Cold Spring Harbor perspectives in biology, Jan 14, 2018
In the past 4 years, because of the advent of new cameras, many ribosome structures have been sol... more In the past 4 years, because of the advent of new cameras, many ribosome structures have been solved by cryoelectron microscopy (cryo-EM) at high, often near-atomic resolution, bringing new mechanistic insights into the processes of translation initiation, peptide elongation, termination, and recycling. Thus, cryo-EM has joined X-ray crystallography as a powerful technique in structural studies of translation. The significance of this new development is that structures of ribosomes in complex with their functional binding partners can now be determined to high resolution in multiple states as they perform their work. The aim of this article is to provide an overview of these new studies and assess the contributions they have made toward an understanding of translation and translational control.
Ribosomal subunit association is a key checkpoint in translation initiation but its structural dy... more Ribosomal subunit association is a key checkpoint in translation initiation but its structural dynamics are poorly understood. Here, we used a recently developed mixing-spraying, time-resolved, cryogenic electron microscopy (cryo-EM) method to study ribosomal subunit association in the sub-second time range. We have improved this method and increased the cryo-EM data yield by tenfold. Pre-equilibrium states of the association reaction were captured by reacting the mixture of ribosomal subunits for 60 ms and 140 ms. We also identified three distinct ribosome conformations in the associated ribosomes. The observed proportions of these conformations are the same in these two time points, suggesting that ribosomes equilibrate among the three conformations within less than 60 ms upon formation. Our results demonstrate that the mixing-spraying method can capture multiple states of macromolecules during a sub-second reaction. Other fast processes, such as translation initiation, decoding, ...
Large T antigen is the replicative helicase of simian virus 40. Its speci®c binding to the origin... more Large T antigen is the replicative helicase of simian virus 40. Its speci®c binding to the origin of replication and oligomerization into a double hexamer distorts and unwinds dsDNA. In viral replication, T antigen acts as a functional homolog of the eukaryotic minichromosome maintenance factor MCM. T antigen is also an oncoprotein involved in transformation through interaction with p53 and pRb. We obtained the three-dimensional structure of the fulllength T antigen double hexamer assembled at its origin of replication by cryoelectron microscopy and single-particle reconstruction techniques. The double hexamer shows different degrees of bending along the DNA axis. The two hexamers are differentiated entities rotated relative to each other. Isolated strands of density, putatively assigned to ssDNA, protrude from the hexamer±hexamer junction mainly at two opposite sites. The structure of the T antigen at the origin of replication can be understood as a snapshot of the dynamic events leading to DNA unwinding. Based on these results a model for the initiation of simian virus 40 DNA replication is proposed.
Single particle analysis in electron microscopy allows direct observation of the reconstructed th... more Single particle analysis in electron microscopy allows direct observation of the reconstructed three-dimensional structures of protein molecules. This method enables a more comprehensive study of membrane proteins which have been problematic in structural studies using X-ray crystallography. These membrane proteins include the voltage-sensitive ion channel proteins, which play an important rule in neural activities, and have great medical significance. The method described is supported by the development of cryo-electron microscopy and the angular reconstitution method. This review summarizes certain principles governing single particle analysis employing angular reconstitution. This method was applied to our study of the voltage-sensitive sodium channel, and the results are discussed. With improvements in resolutions and statistical analyses, the single particle technique is considered to be advantageous in studies of the structural changes and molecular interactions of protein molecules.
Abstract: This essay is a reflection on the ways the X‐ray structures of the ribosome are helping... more Abstract: This essay is a reflection on the ways the X‐ray structures of the ribosome are helping in the interpretation of cryogenic electron microscopy (cryo‐EM) density maps showing the translating ribosome in motion. Through advances in classification methods, cryo‐EM and single‐particle reconstruction methods have recently evolved to the point where they can yield an array of structures from a single sample (“story in a sample”), providing snapshots of an entire subprocess of translation, such as translocation or decoding.
