Jacqueline L. S. Milne

2.2 Å resolution cryo-EM structure of β-galactosidase in complex with a cell-permeant inhibitor

Pushing the limits of electron microscopy Recent advances in cryo–electron microscopy (cryo-EM) allow structures of large macromolecules to be determined at near-atomic resolution. So far, though, resolutions approaching 2 Å, where features key to drug design are revealed, remain the province of x-ray crystallography. Bartesaghi et al. achieved a resolution of 2.2 Å for a 465-kD ligand-bound protein complex using cryo-EM. The density map is detailed enough to show close to 800 water molecules, magnesium and sodium ions, and precise side-chain conformations. These results bring routine use of cryo-EM in rational drug design a step closer. Science, this issue p. 1147 Advances in electron microscopy allow protein structure determination at resolutions useful in drug discovery. 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 β-galactosidase and the cell-permeant inhibitor phenylethyl β-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.

Structural Mechanism of Trimeric HIV-1 Envelope Glycoprotein Activation

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.

Molecular Architectures of Trimeric SIV and HIV-1 Envelope Glycoproteins on Intact Viruses: Strain-Dependent Variation in Quaternary Structure

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.

Trimeric HIV-1 glycoprotein gp140 immunogens and native HIV-1 envelope glycoproteins display the same closed and open quaternary molecular architectures

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.

2.3 A Resolution Cryo-Em Structure of Human P97 and Mechanism of Allosteric Inhibition

AAA ATPase conformational high jinks The protein p97 is an AAA adenosine triphosphatase (ATPase) that uses energy from ATP hydrolysis to regulate substrates involved in intracellular protein quality control. Its role in this central process makes it a target for cancer chemotherapy. Banerjee et al. used cryo-electron microscopy to determine high-resolution structures for multiple conformational states of this dynamic macromolecular machine. They also determined the structure of the ADP-bound state bound to an inhibitor. The structures give insight into nucleotide-driven structural changes that drive function and show how inhibitor binding prevents these conformational changes Science, this issue p. 871 Cryo–electron microscopy reveals atomic-resolution structures of a protein complex that is a target for cancer drug development. 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.

Cryo-electron tomography of bacteria: progress, challenges and future prospects

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.

Molecular architecture and mechanism of an icosahedral pyruvate dehydrogenase complex: a multifunctional catalytic machine

Electron cryo‐microscopy of ‘single particles’ is a powerful method to determine the three‐dimensional (3D) architectures of complex cellular assemblies. The pyruvate dehydrogenase multi‐enzyme complex couples the activity of three component enzymes (E1, E2 and E3) in the oxidative decarboxylation of pyruvate to generate acetyl‐CoA, linking glycolysis and the tricarboxylic acid cycle. We report here a 3D model for an 11 MDa, icosahedral pyruvate dehydrogenase sub‐complex, obtained by combining a 28 Å structure derived from electron cryo‐microscopy with previously determined atomic coordinates of the individual E1 and E2 components. A key feature is that the E1 molecules are located on the periphery of the assembly in an orientation that allows each of the 60 mobile lipoyl domains tethered to the inner E2 core to access multiple E1 and E2 active sites from inside the icosahedral complex. This unexpected architecture provides a highly efficient mechanism for active site coupling and catalytic rate enhancement by the motion of the lipoyl domains in the restricted annular region between the inner core and outer shell of the complex.

Ca2+ signalling is not required for chemotaxis in Dictyostelium

Dictyostelium cells can move rapidly towards a source of cyclic‐AMP (cAMP). This chemoattractant is detected by G‐protein‐linked receptors, which trigger a signalling cascade including a rapid influx of Ca2+. We have disrupted an inositol 1,4,5‐trisphosphate (InsP3) receptor‐like gene, iplA, to produce null cells in which Ca2+ entry in response to chemoattractants is abolished, as is the normal increase in free cytosolic Ca2+ ([Ca2+]c) that follows chemotactic stimulation. However, the resting [Ca2+]c is similar to wild type. This mutant provides a test for the role of Ca2+ influx in both chemotaxis and the signalling cascade that controls it. The production of cyclic‐GMP and cAMP, and the activation of the MAP kinase, DdERK2, triggered from the cAMP receptor, are little perturbed in the mutant; mobilization of actin into the cytoskeleton also follows similar kinetics to wild type. Mutant cells chemotax efficiently towards cAMP or folic acid and their sensitivity to cAMP is similar to wild type. Finally, they move at similar speeds to wild‐type cells, with or without chemoattractant. We conclude that Ca2+ signalling is not necessary for chemotaxis to cAMP.

Cryo-electron microscopy: A primer for the non-microscopist

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.

3D Imaging of mammalian cells with ion-abrasion scanning electron microscopy

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.