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13 páginas,1 figura, 3 tablas, 1 apéndice. Se extraen los autores pertenecientes a The CIBERER network que trabajan en Centros del CSIC del Appendix A, CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research., This study has been funded by Instituto de Salud Carlos III (ISCIII) and Spanish Ministry of Science and Innovation

13 páginas,1 figura, 3 tablas, 1 apéndice. Se extraen los autores pertenecientes a The CIBERER network que trabajan en Centros del CSIC del Appendix A CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research. This study has been funded by Instituto de Salud Carlos III (ISCIII) and Spanish Ministry of Science and Innovation

Resumen del trabajo presentado a las II Jornadas Científicas PTI + Salud Global, celebradas los días 5 y 6 de octubre de 2022 en el Auditorio Santiago Grisolía de Valencia (España).

Resumen del póster presentado a las II Jornadas Científicas PTI + Salud Global, celebradas los días 5 y 6 de octubre de 2022 en el Auditorio Santiago Grisolía de Valencia (España).

Para la realización de esta Tesis, José Luis Llácer Guerri ha disfrutado de una Beca de Formación de Personal Investigador (FPI) (BES-2005-9618) del Ministerio de Educación y Ciencia. El trabajo se ha enmarcado dentro de los proyectos “Máquinas Moleculares para el metabolismo de aminoácidos y de pirimidinas: estructura, función, control, arquitectura, patología y organización génica.” (BFU2004-05159) y “Complejos macromoleculares, proteínas multifuncionales, pluriempleo y enfermedades raras en la familia aminoácido quinasa” (BFU2008-05021) financiados por el Ministerio de Ciencia e Innovación.

The S:A222V point mutation, within the G clade, was characteristic of the 20E (EU1) SARS-CoV-2 variant identified in Spain in early summer 2020. This mutation has now reappeared in the Delta subvariant AY.4.2, raising questions about its specific effect on viral infection. We report combined serological, functional, structural and computational studies characterizing the impact of this mutation. Our results reveal that S:A222V promotes an increased RBD opening and slightly increases ACE2 binding as compared to the parent S:D614G clade. Finally, S:A222V does not reduce sera neutralization capacity, suggesting it does not affect vaccine effectiveness., This research work was supported by the European Commission–NextGenerationEU through the CSIC Global Health Platform. Additionally, authors would like to acknowledge economic support from the Spanish Ministry of Science and Innovation through Grants: PID2019-104757RB-I00 funded by MCIN/AEI/ 10.13039/501100011033, RTI2018-094399-A-I00, and “ERDF A way of making Europe”, by the “European Union”, Grant SEV 2017-0712 funded by MCIN/AEI /10.13039/501100011033, the “Comunidad Autónoma de Madrid" through Grant: S2017/BMD3817, and the European Union (EU) and Horizon 2020 through grants: Marie-Curie Fellowship EnLaCES (MSCA IF 2020, Proposal: 101024130) (to JK), HighResCells (ERC - 2018 - SyG, Proposal: 810057), and iNEXT-Discovery (Proposal: 871037). AM, VR, JB and JLL are funded by CIBERER-ISCIII (proposal: COV20/00437), Fondo Supera COVID-19 (proposal: CSICCOVID19-082), Banco Santander (Proposal: BlockAce), and CSIC PTI Salud Global (Proposal: 202080E110). VR is funded by the Spanish Ministry of Science and Innovation through Grant PID2020-120322RB-C21. IC is funded by project PID2019-104477RB-100, Fondo COVID COV20/00140 and ERC CoG 101001038. MC is funded by the RyC program from the Spanish Ministry of Science and Innovation, the Generalitat Valenciana (SEJI/2019/011).

