PubMed | MRC LMB and University of California at San Francisco
Type: Journal Article | Journal: Cell | Year: 2016
In eukaryotes, accurate protein synthesis relies on a family of translational GTPases that pair with specific decoding factors to decipher the mRNA code on ribosomes. We present structures of the mammalian ribosome engaged with decoding factorGTPase complexes representing intermediates of translationelongation (aminoacyl-tRNAeEF1A), termination (eRF1eRF3), and ribosome rescue (PelotaHbs1l). Comparative analyses reveal that each decoding factor exploits the plasticity of the ribosomal decoding center to differentially remodel ribosomal proteins and rRNA. This leads to varying degrees of large-scale ribosome movements and implies distinct mechanisms for communicating information from the decoding center to each GTPase.Additional structural snapshots of the translationtermination pathway reveal the conformational changes that choreograph the accommodation of decoding factors into the peptidyl transferase center. Our results provide a structural framework forhow different states of the mammalian ribosome are selectively recognized by the appropriate decoding factorGTPase complex to ensure translational fidelity.
PubMed | Imperial College London, MRC LMB, University of Manchester and King's College London
Type: | Journal: Scientific reports | Year: 2016
The Melanoma-Associated Antigen A4 (MAGE-A4) protein is a target for cancer therapy. The function of this protein is not well understood. We report the first comprehensive study on key cancer-associated MAGE-A4 mutations and provide analysis on the consequences of these mutations on the structure, folding and stability of the protein. Based on Nuclear Magnetic Resonance and Circular Dichroism, these mutations had no significant effects on the structure and the folding of the protein. Some mutations affected the thermal stability of the protein remarkably. Native mass spectrometry of wild-type MAGE-A4 showed a broad charge state distribution suggestive of a structurally dynamic protein. Significant intensity was found in relatively low charge states, indicative of a predominantly globular form and some population in more extended states. The latter is supported by Ion Mobility measurements. The MAGE-A4 mutants exhibited similar features. These novel molecular insights shed further light on better understanding of these proteins, which are implicated in a wide range of human cancers.
PubMed | Howard Hughes Medical Institute, Lawrence Berkeley National Laboratory, Purdue University, Massachusetts Institute of Technology and 9 more.
Type: Journal Article | Journal: Journal of structural biology | Year: 2015
Image formation in bright field electron microscopy can be described with the help of the contrast transfer function (CTF). In this work the authors describe the CTF Estimation Challenge, called by the Madrid Instruct Image Processing Center (I2PC) in collaboration with the National Center for Macromolecular Imaging (NCMI) at Houston. Correcting for the effects of the CTF requires accurate knowledge of the CTF parameters, but these have often been difficult to determine. In this challenge, researchers have had the opportunity to test their ability in estimating some of the key parameters of the electron microscope CTF on a large micrograph data set produced by well-known laboratories on a wide set of experimental conditions. This work presents the first analysis of the results of the CTF Estimation Challenge, including an assessment of the performance of the different software packages under different conditions, so as to identify those areas of research where further developments would be desirable in order to achieve high-resolution structural information.
News Article | November 9, 2015
To determine the high-resolution structure of some biomolecules, scientists use the well-established technique X-ray crystallography. But for that century-old method to work, they must first grow large crystals of the substances—one-tenth of a millimeter thick and larger, in most cases. Some biomolecules, however, are difficult to work with and form crystals that are too small to analyze this way. A report now shows that a technique called microED (microelectron diffraction) can determine the structures of biomolecules at atomic resolution by probing crystals with only about one-millionth the volume of those needed for X-ray crystallography. In the study, Tamir Gonen of Howard Hughes Medical Institute’s Janelia Research Campus; David S. Eisenberg of the University of California, Los Angeles; and coworkers used microED to determine the structures of aggregates of two peptides from α-synuclein that play key roles in Parkinson’s disease (Nature 2015, DOI: 10.1038/nature15368). The structures they obtained are similar to those uncovered by research teams studying amyloid-forming peptides involved in other neurodegenerative diseases, notes Michel Goedert of the Medical Research Council Laboratory of Molecular Biology (MRC LMB), in Cambridge, England, in a Nature commentary. But they also provide new information that could aid in the development of potential agents to fight Parkinson’s that inhibit α-synuclein aggregate formation. X-ray crystallography requires relatively large crystals because X-rays quickly damage small ones, destroying samples before they can be analyzed effectively. X-ray free-electron laser (XFEL) diffraction instruments use ultrafast pulses that can analyze crystals about one-thousandth the volume of those probed by conventional X-ray crystallography, but XFEL instruments are rare and expensive to use. Single-particle cryoelectron microscopy, which has become increasingly popular in recent years, doesn’t require crystals at all but is limited to analyzing only large biomolecules. In 2013, Gonen’s group at Janelia developed microED, which is in the cryoelectron microscopy family of techniques. The electron beam used by the method interacts with molecules more “softly” than X-rays do but is still damaging. Gonen and coworkers made the technique work by stepping down the power of the electron beam to extremely low levels, permitting sufficient signal to be collected to derive structures. In the new work, the Gonen-Eisenberg team used microED to analyze crystals so small that they can’t be visualized by conventional light microscopy. Gonen’s group has tested microED on enzymes, such as lysozyme, with well-known structures. But the new study is the first to apply the technique to previously unknown structures. MicroED determined the α-synuclein peptide aggregate structures at 1.4-Å resolution—the best resolution ever achieved by any cryoelectron microscopy technique. Cryoelectron microscopy instruments are relatively inexpensive and already widely available in many laboratories, so crystallographers could eventually use microED routinely. Disadvantages of the technique include an inability to analyze large crystals in addition to small ones and problems with phasing, a process needed to calculate structures. This article has been translated into Spanish by Divulgame.org and can be found here.