Mitochondrial diseases produce profound neurological dysfunction via mutations affecting mitochon... more Mitochondrial diseases produce profound neurological dysfunction via mutations affecting mitochondrial energy production, including the relatively common Leigh syndrome (LS). We recently described an LS case caused by a pathogenic mutation in USMG5, encoding a small supernumerary subunit of mitochondrial ATP synthase. This protein is integral for ATP synthase dimerization, and patient fibroblasts revealed an almost total loss of ATP synthase dimers. Here, we utilize in situ cryoelectron tomography (cryo-ET) in a clinical case-control study of mitochondrial disease to directly study mitochondria within cultured fibroblasts from a patient with LS and a healthy human control subject. Through tomographic analysis of patient and control mitochondria, we find that loss of ATP synthase dimerization due to the pathogenic mutation causes profound disturbances of mitochondrial crista ultrastructure. Overall, this work supports the crucial role of ATP synthase in regulating crista architecture...
The three-dinensional structure of the large (50S) ribosomal subunit from Escherichia coli has be... more The three-dinensional structure of the large (50S) ribosomal subunit from Escherichia coli has been determined from electron micrographs of negatively stained specimens. A new method of three-dimensional reconstruction was used which combines many images of individual subunits recorded at a single high tilt angle. A prominent feature of the reconstruction is a large groove on the side of the subunit that interacts with the small ribosomal subunit. This feature is probably of functional significance as it includes the regions where the peptidyl transferase site and the binding locations of the elongation factors have been mapped previously by immunoelectron microscopy.
A previously developed theory of three-dimensional (3-D) variance estimation [J. Opt. Soc. Am. A ... more A previously developed theory of three-dimensional (3-D) variance estimation [J. Opt. Soc. Am. A 12, 2615-2627 (1995)] is applied to the structural study of a hemocyanin-Fab complex with the electron microscope. The precise locations of structurally variable regions of the macromolecule are determined from the 3-D variance maps. The structural differences among different classes of the macromolecular complex are assessed by the use of the statistical t-test, and the 3-D antibody binding sites are revealed. From a model analysis, a rule is demonstrated for visually identifying a 3-D conformational change by the inspection of the 3-D variance map. Our analysis lays the foundation for numerous practical applications of variance estimation in the 3-D imaging of macromolecules.
Fidelity of tomographic reconstructions is improved and reconstruction artifacts are reduced, wit... more Fidelity of tomographic reconstructions is improved and reconstruction artifacts are reduced, without increasing the number of projections, by combining tilt series taken around two orthogonal axes. Test reconstructions were made from high-voltage EM of rat liver mitochondria in a 0.6 pm thick plastic section. A number of schemes for selecting tilt angles for the projections are compared. A new method for aligning fiducial markers is described. It uses an iterative algorithm to determine the shift, scale, in-plane rotation and tilt angle for each tilt image, enforcing agreement of the expected locations of the fiducial markers in 3D space. These 3D locations are used to find the orientation between two tilt series and to merge both sets of projections.
In this paper, we review current practices for establishing the resolution in single-particle rec... more In this paper, we review current practices for establishing the resolution in single-particle reconstructions. The classical Raleigh criterion for the resolution is not applicable in this case, and the resolution is commonly defined by a consistency test, whereby the data set is randomly split in half and the two resulting reconstructions are then compared. Such a procedure, however, may introduce statistical dependence between the two half-sets, which leads to a too optimistic resolution estimate. On the other hand, this overestimation is counteracted by the diminished statistical properties of a mere half of the data set. The ''true'' resolution of the whole data set can be estimated when the functional relationship between the data size and the resolution is known. We are able to estimate this functional by taking into account the B-factor and the geometry of data collection. Finally, the drawbacks of resolution estimation are entirely avoided by computing the correlation of neighboring voxels in the Fourier domain.
This Meeting Review describes the proceedings and conclusions from the inaugural meeting of the E... more This Meeting Review describes the proceedings and conclusions from the inaugural meeting of the Electron Microscopy Validation Task Force organized by the Unified Data Resource for 3DEM (http://www. emdatabank.org) and held at Rutgers University in New Brunswick, NJ on September 28 and 29, 2010. At the workshop, a group of scientists involved in collecting electron microscopy data, using the data to determine three-dimensional electron microscopy (3DEM) density maps, and building molecular models into the maps explored how to assess maps, models, and other data that are deposited into the Electron Microscopy Data Bank and Protein Data Bank public data archives. The specific recommendations resulting from the workshop aim to increase the impact of 3DEM in biology and medicine.