The S:A222V point mutation, within the G clade, was characteristic of the 20E (EU1) SARS-CoV-2 variant identified in Spain in early summer 2020. This mutation has since reappeared in the Delta subvariant AY.4.2, raising questions about its specific effect on viral infection. We report combined serological, functional, structural and computational studies characterizing the impact of this mutation. Our results reveal that S:A222V promotes an increased RBD opening and slightly increases ACE2 binding as compared to the parent S:D614G clade. Finally, S:A222V does not reduce sera neutralization capacity, suggesting it does not affect vaccine effectiveness., In this research work, PRR and TG were supported by salaries from the European Commission–NextGenerationEU through the CSIC Global Health Platform established by EU Council Regulation 2020/2094. The authors would like to acknowledge economic support from: (a) the Spanish Ministry of Science and Innovation through grants: SEV-2017-0712 funded by MCIN/AEI/10.13039/501100011033 (CPM, JK, RM, COSS, MM, RA, JMC); 116880GB-I00 (JLL) and 120322RB-C21 (VR) funded within PID 2020; PID2019-104757RB-I00 funded by MCIN/AEI/10.13039/501100011033/ and “ERDF A way of making Europe”, by the “European Union” (JMC, COSS, RM, MM, CPM) and 104477RB-100 (IC) funded within PID 2019; RYC-2015-18213 (salary of MC) and RYC-2015-17517 (salary of RG) funded by the Ramon y Cajal fellowship program; RTI2018-094399-A-I00 (JMC, COSS, RM, CPM, MC) funded within the Retos Investigación program; the CRIOMECORR project funded as ESFRI-2019-01-CSIC-16; (b) the Horizon 2020 program through Marie Skłodowska-Curie Individual Fellowships EnLaCES (Proposal 101024130, salary of JK) and EPIDNA (Grant ID: 894498, salary of MW); (c) the European Research Council (ERC) through grants: ERC-2018-SyG-810057 HighResCells funded within the EXCELLENT SCIENCE program (JMC, MM, COSS, salary of RM), Grant 871037 iNEXT DISCOVERY (JMC, MM, COSS) and grant CoG-101001038 funded within the Consolidator Grants program (IC); (d) Generalitat Valenciana through grants: SEJI/2019/011 (MC) and SEJI/2019/030 (JLL); (e) Comunidad de Madrid through grant: S2017/BMD-3817 (JMC); (f) Santander Bank through grant: CSIC-COVID19-082 (AM, VR, JB, JLL, RG) funded through the Fondo Supera Covid-19; (g) Instituto de Salud Carlos III through grants: COV20/00437 (AM, VR, JB, JLL) and COV20/00140 (IC), both funded through the Fondos Covid-19 initiative; (h) Spanish National Research Council through grant: 202080E110 funded within the PTI Salud Global initiative (AM, VR, JB, JLL, RG).

21 páginas, 7 figuras, 2 tablas. Funding for open access charge: Consejo Superior de Investigaciones Científicas/SpanishNational Research Council, The eukaryotic initiation factor 3 (eIF3) complex is involved in every step of translation initiation, but there is limited understanding of its molecular functions. Here, we present a single particle electron cryomicroscopy (cryo-EM) reconstruction of yeast 48S ribosomal preinitiation complex (PIC) in an open conformation conducive to scanning, with core subunit eIF3b bound on the 40S interface near the decoding center in contact with the ternary complex eIF2·GTP·initiator tRNA. eIF3b is relocated together with eIF3i from their solvent interface locations observed in other PIC structures, with eIF3i lacking 40S contacts. Re-processing of micrographs of our previous 48S PIC in a closed state also suggests relocation of the entire eIF3b-3i-3g-3a-Cter module during the course of initiation. Genetic analysis indicates that high fidelity initiation depends on eIF3b interactions at the 40S subunit interface that promote the closed PIC conformation, or facilitate the relocation of eIF3b/eIF3i to the solvent interface, on start codon selection., Spanish government [BFU2017-85814-P]; Generalitat Valenciana [SEJI/2019/030 to J.L.L.]; start-up funds [R(IV)090/1076/2017-4252] from the Indian Institute of Science, Bangalore, India; Wellcome Trust/DBT India Alliance Fellowship [IA/I/17/2/503313 to T.H.]; Human Frontiers in Science Program [RGP-0028/2009]; Intramural Research Program of the NIH (to A.G.H.); T.H. also thanks DST-FIST [SR/FST/LS11-036/2014(C)], UGCSAP [F.4.13/2018/DRS-III (SAP-II)]; DBT-IISc Partnership Program Phase-II [BT/PR27952-NF/22/212/2018] for infrastructure and financial support. Funding for open access charge: Consejo Superior de Investigaciones Cient´ıficas/SpanishNational Research Council.

