Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 26
Filter
Add more filters










Publication year range
1.
Structure ; 26(6): 848-856.e3, 2018 06 05.
Article in English | MEDLINE | ID: mdl-29754826

ABSTRACT

The advent of direct electron detectors has enabled the routine use of single-particle cryo-electron microscopy (EM) approaches to determine structures of a variety of protein complexes at near-atomic resolution. Here, we report the development of methods to account for local variations in defocus and beam-induced drift, and the implementation of a data-driven dose compensation scheme that significantly improves the extraction of high-resolution information recorded during exposure of the specimen to the electron beam. These advances enable determination of a cryo-EM density map for ß-galactosidase bound to the inhibitor phenylethyl ß-D-thiogalactopyranoside where the ordered regions are resolved at a level of detail seen in X-ray maps at ∼ 1.5 Å resolution. Using this density map in conjunction with constrained molecular dynamics simulations provides a measure of the local flexibility of the non-covalently bound inhibitor and offers further opportunities for structure-guided inhibitor design.


Subject(s)
Thiogalactosides/pharmacology , beta-Galactosidase/chemistry , beta-Galactosidase/metabolism , Binding Sites , Cryoelectron Microscopy/methods , Crystallography, X-Ray , Drug Design , Models, Molecular , Molecular Dynamics Simulation , Protein Conformation
2.
Cell ; 165(7): 1698-1707, 2016 Jun 16.
Article in English | MEDLINE | ID: mdl-27238019

ABSTRACT

Recent advances in single-particle cryoelecton microscopy (cryo-EM) are enabling generation of numerous near-atomic resolution structures for well-ordered protein complexes with sizes ≥ ∼200 kDa. Whether cryo-EM methods are equally useful for high-resolution structural analysis of smaller, dynamic protein complexes such as those involved in cellular metabolism remains an important question. Here, we present 3.8 Å resolution cryo-EM structures of the cancer target isocitrate dehydrogenase (93 kDa) and identify the nature of conformational changes induced by binding of the allosteric small-molecule inhibitor ML309. We also report 2.8-Å- and 1.8-Å-resolution structures of lactate dehydrogenase (145 kDa) and glutamate dehydrogenase (334 kDa), respectively. With these results, two perceived barriers in single-particle cryo-EM are overcome: (1) crossing 2 Å resolution and (2) obtaining structures of proteins with sizes < 100 kDa, demonstrating that cryo-EM can be used to investigate a broad spectrum of drug-target interactions and dynamic conformational states.


Subject(s)
Drug Discovery , Glutamate Dehydrogenase/ultrastructure , Isocitrate Dehydrogenase/ultrastructure , L-Lactate Dehydrogenase/ultrastructure , Aminoquinolines/chemistry , Aminoquinolines/pharmacology , Animals , Cattle , Chickens , Cryoelectron Microscopy , Crystallography, X-Ray , Glutamate Dehydrogenase/antagonists & inhibitors , Glutamate Dehydrogenase/chemistry , Humans , Isocitrate Dehydrogenase/antagonists & inhibitors , Isocitrate Dehydrogenase/chemistry , L-Lactate Dehydrogenase/antagonists & inhibitors , L-Lactate Dehydrogenase/chemistry , Models, Molecular , Protein Conformation , Sulfonamides/chemistry , Sulfonamides/pharmacology
3.
Mol Pharmacol ; 89(6): 645-51, 2016 06.
Article in English | MEDLINE | ID: mdl-27036132

ABSTRACT

Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an "open" or "closed" state. Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes.


Subject(s)
Cryoelectron Microscopy/methods , Glutamate Dehydrogenase/ultrastructure , Animals , Cattle , Crystallography, X-Ray , Glutamate Dehydrogenase/chemistry , Guanosine Triphosphate/chemistry , Guanosine Triphosphate/metabolism , Ligands , Mammals , Models, Molecular , NAD/chemistry , NAD/metabolism , Protein Structure, Quaternary
4.
Science ; 351(6275): 871-5, 2016 Feb 19.
Article in English | MEDLINE | ID: mdl-26822609