Initiation of protein synthesis in eukaryotes requires recruitment of the 40 S ribosomal subunit ... more Initiation of protein synthesis in eukaryotes requires recruitment of the 40 S ribosomal subunit to the messenger RNA (mRNA). In most cases, this depends on recognition of a modified nucleotide cap on the 5′ end of the mRNA. However, an alternate pathway uses a structured RNA element in the 5′ untranslated region of the messenger or viral RNA called an internal ribosomal entry site (IRES). Here, we present a cryo-electron microscopy map of the hepatitis C virus (HCV) IRES bound to the 40 S ribosomal subunit at about 20 Å resolution. IRES binding induces a pronounced conformational change in the 40 S subunit and closes the mRNA binding cleft, suggesting a mechanism for IRES-mediated positioning of mRNA in the ribosomal decoding center.
Proceedings of the National Academy of Sciences, 1983
The outer membrane of mitochondria contains proteins that form channels called VDAC (voltage-depe... more The outer membrane of mitochondria contains proteins that form channels called VDAC (voltage-dependent anion-selective channels). Two independent lines of evidence suggest that these channels occur in specific complexes in the outer membrane of Neurospora mitochondria. Electron microscopic images of these outer membranes reveal polymorphic crystalline arrays of putative pores. These arrays can be shown to be interrelated by movement in the membrane plane of a particular rigid channel triplet or of regular aggregates of this triplet. Detergent extracts of the same outer membranes induce single- and multiple-step conductances in planar phospholipid membranes, with a marked preference for the insertion of triplets and multiples of triplets. The tendency of the mitochondrial channels to occur as extended arrays, apparently built up from triplets, may have important functional and evolutionary implications.
Proceedings of the National Academy of Sciences, 2012
Eukaryotic translation termination results from the complex functional interplay between two euka... more Eukaryotic translation termination results from the complex functional interplay between two eukaryotic release factors, eRF1 and eRF3, and the ribosome, in which GTP hydrolysis by eRF3 couples codon recognition with peptidyl-tRNA hydrolysis by eRF1. Here, using cryo-electron microscopy (cryo-EM) and flexible fitting, we determined the structure of eRF1–eRF3–guanosine 5′-[β,γ-imido]triphosphate (GMPPNP)-bound ribosomal pretermination complex (pre-TC), which corresponds to the initial, pre-GTP hydrolysis stage of factor attachment. Our results show that eukaryotic translation termination involves a network of interactions between the two release factors and the ribosome. Our structure provides mechanistic insight into the coordination between GTP hydrolysis by eRF3 and subsequent peptide release by eRF1.
Group II self-splicing introns are phylogenetically diverse retroelements that are widely held to... more Group II self-splicing introns are phylogenetically diverse retroelements that are widely held to be the ancestors of spliceosomal introns and retrotransposons that insert into DNA. Folding of group II intron RNA is often guided by an intron-encoded protein to form a catalytically active ribonucleoprotein (RNP) complex that plays a key role in the activity of the intron. To date, possible structural differences between the intron RNP in its precursor and spliced forms remain unexplored. In this work, we have trapped the native Lactococcus lactis group II intron RNP complex in its precursor form, by deleting the adenosine nucleophile that initiates splicing. Sedimentation velocity, size-exclusion chromatography and cryo-electron microscopy provide the first glimpse of the intron RNP precursor as a large, loosely packed structure. The dimensions contrast with those of compact spliced introns, implying that the RNP undergoes a dramatic conformational change to achieve the catalytically active state.
This protocol details the steps used for visualizing the frozen-hydrated grids as prepared follow... more This protocol details the steps used for visualizing the frozen-hydrated grids as prepared following the accompanying protocol entitled 'Preparation of macromolecular complexes for visualization using cryo-electron microscopy.' This protocol describes how to transfer the grid to the microscope using a standard cryo-transfer holder or, alternatively, using a cryo-cartridge loading system, and how to collect low-dose data using an FEI Tecnai transmission electron microscope. This protocol also summarizes and compares the various options that are available in data collection for threedimensional (3D) single-particle reconstruction. These options include microscope settings, choice of detectors and data collection strategies both in situations where a 3D reference is available and in the absence of such a reference (random-conical and common lines).