Tesina de máter, 50 páginas, figuras y tablas., [EN] In cyanobacteria, transcriptional regulation of nitrogen metabolism-related genes is mainly mediated by the transcription factor NtcA. This transcription factor, which belongs to the CRP/FNR family, interacts with a consensus sequence in the promoter of the genes that regulates in order to recruit RNA polymerase (RNAP) to the transcription start site and activate transcription. The interaction mode and the mechanism by which NtcA activates RNAP is unknown. In very poor nitrogen conditions, NtcA is co-activated by the PipX protein. We know that this co-activation is partly due to PipX stabilization of the active form of NtcA, but we do not know if PipX also interacts with the RNAP and/or plays a role in its activation. The overall aim of the host group, specialized in determination of macromolecular structures is to study the transcription activation by the global nitrogen metabolism regulators NtcA and PipX by electron cryomicroscopy (cryo-EM), obtaining for this purpose the structure of these two proteins bound simultaneously to the RNAP and to a gene promoter. The current project is part of this aim, being its main objective to obtain three variants of a recombinant cyanobacteria Synechococcus elongatus RNAP in Escherichia coli, using polycistronic vectors, suitable for co-expression, that carry the genes that encode the different subunits of RNAP and subsequent purification. Furthermore, we have expressed and purified the proteins NtcA and PipX., [ES] En cianobacterias, la regulación de la transcripción de genes relacionados con el metabolismo del nitrógeno está controlada, generalmente, por el factor de transcripción NtcA. Este factor de transcripción, perteneciente a la familia CRP/FNR, interacciona con una secuencia consenso en el promotor de los genes a los que regula a fin de reclutar a la RNA polimerasa (RNAP) al lugar de inicio de la transcripción y activarla. El modo de interacción y el mecanismo por el cual activa a la RNAP es desconocido. En condiciones de pobreza de nitrógeno, NtcA es co-activada por la proteína PipX. Se conoce que dicha co-activación se debe a la estabilización por parte de PipX de la forma activa del factor NtcA, pero no se sabe si PipX también interacciona con la RNAP y/o juega un papel activándola. El objetivo global del grupo de acogida, experto en determinación de estructuras macromoleculares, es estudiar mediante criomicroscopía electrónica (cryo-EM) la activación de la transcripción por los reguladores globales del metabolismo del nitrógeno NtcA y PipX obteniendo para ello la estructura de estas dos proteínas unidas simultáneamente a la RNAP y al promotor de un gen. Dentro de este plan se enmarca el proyecto de final de máster, cuyo objetivo principal será la obtención de tres variantes de la RNAP de la cianobacteria Synechococcus elongatus de forma recombinante en Escherichia coli utilizando vectores policistrónicos, compatibles para la co-expresión, que portan los genes que codifican las distintas subunidades de la RNAP y su posterior purificación. Además se expresarán y purificarán las proteínas NtcA y PipX.

