scholarly journals Structural and mechanistic bases for a potent HIV-1 capsid inhibitor

Science ◽  
2020 ◽  
Vol 370 (6514) ◽  
pp. 360-364 ◽  
Author(s):  
Stephanie M. Bester ◽  
Guochao Wei ◽  
Haiyan Zhao ◽  
Daniel Adu-Ampratwum ◽  
Naseer Iqbal ◽  
...  
Keyword(s):  
Principal Component ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Capsid Shell ◽  
Hydrogen Deuterium ◽  
Infected Cells ◽  
Long Acting ◽  
Structural Insights ◽  
Hiv 1

The potent HIV-1 capsid inhibitor GS-6207 is an investigational principal component of long-acting antiretroviral therapy. We found that GS-6207 inhibits HIV-1 by stabilizing and thereby preventing functional disassembly of the capsid shell in infected cells. X-ray crystallography, cryo–electron microscopy, and hydrogen-deuterium exchange experiments revealed that GS-6207 tightly binds two adjoining capsid subunits and promotes distal intra- and inter-hexamer interactions that stabilize the curved capsid lattice. In addition, GS-6207 interferes with capsid binding to the cellular HIV-1 cofactors Nup153 and CPSF6 that mediate viral nuclear import and direct integration into gene-rich regions of chromatin. These findings elucidate structural insights into the multimodal, potent antiviral activity of GS-6207 and provide a means for rationally developing second-generation therapies.

scholarly journals Structural Conservation and Adaptation of the Bacterial Flagella Motor

Biomolecules ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1492
Author(s):  
Brittany L. Carroll ◽  
Jun Liu
Keyword(s):  
Electron Tomography ◽  
X Ray ◽  
Bacterial Flagella ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Cryo Electron Tomography ◽  
Flagellar Assembly ◽  
Structural Conservation ◽  
And Function ◽  
Structural Insights

Many bacteria require flagella for the ability to move, survive, and cause infection. The flagellum is a complex nanomachine that has evolved to increase the fitness of each bacterium to diverse environments. Over several decades, molecular, biochemical, and structural insights into the flagella have led to a comprehensive understanding of the structure and function of this fascinating nanomachine. Notably, X-ray crystallography, cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET) have elucidated the flagella and their components to unprecedented resolution, gleaning insights into their structural conservation and adaptation. In this review, we focus on recent structural studies that have led to a mechanistic understanding of flagellar assembly, function, and evolution.

scholarly journals Strain and crack propagation of HIV-1 capsids during uncoating

2021 ◽  
Author(s):  
Alvin Yu ◽  
Elizabeth M.Y Lee ◽  
John A.G. Briggs ◽  
Barbie K Ganser-Pornillos ◽  
Owen Pornillos ◽  
...  
Keyword(s):  
Crack Propagation ◽  
Electron Tomography ◽  
Genetic Material ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Cryo Electron Tomography ◽  
Dynamics Simulations ◽  
Capsid Stability ◽  
Hiv 1

Viral replication in HIV-1 relies on a fullerene-shaped capsid to transport genetic material deep into the nucleus of an infected cell. Capsid stability is linked to the presence of cofactors, including inositol hexakisphosphate (IP6) that bind to pores found in the capsid. Using extensive all-atom molecular dynamics simulations of HIV-1 cores imaged from cryo-electron tomography (cryo-ET) in intact virions, which contain IP6 and a ribonucleoprotein complex, we find markedly striated patterns of strain on capsid lattices. The presence of these cofactors also increases rigidity of the capsid. Conformational analysis of capsid (CA) proteins show CA accommodates strain by locally flexing away from structures resolved using x-ray crystallography and cryo-electron microscopy. Then, cryo-ET of HIV-1 cores undergoing endogenous reverse transcription demonstrate that lattice strain increases in the capsid prior to mechanical failure and that the capsid ruptures by crack propagation along regions of high strain. These results uncover HIV-1 capsid properties involved in their critical disassembly process.

scholarly journals High-resolution cryo-EM structure of photosystem II reveals damage from high-dose electron beams

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Koji Kato ◽  
Naoyuki Miyazaki ◽  
Tasuku Hamaguchi ◽  
Yoshiki Nakajima ◽  
Fusamichi Akita ◽  
...  
Keyword(s):  
Photosystem Ii ◽  
Physiological State ◽  
High Dose ◽  
Final State ◽  
X Ray ◽  
Redox States ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Redox Active ◽  
Beam Damage

AbstractPhotosystem II (PSII) plays a key role in water-splitting and oxygen evolution. X-ray crystallography has revealed its atomic structure and some intermediate structures. However, these structures are in the crystalline state and its final state structure has not been solved. Here we analyzed the structure of PSII in solution at 1.95 Å resolution by single-particle cryo-electron microscopy (cryo-EM). The structure obtained is similar to the crystal structure, but a PsbY subunit was visible in the cryo-EM structure, indicating that it represents its physiological state more closely. Electron beam damage was observed at a high-dose in the regions that were easily affected by redox states, and reducing the beam dosage by reducing frames from 50 to 2 yielded a similar resolution but reduced the damage remarkably. This study will serve as a good indicator for determining damage-free cryo-EM structures of not only PSII but also all biological samples, especially redox-active metalloproteins.