This protocol describes the preparation of frozen-hydrated single-particle specimens of macromole... more This protocol describes the preparation of frozen-hydrated single-particle specimens of macromolecular complexes. First, it describes how to create a grid surface coated with holey carbon by first inducing holes in a Formvar film to act as a template for the holey carbon that is stable under cryo-electron microscopy (cryo-EM) conditions and is sample-friendly. The protocol then describes the steps required to deposit the homogeneous sample on the grid and to plunge-freeze the grid into liquid ethane at the temperature of liquid nitrogen, so that it is suitable for cryo-EM visualization. It takes 4-5 h to make several hundred holey carbon grids and about 1 h to make the frozen-hydrated grids. The time required for sample purification varies from hours to days, depending on the sample and the specific procedure required. A companion protocol details how to collect cryo-EM data using an FEI Tecnai transmission electron microscope that can subsequently be processed to obtain a threedimensional reconstruction of the macromolecular complex.
This protocol describes the reconstruction of biological molecules from the electron micrographs ... more This protocol describes the reconstruction of biological molecules from the electron micrographs of single particles. Computation here is performed using the image-processing software SPIDER and can be managed using a graphical user interface, termed the SPIDER Reconstruction Engine. Two approaches are described to obtain an initial reconstruction: random-conical tilt and common lines. Once an existing model is available, reference-based alignment can be used, a procedure that can be iterated. Also described is supervised classification, a method to look for homogeneous subsets when multiple known conformations of the molecule may coexist.
occupancy of RF2(GAQ) factor was assumed to be the same as for the wild type, because the factors... more occupancy of RF2(GAQ) factor was assumed to be the same as for the wild type, because the factors have similar dissociation constant (K d) values of 0.7 and 1.8 nM, respectively. Complex A served as the control for complex C, whereas the control for complex B was obtained by incubation of complex A (0.13 mM) with puromycin (0.4 mM) in the same polymix buffer for 2 min at 37 8C (complex D). Immediately after the incubation of each complex, the samples were stored on ice and then applied to the cryo-grids at a concentration of 32 nM in a cold room at 4 8C. The micrographs were recorded on a Philips FEI (Eindhoven, The Netherlands) Tecnai F20 field emission gun electron microscope at a magnification of 50,000 £ (^2%) and at 200 kV with an electron dose of ,20e 2 Å 22. Micrographs having Thon rings that showed an absence of astigmatism and drift were selected and scanned on a Zeiss-Imaging scanner (Z/I Imaging Corporation, Huntsville, Alabama, USA) with a sampling step corresponding to a pixel size of 2.82 Å on the object scale. After an automatic particle picking procedure followed by manual and cross-correlation-based selection, totals of 14,965, 18,199, 18,958 and 13,157 ribosome particles were selected for complexes A, B, C and D, respectively. Each data set was subdivided into defocus groups and analysed with the SPIDER system 21 to obtain refined three-dimensional cryo-density maps. The final resolutions of the CTF-corrected volumes were estimated by using the Fourier shell correlation coefficient with a cutoff value of 0.5, and resolutions of 11.25, 12.8, 10.9 and 14.1 Å were obtained for complexes A, B, C and D, respectively. When assessed by the 3j criterion 22 , the resolutions were in the range 7.4-9.5 Å. The final maps were amplitude-corrected by using low-angle X-ray scattering data 23 , and displayed by using IRIS Explorer (Numerical Algorithms Group Inc., Downers Grove, Illinois, USA). SPIDER was used for the computational isolation of the RF2 cryo-EM density. Docking of the X-ray structure of RF2 (pdb code 1GQE), ribosomal proteins and rRNA into the corresponding cryo-densities was performed with O (ref. 24). Individual domains of the RF2 X-ray structure were fitted into the density mass attributable to RF2, and the resulting coordinates were energy-minimized using Insight II to relieve strain from unfavourable steric interactions. Coordinates for the ribosomal proteins and rRNAs were taken from the published atomic structures for the 50S subunit 25 (pdb code 1FFK) and the 30S subunit 26 (pdb code 1FJF). Atomic coordinates for the A-site codon were obtained from ref. 27 (pdb code 1JGO). The figures were generated using IRIS Explorer, Ribbons
Cold Spring Harbor perspectives in biology, Jan 14, 2018
In the past 4 years, because of the advent of new cameras, many ribosome structures have been sol... more In the past 4 years, because of the advent of new cameras, many ribosome structures have been solved by cryoelectron microscopy (cryo-EM) at high, often near-atomic resolution, bringing new mechanistic insights into the processes of translation initiation, peptide elongation, termination, and recycling. Thus, cryo-EM has joined X-ray crystallography as a powerful technique in structural studies of translation. The significance of this new development is that structures of ribosomes in complex with their functional binding partners can now be determined to high resolution in multiple states as they perform their work. The aim of this article is to provide an overview of these new studies and assess the contributions they have made toward an understanding of translation and translational control.
