Multi-domain Architecture Research Articles

NetChain: A Blockchain-Enabled Privacy-Preserving Multi-Domain Network Slice Orchestration Architecture

Multi-domain networking slice orchestration is an essential technology for the programmable and cloud-native 5G network. However, existing research solutions are either based on the impractical assumption that operators will reveal all the private network information or time-consuming secure multi-party computation which is only applicable to limited computation scenarios. To provide agile and privacy-preserving end-to-end network slice orchestration services, this paper proposes NetChain, a multi-domain network slice orchestration architecture based on blockchain and trusted execution environment. Correspondingly, we design a novel consensus algorithm CoNet to ensure the strong security, scalability, and information consistency of NetChain. In addition, a bilateral evaluation mechanism based on game theory is proposed to guarantee fairness and Quality of Experience by suppressing the malicious behaviors during multi-domain network slice orchestration. Finally, the prototype of NetChain is implemented and evaluated on the Microsoft Azure Cloud with confidential computing. Experiment results show that NetChain has good performance and security under the premise of privacy-preserving.

The toposiomerase IIIalpha-RMI1-RMI2 complex orients human Bloom\u2019s syndrome helicase for efficient disruption of D-loops

Homologous recombination (HR) is a ubiquitous and efficient process that serves the repair of severe forms of DNA damage and the generation of genetic diversity during meiosis. HR can proceed via multiple pathways with different outcomes that may aid or impair genome stability and faithful inheritance, underscoring the importance of HR quality control. Human Bloom’s syndrome (BLM, RecQ family) helicase plays central roles in HR pathway selection and quality control via unexplored molecular mechanisms. Here we show that BLM’s multi-domain structural architecture supports a balance between stabilization and disruption of displacement loops (D-loops), early HR intermediates that are key targets for HR regulation. We find that this balance is markedly shifted toward efficient D-loop disruption by the presence of BLM’s interaction partners Topoisomerase IIIα-RMI1-RMI2, which have been shown to be involved in multiple steps of HR-based DNA repair. Our results point to a mechanism whereby BLM can differentially process D-loops and support HR control depending on cellular regulatory mechanisms.

Expression of Large Full-Length PfEMP1 Proteins in HEK293 Cells.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a family of proteins expressed on the surface of red blood cells infected by Plasmodium falciparum. PfEMP1 proteins play a vital role in parasite virulence, and thus are important vaccine candidates to prevent severe disease. VAR2CSA is one specific PfEMP1 essential for pregnancy malaria pathogenesis, and the primary target in pregnancy malaria vaccine development. However, similar to other PfEMP1 proteins, expression of recombinant full-length VAR2CSA is difficult due to its large size, multidomain architecture and high cysteine content. To date, there has been success using higher ordered expression systems (such as mammalian and insect cells) to generate folded and active VAR2CSA. However, recent improvements with mammalian expression systems including cell lines and promoters have pushed the boundaries of yields. Here, we describe a modified protocol beyond current systems that enhances yields of full-length VAR2CSA and can generate higher quantities of material for protein structural and functional characterization.

A rational blueprint for the design of chemically-controlled protein switches

Small-molecule responsive protein switches are crucial components to control synthetic cellular activities. However, the repertoire of small-molecule protein switches is insufficient for many applications, including those in the translational spaces, where properties such as safety, immunogenicity, drug half-life, and drug side-effects are critical. Here, we present a computational protein design strategy to repurpose drug-inhibited protein-protein interactions as OFF- and ON-switches. The designed binders and drug-receptors form chemically-disruptable heterodimers (CDH) which dissociate in the presence of small molecules. To design ON-switches, we converted the CDHs into a multi-domain architecture which we refer to as activation by inhibitor release switches (AIR) that incorporate a rationally designed drug-insensitive receptor protein. CDHs and AIRs showed excellent performance as drug responsive switches to control combinations of synthetic circuits in mammalian cells. This approach effectively expands the chemical space and logic responses in living cells and provides a blueprint to develop new ON- and OFF-switches.

