New York Structural Biology Center

New York City, NY, United States

New York Structural Biology Center

New York City, NY, United States
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Catanese M.T.,Rockefeller University | Uryu K.,Rockefeller University | Kopp M.,Rockefeller University | Kopp M.,Amgen | And 7 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2013

Hepatitis C virus (HCV) is amajor cause of chronic liver disease, with an estimated 170 million people infected worldwide. Low yields, poor stability, and inefficient binding to conventional EM grids have posed significant challenges to the purification and structural analysis of HCV. In this report, we generated an infectious HCV genome with an affinity tag fused to the E2 envelope glycoprotein. Using affinity grids, previously described to isolate proteins and macromolecular complexes for single-particle EM, we were able to purify enveloped particles directly from cell culture media. This approach allowed for rapid in situ purification of virions and increased particle density that were instrumental for cryo-EM and cryoelectron tomography (cryo-ET). Moreover, it enabled ultrastructural analysis of virions produced by primary human hepatocytes. HCV appears to be the most structurally irregular member of the Flaviviridae family. Particles are spherical, with spike-like projections, and heterogeneous in size ranging from 40to100nmin diameter.Exosomes, althoughisolated fromunfractionated culture media, were absent in highly infectious, purified virus preparations. Cryo-ET studies provided low-resolution 3D structural information of highly infectious virions. In addition to apolipoprotein (apo)E, HCV particles also incorporate apoB and apoA-I. In general, host apolipoproteins were more readily accessible to antibody labeling than HCV glycoproteins, suggesting either lower abundance or masking by host proteins.


Stokes D.L.,New York University | Stokes D.L.,New York Structural Biology Center
Current Opinion in Structural Biology | Year: 2012

During the past year, electron crystallography of membrane proteins has provided structural insights into the mechanism of several different transporters and into their interactions with lipid molecules within the bilayer. From a technical perspective there have been important advances in high-throughput screening of crystallization trials and in automated imaging of membrane crystals with the electron microscope. There have also been key developments in software, and in molecular replacement and phase extension methods designed to facilitate the process of structure determination. © 2012 Elsevier Ltd.


Lin W.,Brookhaven National Laboratory | Chai J.,Brookhaven National Laboratory | Love J.,New York Structural Biology Center | Fu D.,Brookhaven National Laboratory
Journal of Biological Chemistry | Year: 2010

All living cells need zinc ions to support cell growth. Zrt-, Irt-like proteins (ZIPs) represent a major route for entry of zinc ions into cells, but how ZIPs promote zinc uptake has been unclear. Here we report the molecular characterization of ZIPB from Bordetella bronchiseptica, the first ZIP homolog to be purified and functionally reconstituted into proteoliposomes. Zinc flux through ZIPB was found to be nonsaturable and electrogenic, yielding membrane potentials as predicted by the Nernst equation. Conversely, membrane potentials drove zinc fluxes with a linear voltage-flux relationship. Direct measurements of metal uptake by inductively coupled plasma mass spectroscopy demonstrated that ZIPB is selective for two group 12 transition metal ions, Zn2+ and Cd2+, whereas rejecting transition metal ions in groups 7 through 11. Our results provide the molecular basis for cellular zinc acquisition by a zinc-selective channel that exploits in vivo zinc concentration gradients to move zinc ions into the cytoplasm.