Tesis de máster, 45 p. imágenes y tablas., [EN] Several initiation factors (eIFs) play key roles during the translation initiation process. Not surprisingly, its deregulation is associated with the development of various diseases in humans. Therefore, it is important to understand their function at molecular detail in order to decipher the mechanism of gene expression regulation at a global level. In this work, we study initiation factors eIF4A (TIF1) and eIF4B (TIF3) from Saccharomyces cerevisiae. These two eIFs are involved in the binding of mRNA to the small subunit of the ribosome at the beginning of translation. eIF4A is an RNA helicase that is responsible for the unwinding of the 5´non-translated end of the mRNA, and eIF4B acts as an accessory protein that stimulates the activity of eIF4A. Although both participate in the same stage of the translation process, experimental evidence of a possible direct interaction between the two proteins has not yet been obtained. eIF4B is a highly unstructured protein and therefore difficult to obtain in a functional state in sufficient amounts for in vitro interactions assays with eIF4A. In the present work, we stablish the components and the most appropriate condition to favour the formation of the eIF4-eIF4B complex. This study provides the groundwork for obtaining a stable complex, which would allow a structural approach using X-ray crystallography, with the ultimate aim of elucidating the structure of the complex and thus obtaining important clues about some particular aspects of translation initiation in eukaryotes., [ES] En el proceso de iniciación de la traducción en eucariotas participan una serie de factores de iniciación con diversas funciones clave para que el proceso se pueda llevar a cabo. La actividad y expresión de estos factores están altamente reguladas y, por tanto, no es ninguna sorpresa que la desregulación de estos esté asociada al desarrollo de varias enfermedades en humanos. Por tanto, es de gran importancia el poder comprender la función de estos en detalle, lo que nos daría pistas para entender el mecanismo de regulación de la expresión génica a nivel global. En este trabajo, concretamente, se estudian dos factores de iniciación de Saccharomyces cerevisiae, eIF4A (TIF1) y eIF4B (TIF3), implicados en la unión del RNAm a la subunidad pequeña del ribosoma al inicio de la traducción. eIF4A es una RNA helicasa que se encarga del desenrollamiento del extremo 5´no traducido del RNAm, y eIF4B actúa como una proteína accesoria que estimula la actividad de eIF4A. A pesar de que ambas participan en la misma etapa del proceso de traducción, todavía no se han obtenido evidencias experimentales de una posible interacción directa entre ambas. eIF4B es una proteína altamente desestructurada, lo que dificulta su obtención para por ejemplo estudios de interacción con eIF4A. En este trabajo se trata de obtener qué componentes son necesarios y cuáles son las condiciones más adecuadas para favorecer la formación del complejo. Este estudio sienta las bases para la formación de un complejo estable, lo que permitiría un abordaje estructural mediante cristalografía de rayos X con el objetivo de elucidar la estructura del complejo, y así obtener importantes pistas sobre algunos aspectos particulares de la iniciación de la traducción en eucariotas.

11 páginas, 5 figuras, 1 tabla., One of the responses to stress by eukaryotic cells is the down-regulation of protein synthesis by phosphorylation of translation initiation factor eIF2. Phosphorylation results in low availability of the eIF2 ternary complex (eIF2-GTP-tRNAi) by affecting the interaction of eIF2 with its GTP-GDP exchange factor eIF2B. We have determined the cryo-EM structure of yeast eIF2B in complex with phosphorylated eIF2 at an overall resolution of 4.2 Å. Two eIF2 molecules bind opposite sides of an eIF2B hetero-decamer through eIF2α-D1, which contains the phosphorylated Ser51. eIF2α-D1 is mainly inserted between the N-terminal helix bundle domains of δ and α subunits of eIF2B. Phosphorylation of Ser51 enhances binding to eIF2B through direct interactions of phosphate groups with residues in eIF2Bα and indirectly by inducing contacts of eIF2α helix 58-63 with eIF2Bδ leading to a competition with Met-tRNAi., This study was supported by the MRC-LMB EM Facility. This work was supported by grants from the Medical Research Council (MC_U105184332) and the Wellcome Trust (WT096570) to V.R. and by a grant BFU2017-85814-P from the Spanish government to J.L.L.