Ribosomal subunit association is a key checkpoint in translation initiation but its structural dy... more Ribosomal subunit association is a key checkpoint in translation initiation but its structural dynamics are poorly understood. Here, we used a recently developed mixing-spraying, time-resolved, cryogenic electron microscopy (cryo-EM) method to study ribosomal subunit association in the sub-second time range. We have improved this method and increased the cryo-EM data yield by tenfold. Pre-equilibrium states of the association reaction were captured by reacting the mixture of ribosomal subunits for 60 ms and 140 ms. We also identified three distinct ribosome conformations in the associated ribosomes. The observed proportions of these conformations are the same in these two time points, suggesting that ribosomes equilibrate among the three conformations within less than 60 ms upon formation. Our results demonstrate that the mixing-spraying method can capture multiple states of macromolecules during a sub-second reaction. Other fast processes, such as translation initiation, decoding, ...
Large T antigen is the replicative helicase of simian virus 40. Its speci®c binding to the origin... more Large T antigen is the replicative helicase of simian virus 40. Its speci®c binding to the origin of replication and oligomerization into a double hexamer distorts and unwinds dsDNA. In viral replication, T antigen acts as a functional homolog of the eukaryotic minichromosome maintenance factor MCM. T antigen is also an oncoprotein involved in transformation through interaction with p53 and pRb. We obtained the three-dimensional structure of the fulllength T antigen double hexamer assembled at its origin of replication by cryoelectron microscopy and single-particle reconstruction techniques. The double hexamer shows different degrees of bending along the DNA axis. The two hexamers are differentiated entities rotated relative to each other. Isolated strands of density, putatively assigned to ssDNA, protrude from the hexamer±hexamer junction mainly at two opposite sites. The structure of the T antigen at the origin of replication can be understood as a snapshot of the dynamic events leading to DNA unwinding. Based on these results a model for the initiation of simian virus 40 DNA replication is proposed.
Single particle analysis in electron microscopy allows direct observation of the reconstructed th... more Single particle analysis in electron microscopy allows direct observation of the reconstructed three-dimensional structures of protein molecules. This method enables a more comprehensive study of membrane proteins which have been problematic in structural studies using X-ray crystallography. These membrane proteins include the voltage-sensitive ion channel proteins, which play an important rule in neural activities, and have great medical significance. The method described is supported by the development of cryo-electron microscopy and the angular reconstitution method. This review summarizes certain principles governing single particle analysis employing angular reconstitution. This method was applied to our study of the voltage-sensitive sodium channel, and the results are discussed. With improvements in resolutions and statistical analyses, the single particle technique is considered to be advantageous in studies of the structural changes and molecular interactions of protein molecules.
Abstract: This essay is a reflection on the ways the X‐ray structures of the ribosome are helping... more Abstract: This essay is a reflection on the ways the X‐ray structures of the ribosome are helping in the interpretation of cryogenic electron microscopy (cryo‐EM) density maps showing the translating ribosome in motion. Through advances in classification methods, cryo‐EM and single‐particle reconstruction methods have recently evolved to the point where they can yield an array of structures from a single sample (“story in a sample”), providing snapshots of an entire subprocess of translation, such as translocation or decoding.
