scholarly journals 3D Structural Fluctuation of IgG1 Antibody Revealed by Individual Particle Electron Tomography

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Xing Zhang ◽  
Lei Zhang ◽  
Huimin Tong ◽  
Bo Peng ◽  
Matthew J. Rames ◽  
...  
Keyword(s):  
Protein Dynamics ◽  
Electron Tomography ◽  
3D Structure ◽  
Three Dimensional ◽  
Individual Particle ◽  
Single Particle Reconstruction ◽  
X Ray Crystallography ◽  
Igg1 Antibody ◽  
Structural Fluctuation ◽  
Dynamics Simulations

Abstract Commonly used methods for determining protein structure, including X-ray crystallography and single-particle reconstruction, often provide a single and unique three-dimensional (3D) structure. However, in these methods, the protein dynamics and flexibility/fluctuation remain mostly unknown. Here, we utilized advances in electron tomography (ET) to study the antibody flexibility and fluctuation through structural determination of individual antibody particles rather than averaging multiple antibody particles together. Through individual-particle electron tomography (IPET) 3D reconstruction from negatively-stained ET images, we obtained 120 ab-initio 3D density maps at an intermediate resolution (~1–3 nm) from 120 individual IgG1 antibody particles. Using these maps as a constraint, we derived 120 conformations of the antibody via structural flexible docking of the crystal structure to these maps by targeted molecular dynamics simulations. Statistical analysis of the various conformations disclosed the antibody 3D conformational flexibility through the distribution of its domain distances and orientations. This blueprint approach, if extended to other flexible proteins, may serve as a useful methodology towards understanding protein dynamics and functions.

scholarly journals Erratum: Corrigendum: 3D Structural Fluctuation of IgG1 Antibody Revealed by Individual Particle Electron Tomography

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Xing Zhang ◽  
Lei Zhang ◽  
Huimin Tong ◽  
Bo Peng ◽  
Matthew J. Rames ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Individual Particle ◽  
Igg1 Antibody ◽  
Structural Fluctuation

New structural insights into anomeric carbohydrate recognition by frutalin: an α-d-galactose-binding lectin from breadfruit seeds

2019 ◽  
Vol 476 (1) ◽  
pp. 101-113 ◽  
Author(s):  
Antonio Eufrásio Vieira Neto ◽  
Felipe Domingos de Sousa ◽  
Humberto D'Muniz Pereira ◽  
Frederico Bruno Mendes Batista Moreno ◽  
Marcos Roberto Lourenzoni ◽  
...  
Keyword(s):  
Amino Acids ◽  
Biological Activities ◽  
3D Structure ◽  
Carbohydrate Binding ◽  
X Ray Crystallography ◽  
Multiple Binding Sites ◽  
Multiple Binding ◽  
New Perspective ◽  
Dynamics Simulations ◽  
Binding Lectin

Abstract Frutalin (FTL) is a multiple-binding lectin belonging to the jacalin-related lectin (JRL) family and derived from Artocarpus incisa (breadfruit) seeds. This lectin specifically recognizes and binds α-d-galactose. FTL has been successfully used in immunobiological research for the recognition of cancer-associated oligosaccharides. However, the molecular bases by which FTL promotes these specific activities remain poorly understood. Here, we report the whole 3D structure of FTL for the first time, as determined by X-ray crystallography. The obtained crystals diffracted to 1.81 Å (Apo-frutalin) and 1.65 Å (frutalin–d-Gal complex) of resolution. The lectin exhibits post-translational cleavage yielding an α- (133 amino acids) and β-chain (20 amino acids), presenting a homotetramer when in solution, with a typical JRL β-prism. The β-prism was composed of three 4-stranded β-sheets forming three antiparallel Greek key motifs. The carbohydrate-binding site (CBS) involved the N-terminus of the α-chain and was formed by four key residues: Gly25, Tyr146, Trp147 and Asp149. Together, these results were used in molecular dynamics simulations in aqueous solutions to shed light on the molecular basis of FTL-ligand binding. The simulations suggest that Thr-Ser-Ser-Asn (TSSN) peptide excision reduces the rigidity of the FTL CBS, increasing the number of interactions with ligands and resulting in multiple-binding sites and anomeric recognition of α-d-galactose sugar moieties. Our findings provide a new perspective to further elucidate the versatility of FTL in many biological activities.