33 páginas, 10 figuras, 5 tablas, In eukaryotic translation initiation, AUG recognition of the mRNA requires accommodation of Met-tRNAi in a 'PIN' state, which is antagonized by the factor eIF1. eIF5 is a GTPase activating protein (GAP) of eIF2 that additionally promotes stringent AUG selection, but the molecular basis of its dual function was unknown. We present a cryo-electron microscopy (cryo-EM) reconstruction of a yeast 48S pre-initiation complex (PIC), at an overall resolution of 3.0 Å, featuring the N-terminal domain (NTD) of eIF5 bound to the 40S subunit at the location vacated by eIF1. eIF5 interacts with and allows a more accommodated orientation of Met-tRNAi. Substitutions of eIF5 residues involved in the eIF5-NTD/tRNAi interaction influenced initiation at near-cognate UUG codonsin vivo, and the closed/open PIC conformation in vitro, consistent with direct stabilization of the codon:anticodon duplex by the wild-type eIF5-NTD. The present structure reveals the basis for a key role of eIF5 in start-codon selection., We are grateful to CG Savva, G McMullan for technical support with cryo-EM, T Darling and J Grimmett for help with computing and J Brasa for help with figures/movies. JL was supported by a FEBS postdoctoral fellowship. TH acknowledges start-up funds from the Indian Institute of Science. This work was funded by grants to from the UK Medical Research Council (MC_U105184332), Wellcome Trust Senior Investigator award (WT096570), the Agouron Institute and the Jeantet Foundation to VR; by the Department of Science and Technology, Government of India Grant [Int/NZ/P-2/13] to AKS; from the NIH (GM62128) formerly to JRL; the Human Frontiers in Science Program (RGP-0028/ 2009) to AGH, JRL and VR; and by the Intramural Research Program of the NIH (AGH, JRL).

Summary Translation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) containing the 40S ribosomal subunit, eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNAi), and eIF5. The PIC, in an open conformation, attaches to the 5′ end of the mRNA and scans to locate the start codon, whereupon it closes to arrest scanning. We present single particle cryo-electron microscopy (cryo-EM) reconstructions of 48S PICs from yeast in these open and closed states, at 6.0 Å and 4.9 Å, respectively. These reconstructions show eIF2β as well as a configuration of eIF3 that appears to encircle the 40S, occupying part of the subunit interface. Comparison of the complexes reveals a large conformational change in the 40S head from an open mRNA latch conformation to a closed one that constricts the mRNA entry channel and narrows the P site to enclose tRNAi, thus elucidating key events in start codon recognition., Graphical Abstract, Highlights • Structures of eukaryotic translation initiation complexes in open and closed states • In the open complex the 40S head moves upward to open the mRNA entry channel latch • Transition to closed state locks initiator tRNA in the P site base-paired with AUG • The structures show how eIF3 contacts eIF2 and eIF1 on the 40S subunit interface, The small ribosomal subunit latch has to open to allow mRNA loading. Llacer et al. report cryo-EM reconstructions of yeast translation pre-initiation complexes in both open and closed conformations, highlighting structural differences and elucidating key events in start codon recognition.

6 páginas, 6 figuras. Contiete material suplementario, Mitochondria have specialized ribosomes that have diverged from their bacterial and cytoplasmic counterparts. We have solved the structure of the yeast mitoribosomal large subunit using single-particle cryo-electron microscopy. The resolution of 3.2 angstroms enabled a nearly complete atomic model to be built de novo and refined, including 39 proteins, 13 of which are unique to mitochondria, as well as expansion segments of mitoribosomal RNA. The structure reveals a new exit tunnel path and architecture, unique elements of the E site, and a putative membrane docking site., This work was funded by grants from the UK Medical Research Council (MC_U105184332 to VR, MC_UP_A025_1013 to SHWS and MC_UP_A025_1012 to GM); a Wellcome Trust Senior Investigator award (WT096570), the Agouron Institute and the Jeantet Foundation (VR); fellowships from Human Frontiers Science Programme (AA), EU FP7 Marie Curie (XB), FEBS (JLL), and EMBO (TH). Cryo-EM density maps have been deposited with the EMDB (accession number EMD-2566) and coordinates have been deposited with the PDB (entry code 3J6B).