News Article | September 28, 2016
No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Mitochondria were isolated from ovine heart tissue according to procedure 3 of ref. 44 and stored at −80 °C. Before supercomplex extraction with digitonin, frozen mitochondria were thawed on ice and washed by resuspension to a final concentration of ~6 mg protein per ml by manual homogenization in milliQ (18 MΩ) water to which KCl was added to a final concentration of 150 mM. Next, the membranes were pelleted by centrifugation at 32,000g for 45 min, followed by a second wash with resuspension in buffer M (20 mM Tris-HCl pH 7.4, 50 mM NaCl, 1 mM EDTA, 10% v/v glycerol, 2 mM DTT and 0.005% PMSF) by manual homogenization to a concentration of ~4 mg protein per ml and centrifugation at 32,000g for 45 min. Finally, membranes were resuspended in buffer M at ~10 mg protein per ml and either refrozen for storage at −80 °C or used directly for preparation of supercomplexes. Supercomplexes were isolated from the washed mitochondrial membranes by digitonin extraction followed by sucrose gradient ultracentrifugation, as described previously18, 45 with slight modifications. Briefly, an aliquot of washed mitochondrial membranes, containing ~5 mg of total protein, was resuspended in 2 ml buffer MX (150 mM potassium acetate, 30 mM HEPES pH 7.7, 10% glycerol and 0.002% PMSF) by manual homogenization. To the mitochondrial suspension, 1 ml of a 3% digitonin solution was added giving a final detergent concentration of 1% and a detergent to protein ratio of 6:1 (w:w). The sample was then agitated by rotation at 4 °C for 30 min before centrifugation at ~16,000g for 10 min. The supernatant was then concentrated to 0.5 ml and applied to linear sucrose gradients (10–45% sucrose in 15 mM HEPES pH 7.7, 20 mM KCl) prepared on a BioComp Gradient Station. The gradients were spun at 130,000g for 21 h, then fractionated. The fractions were run on BN–PAGE using linear gradient gels (4–20% polyacrylamide) in order to visualize the protein content (Extended Data Fig. 1). Fractions containing respirasomes were run over a PD-10 desalting column equilibrated in 100 mM NaCl, 20 mM HEPES pH 7.7, 0.1% digitonin and allowed to stand for ~1 h at room temperature until a white precipitate formed from the excess digitonin. The precipitate was pelleted by centrifugation and the supernatant was concentrated to an absorbance of ~2.8 absorbance units (AU) at 480 nm, which corresponds to about ~2.0 mg ml−1. Aliquots of 2.7 μl of the isolated supercomplexes were applied to Quantifoil Cu R0.6/1, 300 mesh holey carbon film grids, which were glow-discharged in air for 120 s at 25 mA. Using FEI Vitrobot MKIII, the grids were blotted for 32 s at 4 °C at 100% humidity, plunged into liquid ethane and stored in liquid nitrogen. The grids were loaded onto a FEI Titan Krios transmission electron microscope (MRC LMB, Cambridge, UK) operated at 300 kV. Images were collected using EPU software on a Falcon-II detector at a calibrated magnification of ×81,395 (pixel size of 1.72 Å) and a dose rate of 17.0 electrons per Å2 per second (Extended Data Fig. 1b). Each image was exposed for a total of 4 s and dose-fractionated into 69 movie frames. A defocus range of 1.0–5.0 μm was used. Three datasets were collected, two datasets of freshly prepared supercomplexes comprising 1,178 micrographs and one dataset of overnight incubated supercomplexes of 430 micrographs for a total of 1,608 micrographs. The dataset for supercomplex particles prepared in the same fashion but with an overnight incubation at 4 °C before grid preparations was collected in order to determine if the ratio of the tight-to-loose respirasomes changes with time. In the original datasets, the distribution of particles was 51% tight, 26% loose and 22% CICIII (of 29,659 total good particles after 3D classification). For the particles incubated overnight, the distribution was 26% tight, 49% loose and 25% CICIII (of 12,474 total good particles). Both supercomplex datasets were combined for the calculation of the final structures. All processing steps were performed using RELION v.1.4 (ref. 46) unless otherwise stated. A subset of ~2,000 particles were picked manually from 4-s averaged images, extracted using 2962 pixel box and subjected to reference-free 2D classification. Representative 2D classes were then used as reference images for automatic particle picking of all micrographs. The automatically picked particles were manually screened to