Edge In-Network Computing Meets Blockchain: A Multi-Domain Heterogeneous Resource Trust Management Architecture

In-network computing indicates the convergence of computing and network, which can be further considered as computing-based networking and network-enabled computing, from which comes the edge in-network computing paradigm. In this newly proposed paradigm, the computing, caching, and communication resources are deployed on in-transit edge nodes, such as routers or switches; hence, services are provided with less delay and energy consumption by the deep aggregation of edge-side resources. However, open challenges still exist in dealing with heterogeneous resource sharing, scheduling, and ensuring credibility of service processing. Given this background, this article aims to address the edge-side heterogeneous resource trust management in multi-domain scenarios. A multi-domain heterogeneous resource trust management architecture is first constructed to achieve trusted resource sharing and transaction. Then, under the proposed architecture, a smart-contract-based resource sharing mechanism is formulated, including an incentive mechanism based on end-side node contribution. Furthermore, a differential-evolution-enabled containerized microservice orchestration algorithm is finally designed to minimize operation delay and load imbalance during the container deployment. The application of this platform in a highly elastic power grid scenario has been tested as an example.

Achieving Efficient and Privacy-Preserving Multi-Domain Big Data Deduplication in Cloud

Secure data deduplication, as it can eliminate redundancies over encrypted data, has been widely developed in cloud storage to reduce storage space and communication overheads. Among them, the convergent encryption has been extensively adopted. However, it is vulnerable to brute-force attacks that can determine which plaintext in a message space corresponds to a given ciphertext. Many existing schemes have to sacrifice efficiency to resist brute-force attacks, especially for cross-domain deduplication, which is inevitably contrary to practical applications. Moreover, few existing schemes consider protecting the message equality information (i.e., whether two different ciphertexts correspond to an identical plaintext). To address the above challenges, in this paper, we propose an efficient and privacy-preserving big data deduplication scheme for a two-level multi-domain architecture. Specifically, by generating a random tag and a constant number of random ciphertexts for each data, our scheme not only ensures data confidentiality under multi-domain deduplication but also resists brute-force attacks. By allowing only the agent and cloud service provider to perform intra-deduplication and inter-deduplication, respectively, our scheme can protect the message equality information from disclosure as much as possible. Detailed security analysis shows that our scheme achieves privacy-preservation for both data content and the message equality information and data integrity while resisting brute-force attacks. Furthermore, extensive simulations demonstrate that our scheme significantly outperforms the existing competing schemes, especially the computational cost and the time complexity of the duplicate search.

The large bat Helitron DNA transposase forms a compact monomeric assembly that buries and protects its covalently bound 5′-transposon end

Helitrons are widespread eukaryotic DNA transposons that have significantly contributed to genome variability and evolution, in part because of their distinctive, replicative rolling-circle mechanism, which often mobilizes adjacent genes. Although most eukaryotic transposases form oligomers and use RNase H-like domains to break and rejoin double-stranded DNA (dsDNA), Helitron transposases contain a single-stranded DNA (ssDNA)-specific HUH endonuclease domain. Here, we report the cryo-electron microscopy structure of a Helitron transposase bound to the 5'-transposon end, providing insight into its multidomain architecture and function. The monomeric transposase forms a tightly packed assembly that buries the covalently attached cleaved end, protecting it until the second end becomes available. The structure reveals unexpected architectural similarity to TraI, a bacterial relaxase that also catalyzes ssDNA movement. The HUH active site suggests how two juxtaposed tyrosines, a feature of many replication initiators that use HUH nucleases, couple the conformational shift of an α-helix to control strand cleavage and ligation reactions.

Identification of a novel tailor-made chitinase from white shrimp Fenneropenaeus merguiensis

Fenneropenaeus merguiensis (commonly named banana shrimp) is one of the most important farmed crustacean worldwide species for the fisheries and aquaculture industry. Besides its nutritional value, it is a good source of chitinase, an enzyme with excellent biological and catalytic properties for many industrial applications. In the present study, a putative chitinase-encoding cDNA was synthesized from mRNA from F. merguiensis hepatopancreas tissue. Subsequently, the corresponding cDNA was cloned, sequenced and functionally expressed in Escherichia coli, and the recombinant F. merguiensis chitinase (rFmCHI) was purified by His-tag affinity chromatography. The bioinformatics analysis of aminoacid sequence of rFmCHI displayed a cannonical multidomain architecture in chitinases which belongs to glycoside hydrolase family 18 (GH18 chitinase). Biochemical characterization revealed rFmCHI as a monomeric enzyme of molecular weight 52 kDa with maximum activity at 40 °C and pH 6.0 Moreover, the recombinant enzyme is also stable up to 60 °C, and in the pH range 5.0-8.0. Steady-state kinetic studies for colloidal chitin revealed KM, Vmax and kcat values of 78.18 μM, 0.07261 μM. min−1 and 43.37 s−1, respectively. Overall, our results aim to demonstrate the potential of rFmCHI as suitable catalyst for bioconversion of chitin waste.

