...  
Keyword(s):  
Rapid Determination ◽  
Data Bank ◽  
Molecular Replacement ◽  
Structure Quality ◽  
Space Groups ◽  
Important Target ◽  
Density Maps ◽  
Main Protease ◽  
Experimental Approaches

The appearance at the end of 2019 of the new SARS-CoV-2 coronavirus led to an unprecedented response by the structural biology community, resulting in the rapid determination of many hundreds of structures of proteins encoded by the virus. As part of an effort to analyze and, if necessary, remediate these structures as deposited in the Protein Data Bank (PDB), this work presents a detailed analysis of 81 crystal structures of the main protease 3CLpro, an important target for the design of drugs against COVID-19. The structures of the unliganded enzyme and its complexes with a number of inhibitors were determined by multiple research groups using different experimental approaches and conditions; the resulting structures span 13 different polymorphs representing seven space groups. The structures of the enzyme itself, all determined by molecular replacement, are highly similar, with the exception of one polymorph with a different inter-domain orientation. However, a number of complexes with bound inhibitors were found to pose significant problems. Some of these could be traced to faulty definitions of geometrical restraints for ligands and to the general problem of a lack of such information in the PDB depositions. Several problems with ligand definition in the PDB itself were also noted. In several cases extensive corrections to the models were necessary to adhere to the evidence of the electron-density maps. Taken together, this analysis of a large number of structures of a single, medically important protein, all determined within less than a year using modern experimental tools, should be useful in future studies of other systems of high interest to the biomedical community.

Phasing in Crystallography

2013 ◽  
Author(s):  
Carmelo Giacovazzo
Keyword(s):  
Protein Structures ◽  
Data Bank ◽  
Molecular Replacement ◽  
Main Research ◽  
Elite Group ◽  
Isomorphous Replacement ◽  
Technological Advances ◽  
Data Resolution ◽  
Rational Classification

Modern crystallographic methods originate from the synergy of two main research streams, the small-molecule and the macro-molecular streams. The first stream was able to definitively solve the phase problem for molecules up to 200 atoms in the asymmetric unit. The achievements obtained by the macromolecular stream are also impressive. A huge number of protein structures have been deposited in the Protein Data Bank. The solution of them is no longer reserved to an elite group of scientists, but may be attained in a large number of laboratories around the world, even by young scientists. New probabilistic approaches have been tailored to deal with larger structures, errors in the experimental data, and modest data resolution. Traditional phasing techniques like ab initio, molecular replacement, isomorphous replacement, and anomalous dispersion techniques have been revisited. The new approaches have been implemented in robust phasing programs, which have been organized in automatic pipelines usable even by non-experts. Protein structures, which 50 years ago could take months or even years to solve, can now be solved in a matter of hours, partly also due to technological advances in computer science. This book describes all modern crystallographic phasing methods, and introduces a new rational classification of them. A didactic approach is used, with the techniques described simply and logically in the main text, and further mathematical details confined to the Appendices for motivated readers. Numerous figures and applicative details illustrate the text.

scholarly journals Detection of translational noncrystallographic symmetry in Patterson functions

2021 ◽  
Vol 77 (2) ◽  
pp. 131-141
Author(s):  
Iracema Caballero ◽  
Massimo D. Sammito ◽  
Pavel V. Afonine ◽  
Isabel Usón ◽  
Randy J. Read ◽  
...  
Keyword(s):  
Maximum Likelihood ◽  
Protein Structures ◽  
Data Bank ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
Intensity Factors ◽  
Patterson Function ◽  
Likelihood Functions ◽  
Structure Solution ◽  
Ranked List

Detection of translational noncrystallographic symmetry (TNCS) can be critical for success in crystallographic phasing, particularly when molecular-replacement models are poor or anomalous phasing information is weak. If the correct TNCS is detected then expected intensity factors for each reflection can be refined, so that the maximum-likelihood functions underlying molecular replacement and single-wavelength anomalous dispersion use appropriate structure-factor normalization and variance terms. Here, an analysis of a curated database of protein structures from the Protein Data Bank to investigate how TNCS manifests in the Patterson function is described. These studies informed an algorithm for the detection of TNCS, which includes a method for detecting the number of vectors involved in any commensurate modulation (the TNCS order). The algorithm generates a ranked list of possible TNCS associations in the asymmetric unit for exploration during structure solution.