ABSTRACT

p97 is a hexameric AAA+ adenosine triphosphatase (ATPase) that is an attractive target for cancer drug development. We report cryo-electron microscopy (cryo-EM) structures for adenosine diphosphate (ADP)-bound, full-length, hexameric wild-type p97 in the presence and absence of an allosteric inhibitor at resolutions of 2.3 and 2.4 angstroms, respectively. We also report cryo-EM structures (at resolutions of ~3.3, 3.2, and 3.3 angstroms, respectively) for three distinct, coexisting functional states of p97 with occupancies of zero, one, or two molecules of adenosine 5'-O-(3-thiotriphosphate) (ATPγS) per protomer. A large corkscrew-like change in molecular architecture, coupled with upward displacement of the N-terminal domain, is observed only when ATPγS is bound to both the D1 and D2 domains of the protomer. These cryo-EM structures establish the sequence of nucleotide-driven structural changes in p97 at atomic resolution. They also enable elucidation of the binding mode of an allosteric small-molecule inhibitor to p97 and illustrate how inhibitor binding at the interface between the D1 and D2 domains prevents propagation of the conformational changes necessary for p97 function.


Subject(s)
Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphatases/chemistry , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/chemistry , Adenosine Diphosphate/chemistry , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/chemistry , Allosteric Regulation , Binding Sites , Cryoelectron Microscopy , Enzyme Inhibitors , Humans , Models, Molecular , Protein Structure, Tertiary
5.
Science ; 348(6239): 1147-51, 2015 Jun 05.
Article in English | MEDLINE | ID: mdl-25953817

ABSTRACT

Cryo-electron microscopy (cryo-EM) is rapidly emerging as a powerful tool for protein structure determination at high resolution. Here we report the structure of a complex between Escherichia coli ß-galactosidase and the cell-permeant inhibitor phenylethyl ß-D-thiogalactopyranoside (PETG), determined by cryo-EM at an average resolution of ~2.2 angstroms (Å). Besides the PETG ligand, we identified densities in the map for ~800 water molecules and for magnesium and sodium ions. Although it is likely that continued advances in detector technology may further enhance resolution, our findings demonstrate that preparation of specimens of adequate quality and intrinsic protein flexibility, rather than imaging or image-processing technologies, now represent the major bottlenecks to routinely achieving resolutions close to 2 Å using single-particle cryo-EM.


Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Thiogalactosides/chemistry , beta-Galactosidase/chemistry , Catalytic Domain , Cryoelectron Microscopy , Crystallography, X-Ray , Water/chemistry
6.
Nat Struct Mol Biol ; 20(12): 1352-7, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24154805

ABSTRACT

The activation of trimeric HIV-1 envelope glycoprotein (Env) by its binding to the cell-surface receptor CD4 and co-receptors (CCR5 or CXCR4) represents the first of a series of events that lead to fusion between viral and target-cell membranes. Here, we present the cryo-EM structure, at subnanometer resolution (~6 Å at 0.143 FSC), of the 'closed', prefusion state of trimeric HIV-1 Env complexed to the broadly neutralizing antibody VRC03. We show that three gp41 helices at the core of the trimer serve as an anchor around which the rest of Env is reorganized upon activation to the 'open' quaternary conformation. The architecture of trimeric HIV-1 Env in the prefusion state and in the activated intermediate state resembles the corresponding states of influenza hemagglutinin trimers, thus providing direct evidence for the similarity in entry mechanisms used by HIV-1, influenza and related enveloped viruses.


Subject(s)
HIV Envelope Protein gp41/chemistry , HIV-1 , Antibodies, Neutralizing/chemistry , Cryoelectron Microscopy , Models, Molecular , Protein Multimerization , Protein Structure, Tertiary , Structure-Activity Relationship , Virus Internalization
7.
J Virol ; 87(12): 7191-6, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23596305

ABSTRACT

We describe cryo-electron microscopic studies of the interaction between the ectodomain of the trimeric HIV-1 envelope glycoprotein (Env) and Z13e1, a broadly neutralizing antibody that targets the membrane-proximal external region (MPER) of the gp41 subunit. We show that Z13e1-bound Env displays an open quaternary conformation similar to the CD4-bound conformation. Our results support the idea that MPER-directed antibodies, such as Z13e1, block viral entry by interacting with Env at a step after CD4 activation.