GAN-Based Multi-Style Photo Cartoonization.

Cartoon is a common form of art in our daily life and automatic generation of cartoon images from photos is highly desirable. However, state-of-the-art single-style methods can only generate one style of cartoon images from photos and existing multi-style image style transfer methods still struggle to produce high-quality cartoon images due to their highly simplified and abstract nature. In this article, we propose a novel multi-style generative adversarial network (GAN) architecture, called MS-CartoonGAN, which can transform photos into multiple cartoon styles. MS-CartoonGAN uses only unpaired photos and cartoon images of multiple styles for training. To achieve this, we propose to use (1) a hierarchical semantic loss with sparse regularization to retain semantic content and recover flat shading in different abstract levels, (2) a new edge-promoting adversarial loss for producing fine edges, and (3) a style loss to enhance the difference between output cartoon styles and make training process more stable. We also develop a multi-domain architecture, where the generator consists of a shared encoder and multiple decoders for different cartoon styles, along with multiple discriminators for individual styles. By observing that cartoon images drawn by different artists have their unique styles while sharing some common characteristics, our shared network architecture exploits the common characteristics of cartoon styles, achieving better cartoonization and being more efficient than single-style cartoonization. We show that our multi-domain architecture can theoretically guarantee to output desired multiple cartoon styles. Through extensive experiments including a user study, we demonstrate the superiority of the proposed method, outperforming state-of-the-art single-style and multi-style image style transfer methods.

Multi-domain inspection of offshore wind farms using an autonomous surface vehicle

The offshore wind power industry is an emerging and exponentially growing sector, which calls to a necessity for a cyclical monitoring and inspection to ensure the safety and efficiency of the wind farm facilities. Thus, the emersed (aerial) and immersed (underwater) scenarios must be reconstructed to create a more complete and reliable map that maximizes the observability of all the offshore structures from the wind turbines to the cable arrays, presenting a multi domain scenario.This work proposes the use of an Autonomous Surface Vehicle (ASV) to map both domains simultaneously. As such, it will produce a multi-domain map through the fusion of navigational sensors, GPS and IMU, to localize the vehicle and aid the registration process for the perception sensors, 3D Lidar and Multibeam echosounder sonar. The performed experiments demonstrate the ability of the multi-domain mapping architecture to provide an accurate reconstruction of both scenarios into a single representation using the odometry system as the initial seed to further improve the map with data filtering and registration processes. An error of 0.049 m for the odometry estimation is observed with the GPS/IMU fusion for simulated data and 0.07 m for real field tests. The multi-domain map methodology requires an average of 300 ms per iteration to reconstruct the environment, with an error of at most 0.042 m in simulation.

Structure and dynamics analysis of multi-domain putative β-1,4-glucosidase of family 3 glycoside hydrolase (PsGH3) from Pseudopedobacter saltans.

Structure and conformational behaviour of a putative β-1,4-glucosidase of glycoside hydrolase family 3 (PsGH3) from Pseudopedobacter saltans was predicted by using in-silico tools. PsGH3 modeled structure constructed using Phyre2 displayed multidomain architecture comprising an N-terminal (β/α)8-fold domain followed by (α/β)6-sandwich domain, PA14 domain, and a C-terminal domain resembling an immunoglobulin fold. Ramachandran plot displayed 99.3% of amino acids in the allowed region and 0.7% residues in the disallowed region. Multiple sequence alignment (MSA) and structure superposition of PsGH3 with other homologues from GH3 family revealed the conserved residues, Asp274 and Glu624 present in loops LA and LB, respectively originating from N-terminal domain act as catalytic residues. The volume and area calculated for PsGH3 displayed a deep active-site conformation comparable with its homologues, β-1,4-glucosidases (GH3) of Kluyveromyces marxianus and Streptomyces venezuelae. Molecular dynamic (MD) simulation of PsGH3 structure for 80 ns suggested stable and compact structure. Molecular docking studies revealed deeper active site conformation of PsGH3 that could house larger cellooligosaccharides up to 7° of polymerization (DP7). The amino acid residues, Ala86, Leu88, Cys275, Pro483, Phe493, Asn417, Asn491, Pro492, and Leu495 created a binding pocket near the catalytic cleft, crucial for ligand binding. MD simulation of PsGH3 in the presence of cellooligosaccharides, viz., cellobiose and celloheptaose showed stability in terms of RMSD, Rg, and SASA values till 80 ns. The calculation of average number of hydrogen bond (H-bond), interaction energy, and binding free energy confirmed the stronger binding affinity of the larger cellooligosaccharides such as celloheptaose in the binding cavity of PsGH3.

