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.

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 Observation of unphosphorylated STAT3 core protein binding to targetdsDNA by PEMSA and X-ray crystallography

FEBS Letters ◽  
2013 ◽  
Vol 587 (7) ◽  
pp. 833-839 ◽  
Author(s):  
Edwin Nkansah ◽  
Rahi Shah ◽  
Gavin W. Collie ◽  
Gary N. Parkinson ◽  
Jonathan Palmer ◽  
...  
Keyword(s):  
Protein Binding ◽  
Core Protein ◽  
X Ray ◽  
X Ray Crystallography

Using cryo-electron microscopy maps for X-ray structure determination of homologues

2020 ◽  
Vol 76 (1) ◽  
pp. 63-72
Author(s):  
Lingxiao Zeng ◽  
Wei Ding ◽  
Quan Hao
Keyword(s):  
Electron Microscopy ◽  
Hybrid Method ◽  
Model Building ◽  
Test Cases ◽  
X Ray ◽  
X Ray Crystallography ◽  
Sequence Identity ◽  
Whole Process ◽  
Cryo Electron Microscopy

The combination of cryo-electron microscopy (cryo-EM) and X-ray crystallography reflects an important trend in structural biology. In a previously published study, a hybrid method for the determination of X-ray structures using initial phases provided by the corresponding parts of cryo-EM maps was presented. However, if the target structure of X-ray crystallography is not identical but homologous to the corresponding molecular model of the cryo-EM map, then the decrease in the accuracy of the starting phases makes the whole process more difficult. Here, a modified hybrid method is presented to handle such cases. The whole process includes three steps: cryo-EM map replacement, phase extension by NCS averaging and dual-space iterative model building. When the resolution gap between the cryo-EM and X-ray crystallographic data is large and the sequence identity is low, an intermediate stage of model building is necessary. Six test cases have been studied with sequence identity between the corresponding molecules in the cryo-EM and X-ray structures ranging from 34 to 52% and with sequence similarity ranging from 86 to 91%. This hybrid method consistently produced models with reasonable R work and R free values which agree well with the previously determined X-ray structures for all test cases, thus indicating the general applicability of the method for X-ray structure determination of homologues using cryo-EM maps as a starting point.

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 Structure of Aichi Virus 1 and Its Empty Particle: Clues to Kobuvirus Genome Release Mechanism

2016 ◽  
Vol 90 (23) ◽  
pp. 10800-10810 ◽  
Author(s):  
Charles Sabin ◽  
Tibor Füzik ◽  
Karel Škubník ◽  
Lenka Pálková ◽  
A. Michael Lindberg ◽  
...  
Keyword(s):  
Aichi Virus ◽  
Release Mechanism ◽  
Human Pathogen ◽  
Content Type ◽  
X Ray ◽  
X Ray Crystallography ◽  
Antiviral Compounds ◽  
Cryo Electron Microscopy ◽  
Empty Capsid

ABSTRACTAichi virus 1(AiV-1) is a human pathogen from theKobuvirusgenus of thePicornaviridaefamily. Worldwide, 80 to 95% of adults have antibodies against the virus. AiV-1 infections are associated with nausea, gastroenteritis, and fever. Unlike most picornaviruses, kobuvirus capsids are composed of only three types of subunits: VP0, VP1, and VP3. We present here the structure of the AiV-1 virion determined to a resolution of 2.1 Å using X-ray crystallography. The surface loop puff of VP0 and knob of VP3 in AiV-1 are shorter than those in other picornaviruses. Instead, the 42-residue BC loop of VP0 forms the most prominent surface feature of the AiV-1 virion. We determined the structure of AiV-1 empty particle to a resolution of 4.2 Å using cryo-electron microscopy. The empty capsids are expanded relative to the native virus. The N-terminal arms of capsid proteins VP0, which mediate contacts between the pentamers of capsid protein protomers in the native AiV-1 virion, are disordered in the empty capsid. Nevertheless, the empty particles are stable, at leastin vitro, and do not contain pores that might serve as channels for genome release. Therefore, extensive and probably reversible local reorganization of AiV-1 capsid is required for its genome release.IMPORTANCEAichi virus 1 (AiV-1) is a human pathogen that can cause diarrhea, abdominal pain, nausea, vomiting, and fever. AiV-1 is identified in environmental screening studies with higher frequency and greater abundance than other human enteric viruses. Accordingly, 80 to 95% of adults worldwide have suffered from AiV-1 infections. We determined the structure of the AiV-1 virion. Based on the structure, we show that antiviral compounds that were developed against related enteroviruses are unlikely to be effective against AiV-1. The surface of the AiV-1 virion has a unique topology distinct from other related viruses from thePicornaviridaefamily. We also determined that AiV-1 capsids form compact shells even after genome release. Therefore, AiV-1 genome release requires large localized and probably reversible reorganization of the capsid.

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