Domain topology of human Rasal

2017 ◽  
Vol 399 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Jorge Cuellar ◽  
José María Valpuesta ◽  
Alfred Wittinghofer ◽  
Begoña Sot
Keyword(s):  
Electron Microscopy ◽  
Small Gtpase ◽  
Full Length ◽  
Gtpase Activating Protein ◽  
Ras Gtpase ◽  
Cryo Electron Microscopy ◽  
Domain Protein ◽  
Negative Staining Electron Microscopy ◽  
Resolution Structure

AbstractRasal is a modular multi-domain protein of the GTPase-activating protein 1 (GAP1) family; its four known members, GAP1m, Rasal, GAP1IP4BPand Capri, have a Ras GTPase-activating domain (RasGAP). This domain supports the intrinsically slow GTPase activity of Ras by actively participating in the catalytic reaction. In the case of Rasal, GAP1IP4BPand Capri, their remaining domains are responsible for converting the RasGAP domains into dual Ras- and Rap-GAPs, via an incompletely understood mechanism. Although Rap proteins are small GTPase homologues of Ras, their catalytic residues are distinct, which reinforces the importance of determining the structure of full-length GAP1 family proteins. To date, these proteins have not been crystallized, and their size is not adequate for nuclear magnetic resonance (NMR) or for high-resolution cryo-electron microscopy (cryoEM). Here we present the low resolution structure of full-length Rasal, obtained by negative staining electron microscopy, which allows us to propose a model of its domain topology. These results help to understand the role of the different domains in controlling the dual GAP activity of GAP1 family proteins.

scholarly journals Structure of the full-length human Pannexin1 channel and insights into its role in pyroptosis

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Sensen Zhang ◽  
Baolei Yuan ◽  
Jordy Homing Lam ◽  
Jun Zhou ◽  
Xuan Zhou ◽  
...  
Keyword(s):  
Md Simulation ◽  
Potential Role ◽  
Md Simulations ◽  
Full Length ◽  
Inflammasome Activation ◽  
Simulation Studies ◽  
Cryo Electron Microscopy ◽  
Gating Mechanism ◽  
Atp Efflux

AbstractPannexin1 (PANX1) is a large-pore ATP efflux channel with a broad distribution, which allows the exchange of molecules and ions smaller than 1 kDa between the cytoplasm and extracellular space. In this study, we show that in human macrophages PANX1 expression is upregulated by diverse stimuli that promote pyroptosis, which is reminiscent of the previously reported lipopolysaccharide-induced upregulation of PANX1 during inflammasome activation. To further elucidate the function of PANX1, we propose the full-length human Pannexin1 (hPANX1) model through cryo-electron microscopy (cryo-EM) and molecular dynamics (MD) simulation studies, establishing hPANX1 as a homo-heptamer and revealing that both the N-termini and C-termini protrude deeply into the channel pore funnel. MD simulations also elucidate key energetic features governing the channel that lay a foundation to understand the channel gating mechanism. Structural analyses, functional characterizations, and computational studies support the current hPANX1-MD model, suggesting the potential role of hPANX1 in pyroptosis during immune responses.

scholarly journals A resolution record for cryoEM

2021 ◽  
Vol 10 ◽  
Author(s):  
Jonathan Ashmore ◽  
Bridget Carragher ◽  
Peter B Rosenthal ◽  
William Weis
Keyword(s):  
Electron Microscopy ◽  
Image Analysis ◽  
Structure Determination ◽  
Test Specimen ◽  
Structural Information ◽  
Atomic Resolution ◽  
Fast Growing ◽  
Cryo Electron Microscopy ◽  
New Instrument ◽  
Resolution Structure

Cryo electron microscopy (cryoEM) is a fast-growing technique for structure determination. Two recent papers report the first atomic resolution structure of a protein obtained by averaging images of frozen-hydrated biomolecules. They both describe maps of symmetric apoferritin assemblies, a common test specimen, in unprecedented detail. New instrument improvements, different in the two studies, have contributed better images, and image analysis can extract structural information sufficient to resolve individual atomic positions. While true atomic resolution maps will not be routine for most proteins, the studies suggest structures determined by cryoEM will continue to improve, increasing their impact on biology and medicine.

scholarly journals Rapid high-resolution structure analysis of small, biotechnologically relevant enzymes by cryo-electron microscopy

2021 ◽  
Author(s):  
Nicole Dimos ◽  
Carl P.O. Helmer ◽  
Andrea M. Chanique ◽  
Markus C. Wahl ◽  
Robert Kourist ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Structural Biology ◽  
Structure Analysis ◽  
Cryo Electron Microscopy ◽  
High Resolution Structure ◽  
Fast Development ◽  
Pharmaceutical Industries ◽  
Exquisite Specificity ◽  
Resolution Structure

Enzyme catalysis has emerged as a key technology for developing efficient, sustainable processes in the chemical, biotechnological and pharmaceutical industries. Plants provide large and diverse pools of biosynthetic enzymes that facilitate complex reactions, such as the formation of intricate terpene carbon skeletons, with exquisite specificity. High-resolution structural analysis of these enzymes is crucial to understand their mechanisms and modulate their properties by targeted engineering. Although cryo-electron microscopy (cryo-EM) has revolutionized structural biology, its applicability to high-resolution structure analysis of comparatively small enzymes is so far largely unexplored. Here, we show that cryo-EM can reveal the structures of ~120 kDa plant borneol dehydrogenases at or below 2 Å resolution, paving the way for the fast development of new biocatalysts that provide access to bioactive terpenes and terpenoids.