remove false positives and pick any additional particles that were missed, resulting in an initial dataset of 67,650. MOTIONCORR47 was used for whole-image drift correction of movie frames 1–32 of each micrograph (the remaining frames were not used subsequently). Contrast transfer function (CTF) parameters of the corrected micrographs were estimated using Gctf and refined locally for each particle48. The particles were extracted using 2962 pixel box and sorted by reference-free 2D classification. We selected 54,484 particles from good 2D classes for the 3D classification (Extended Data Fig. 1c, d), which was run for 15 iterations, using an angular sampling of 1.8°, a regularization parameter T of 8 and a 30 Å low-pass filtered initial model from a previous low-resolution structure of the respirasome19, with a soft mask around the model. 3D classification was then continued as above for 15 iterations with an angular sampling of 0.9° and finally for 20 iterations with an angular sampling of 0.5°. This resulted in three good classes (tight, loose and supercomplex I–III) and a class of bad particles with no clearly resolved features (Extended Data Fig. 1d). A subset of 42,133 particles including the three good classes was selected for the first 3D auto-refinement. This subset of particles were re-extracted from the motion corrected micrographs with a 4962 pixel box (to allow for high-resolution CTF correction49), and all further refinement was performed using this box size. After initial auto-refinement, a particle-based beam-induced motion correction and radiation-damage weighing (particle polishing) was performed (Extended Data Fig. 2e, f)50. Auto-refinement of all the polished particles together resulted in a reconstruction at 6.1 Å overall resolution with an estimated angular accuracy of 0.8°. The particles were then split into their respective 3D classes (tight, 18,397 particles; loose, 13,910 particles; and CICIII , 9,844 particles; Extended Data Fig. 1d) and auto-refinement was run for each class independently, resulting in final reconstructions at 5.8, 6.7 and 7.8 Å resolution with angular accuracies of 0.6°, 0.7° and 1.0°, respectively. All resolutions are based on the gold-standard (two halves of data refined independently) Fourier shell correlation (FSC) = 0.143 criterion51 (Extended Data Fig. 2d). FSC curves were calculated using soft masks around the protein and high-resolution noise substitution was used to correct for convolution effects of the masks on the FSC curves52. Prior to visualization, all maps were corrected for the modulation transfer function of the detector. Local resolution analysis by Resmap53 revealed a range of resolution for each supercomplex reconstruction with the highest resolution in the core of complexes I and III (Extended Data Fig. 2). The tight respirasome architecture shows resolutions ranging from 5.0 Å in the core of CI to ~8–9 Å resolution on the distal end of CIV (Extended Data Fig. 2a). The loose respirasome architecture shows resolutions ranging from 6.0 Å in the core of CI to ~10–12 Å resolution for the distal end of CIV (Extended Data Fig. 2b). Supercomplex CICIII shows 7.0 Å resolution at the core of CI ranging to ~15 Å at the peripheries of the CI and CIII matrix domains (Extended Data Fig. 2c). In Fig. 1 and Extended Data Figs 3, 4, 5, the maps have been carved in order to remove the detergent micelle and give a clear view of the transmembrane helices. Due to weaker density for CIV in Fig. 1a and Extended Data Fig. 3, it was contoured at a lower level and in Fig. 1b and Extended Data Fig. 4 it was filtered to 8 Å, whereas the maps for CI and CIII are filtered at 6.7 Å. For the CIII and CIV models, available high-resolution structures of the bovine enzymes (PDB accession codes 1BGY (ref. 54) 1NTM (ref. 43) and 1V54 (ref. 39)) were used as starting models. Mutations to the ovine sequences were made in COOT55 manually, as only few changes were needed. Sequences for ovine COX7c and COX8b were not available in the online databases and hence the bovine sequences were used. The amino acid residues in these models were truncated at the beta-carbon using CHAINSAW56 in the CCP4 program suite57 to generate a ‘poly-alanine’ model and fit into the cryo-EM density map for the tight respirasomes as a rigid body. For CI, the low-resolution poly-alanine model of the bovine enzyme was used as a starting model (PDB accession code 4UQ8 (ref. 23)). This model was fit into the tight respirasome map and