Subject(s)
Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/metabolism , HIV Envelope Protein gp41/immunology , env Gene Products, Human Immunodeficiency Virus/chemistry , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Cryoelectron Microscopy , Humans , Models, Molecular , Neutralization Tests , Protein Binding , Protein Conformation , Protein Multimerization , env Gene Products, Human Immunodeficiency Virus/immunology , env Gene Products, Human Immunodeficiency Virus/metabolism
8.
FEBS J ; 280(1): 28-45, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23181775

ABSTRACT

Cryo-electron microscopy (cryo-EM) is increasingly becoming a mainstream technology for studying the architecture of cells, viruses and protein assemblies at molecular resolution. Recent developments in microscope design and imaging hardware, paired with enhanced image processing and automation capabilities, are poised to further advance the effectiveness of cryo-EM methods. These developments promise to increase the speed and extent of automation, and to improve the resolutions that may be achieved, making this technology useful to determine a wide variety of biological structures. Additionally, established modalities for structure determination, such as X-ray crystallography and nuclear magnetic resonance spectroscopy, are being routinely integrated with cryo-EM density maps to achieve atomic-resolution models of complex, dynamic molecular assemblies. In this review, which is directed towards readers who are not experts in cryo-EM methodology, we provide an overview of emerging themes in the application of this technology to investigate diverse questions in biology and medicine. We discuss the ways in which these methods are being used to study structures of macromolecular assemblies that range in size from whole cells to small proteins. Finally, we include a description of how the structural information obtained by cryo-EM is deposited and archived in a publicly accessible database.


Subject(s)
Cryoelectron Microscopy/methods , Animals , Electron Microscope Tomography , Fourier Analysis , Humans , Imaging, Three-Dimensional , Macromolecular Substances/ultrastructure , Proteins/ultrastructure
9.
PLoS Pathog ; 8(7): e1002797, 2012.
Article in English | MEDLINE | ID: mdl-22807678

ABSTRACT

HIV-1 infection begins with the binding of trimeric viral envelope glycoproteins (Env) to CD4 and a co-receptor on target T-cells. Understanding how these ligands influence the structure of Env is of fundamental interest for HIV vaccine development. Using cryo-electron microscopy, we describe the contrasting structural outcomes of trimeric Env binding to soluble CD4, to the broadly neutralizing, CD4-binding site antibodies VRC01, VRC03 and b12, or to the monoclonal antibody 17b, a co-receptor mimic. Binding of trimeric HIV-1 BaL Env to either soluble CD4 or 17b alone, is sufficient to trigger formation of the open quaternary conformation of Env. In contrast, VRC01 locks Env in the closed state, while b12 binding requires a partial opening in the quaternary structure of trimeric Env. Our results show that, despite general similarities in regions of the HIV-1 gp120 polypeptide that contact CD4, VRC01, VRC03 and b12, there are important differences in quaternary structures of the complexes these ligands form on native trimeric Env, and potentially explain differences in the neutralizing breadth and potency of antibodies with similar specificities. From cryo-electron microscopic analysis at ∼9 Å resolution of a cleaved, soluble version of trimeric Env, we show that a structural signature of the open Env conformation is a three-helix motif composed of α-helical segments derived from highly conserved, non-glycosylated N-terminal regions of the gp41 trimer. The three N-terminal gp41 helices in this novel, activated Env conformation are held apart by their interactions with the rest of Env, and are less compactly packed than in the post-fusion, six-helix bundle state. These findings suggest a new structural template for designing immunogens that can elicit antibodies targeting HIV at a vulnerable, pre-entry stage.


Subject(s)
HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/metabolism , HIV-1/metabolism , Receptors, HIV/metabolism , Antibodies, Monoclonal/metabolism , Binding Sites , Binding Sites, Antibody , CD4 Antigens/metabolism , Cryoelectron Microscopy , HIV Envelope Protein gp120/immunology , HIV-1/ultrastructure , Ligands , Models, Molecular , Molecular Mimicry , Protein Binding , Protein Multimerization , Protein Structure, Quaternary , Protein Structure, Secondary , Recombinant Proteins/metabolism
10.
J Virol ; 85(23): 12114-23, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21937655