Examining the structure of the mature amyloid fibril

2002 ◽  
Vol 30 (4) ◽  
pp. 521-525 ◽  
Author(s):  
O. S. Makin ◽  
L. C. Serpell
Keyword(s):  
Self Assembly ◽  
Rational Design ◽  
Amyloid Fibrils ◽  
Structural Information ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Fibre Diffraction ◽  
Complementary Techniques ◽  
Mature Fibril

The pathogenesis of the group of diseases known collectively as the amyloidoses is characterized by the deposition of insoluble amyloid fibrils. These are straight, unbranching structures about 70–120 å (1 å = 0.1 nm) in diameter and of indeterminate length formed by the self-assembly of a diverse group of normally soluble proteins. Knowledge of the structure of these fibrils is necessary for the understanding of their abnormal assembly and deposition, possibly leading to the rational design of therapeutic agents for their prevention or disaggregation. Structural elucidation is impeded by fibril insolubility and inability to crystallize, thus preventing the use of X-ray crystallography and solution NMR. CD, Fourier-transform infrared spectroscopy and light scattering have been used in the study of the mechanism of fibril formation. This review concentrates on the structural information about the final, mature fibril and in particular the complementary techniques of cryo-electron microscopy, solid-state NMR and X-ray fibre diffraction.

scholarly journals PDBrenum: a webserver and program providing Protein Data Bank files renumbered according to their UniProt sequences

2021 ◽  
Author(s):  
Bulat Faezov ◽  
Roland L. Dunbrack
Keyword(s):  
Protein Data Bank ◽  
Data Bank ◽  
Post Translational Modifications ◽  
X Ray ◽  
X Ray Crystallography ◽  
Link Type ◽  
Binding Partners ◽  
Cryo Electron Microscopy ◽  
Comparative Structure ◽  
In The Beginning

AbstractThe Protein Data Bank (PDB) was established at Brookhaven National Laboratories in 1971 as an archive for biological macromolecular crystal structures. In the beginning the archive held only seven structures but in early 2021, the database has more than 170,000 structures solved by X-ray crystallography, nuclear magnetic resonance, cryo-electron microscopy, and other methods. Many proteins have been studied under different conditions (e.g., binding partners such as ligands, nucleic acids, or other proteins; mutations and post-translational modifications), thus enabling comparative structure-function studies. However, these studies are made more difficult because authors are allowed by the PDB to number the amino acids in each protein sequence in any manner they wish. This results in the same protein being numbered differently in the available PDB entries. In addition to the coordinates, there are many fields that contain information regarding specific residues in the sequence of each protein in the entry. Here we provide a webserver and Python3 application that fixes the PDB sequence numbering problem by replacing the author numbering with numbering derived from the corresponding UniProt sequences. We obtain this correspondence from the SIFTS database from PDBe. The server and program can take a list of PDB entries and provide renumbered files in mmCIF format and the legacy PDB format for both asymmetric unit files and biological assembly files provided by PDBe. The server can also take a list of UniProt identifiers (“P04637” or “P53_HUMAN”) and return the desired files.AvailabilitySource code is freely available at https://github.com/Faezov/PDBrenum. The webserver is located at: http://dunbrack3.fccc.edu/[email protected] or [email protected].

scholarly journals Homogeneously N-glycosylated proteins derived from the GlycoDelete HEK293 cell line enable diffraction-quality crystallogenesis

2020 ◽  
Vol 76 (12) ◽  
pp. 1244-1255
Author(s):  
Sandra Kozak ◽  
Yehudi Bloch ◽  
Steven De Munck ◽  
Aleksandra Mikula ◽  
Isabel Bento ◽  
...  
Keyword(s):  
Cell Line ◽  
Protein Interactions ◽  
Successful Implementation ◽  
Structural Studies ◽  
Protein Fragments ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Acid Protein ◽  
Stimulating Factor