Mitochondrial diseases produce profound neurological dysfunction via mutations affecting mitochon... more Mitochondrial diseases produce profound neurological dysfunction via mutations affecting mitochondrial energy production, including the relatively common Leigh syndrome (LS). We recently described an LS case caused by a pathogenic mutation in USMG5, encoding a small supernumerary subunit of mitochondrial ATP synthase. This protein is integral for ATP synthase dimerization, and patient fibroblasts revealed an almost total loss of ATP synthase dimers. Here, we utilize in situ cryoelectron tomography (cryo-ET) in a clinical case-control study of mitochondrial disease to directly study mitochondria within cultured fibroblasts from a patient with LS and a healthy human control subject. Through tomographic analysis of patient and control mitochondria, we find that loss of ATP synthase dimerization due to the pathogenic mutation causes profound disturbances of mitochondrial crista ultrastructure. Overall, this work supports the crucial role of ATP synthase in regulating crista architecture...
The three-dinensional structure of the large (50S) ribosomal subunit from Escherichia coli has be... more The three-dinensional structure of the large (50S) ribosomal subunit from Escherichia coli has been determined from electron micrographs of negatively stained specimens. A new method of three-dimensional reconstruction was used which combines many images of individual subunits recorded at a single high tilt angle. A prominent feature of the reconstruction is a large groove on the side of the subunit that interacts with the small ribosomal subunit. This feature is probably of functional significance as it includes the regions where the peptidyl transferase site and the binding locations of the elongation factors have been mapped previously by immunoelectron microscopy.
A previously developed theory of three-dimensional (3-D) variance estimation [J. Opt. Soc. Am. A ... more A previously developed theory of three-dimensional (3-D) variance estimation [J. Opt. Soc. Am. A 12, 2615-2627 (1995)] is applied to the structural study of a hemocyanin-Fab complex with the electron microscope. The precise locations of structurally variable regions of the macromolecule are determined from the 3-D variance maps. The structural differences among different classes of the macromolecular complex are assessed by the use of the statistical t-test, and the 3-D antibody binding sites are revealed. From a model analysis, a rule is demonstrated for visually identifying a 3-D conformational change by the inspection of the 3-D variance map. Our analysis lays the foundation for numerous practical applications of variance estimation in the 3-D imaging of macromolecules.
Fidelity of tomographic reconstructions is improved and reconstruction artifacts are reduced, wit... more Fidelity of tomographic reconstructions is improved and reconstruction artifacts are reduced, without increasing the number of projections, by combining tilt series taken around two orthogonal axes. Test reconstructions were made from high-voltage EM of rat liver mitochondria in a 0.6 pm thick plastic section. A number of schemes for selecting tilt angles for the projections are compared. A new method for aligning fiducial markers is described. It uses an iterative algorithm to determine the shift, scale, in-plane rotation and tilt angle for each tilt image, enforcing agreement of the expected locations of the fiducial markers in 3D space. These 3D locations are used to find the orientation between two tilt series and to merge both sets of projections.
In this paper, we review current practices for establishing the resolution in single-particle rec... more In this paper, we review current practices for establishing the resolution in single-particle reconstructions. The classical Raleigh criterion for the resolution is not applicable in this case, and the resolution is commonly defined by a consistency test, whereby the data set is randomly split in half and the two resulting reconstructions are then compared. Such a procedure, however, may introduce statistical dependence between the two half-sets, which leads to a too optimistic resolution estimate. On the other hand, this overestimation is counteracted by the diminished statistical properties of a mere half of the data set. The ''true'' resolution of the whole data set can be estimated when the functional relationship between the data size and the resolution is known. We are able to estimate this functional by taking into account the B-factor and the geometry of data collection. Finally, the drawbacks of resolution estimation are entirely avoided by computing the correlation of neighboring voxels in the Fourier domain.
This Meeting Review describes the proceedings and conclusions from the inaugural meeting of the E... more This Meeting Review describes the proceedings and conclusions from the inaugural meeting of the Electron Microscopy Validation Task Force organized by the Unified Data Resource for 3DEM (http://www. emdatabank.org) and held at Rutgers University in New Brunswick, NJ on September 28 and 29, 2010. At the workshop, a group of scientists involved in collecting electron microscopy data, using the data to determine three-dimensional electron microscopy (3DEM) density maps, and building molecular models into the maps explored how to assess maps, models, and other data that are deposited into the Electron Microscopy Data Bank and Protein Data Bank public data archives. The specific recommendations resulting from the workshop aim to increase the impact of 3DEM in biology and medicine.