ABSTRACT

The trimeric envelope glycoprotein (Env) spikes displayed on the surfaces of simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) virions are composed of three heterodimers of the viral glycoproteins gp120 and gp41. Although binding of gp120 to cell surface CD4 and a chemokine receptor is known to elicit conformational changes in gp120 and gp41, changes in quaternary structure of the trimer have only recently been elucidated. For the HIV-1 BaL isolate, CD4 attachment results in a striking rearrangement of the trimer from a "closed" to an "open" conformation. The effect of CD4 on SIV trimers, however, has not been described. Using cryo-electron tomography, we have now determined molecular architectures of the soluble CD4 (sCD4)-bound states of SIV Env trimers for three different strains (SIVmneE11S, SIVmac239, and SIV CP-MAC). In marked contrast to HIV-1 BaL, SIVmneE11S and SIVmac239 Env showed only minor conformational changes following sCD4 binding. In SIV CP-MAC, where trimeric Env displays a constitutively "open" conformation similar to that seen for HIV-1 BaL Env in the sCD4-complexed state, we show that there are no significant further changes in conformation upon the binding of either sCD4 or 7D3 antibody. The density maps also show that 7D3 and 17b antibodies target epitopes on gp120 that are on opposites sides of the coreceptor binding site. These results provide new insights into the structural diversity of SIV Env and show that there are strain-dependent variations in the orientation of sCD4 bound to trimeric SIV Env.


Subject(s)
CD4 Antigens/chemistry , CD4 Antigens/metabolism , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/metabolism , Animals , Antibodies, Neutralizing/immunology , CD4 Antigens/immunology , Cryoelectron Microscopy , Crystallography, X-Ray , Electron Microscope Tomography , Humans , Membrane Glycoproteins/immunology , Models, Molecular , Protein Structure, Quaternary , Receptors, CCR5/immunology , Receptors, CCR5/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Viral Envelope Proteins/immunology , Virus Internalization
11.
Proc Natl Acad Sci U S A ; 108(28): 11440-5, 2011 Jul 12.
Article in English | MEDLINE | ID: mdl-21709254

ABSTRACT

The initial step in HIV-1 infection occurs with the binding of cell surface CD4 to trimeric HIV-1 envelope glycoproteins (Env), a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120). The design of soluble versions of trimeric Env that display structural and functional properties similar to those observed on intact viruses is highly desirable from the viewpoint of designing immunogens that could be effective as vaccines against HIV/AIDS. Using cryoelectron tomography combined with subvolume averaging, we have analyzed the structure of SOSIP gp140 trimers, which are cleaved, solubilized versions of the ectodomain of trimeric HIV-1 Env. We show that unliganded gp140 trimers adopt a quaternary arrangement similar to that displayed by native unliganded trimers on the surface of intact HIV-1 virions. When complexed with soluble CD4, Fab 17b, which binds to gp120 at its chemokine coreceptor binding site, or both soluble CD4 and 17b Fab, gp140 trimers display an open conformation in which there is an outward rotation and displacement of each gp120 protomer. We demonstrate that the molecular arrangements of gp120 trimers in the closed and open conformations of the soluble trimer are the same as those observed for the closed and open states, respectively, of trimeric gp120 on intact HIV-1 BaL virions, establishing that soluble gp140 trimers can be designed to mimic the quaternary structural transitions displayed by native trimeric Env.


Subject(s)
HIV-1/chemistry , HIV-1/immunology , env Gene Products, Human Immunodeficiency Virus/chemistry , env Gene Products, Human Immunodeficiency Virus/immunology , AIDS Vaccines/chemistry , AIDS Vaccines/immunology , Biophysical Phenomena , CD4 Antigens/chemistry , Cryoelectron Microscopy , HIV Antibodies/chemistry , HIV Antigens/chemistry , HIV Antigens/immunology , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/immunology , Humans , Immunoglobulin Fab Fragments/chemistry , Ligands , Models, Molecular , Protein Structure, Quaternary , env Gene Products, Human Immunodeficiency Virus/ultrastructure
12.
J Bacteriol ; 193(6): 1341-50, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21148724