scholarly journals Near-Atomic Resolution Structure of a Highly Neutralizing Fab Bound to Canine Parvovirus

2016 ◽  
Vol 90 (21) ◽  
pp. 9733-9742 ◽  
Author(s):  
Lindsey J. Organtini ◽  
Hyunwook Lee ◽  
Sho Iketani ◽  
Kai Huang ◽  
Robert E. Ashley ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Receptor Binding ◽  
Conformational Changes ◽  
De Novo ◽  
Mutational Analysis ◽  
Antibody Fragment ◽  
Canine Parvovirus ◽  
Single Chain ◽  
Cryo Electron Microscopy ◽  
Resolution Structure

ABSTRACT Canine parvovirus (CPV) is a highly contagious pathogen that causes severe disease in dogs and wildlife. Previously, a panel of neutralizing monoclonal antibodies (MAb) raised against CPV was characterized. An antibody fragment (Fab) of MAb E was found to neutralize the virus at low molar ratios. Using recent advances in cryo-electron microscopy (cryo-EM), we determined the structure of CPV in complex with Fab E to 4.1 Å resolution, which allowed de novo building of the Fab structure. The footprint identified was significantly different from the footprint obtained previously from models fitted into lower-resolution maps. Using single-chain variable fragments, we tested antibody residues that control capsid binding. The near-atomic structure also revealed that Fab binding had caused capsid destabilization in regions containing key residues conferring receptor binding and tropism, which suggests a mechanism for efficient virus neutralization by antibody. Furthermore, a general technical approach to solving the structures of small molecules is demonstrated, as binding the Fab to the capsid allowed us to determine the 50-kDa Fab structure by cryo-EM. IMPORTANCE Using cryo-electron microscopy and new direct electron detector technology, we have solved the 4 Å resolution structure of a Fab molecule bound to a picornavirus capsid. The Fab induced conformational changes in regions of the virus capsid that control receptor binding. The antibody footprint is markedly different from the previous one identified by using a 12 Å structure. This work emphasizes the need for a high-resolution structure to guide mutational analysis and cautions against relying on older low-resolution structures even though they were interpreted with the best methodology available at the time.

scholarly journals The chemotactic response to PDGF-BB: evidence of a role for Ras.

1995 ◽  
Vol 130 (3) ◽  
pp. 725-731 ◽  
Author(s):  
V Kundra ◽  
B Anand-Apte ◽  
L A Feig ◽  
B R Zetter
Keyword(s):  
Chemotactic Response ◽  
Dominant Negative ◽  
Pdgf Receptor ◽  
Gtpase Activating Protein ◽  
Ras Gtpase ◽  
Downstream Effector ◽  
Ras Inhibition ◽  
Receptor Beta ◽  
Constitutively Active

The PDGF receptor-beta mediates both mitogenic and chemotactic responses to PDGF-BB. Although the role of Ras in tyrosine kinase-mediated mitogenesis has been characterized extensively, its role in PDGF-stimulated chemotaxis has not been defined. Using cells expressing a dominant-negative ras, we find that Ras inhibition suppresses migration toward PDGF-BB. Overexpression of either Ras-GTPase activating protein (Ras-GAP) or a Ras guanine releasing factor (GRF) also inhibited PDGF-stimulated chemotaxis. In addition, cells producing excess constitutively active Ras failed to migrate toward PDGF-BB, consistent with the observation that either excess ligand or excess signaling intermediate can suppress the chemotactic response. These results suggest that Ras can function in normal cells to support chemotaxis toward PDGF-BB and that either too little or too much Ras activity can abrogate the chemotactic response. In contrast to Ras overexpression, cells producing excess constitutively active Raf, a downstream effector of Ras, did migrate toward PDGF-BB. Cells expressing dominant-negative Ras were able to migrate toward soluble fibronectin demonstrating that these cells retained the ability to migrate. These results suggest that Ras is an intermediate in PDGF-stimulated chemotaxis but may not be required for fibronectin-stimulated cell motility.

scholarly journals Intelligent Resolution: Integrating Cryo-EM with AI-driven Multi-resolution Simulations to Observe the SARS-CoV-2 Replication-Transcription Machinery in Action

2021 ◽  
Author(s):  
Anda Trifan ◽  
Defne Gorgun ◽  
Zongyi Li ◽  
Alexander Brace ◽  
Maxim Zvyagin ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Molecular Dynamics ◽  
Finite Element Analysis ◽  
Finite Element ◽  
Pharmaceutical Compounds ◽  
Viral Mrna ◽  
Element Analysis ◽  
Cryo Electron Microscopy ◽  
Optimal Resource ◽  
Domain Protein

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) replication transcription complex (RTC) is a multi-domain protein responsible for replicating and transcribing the viral mRNA inside a human cell. Attacking RTC function with pharmaceutical compounds is a pathway to treating COVID-19. Conventional tools, e.g., cryo-electron microscopy and all-atom molecular dynamics (AAMD), do not provide sufficiently high resolution or timescale to capture important dynamics of this molecular machine. Consequently, we develop an innovative workflow that bridges the gap between these resolutions, using mesoscale fluctuating finite element analysis (FFEA) continuum simulations and a hierarchy of AI-methods that continually learn and infer features for maintaining consistency between AAMD and FFEA simulations. We leverage a multi-site distributed workflow manager to orchestrate AI, FFEA, and AAMD jobs, providing optimal resource utilization across HPC centers. Our study provides unprecedented access to study the SARS-CoV-2 RTC machinery, while providing general capability for AI-enabled multi-resolution simulations at scale.

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