Initiation of protein synthesis in eukaryotes requires recruitment of the 40 S ribosomal subunit ... more Initiation of protein synthesis in eukaryotes requires recruitment of the 40 S ribosomal subunit to the messenger RNA (mRNA). In most cases, this depends on recognition of a modified nucleotide cap on the 5′ end of the mRNA. However, an alternate pathway uses a structured RNA element in the 5′ untranslated region of the messenger or viral RNA called an internal ribosomal entry site (IRES). Here, we present a cryo-electron microscopy map of the hepatitis C virus (HCV) IRES bound to the 40 S ribosomal subunit at about 20 Å resolution. IRES binding induces a pronounced conformational change in the 40 S subunit and closes the mRNA binding cleft, suggesting a mechanism for IRES-mediated positioning of mRNA in the ribosomal decoding center.
Proceedings of the National Academy of Sciences, 1983
The outer membrane of mitochondria contains proteins that form channels called VDAC (voltage-depe... more The outer membrane of mitochondria contains proteins that form channels called VDAC (voltage-dependent anion-selective channels). Two independent lines of evidence suggest that these channels occur in specific complexes in the outer membrane of Neurospora mitochondria. Electron microscopic images of these outer membranes reveal polymorphic crystalline arrays of putative pores. These arrays can be shown to be interrelated by movement in the membrane plane of a particular rigid channel triplet or of regular aggregates of this triplet. Detergent extracts of the same outer membranes induce single- and multiple-step conductances in planar phospholipid membranes, with a marked preference for the insertion of triplets and multiples of triplets. The tendency of the mitochondrial channels to occur as extended arrays, apparently built up from triplets, may have important functional and evolutionary implications.
Proceedings of the National Academy of Sciences, 2012
Eukaryotic translation termination results from the complex functional interplay between two euka... more Eukaryotic translation termination results from the complex functional interplay between two eukaryotic release factors, eRF1 and eRF3, and the ribosome, in which GTP hydrolysis by eRF3 couples codon recognition with peptidyl-tRNA hydrolysis by eRF1. Here, using cryo-electron microscopy (cryo-EM) and flexible fitting, we determined the structure of eRF1–eRF3–guanosine 5′-[β,γ-imido]triphosphate (GMPPNP)-bound ribosomal pretermination complex (pre-TC), which corresponds to the initial, pre-GTP hydrolysis stage of factor attachment. Our results show that eukaryotic translation termination involves a network of interactions between the two release factors and the ribosome. Our structure provides mechanistic insight into the coordination between GTP hydrolysis by eRF3 and subsequent peptide release by eRF1.
Group II self-splicing introns are phylogenetically diverse retroelements that are widely held to... more Group II self-splicing introns are phylogenetically diverse retroelements that are widely held to be the ancestors of spliceosomal introns and retrotransposons that insert into DNA. Folding of group II intron RNA is often guided by an intron-encoded protein to form a catalytically active ribonucleoprotein (RNP) complex that plays a key role in the activity of the intron. To date, possible structural differences between the intron RNP in its precursor and spliced forms remain unexplored. In this work, we have trapped the native Lactococcus lactis group II intron RNP complex in its precursor form, by deleting the adenosine nucleophile that initiates splicing. Sedimentation velocity, size-exclusion chromatography and cryo-electron microscopy provide the first glimpse of the intron RNP precursor as a large, loosely packed structure. The dimensions contrast with those of compact spliced introns, implying that the RNP undergoes a dramatic conformational change to achieve the catalytically active state.
This protocol details the steps used for visualizing the frozen-hydrated grids as prepared follow... more This protocol details the steps used for visualizing the frozen-hydrated grids as prepared following the accompanying protocol entitled 'Preparation of macromolecular complexes for visualization using cryo-electron microscopy.' This protocol describes how to transfer the grid to the microscope using a standard cryo-transfer holder or, alternatively, using a cryo-cartridge loading system, and how to collect low-dose data using an FEI Tecnai transmission electron microscope. This protocol also summarizes and compares the various options that are available in data collection for threedimensional (3D) single-particle reconstruction. These options include microscope settings, choice of detectors and data collection strategies both in situations where a 3D reference is available and in the absence of such a reference (random-conical and common lines).