manual building was performed in COOT55 using Ramachandran and secondary-structure restraints. Mammalian CI consists of 14 conserved core subunits present throughout the species, including bacteria, and 31 mitochondria-specific supernumerary subunits (29 unique supernumerary subunits and two copies of the acyl carrier protein SDAP). As noted before23, core subunits retain the fold and architecture originally characterized in bacteria29, 58, 59. Building was guided largely by secondary structure predictions and homology structure predictions generated using the programs PSIPRED60, 61 and Phyre2 (ref. 62). We improved the completeness of the poly-alanine model for all currently assigned subunits (with the exception of the B8 and SDAP subunits whose structures are known from homologues63, 64) and assigned three additional subunits (Extended Data Fig. 7). B18 and PDSW are both found on the intermembrane space side of CI and both contain strong secondary structure predictions for helix-turn-helix motifs with B18 containing a double CX C CHCH domain65. Two regions of density were previously identified as probable candidates for either of these subunits, but a definitive assignment between the two was not made66. Based on secondary structure prediction, which differentiates the subunits by their helical structure at the C terminus, we assigned B18 to the density near the interface of CI and IV that extends a long (~30 amino acid residues) helix into the interface of the three complexes (Fig. 2 and Extended Data Fig. 7). This means that the helical density found underneath ND4 and ND5 corresponds to PDSW, which is predicted to have an additional shorter C-terminal α-helix (15–20 amino acid residues) following its predicted helix-turn-helix motif. Density for an α-helix (which we suggest belongs to the PDSW subunit) can be seen near the end of the PDSW helix-turn-helix motif that protrudes from CI towards CIII in the supercomplex structures. Additionally, we assigned and built subunit B17.2. B17.2 contains strong secondary structure prediction on its N terminus for two short helices followed by a 3–4 strand β-sheet, then by a long coil (~70 amino acid residues) with no predicted secondary structure. Starting from a model generated by homology structure prediction using Phyre2 (ref. 62), we were able to fit B17.2 into density adjacent to the 49-kDa, TYKY and PSST subunits of the CI Q-modules. After manual adjustment of the secondary structure elements from the predicted homology model, density could be seen extending away from the C terminus, which clearly belonged to the B17.2 C-terminal coil. These C-terminal residues snake along the surface of the peripheral arm and extend towards the N-module. This extended coil connecting the N- and Q-modules of the hydrophilic arm speaks to the role of B17.2 and its homologue B17.2L in CI assembly67. In subsequent work with isolated ovine CI, we assigned all remaining CI subunits32 and, where relevant, these assignments (B15, B12 and AGGG) are used here. A single round of real space refinement (morphing plus minimization) was performed using PHENIX real space refine68 for each complex individually in the tight respirasome map. Each of the refined complexes were fit into the lower resolution loose respirasome and CICIII maps as rigid bodies. Figures showing the fits of each complex into the density are shown in Extended Data Figs 3, 4, 5 for each map.
Shvets E.,MRC LMB |
Ludwig A.,Nanyang Technological University |
Nichols B.J.,MRC LMB
Current Opinion in Cell Biology | Year: 2014
Recent data from the study of the cell biology of caveolae have provided insights both into how these flask-shaped invaginations of the plasma membrane are formed and how they may function in different contexts. This review discusses experiments that analyse the composition and ultrastructural distribution of protein complexes responsible for generating caveolae, that suggest functions for caveolae in response to mechanical stress or damage to the plasma membrane, that show that caveolae may have an important role during the signalling events for regulation of metabolism, and that imply that caveolae can act as endocytic vesicles at the plasma membrane. We also highlight unexpected roles for caveolar proteins in regulating circadian rhythms and new insights into the way in which caveolae may be involved in fatty acid uptake in the intestine. Current outstanding questions in the field are emphasised. © 2014 Elsevier Ltd.