scholarly journals Molecular replacement with a large number of molecules in the asymmetric unit

2014 ◽  
Vol 70 (9) ◽  
pp. 1296-1302 ◽  
Author(s):  
Chacko Jobichen ◽  
Kunchithapadam Swaminathan
Keyword(s):  
Protein Data Bank ◽  
Structure Determination ◽  
Protein Structures ◽  
Data Bank ◽  
Lessons Learned ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
Exponential Increase ◽  
Crystallographic Symmetry ◽  
Input Model

The exponential increase in protein structures deposited in the Protein Data Bank (PDB) has resulted in the elucidation of most, if not all, protein folds, thus making molecular replacement (MR) the most frequently used method for structure determination. A survey of the PDB shows that most of the structures determined by molecular replacement contain less than ten molecules in the asymmetric unit and that it is predominantly virus and ribosome structures that contain more than 20 molecules in the asymmetric unit. While the success of the MR method depends on several factors, such as the homology and the size of an input model, it is also a well known fact that this method can become significantly difficult in cases with a large number of molecules in the asymmetric unit, higher crystallographic symmetry and tight packing. In this paper, five representative structures containing 16–18 homomeric molecules in the asymmetric unit and the strategies that have been used to solve these structures are described. The difficulties faced and the lessons learned from these structure-determination efforts will be useful for selected and similar future situations with a large number of molecules in the asymmetric unit.

scholarly journals MicroED: Three Dimensional Electron Crystallography of Protein Micro-Crystals

2014 ◽  
Vol 70 (a1) ◽  
pp. C1063-C1063
Author(s):  
Tamir Gonen
Keyword(s):  
Electron Diffraction ◽  
Protein Structures ◽  
Three Dimensional ◽  
Electron Diffraction Data ◽  
Molecular Replacement ◽  
Electron Crystallography ◽  
Electron Dose ◽  
Tilt Series ◽  
Diffraction Patterns

We demonstrate that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7Å resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1 - 10and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9Å resolution. In this seminar I will present our initial proof of principle study and highlight the major advances since the first publication.

scholarly journals Iditis: Protein Structure Database

1998 ◽  
Vol 54 (6) ◽  
pp. 1071-1077 ◽  
Author(s):  
Stephen Gardner ◽  
Janet Thornton
Keyword(s):  
User Interface ◽  
Protein Structure ◽  
Experimental Design ◽  
Graphical User Interface ◽  
Molecular Interactions ◽  
Protein Structures ◽  
Structure Database ◽  
Wide Range ◽  
Database Engine

The validation, enrichment and organization of the data stored in PDB files is essential for those data to be used accurately and efficiently for modelling, experimental design and the determination of molecular interactions. TheIditisprotein structure database has been designed to allow the widest possible range of queries to be performed across all available protein structures. TheIditisdatabase is the most comprehensive protein structure resource currently available, and contains over 500 fields of information describing all publicly deposited protein structures. A custom-written database engine and graphical user interface provide a natural and simple environment for the construction of searches for complex sequence- and structure-based motifs. Extensions and specialized interfaces allow the data generated by the database to used in conjunction with a wide range of applications.

Concentration determination of chromatin in unstained resin-embedded sections by means of Z-contrast in STEM

1990 ◽  
Vol 48 (2) ◽  
pp. 186-187
Author(s):  
M. Haider ◽  
B. Bohrmann
Keyword(s):  
Energy Loss ◽  
Freeze Substitution ◽  
Biological Macromolecules ◽  
Local Concentration ◽  
E Coli ◽  
Concentration Determination ◽  
Phosphorous Content ◽  
Z Contrast ◽  
Electron Energy Loss

The technique of Z-contrast in STEM offers the possibility to determine the local concentration of macromolecules like lipids, proteins or DNA. Contrast formation depends on the atomic composition of the particular structure. In the case of DNA, its phosphorous content discriminates it from other biological macromolecules. In our studies, sections of E. coli, the dinoflagellate Amphidinium carterae and Euglena spec. cells were used which were obtained by cryofixation followed by freeze-substitution into acetone with 3% glutaraldehyde. The samples were then embedded either in Lowicryl HM20 at low temperature or in Epon at high temperature. Sections were coated on both sides with 30Å carbon.The DF- and the inelastic image have been recorded simultaneously with a Cryo-STEM. This Cryo-STEM is equipped with a highly dispersive Electron Energy Loss Spectrometer. With this instrument pure Z-contrast can be achieved either with a Filtered DF-image divided by the inelastic image or, as is used in this paper, by dividing the conventional DF-image by an inelastic image which has been recorded with an inelastic detector whose response is dependent on the total energy loss of the inelastically scattered electrons.

Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with sumo and chaperones in Escherichia coli

2018 ◽  
Vol 40 (4) ◽  
Author(s):  
Dang Thi Ngoc Ha ◽  
Le Thi Thu Hong ◽  
Truong Nam Hai
Keyword(s):  
Recombinant Antibody ◽  
Blood Type ◽  
Soluble Form ◽  
Supernatant Fraction ◽  
Blood Group Antigens ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Scfv ◽  
Single Chain Variable Fragments

Single chain variable fragments (scFv) have widely been used in research, diagnosis and treatment, but the scFv is considered as difficult protein for expression in E. coli. In previous studies, we expressed a construction of recombinant single chain variable fragments again antigen specific for blood type A (antiA-scFv) individually or fused with Trx or SUMO. However, soluble fraction was low abandant and only approximately 40% when fused with Trx, the other cases were expressed in form of inclusion body. Therefore, it was difficult for purification, refolding and activity assesment. In thispaper, we demonstrated a suitable construction for soluble production of antiA-scFv fused with SUMO (SM/antiA-scFv) in presence of chaparones. Under fermentation with 0.1 mM IPTG at 20oC, the SM/antiA-scFv was entirely expressed in soluble form. Importantly, after cleavage from SUMO with SUMOprotease, antiA-scFv was still maintained in the supernatant fraction. Therefore, it can help ensure bioactivity and is useful for purification process. To the best of our knowledge, this is the first report showing soluble recombinant scFv fused with SUMO in presence of chaperone for determination of blood group antigens. Thus, this result facilitates the optimal study of soluble expression, purification and bioactivity determination of the antiA-scFv recombinant antibody. 

Identification of N-Substituted Triazolo-azetidines as Novel Antibacterials using pDualrep2 HTS Platform

2019 ◽  
Vol 22 (5) ◽  
pp. 346-354
Author(s):  
Yan A. Ivanenkov ◽  
Renat S. Yamidanov ◽  
Ilya A. Osterman ◽  
Petr V. Sergiev ◽  
Vladimir A. Aladinskiy ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Large Scale ◽  
Underlying Mechanism ◽  
Luciferase Assay ◽  
Reporter System ◽  
E Coli ◽  
Starting Point ◽  
High Concentrations ◽  
Mic Value

Aim and Objective: Antibiotic resistance is a serious constraint to the development of new effective antibacterials. Therefore, the discovery of the new antibacterials remains one of the main challenges in modern medicinal chemistry. This study was undertaken to identify novel molecules with antibacterial activity. Materials and Methods: Using our unique double-reporter system, in-house large-scale HTS campaign was conducted for the identification of antibacterial potency of small-molecule compounds. The construction allows us to visually assess the underlying mechanism of action. After the initial HTS and rescreen procedure, luciferase assay, C14-test, determination of MIC value and PrestoBlue test were carried out. Results: HTS rounds and rescreen campaign have revealed the antibacterial activity of a series of Nsubstituted triazolo-azetidines and their isosteric derivatives that has not been reported previously. Primary hit-molecule demonstrated a MIC value of 12.5 µg/mL against E. coli Δ tolC with signs of translation blockage and no SOS-response. Translation inhibition (26%, luciferase assay) was achieved at high concentrations up to 160 µg/mL, while no activity was found using C14-test. The compound did not demonstrate cytotoxicity in the PrestoBlue assay against a panel of eukaryotic cells. Within a series of direct structural analogues bearing the same or bioisosteric scaffold, compound 2 was found to have an improved antibacterial potency (MIC=6.25 µg/mL) close to Erythromycin (MIC=2.5-5 µg/mL) against the same strain. In contrast to the parent hit, this compound was more active and selective, and provided a robust IP position. Conclusion: N-substituted triazolo-azetidine scaffold may be used as a versatile starting point for the development of novel active and selective antibacterial compounds.

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