Structural studies of glycoproteins and their complexes provide critical insights into their roles in normal physiology and disease. Most glycoproteins contain N-linked glycosylation, a key post-translation modification that critically affects protein folding and stability and the binding kinetics underlying protein interactions. However, N-linked glycosylation is often an impediment to yielding homogeneous protein preparations for structure determination by X-ray crystallography or other methods. In particular, obtaining diffraction-quality crystals of such proteins and their complexes often requires modification of both the type of glycosylation patterns and their extent. Here, we demonstrate the benefits of producing target glycoproteins in the GlycoDelete human embryonic kidney 293 cell line that has been engineered to produce N-glycans as short glycan stumps comprising N-acetylglucosamine, galactose and sialic acid. Protein fragments of human Down syndrome cell-adhesion molecule and colony-stimulating factor 1 receptor were obtained from the GlycoDelete cell line for crystallization. The ensuing reduction in the extent and complexity of N-glycosylation in both protein molecules compared with alternative glycoengineering approaches enabled their productive deployment in structural studies by X-ray crystallography. Furthermore, a third successful implementation of the GlycoDelete technology focusing on murine IL-12B is shown to lead to N-glycosylation featuring an immature glycan in diffraction-quality crystals. It is proposed that the GlycoDelete cell line could serve as a valuable go-to option for the production of homogeneous glycoproteins and their complexes for structural studies by X-ray crystallography and cryo-electron microscopy.

scholarly journals Neutralizing antibodies induced by first-generation gp41-stabilized HIV-1 envelope trimers and nanoparticles

2020 ◽  
Author(s):  
Sonu Kumar ◽  
Xiaohe Lin ◽  
Timothy Ngo ◽  
Benjamin Shapero ◽  
Cindy Sou ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Neutralizing Antibodies ◽  
First Generation ◽  
Heptad Repeat ◽  
X Ray ◽  
X Ray Crystallography ◽  
Clade C ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Hiv 1

ABSTRACTAntigen-specific B-cell sorting and next-generation sequencing (NGS) were combined to isolate HIV-1 neutralizing antibodies (NAbs) from mice and rabbits immunized with BG505 trimers and nanoparticles. Three mouse NAbs potently neutralize BG505.T332N and recognize a glycan epitope centered at the C3/V4 region, as revealed by electron microscopy (EM), x-ray crystallography, and epitope mapping. Three potent NAbs were sorted from rabbit B cells that target glycan holes on the BG505 envelope glycoprotein (Env) and account for a significant portion of autologous NAb response. We then determined a 3.4Å-resolution crystal structure for the clade C transmitted/founder Du172.17 Env with a redesigned heptad repeat 1 (HR1) bend. This clade C Env, as a soluble trimer and attached to a ferritin nanoparticle, along with a clade A Q482-d12 Env trimer, elicited distinct NAb responses in rabbits. Our study demonstrates that nanoparticles presenting gp41-stabilized trimers can induce potent NAb responses in mice and rabbits with Env-dependent breadth.TEASERMouse and rabbit NAbs elicited by gp41-stabilized trimers and nanoparticles neutralize autologous HIV-1 by targeting different epitopes

scholarly journals 2.7 Å cryo-EM structure of rotavirus core protein VP3, a unique capping machine with a helicase activity

2020 ◽  
Vol 6 (16) ◽  
pp. eaay6410 ◽  
Author(s):  
Dilip Kumar ◽  
Xinzhe Yu ◽  
Sue E. Crawford ◽  
Rodolfo Moreno ◽  
Joanita Jakana ◽  
...  
Keyword(s):  
Core Protein ◽  
Messenger Rnas ◽  
Helicase Activity ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Rna Triphosphatase ◽  
Modular Domain ◽  
Biochemical Analyses ◽  
Nascent Transcripts

In many viruses, including rotavirus (RV), the major pathogen of infantile gastroenteritis, capping of viral messenger RNAs is a pivotal step for efficient translation of the viral genome. In RV, VP3 caps the nascent transcripts synthesized from the genomic dsRNA segments by the RV polymerase VP1 within the particle core. Here, from cryo–electron microscopy, x-ray crystallography, and biochemical analyses, we show that VP3 forms a stable tetrameric assembly with each subunit having a modular domain organization, which uniquely integrates five distinct enzymatic steps required for capping the transcripts. In addition to the previously known guanylyl- and methyltransferase activities, we show that VP3 exhibits hitherto unsuspected RNA triphosphatase activity necessary for initiating transcript capping and RNA helicase activity likely required for separating the RNA duplex formed transiently during endogenous transcription. From our studies, we propose a new mechanism for how VP3 inside the virion core caps the nascent transcripts exiting from the polymerase.

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