ABSTRACT

We present a cryo-electron tomographic analysis of the three-dimensional architecture of a strain of the Gram-negative bacterium Bdellovibrio bacteriovorus in which endogenous MreB2 was replaced with monomeric teal fluorescent protein (mTFP)-labeled MreB2. In contrast to wild-type Bdellovibrio cells that predominantly displayed a compact nucleoid region, cells expressing mTFP-labeled MreB2 displayed a twisted spiral organization of the nucleoid. The more open structure of the MreB2-mTFP nucleoids enabled clear in situ visualization of ribosomes decorating the periphery of the nucleoid. Ribosomes also bordered the edges of more compact nucleoids from both wild-type cells and mutant cells. Surprisingly, MreB2-mTFP localized to the interface between the spiral nucleoid and the cytoplasm, suggesting an intimate connection between nucleoid architecture and MreB arrangement. Further, in contrast to wild-type cells, where a single tight chemoreceptor cluster localizes close to the single polar flagellum, MreB2-mTFP cells often displayed extended chemoreceptor arrays present at one or both poles and displayed multiple or inaccurately positioned flagella. Our findings provide direct structural evidence for spiral organization of the bacterial nucleoid and suggest a possible role for MreB in regulation of nucleoid architecture and localization of the chemotaxis apparatus.


Subject(s)
Bdellovibrio/ultrastructure , Chromosomes, Bacterial/ultrastructure , Cryoelectron Microscopy , Genes, Reporter , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Ribosomes/ultrastructure , Staining and Labeling/methods
13.
PLoS Pathog ; 6(12): e1001249, 2010 Dec 23.
Article in English | MEDLINE | ID: mdl-21203482

ABSTRACT

The initial step in target cell infection by human, and the closely related simian immunodeficiency viruses (HIV and SIV, respectively) occurs with the binding of trimeric envelope glycoproteins (Env), composed of heterodimers of the viral transmembrane glycoprotein (gp41) and surface glycoprotein (gp120) to target T-cells. Knowledge of the molecular structure of trimeric Env on intact viruses is important both for understanding the molecular mechanisms underlying virus-cell interactions and for the design of effective immunogen-based vaccines to combat HIV/AIDS. Previous analyses of intact HIV-1 BaL virions have already resulted in structures of trimeric Env in unliganded and CD4-liganded states at ~20 Å resolution. Here, we show that the molecular architectures of trimeric Env from SIVmneE11S, SIVmac239 and HIV-1 R3A strains are closely comparable to that previously determined for HIV-1 BaL, with the V1 and V2 variable loops located at the apex of the spike, close to the contact zone between virus and cell. The location of the V1/V2 loops in trimeric Env was definitively confirmed by structural analysis of HIV-1 R3A virions engineered to express Env with deletion of these loops. Strikingly, in SIV CP-MAC, a CD4-independent strain, trimeric Env is in a constitutively "open" conformation with gp120 trimers splayed out in a conformation similar to that seen for HIV-1 BaL Env when it is complexed with sCD4 and the CD4i antibody 17b. Our findings suggest a structural explanation for the molecular mechanism of CD4-independent viral entry and further establish that cryo-electron tomography can be used to discover distinct, functionally relevant quaternary structures of Env displayed on intact viruses.


Subject(s)
HIV-1/chemistry , Protein Structure, Quaternary , Simian Immunodeficiency Virus/chemistry , Viral Envelope Proteins/chemistry , CD4 Antigens , Electron Microscope Tomography , HIV Envelope Protein gp120/chemistry , Humans , Species Specificity , Virus Internalization
14.
Nat Rev Microbiol ; 7(9): 666-75, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19668224

ABSTRACT

Recent advances in three-dimensional electron microscopy provide remarkable tools to image the interior of bacterial cells. Glimpses of cells at resolutions that are 1-2 orders of magnitude higher than those currently attained with light microscopy can now be obtained with cryo-electron tomography, especially when used in combination with new tools for image averaging. This Review highlights recent advances in this area and provides an assessment of the general applicability, current limitations and type of structural information that can be obtained about the organization of intact cells using tomography. Possible future directions for whole cell imaging are also discussed.