The multidomain architecture of a bacteriophage endolysin enables intramolecular synergism and regulation of bacterial lysis

Endolysins are peptidoglycan hydrolases produced at the end of the bacteriophage (phage) replication cycle to lyse the host cell. Endolysins in Gram-positive phages come in a variety of multimodular forms that combine different catalytic and cell wall binding domains. However, the reason why phages adopt endolysins with such complex multidomain architecture is not well understood. In this study, we used the Streptococcus dysgalactiae phage endolysin PlySK1249 as a model to investigate the role of multidomain architecture in phage-induced bacterial lysis and lysis regulation. PlySK1249 consists of an amidase (Ami) domain that lyses bacterial cells, a nonbacteriolytic endopeptidase (CHAP) domain that acts as a dechaining enzyme, and a central LysM cell wall binding domain. We observed that the Ami and CHAP domains synergized for peptidoglycan digestion and bacteriolysis in the native enzyme or when expressed individually and reunified. The CHAP endopeptidase resolved complex polymers of stem-peptides to dimers and helped the Ami domain to digest peptidoglycan to completion. We also found that PlySK1249 was subject to proteolytic cleavage by host cell wall proteases both in vitro and after phage induction. Cleavage disconnected the different domains by hydrolyzing their linker regions, thus hindering their bacteriolytic cooperation and possibly modulating the lytic activity of the enzyme. PlySK1249 cleavage by cell-wall-associated proteases may represent another example of phage adaptation toward the use of existing bacterial regulation mechanism for their own advantage. In addition, understanding more thoroughly the multidomain interplay of PlySK1249 broadens our knowledge on the ideal architecture of therapeutic antibacterial endolysins.

Crystal structure and mutational analysis of the human TRIM7 B30.2 domain provide insights into the molecular basis of its binding to glycogenin-1

Tripartite motif (TRIM)7 is an E3 ubiquitin ligase that was first identified through its interaction with glycogenin-1 (GN1), the autoglucosyltransferase that initiates glycogen biosynthesis. A growing body of evidence indicates that TRIM7 plays an important role in cancer development, viral pathogenesis, and atherosclerosis and, thus, represents a potential therapeutic target. TRIM family proteins share a multidomain architecture with a conserved N-terminal TRIM and a variable C-terminal domain. Human TRIM7 contains the canonical TRIM motif and a B30.2 domain at the C terminus. To contribute to the understanding of the mechanism of action of TRIM7, we solved the X-ray crystal structure of its B30.2 domain (TRIM7B30.2) in two crystal forms at resolutions of 1.6 Å and 1.8 Å. TRIM7B30.2 exhibits the typical B30.2 domain fold, consisting of two antiparallel β-sheets of seven and six strands, arranged as a distorted β-sandwich. Furthermore, two long loops partially cover the concave face of the β-sandwich defined by the β-sheet of six strands, thus forming a positively charged cavity. We used sequence conservation and mutational analyses to provide evidence of a putative binding interface for GN1. These studies showed that Leu423, Ser499, and Cys501 of TRIM7B30.2 and the C-terminal 33 amino acids of GN1 are critical for this binding interaction. Molecular dynamics simulations also revealed that hydrogen bond and hydrophobic interactions play a major role in the stability of a modeled TRIM7B30.2-GN1 C-terminal peptide complex. These data provide useful information that could be used to target this interaction for the development of potential therapeutic agents.

Role of NMR in High Ordered Structure Characterization of Monoclonal Antibodies.

Obtaining high ordered structure (HOS) information is of importance to guarantee the efficacy and safety of monoclonal antibodies (mAbs) in clinical application. Assessment of HOS should ideally be performed in a non-invasive manner under their formulated storage conditions, as any perturbation can introduce unexpected detritions. However, most of the currently available techniques only indirectly report HOS of mAbs and/or require a certain condition to conduct the analyses. Besides, the flexible multidomain architecture of mAbs has hampered atomic-resolution structural analyses using X-ray crystallography and cryo-electron microscopy. In contrast, the ability of nuclear magnetic resonance (NMR) spectroscopy to structurally analyze biomolecules in various conditions in a non-invasive and quantitative manner is suitable to meet the needs. However, the application of NMR to mAbs is not straightforward due to the high molecular weight of the system. In this review, we will discuss how NMR techniques have been applied to HOS analysis of mAbs, along with the recent advances of the novel 15N direct detection NMR strategy that allows for obtaining the structural fingerprint of mAbs at lower temperatures under multiple formulation conditions. The potential application of these NMR strategies will benefit next-generation mAbs, such as antibody-drug conjugates and bispecific antibodies.