11 páginas, 7 figuras. Dispone de material suplementario en: t http://dx.doi.org/10.1016/j.cell.2014.10.001., During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit. This conformation (POUT) is compatible with scanning mRNA for the AUG start codon. Base pairing with AUG is thought to promote isomerization to a more stable conformation (PIN) that arrests scanning and promotes dissociation of eIF1 from the 40S subunit. Here, we present a cryoEM reconstruction of a yeast preinitiation complex at 4.0 Å resolution with initiator tRNA in the PIN state, prior to eIF1 release. The structure reveals stabilization of the codon-anticodon duplex by the N-terminal tail of eIF1A, changes in the structure of eIF1 likely instrumental in its subsequent release, and changes in the conformation of eIF2. The mRNA traverses the entire mRNA cleft and makes connections to the regulatory domain of eIF2?, eIF1A, and ribosomal elements that allow recognition of context nucleotides surrounding the AUG codon., This work was funded by grants to V.R. from the UK Medical Research Council (MC_U105184332), Wellcome Trust Senior Investigator award (WT096570), the Agouron Institute and the Jeantet Foundation, from the NIH (GM62128) previously to J.R.L., and from the Human Frontiers in Science Program (RGP-0028/2009) to A.G.H., J.R.L., and V.R., and by the Intramural Research Program of the NIH (A.G.H., P.M.-M., J.R.L., and A.M.)

To obtain insights into archaeal nitrogen signaling and haloadaptation of the nitrogen/carbon/energy-signaling protein PII, we determined crystal structures of recombinantly produced GlnK2 from the extreme halophilic archaeon Haloferax mediterranei, complexed with AMP or with the PII effectors ADP or ATP, at respective resolutions of 1.49 Å, 1.45 Å, and 2.60 Å. A unique trait of these structures was a three-tongued crown protruding from the trimer body convex side, formed by an 11-residue, N-terminal, highly acidic extension that is absent from structurally studied PII proteins. This extension substantially contributed to the very low pI value, which is a haloadaptive trait of H. mediterranei GlnK2, and participated in hexamer-forming contacts in one crystal. Similar acidic N-extensions are shown here to be common among PII proteins from halophilic organisms. Additional haloadaptive traits prominently represented in H. mediterranei GlnK2 are a very high ratio of small residues to large hydrophobic aliphatic residues, and the highest ratio of polar to nonpolar exposed surface for any structurally characterized PII protein. The presence of a dense hydration layer in the region between the three T-loops might also be a haloadaptation. Other unique findings revealed by the GlnK2 structure that might have functional relevance are: the adoption by its T-loop of a three-turn α-helical conformation, perhaps related to the ability of GlnK2 to directly interact with glutamine synthetase; and the firm binding of AMP, confirmed by biochemical binding studies with ATP, ADP, and AMP, raising the possibility that AMP could be an important PII effector, at least in archaea., This work was supported by grants from the Spanish government (BFU2011- 30407 and BIO2008_00082, to V.R. and M.J.B, respectively) and from the Valencian government (Prometeo 2009/51 to V.R.). C.P. is a JAE-Predoc fellow of the CSIC and during this work, L.P.R was a FPU fellow of the Spanish Ministry of Education. We thank Diamond Light Source for access to beamlines I-03 and I-04 (mx8035) that contributed to the results presented here. We also thank the ESRF beamlines BM30 and ID23-2 and the personnel assisting these lines. The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement N°283570).

Summary In bacterial translational initiation, three initiation factors (IFs 1–3) enable the selection of initiator tRNA and the start codon in the P site of the 30S ribosomal subunit. Here, we report 11 single-particle cryo-electron microscopy (cryoEM) reconstructions of the complex of bacterial 30S subunit with initiator tRNA, mRNA, and IFs 1–3, representing different steps along the initiation pathway. IF1 provides key anchoring points for IF2 and IF3, thereby enhancing their activities. IF2 positions a domain in an extended conformation appropriate for capturing the formylmethionyl moiety charged on tRNA. IF3 and tRNA undergo large conformational changes to facilitate the accommodation of the formylmethionyl-tRNA (fMet-tRNAfMet) into the P site for start codon recognition., Graphical Abstract, Highlights • Structures of the 30S ribosomal subunit with initiation factors, tRNA and mRNA • IF3 helps to position the correct start codon in the P site before binding of tRNA • Large-scale conformational changes of IF3 and tRNA are observed • IF3 movements facilitate the accommodation of initiator tRNA in P site, Bacterial ribosomes can start scanning for start codons only after undergoing large scale conformational changes governed by three key initiation factors.