Subject(s)
Bacteria/ultrastructure , Cryoelectron Microscopy/methods , Electron Microscope Tomography/methods , Imaging, Three-Dimensional/methods
15.
J Struct Biol ; 166(1): 1-7, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19116171

ABSTRACT

Understanding the hierarchical organization of molecules and organelles within the interior of large eukaryotic cells is a challenge of fundamental interest in cell biology. We are using ion-abrasion scanning electron microscopy (IA-SEM) to visualize this hierarchical organization in an approach that combines focused ion-beam milling with scanning electron microscopy. Here, we extend our previous studies on imaging yeast cells to image subcellular architecture in human melanoma cells and melanocytes at resolutions as high as approximately 6 and approximately 20 nm in the directions parallel and perpendicular, respectively, to the direction of ion-beam milling. The 3D images demonstrate the striking spatial relationships between specific organelles such as mitochondria and membranes of the endoplasmic reticulum, and the distribution of unique cellular components such as melanosomes. We also show that 10nm-sized gold particles and quantum dot particles with 7 nm-sized cores can be detected in single cross-sectional images. IA-SEM is thus a useful tool for imaging large mammalian cells in their entirety at resolutions in the nanometer range.


Subject(s)
Eukaryotic Cells/ultrastructure , Imaging, Three-Dimensional/methods , Microscopy, Electron, Scanning/methods , Cell Line, Tumor , Cytological Techniques/methods , Gold , Humans , Intracellular Membranes/ultrastructure , Melanocytes/ultrastructure , Melanoma/ultrastructure , Melanosomes/ultrastructure , Microscopy, Electron, Transmission , Mitochondria/ultrastructure , Organelles/ultrastructure , Quantum Dots , Staining and Labeling
16.
J Struct Biol ; 164(1): 7-17, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18585059

ABSTRACT

Tomograms of biological specimens derived using transmission electron microscopy can be intrinsically noisy due to the use of low electron doses, the presence of a "missing wedge" in most data collection schemes, and inaccuracies arising during 3D volume reconstruction. Before tomograms can be interpreted reliably, for example, by 3D segmentation, it is essential that the data be suitably denoised using procedures that can be individually optimized for specific data sets. Here, we implement a systematic procedure to compare various nonlinear denoising techniques on tomograms recorded at room temperature and at cryogenic temperatures, and establish quantitative criteria to select a denoising approach that is most relevant for a given tomogram. We demonstrate that using an appropriate denoising algorithm facilitates robust segmentation of tomograms of HIV-infected macrophages and Bdellovibrio bacteria obtained from specimens at room and cryogenic temperatures, respectively. We validate this strategy of automated segmentation of optimally denoised tomograms by comparing its performance with manual extraction of key features from the same tomograms.


Subject(s)
Algorithms , Artifacts , Electron Microscope Tomography/methods , Image Processing, Computer-Assisted/methods , Animals , Artificial Intelligence , Bdellovibrio/cytology , Electron Microscope Tomography/standards , HIV Infections/pathology , Humans , Image Processing, Computer-Assisted/standards , Macrophages/pathology , Macrophages/virology
17.
Nanomedicine (Lond) ; 3(1): 125-31, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18393671

ABSTRACT

A wide range of medically important nanosized biological assemblies are not amenable to study by standard structural techniques, such as x-ray crystallography or nuclear magnetic resonance spectroscopy, either owing to their large size or the intrinsic heterogeneity of the specimen. The emerging technique of cryo-electron tomography is being applied actively to study these nanoparticles and has the potential of providing high-resolution structural information on these heterogeneous assemblies. Although the majority of structural methods involve the averaging of large numbers of structurally homogeneous molecules, tomography enables the visualization and quantitation of variation in a mixed population. Here, we present a review of the principles of cryo-electron tomography as applied to the 3D analysis of nanoparticles and illustrate applications where it can be used for visualizing the architecture of enveloped viruses and for the analysis of size and compositional variation of Doxil, a commonly used, US FDA-approved nanomedicine.