This protocol describes the preparation of frozen-hydrated single-particle specimens of macromole... more This protocol describes the preparation of frozen-hydrated single-particle specimens of macromolecular complexes. First, it describes how to create a grid surface coated with holey carbon by first inducing holes in a Formvar film to act as a template for the holey carbon that is stable under cryo-electron microscopy (cryo-EM) conditions and is sample-friendly. The protocol then describes the steps required to deposit the homogeneous sample on the grid and to plunge-freeze the grid into liquid ethane at the temperature of liquid nitrogen, so that it is suitable for cryo-EM visualization. It takes 4-5 h to make several hundred holey carbon grids and about 1 h to make the frozen-hydrated grids. The time required for sample purification varies from hours to days, depending on the sample and the specific procedure required. A companion protocol details how to collect cryo-EM data using an FEI Tecnai transmission electron microscope that can subsequently be processed to obtain a threedimensional reconstruction of the macromolecular complex.
This protocol describes the reconstruction of biological molecules from the electron micrographs ... more This protocol describes the reconstruction of biological molecules from the electron micrographs of single particles. Computation here is performed using the image-processing software SPIDER and can be managed using a graphical user interface, termed the SPIDER Reconstruction Engine. Two approaches are described to obtain an initial reconstruction: random-conical tilt and common lines. Once an existing model is available, reference-based alignment can be used, a procedure that can be iterated. Also described is supervised classification, a method to look for homogeneous subsets when multiple known conformations of the molecule may coexist.
occupancy of RF2(GAQ) factor was assumed to be the same as for the wild type, because the factors... more occupancy of RF2(GAQ) factor was assumed to be the same as for the wild type, because the factors have similar dissociation constant (K d) values of 0.7 and 1.8 nM, respectively. Complex A served as the control for complex C, whereas the control for complex B was obtained by incubation of complex A (0.13 mM) with puromycin (0.4 mM) in the same polymix buffer for 2 min at 37 8C (complex D). Immediately after the incubation of each complex, the samples were stored on ice and then applied to the cryo-grids at a concentration of 32 nM in a cold room at 4 8C. The micrographs were recorded on a Philips FEI (Eindhoven, The Netherlands) Tecnai F20 field emission gun electron microscope at a magnification of 50,000 £ (^2%) and at 200 kV with an electron dose of ,20e 2 Å 22. Micrographs having Thon rings that showed an absence of astigmatism and drift were selected and scanned on a Zeiss-Imaging scanner (Z/I Imaging Corporation, Huntsville, Alabama, USA) with a sampling step corresponding to a pixel size of 2.82 Å on the object scale. After an automatic particle picking procedure followed by manual and cross-correlation-based selection, totals of 14,965, 18,199, 18,958 and 13,157 ribosome particles were selected for complexes A, B, C and D, respectively. Each data set was subdivided into defocus groups and analysed with the SPIDER system 21 to obtain refined three-dimensional cryo-density maps. The final resolutions of the CTF-corrected volumes were estimated by using the Fourier shell correlation coefficient with a cutoff value of 0.5, and resolutions of 11.25, 12.8, 10.9 and 14.1 Å were obtained for complexes A, B, C and D, respectively. When assessed by the 3j criterion 22 , the resolutions were in the range 7.4-9.5 Å. The final maps were amplitude-corrected by using low-angle X-ray scattering data 23 , and displayed by using IRIS Explorer (Numerical Algorithms Group Inc., Downers Grove, Illinois, USA). SPIDER was used for the computational isolation of the RF2 cryo-EM density. Docking of the X-ray structure of RF2 (pdb code 1GQE), ribosomal proteins and rRNA into the corresponding cryo-densities was performed with O (ref. 24). Individual domains of the RF2 X-ray structure were fitted into the density mass attributable to RF2, and the resulting coordinates were energy-minimized using Insight II to relieve strain from unfavourable steric interactions. Coordinates for the ribosomal proteins and rRNAs were taken from the published atomic structures for the 50S subunit 25 (pdb code 1FFK) and the 30S subunit 26 (pdb code 1FJF). Atomic coordinates for the A-site codon were obtained from ref. 27 (pdb code 1JGO). The figures were generated using IRIS Explorer, Ribbons
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