Structure, dynamics and functions of UBQLNs: at the crossroads of protein quality control machinery.

Cells rely on protein homeostasis to maintain proper biological functions. Dysregulation of protein homeostasis contributes to the pathogenesis of many neurodegenerative diseases and cancers. Ubiquilins (UBQLNs) are versatile proteins that engage with many components of protein quality control (PQC) machinery in cells. Disease-linked mutations of UBQLNs are most commonly associated with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerative disorders. UBQLNs play well-established roles in PQC processes, including facilitating degradation of substrates through the ubiquitin-proteasome system (UPS), autophagy, and endoplasmic-reticulum-associated protein degradation (ERAD) pathways. In addition, UBQLNs engage with chaperones to sequester, degrade, or assist repair of misfolded client proteins. Furthermore, UBQLNs regulate DNA damage repair mechanisms, interact with RNA-binding proteins (RBPs), and engage with cytoskeletal elements to regulate cell differentiation and development. Important to the myriad functions of UBQLNs are its multidomain architecture and ability to self-associate. UBQLNs are linked to numerous types of cellular puncta, including stress-induced biomolecular condensates, autophagosomes, aggresomes, and aggregates. In this review, we focus on deciphering how UBQLNs function on a molecular level. We examine the properties of oligomerization-driven interactions among the structured and intrinsically disordered segments of UBQLNs. These interactions, together with the knowledge from studies of disease-linked mutations, provide significant insights to UBQLN structure, dynamics and function.

MetaGeneHunt for protein domain annotation in short-read metagenomes

The annotation of short-reads metagenomes is an essential process to understand the functional potential of sequenced microbial communities. Annotation techniques based solely on the identification of local matches tend to confound local sequence similarity and overall protein homology and thus don’t mirror the complex multidomain architecture and the shuffling of functional domains in many protein families. Here, we present MetaGeneHunt to identify specific protein domains and to normalize the hit-counts based on the domain length. We used MetaGeneHunt to investigate the potential for carbohydrate processing in the mouse gastrointestinal tract. We sampled, sequenced, and analyzed the microbial communities associated with the bolus in the stomach, intestine, cecum, and colon of five captive mice. Focusing on Glycoside Hydrolases (GHs) we found that, across samples, 58.3% of the 4,726,023 short-read sequences matching with a GH domain-containing protein were located outside the domain of interest. Next, before comparing the samples, the counts of localized hits matching the domains of interest were normalized to account for the corresponding domain length. Microbial communities in the intestine and cecum displayed characteristic GH profiles matching distinct microbial assemblages. Conversely, the stomach and colon were associated with structurally and functionally more diverse and variable microbial communities. Across samples, despite fluctuations, changes in the functional potential for carbohydrate processing correlated with changes in community composition. Overall MetaGeneHunt is a new way to quickly and precisely identify discrete protein domains in sequenced metagenomes processed with MG-RAST. In addition, using the sister program “GeneHunt” to create custom Reference Annotation Table, MetaGeneHunt provides an unprecedented way to (re)investigate the precise distribution of any protein domain in short-reads metagenomes.

The streptococcal multidomain fibrillar adhesin CshA has an elongated polymeric architecture

The cell surfaces of many bacteria carry filamentous polypeptides termed adhesins that enable binding to both biotic and abiotic surfaces. Surface adherence is facilitated by the exquisite selectivity of the adhesins for their cognate ligands or receptors and is a key step in niche or host colonization and pathogenicity. Streptococcus gordonii is a primary colonizer of the human oral cavity and an opportunistic pathogen, as well as a leading cause of infective endocarditis in humans. The fibrillar adhesin CshA is an important determinant of S. gordonii adherence, forming peritrichous fibrils on its surface that bind host cells and other microorganisms. CshA possesses a distinctive multidomain architecture comprising an N-terminal target-binding region fused to 17 repeat domains (RDs) that are each ∼100 amino acids long. Here, using structural and biophysical methods, we demonstrate that the intact CshA repeat region (CshA_RD1-17, domains 1-17) forms an extended polymeric monomer in solution. We recombinantly produced a subset of CshA RDs and found that they differ in stability and unfolding behavior. The NMR structure of CshA_RD13 revealed a hitherto unreported all β-fold, flanked by disordered interdomain linkers. These findings, in tandem with complementary hydrodynamic studies of CshA_RD1-17, indicate that this polypeptide possesses a highly unusual dynamic transitory structure characterized by alternating regions of order and disorder. This architecture provides flexibility for the adhesive tip of the CshA fibril to maintain bacterial attachment that withstands shear forces within the human host. It may also help mitigate deleterious folding events between neighboring RDs that share significant structural identity without compromising mechanical stability.