scholarly journals Role of Computational Methods in Going beyond X-ray Crystallography to Explore Protein Structure and Dynamics

2018 ◽  
Vol 19 (11) ◽  
pp. 3401 ◽  
Author(s):  
Ashutosh Srivastava ◽  
Tetsuro Nagai ◽  
Arpita Srivastava ◽  
Osamu Miyashita ◽  
Florence Tama
Keyword(s):  
Protein Dynamics ◽  
Computational Methods ◽  
Protein Structures ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
X Ray ◽  
X Ray Crystallography ◽  
Insight Into

Protein structural biology came a long way since the determination of the first three-dimensional structure of myoglobin about six decades ago. Across this period, X-ray crystallography was the most important experimental method for gaining atomic-resolution insight into protein structures. However, as the role of dynamics gained importance in the function of proteins, the limitations of X-ray crystallography in not being able to capture dynamics came to the forefront. Computational methods proved to be immensely successful in understanding protein dynamics in solution, and they continue to improve in terms of both the scale and the types of systems that can be studied. In this review, we briefly discuss the limitations of X-ray crystallography in studying protein dynamics, and then provide an overview of different computational methods that are instrumental in understanding the dynamics of proteins and biomacromolecular complexes.

scholarly journals Nonlinear mechanics of lamin filaments and the meshwork topology build an emergent nuclear lamina

2019 ◽  
Author(s):  
K. Tanuj Sapra ◽  
Zhao Qin ◽  
Anna Dubrovsky-Gaupp ◽  
Ueli Aebi ◽  
Daniel J. Müller ◽  
...  
Keyword(s):  
Mechanical Response ◽  
Mechanical Stability ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Small World ◽  
Nuclear Lamina ◽  
Integrative Approach ◽  
Graph Theory Analysis ◽  
Dynamics Simulations

AbstractThe nuclear lamina – a meshwork of intermediate filaments termed lamins – functions as a mechanotransduction interface between the extracellular matrix and the nucleus via the cytoskeleton. Although lamins are primarily responsible for the mechanical stability of the nucleus in multicellular organisms, in situ characterization of lamin filaments under tension has remained elusive. Here, we apply an integrative approach combining atomic force microscopy, cryo-electron tomography, network analysis, and molecular dynamics simulations to directly measure the mechanical response of single lamin filaments in its three-dimensional meshwork. Endogenous lamin filaments portray non-Hookean behavior – they deform reversibly under a force of a few hundred picoNewtons and stiffen at nanoNewton forces. The filaments are extensible, strong and tough, similar to natural silk and superior to the synthetic polymer Kevlar®. Graph theory analysis shows that the lamin meshwork is not a random arrangement of filaments but the meshwork topology follows ‘small world’ properties. Our results suggest that the lamin filaments arrange to form a robust, emergent meshwork that dictates the mechanical properties of individual lamin filaments. The combined approach provides quantitative insights into the structure-function organization of lamins in situ, and implies a role of meshwork topology in laminopathies.

scholarly journals Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion

2021 ◽  
Vol 77 (5) ◽  
pp. 645-662
Author(s):  
Risako Tamura-Sakaguchi ◽  
Rie Aruga ◽  
Mika Hirose ◽  
Toru Ekimoto ◽  
Takuya Miyake ◽  
...  
Keyword(s):  
Complex Formation ◽  
Structure Determination ◽  
Structural Changes ◽  
Insertion Site ◽  
3D Structure ◽  
Binding Pocket ◽  
Crystallographic Analysis ◽  
Stable Complex ◽  
X Ray Crystallography ◽  
Dynamics Simulations

Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, an alternative method has been developed to prepare an antibody complex by inserting an exogenous epitope into the target. It has already been demonstrated that the Fab of the NZ-1 monoclonal antibody can form a stable complex with a target containing a PA12 tag as an inserted epitope. Nevertheless, it was also found that complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Here, an attempt was made to improve the tag-insertion method, and it was consequently discovered that an alternate tag (PA14) could replace various loops on the target without inducing large structural changes. Crystallographic analysis demonstrated that the inserted PA14 tag adopts a loop-like conformation with closed ends in the antigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in the bound conformation, the more optimal PA14 tag had only a minor impact on the target structure. In fact, the PA14 tag could also be inserted into a sterically hindered loop for labeling. Molecular-dynamics simulations also showed a rigid structure for the target regardless of PA14 insertion and complex formation with the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was performed on a bacterial site-2 protease, which enabled an approximation of the domain arrangement based on the docking mode of the NZ-1 Fab.

Structural Analysis of Proteins on Lipid Substrates

2000 ◽  
Vol 6 (S2) ◽  
pp. 1182-1183
Author(s):  
Elizabeth M. Wilson-Kubalek
Keyword(s):  
Structural Information ◽  
Rapid Determination ◽  
3D Structure ◽  
Three Dimensional ◽  
Molecular Complexes ◽  
Structural Data ◽  
X Ray Crystallography ◽  
Helical Protein ◽  
3D Structure Determination

Electron microscopy (EM) has become an increasingly powerful method for the determination of three-dimensional (3D) structures of proteins and macromolecular complexes. EM offers advantages over X-ray crystallography and NMR for obtaining structural information about proteins in physiological conditions, as components of large assemblies, that cannot be obtained in large quantity, or that fail to yield 3D crystals. EM has been used to obtain structural data from images of isolated molecules and molecular complexes, two-dimensional (2D) protein crystals, and helical protein arrays. Helically arranged proteins allow the most rapid determination of 3D maps because they contain a complete range of equally spaced molecular views, therefore no tilting of the sample with respect to the electron beam is required. However, so far 3D structure determination of helical assemblies has been limited to proteins that naturally adopt this organization and to proteins that fortuitously crystallize as helices.

scholarly journals ConoMode, a database for conopeptide binding modes

Database ◽  
2020 ◽  
Vol 2020 ◽  
Author(s):  
Xiao Li ◽  
Hao Liu ◽  
Chunxiao Gao ◽  
Yangyang Li ◽  
Dongning Jia ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Large Family ◽  
High Specificity ◽  
Data Bank ◽  
Binding Modes ◽  
3D Structures ◽  
Target Proteins ◽  
X Ray Crystallography ◽  
Dynamics Simulations ◽  
Coordinate Data

Abstract ConoMode is a database for complex three-dimensional (3D) structures of conopeptides binding with their target proteins. Conopeptides, a large family of peptides from the venom of marine snails of the Conus genus, have exceptionally diverse sequences, and their high specificity to block ion channels makes them crucial as drug leads and tools for physiological studies. ConoMode is a specialized archive for the collection of 3D coordinate data for the conopeptides and their binding target proteins from published literature and the Protein Data Bank. These 3D structures can be determined using experimental methods such as X-ray crystallography and electron microscopy and computational methods including docking, homology modeling and molecular dynamics simulations. The binding modes for the conopeptides determined using computational modeling must be validated based on experimental data. The 3D coordinate data from ConoMode can be searched, visualized, downloaded and uploaded. Currently, ConoMode manages 19 conopeptide sequences (from 10 Conus species), 15 protein sequences and 37 3D structures. ConoMode utilizes a modern technical framework to provide a good user experience on mobile devices with touch interaction features. Furthermore, the database is fully optimized for unstructured data and flexible data models. Database URL: http://conomode.qnlm.ac/conomode/conomode/index

scholarly journals In situ structure of virus capsids within cell nuclei by correlative light and cryo-electron tomography