Shvets E.,MRC LMB |
Bitsikas V.,MRC LMB |
Howard G.,MRC LMB |
Hansen C.G.,University of California at San Diego |
Nichols B.J.,MRC LMB
Nature Communications | Year: 2015
Caveolae have long been implicated in endocytosis. Recent data question this link, and in the absence of specific cargoes the potential cellular function of caveolar endocytosis remains unclear. Here we develop new tools, including doubly genome-edited cell lines, to assay the subcellular dynamics of caveolae using tagged proteins expressed at endogenous levels. We find that around 5% of the cellular pool of caveolae is present on dynamic endosomes, and is delivered to endosomes in a clathrin-independent manner. Furthermore, we show that caveolae are indeed likely to bud directly from the plasma membrane. Using a genetically encoded tag for electron microscopy and ratiometric light microscopy, we go on to show that bulk membrane proteins are depleted within caveolae. Although caveolae are likely to account for only a small proportion of total endocytosis, cells lacking caveolae show fundamentally altered patterns of membrane traffic when loaded with excess glycosphingolipid. Altogether, these observations support the hypothesis that caveolar endocytosis is specialized for transport of membrane lipid. © 2015 Macmillan Publishers Limited. All rights reserved.
Hansen C.G.,MRC LMB |
Nichols B.J.,MRC LMB
Trends in Cell Biology | Year: 2010
Caveolae are ampullate (flask-shaped) invaginations that are abundant in the plasma membrane of many mammalian cell types. Although caveolae are implicated in a wide range of processes including endothelial transcytosis, lipid homeostasis and cellular signalling, a detailed molecular picture of many aspects of their function has been elusive. Until recently, the only extensively characterised protein components of caveolae were the caveolins. Recently, data from several laboratories have demonstrated that a family of four related proteins, termed cavins 1-4, plays key roles in caveolar biogenesis and function. Salient properties of the cavin family include their propensity to form complexes with each other and their different but overlapping tissue distribution. This review summarises recent data on the cavins, and sets them in the context of open questions on the construction and function of caveolae. The discovery of cavins implies that caveolae might have unexpectedly diverse structural properties, in accord with the wide range of functions attributed to these 'little caves'. © 2010 Elsevier Ltd.
Fox A.M.,MRC LMB |
Ciziene D.,MRC LMB |
McLaughlin S.H.,MRC LMB |
Stewart M.,MRC LMB
Journal of Biological Chemistry | Year: 2011
The toroid-shaped nuclear protein export factor CRM1 is constructed from 21 tandem HEAT repeats, each of which contains an inner (B) and outer (A)α-helix joined by loops. Proteins targeted for export have a nuclear export signal (NES) that binds between the A-helices of HEAT repeats 11 and 12 on the outer surface of CRM1. RanGTP binding increases the affinity of CRM1 for NESs. In the absence of RanGTP, the CRM1 C-terminal helix, together with the HEAT repeat 9 loop, modulates its affinity for NESs. Here we show that there is an electrostatic interaction between acidic residues at the extreme distal tip of the C-terminal helix and basic residues on the HEAT repeat 12 B-helix that lies on the inner surface of CRM1 beneath the NES binding site. Small angle x-ray scattering indicates that the increased affinity for NESs generated by mutations in the C-terminal helix is not associated with large scale changes in CRM1 conformation, consistent with the modulation of NES affinity being mediated by a local change in CRM1 near the NES binding site. These data also suggest that in the absence of RanGTP, the C-terminal helix lies across the CRM1 toroid in a position similar to that seen in the CRM1-Snurportin crystal structure. By creating local changes that stabilize the NES binding site in its closed conformation and thereby reducing the affinity of CRM1 for NESs, the C-terminal helix and HEAT 9 loop facilitate release of NES-containing cargo in the cytoplasm and also inhibit their return to the nucleus. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
PubMed | University of California at San Diego and MRC LMB
Type: | Journal: Nature communications | Year: 2015
Caveolae have long been implicated in endocytosis. Recent data question this link, and in the absence of specific cargoes the potential cellular function of caveolar endocytosis remains unclear. Here we develop new tools, including doubly genome-edited cell lines, to assay the subcellular dynamics of caveolae using tagged proteins expressed at endogenous levels. We find that around 5% of the cellular pool of caveolae is present on dynamic endosomes, and is delivered to endosomes in a clathrin-independent manner. Furthermore, we show that caveolae are indeed likely to bud directly from the plasma membrane. Using a genetically encoded tag for electron microscopy and ratiometric light microscopy, we go on to show that bulk membrane proteins are depleted within caveolae. Although caveolae are likely to account for only a small proportion of total endocytosis, cells lacking caveolae show fundamentally altered patterns of membrane traffic when loaded with excess glycosphingolipid. Altogether, these observations support the hypothesis that caveolar endocytosis is specialized for transport of membrane lipid.