Subject(s)
Cryoelectron Microscopy/methods , Cryoelectron Microscopy/trends , Image Enhancement/methods , Imaging, Three-Dimensional/methods , Nanoparticles/ultrastructure , Tomography/methods , Tomography/trends , Cryoelectron Microscopy/instrumentation , Tomography/instrumentation
18.
J Bacteriol ; 190(7): 2588-96, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18203829

ABSTRACT

Bdellovibrio bacteriovorus cells are small deltaproteobacterial cells that feed on other gram-negative bacteria, including human pathogens. Using cryo-electron tomography, we demonstrated that B. bacteriovorus cells are capable of substantial flexibility and local deformation of the outer and inner membranes without loss of cell integrity. These shape changes can occur in less than 2 min, and analysis of the internal architecture of highly bent cells showed that the overall distribution of molecular machines and the nucleoid is similar to that in moderately bent cells. B. bacteriovorus cells appear to contain an extensive internal network of short and long filamentous structures. We propose that rearrangements of these structures, in combination with the unique properties of the cell envelope, may underlie the remarkable ability of B. bacteriovorus cells to find and enter bacterial prey.


Subject(s)
Bdellovibrio/ultrastructure , Cryoelectron Microscopy/methods , Tomography/methods , Bacterial Physiological Phenomena , Bdellovibrio/cytology , Cell Membrane/ultrastructure , Cytoplasm/ultrastructure
19.
Structure ; 16(1): 93-103, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18184587

ABSTRACT

Icosahedral pyruvate dehydrogenase (PDH) enzyme complexes are molecular machines consisting of a central E2 core decorated by a shell of peripheral enzymes (E1 and E3) found localized at a distance of approximately 75-90 A from the core. Using a combination of biochemical, biophysical, and cryo-electron microscopic techniques, we show here that the gap between the E2 core and the shell of peripheral enzymes is maintained by the flexible but extended conformation adopted by 60 linker polypeptides that radiate outwards from the inner E2 core, irrespective of the E1 or E3 occupancy. The constancy of the gap is thus not due to protein-protein interactions in the outer protein shell. The extended nature of the E2 inner-linker regions thereby creates the restricted annular space in which the lipoyl domains of E2 that carry catalytic intermediates shuttle between E1, E2, and E3 active sites, while their conformational flexibility facilitates productive encounters.


Subject(s)
Multienzyme Complexes/chemistry , Peptides/chemistry , Pyruvate Dehydrogenase Complex/chemistry , Amino Acid Sequence , Circular Dichroism , Dimerization , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Peptide Fragments/chemical synthesis , Peptide Fragments/chemistry , Protein Subunits/chemistry , Protein Subunits/metabolism , Spectrophotometry, Ultraviolet , Ultracentrifugation
20.
Mol Cell Proteomics ; 7(2): 442-7, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18029347

ABSTRACT

We have explored a general approach for the determination of absolute amounts and the relative stoichiometry of proteins in a mixture using fluorescence and mass spectrometry. We engineered a gene to express green fluorescent protein (GFP) with a synthetic fusion protein (GAB-GFP) in Escherichia coli to function as a spectroscopic standard for the quantification of an analogous stable isotope-labeled, non-fluorescent fusion protein (GAB*) and for the quantification and stoichiometric analysis of purified transducin, a heterotrimeric G-protein complex. Both GAB-GFP and GAB* contain concatenated sequences of specific proteotypic peptides that are derived from the alpha, beta, and gamma protein subunits of transducin and that are each flanked by spacer regions that maintain the native proteolytic properties for these peptide fragments. Spectroscopic quantification of GAB-GFP provided a molar scale for mass spectrometric ratios from tryptic peptides of GAB* and defined molar responses for mass spectrometric signal intensities from a purified transducin complex. The stoichiometry of transducin subunits alpha, beta, and gamma was measured to be 1:1.1:1.15 over a 5-fold range of labeled internal standard with a relative standard deviation of 9%. Fusing a unique genetically coded spectroscopic signal element with concatenated proteotypic peptides provides a powerful method to accurately quantify and determine the relative stoichiometry of multiple proteins present in complexes or mixtures that cannot be readily assessed using classical gravimetric, enzymatic, or antibody-based technologies.


Subject(s)
Green Fluorescent Proteins/metabolism , Mass Spectrometry , Peptides/metabolism , Proteins/analysis , Amino Acid Sequence , Animals , Cattle , Green Fluorescent Proteins/chemistry , Isotope Labeling , Molecular Sequence Data , Peptides/chemistry , Protein Processing, Post-Translational , Protein Subunits/chemistry , Protein Subunits/metabolism , Proteins/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Reference Standards , Reproducibility of Results , Time Factors , Transducin/chemistry , Transducin/metabolism , Trypsin/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...