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Swetha Vijayakrishnan ◽  
Marion McElwee ◽  
Colin Loney ◽  
Frazer Rixon ◽  
David Bhella
Keyword(s):  
Electron Microscopy ◽  
Structure Determination ◽  
Electron Tomography ◽  
3D Structure ◽  
Three Dimensional ◽  
Cell Nuclei ◽  
Nuclear Egress ◽  
Virus Capsids ◽  
Cryo Electron Tomography

Abstract Cryo electron microscopy (cryo-EM), a key method for structure determination involves imaging purified material embedded in vitreous ice. Images are then computationally processed to obtain three-dimensional structures approaching atomic resolution. There is increasing interest in extending structural studies by cryo-EM into the cell, where biological structures and processes may be imaged in context. The limited penetrating power of electrons prevents imaging of thick specimens (> 500 nm) however. Cryo-sectioning methods employed to overcome this are technically challenging, subject to artefacts or involve specialised and costly equipment. Here we describe the first structure of herpesvirus capsids determined by sub-tomogram averaging from nuclei of eukaryotic cells, achieved by cryo-electron tomography (cryo-ET) of re-vitrified cell sections prepared using the Tokuyasu method. Our reconstructions confirm that the capsid associated tegument complex is present on capsids prior to nuclear egress. We demonstrate that this method is suited to both 3D structure determination and correlative light/electron microscopy, thus expanding the scope of cryogenic cellular imaging.

scholarly journals Interactions between the Trimeric Autotransporter Adhesin EmaA and Collagen Revealed by Three-Dimensional Electron Tomography

2019 ◽  
Vol 201 (16) ◽  
Author(s):  
Fereshteh Azari ◽  
Michael Radermacher ◽  
Keith P. Mintz ◽  
Teresa Ruiz
Keyword(s):  
Bacterial Adhesion ◽  
Electron Tomography ◽  
3D Structure ◽  
Three Dimensional ◽  
Functional Domain ◽  
Clear Understanding ◽  
Collagen Binding ◽  
Receptor Complexes ◽  
3D Electron ◽  
Repulsive Forces

ABSTRACTBacterial adhesion to host tissues is considered the first and critical step of microbial infection. The extracellular matrix protein adhesin A (EmaA) is a collagen-binding adhesin of the periodontal pathogenAggregatibacter actinomycetemcomitans. Three 202-kDa EmaA monomers form antenna-like structures on the bacterial surface with the functional domain located at the apical end. The structure of the 30-nm functional domain has been determined by three-dimensional (3D) electron tomography and subvolume averaging. The region exhibits a complex architecture composed of three subdomains (SI to SIII) and a linker between subdomains SII and SIII. However, the molecular interaction between the adhesin receptor complexes has yet to be revealed. This study provides the first detailed 3D structure of reconstituted EmaA/collagen complexes obtained using 3D electron tomography and image processing techniques. The observed interactions of EmaA with collagen were not to whole, intact fibrils, but rather to individual collagen triple helices dissociated from the fibrils. The majority of the contacts with the EmaA functional domain encompassed subdomains SII and SIII and in some cases the tip of the apical domain, involving SI. These data suggest a multipronged mechanism for the interaction of Gram-negative bacteria with collagen.IMPORTANCEBacterial adhesion is a crucial step for bacterial colonization and infection. In recent years, the number of antibiotic-resistant strains has dramatically increased; therefore, there is a need to search for novel antimicrobial agents. Thus, great efforts are being devoted to develop a clear understanding of the bacterial adhesion mechanism for preventing infections. In host/pathogen interactions, once repulsive forces are overcome, adhesins recognize and tightly bind to specific receptors on the host cell or tissue components. Here, we present the first 3D structure of the interaction between the collagen-binding adhesin EmaA and collagen, which is critical for the development of endocarditis in humans.

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