A complex IRES at the 5'-UTR of a viral mRNA assembles a functional 48S complex via an uAUG intermediate.

Taking control of the cellular apparatus for protein production is a requirement for virus progression. To ensure this control, diverse strategies of cellular mimicry and/or ribosome hijacking have evolved. The initiation stage of translation is especially targeted as it involves multiple steps and the engagement of numerous initiation factors. The use of structured RNA sequences, called Internal Ribosomal Entry Sites (IRES), in viral RNAs is a widespread strategy for the exploitation of eukaryotic initiation. Using a combination of electron cryo-microscopy (cryo-EM) and reconstituted translation initiation assays with native components, we characterized how a novel IRES at the 5'-UTR of a viral RNA assembles a functional initiation complex via an uAUG intermediate. The IRES features a novel extended, multi-domain architecture, that circles the 40S head. The structures and accompanying functional data illustrate the importance of 5'-UTR regions in translation regulation and underline the relevance of the untapped diversity of viral IRESs.

A multidomain virtual network embedding algorithm based on multiobjective optimization for Internet of Drones architecture in Industry 4.0

SummaryUnmanned aerial vehicle (UAV) has a broad application prospect in the future, especially in the Industry 4.0. The development of Internet of Drones (IoD) makes UAV operation more autonomous. Network virtualization technology is a promising technology to support IoD, so the allocation of virtual resources becomes a crucial issue in IoD. How to rationally allocate potential material resources has become an urgent problem to be solved. The main work of this paper is presented as follows: (a) In order to improve the optimization performance and reduce the computation time, we propose a multidomain virtual network embedding algorithm (MP‐VNE) adopting the centralized hierarchical multidomain architecture. The proposed algorithm can avoid the local optimum through incorporating the genetic variation factor into the traditional particle swarm optimization process. (b) In order to simplify the multiobjective optimization problem, we transform the multiobjective problem into a single‐objective problem through weighted summation method. The results prove that the proposed algorithm can rapidly converge to the optimal solution. (c) In order to reduce the mapping cost, we propose an algorithm for selecting candidate nodes based on the estimated mapping cost. Each physical domain calculates the estimated mapping cost of all nodes according to the formula of the estimated mapping cost, and chooses the node with the lowest estimated mapping cost as the candidate node. The simulation results show that the proposed MP‐VNE algorithm has better performance than MC‐VNM, LID‐VNE, and other algorithms in terms of delay, cost and comprehensive indicators.

Long-range interdomain communications in eIF5B regulate GTP hydrolysis and translation initiation

Translation initiation controls protein synthesis by regulating the delivery of the first aminoacyl-tRNA to messenger RNAs (mRNAs). In eukaryotes, initiation is sophisticated, requiring dozens of protein factors and 2 GTP-regulated steps. The GTPase eIF5B gates progression to elongation during the second GTP-regulated step. Using electron cryomicroscopy (cryo-EM), we imaged an in vitro initiation reaction which is set up with purified yeast components and designed to stall with eIF5B and a nonhydrolyzable GTP analog. A high-resolution reconstruction of a "dead-end" intermediate at 3.6 Å allowed us to visualize eIF5B in its ribosome-bound conformation. We identified a stretch of residues in eIF5B, located close to the γ-phosphate of GTP and centered around the universally conserved tyrosine 837 (Saccharomyces cerevisiae numbering), that contacts the catalytic histidine of eIF5B (H480). Site-directed mutagenesis confirmed the essential role that these residues play in regulating ribosome binding, GTP hydrolysis, and translation initiation both in vitro and in vivo. Our results illustrate how eIF5B transmits the presence of a properly delivered initiator aminoacyl-tRNA at the P site to the distant GTPase center through interdomain communications and underscore the importance of the multidomain architecture in translation factors to sense and communicate ribosomal states.