News Article | December 1, 2016
Site: www.eurekalert.org

LA JOLLA--(December 1, 2016) Ribosomes--macromolecular machines consisting of RNA and proteins that twist, fold and turn--are responsible for making all of the protein within a cell and could hold the key to deciphering a range of diseases. Despite the intricacies of ribosomes, cells are able to churn out 100,000 of them every hour. But because they assemble so speedily, researchers haven't been able to figure out how they come together. A collaboration led by Salk Institute for Biological Studies and The Scripps Research Institute in La Jolla, California, deployed a cutting-edge imaging method called single-particle cryo-electron microscopy (cryo-EM) and accompanying analysis tools to decipher some of the key steps for how ribosomes are assembled, a first step in understanding their roles in health and disease. The results are published online December 1, 2016, in the journal Cell. "These new structures we captured with cryo-EM show that it is possible to image and interpret diverse molecular machines in action," says the study's co-senior author Dmitry Lyumkis, a Helmsley-Salk Fellow at Salk. "This is a completely different way of seeing and doing structural biology. This paper is a prime example of the fact that we can do far more intricate analyses than have ever been expected." Understanding molecular structures is important not only for basic research into biology but also for the drug development process to better understand how to make safer and more effective medicines. Researchers traditionally turn to X-ray crystallography, a method that requires its users to extensively purify a molecule and then re-make it in crystal form, but this method has limitations. In the past few years, advances in cryo-EM have allowed scientists to image single particles with resolution comparable to that of traditional X-ray methods. But in single-particle cryo-EM, proteins (the "particles") are flash-frozen and imaged using streams of electrons, meaning the molecules don't need to be crystallized and can retain much of their native structure. Although cryo-EM has been around for a while, new cameras are making it easier to capture proteins at high resolution before the electron spray zaps them. Importantly, computational tools for analyzing cryo-EM data have matured such that researchers can now purify molecules in silico by a computer rather than through traditional biochemical approaches. This becomes a much more powerful approach for separating mixtures of species, allowing the researchers to identify and distinguish structurally distinct populations of particles in greater detail than before. In the new study, co-senior author James Williamson, professor of molecular biology and chemistry at The Scripps Research Institute, and his team developed a method to stall one major component of ribosomes, the 50s subunit, from coming together so quickly. The scientists were able to chemically pause a mixture of different molecules in various stages of assembly. Lyumkis's group then used high-end cryo-EM to image and analyze these stalled structures, which had not been attempted for such a mix of diverse forms of a particular molecule. "Others have shown that you can capture a couple of different structural states of a molecule," Lyumkis says. "But, as far as I know, no one has tried to take this crude mixture of stuff, put it onto a cryo-EM grid, and ask what was in there." The team found that there are at least 15 types of complexes in the mixture, 13 of which are actively assembling 50s subunits. They imaged each of these structures at a resolution high enough to decipher the protein and RNA constituents. They were then able to use computer algorithms to order the complexes according to their assembly pathway. The team's analysis suggests that ribosomes can take several different routes for assembly, which is important to ensure that the process is efficient and can withstand a variety of cell stresses, according to Williamson. "If you imagine an assembly line where every step has to happen in sequential order, and there is a problem at one of those steps, everything grinds to a halt," he says. "If there are parallel pathways, then assembly can proceed through other channels until the problem is resolved. It took the scientists over a year to make sense of the structures, employing relatively new image analysis tools. But they have laid the groundwork to study other large, dynamic, and structurally heterogeneous molecular machines, which, Lyumkis says, will lead to new basic science and translational discoveries. Other authors on the study are Joseph H. Davis of The Scripps Research Institute; and Yong Zi Tan, Bridget Carragher and Clinton Potter of Columbia University and the New York Structural Biology Center. The research was supported by the Jane Coffin Child's Foundation; the National Institute of Aging; The Leona M. and Harry B. Helmsley Charitable Trust; the National Institute of General Medical Sciences; the Simons Foundation; and the Agency for Science, Technology and Research Singapore. About the Salk Institute for Biological Studies: Every cure has a starting point. The Salk Institute embodies Jonas Salk's mission to dare to make dreams into reality. Its internationally renowned and award-winning scientists explore the very foundations of life, seeking new understandings in neuroscience, genetics, immunology and more. The Institute is an independent nonprofit organization and architectural landmark: small by choice, intimate by nature and fearless in the face of any challenge. Be it cancer or Alzheimer's, aging or diabetes, Salk is where cures begin. Learn more at: salk.edu.


Mistry J.,European Bioinformatics Institute | Kloppmann E.,TU Munich | Kloppmann E.,New York Structural Biology Center | Rost B.,TU Munich | And 3 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2013

High-resolution structural knowledge is key to understanding how proteins function at the molecular level. The number of entries in the Protein Data Bank (PDB), the repository of all publicly available protein structures, continues to increase, with more than 8000 structures released in 2012 alone. The authors of this article have studied how structural coverage of the protein-sequence space has changed over time by monitoring the number of Pfam families that acquired their first representative structure each year from 1976 to 2012. Twenty years ago, for every 100 new PDB entries released, an estimated 20 Pfam families acquired their first structure. By 2012, this decreased to only about five families per 100 structures. The reasons behind the slower pace at which previously uncharacterized families are being structurally covered were investigated. It was found that although more than 50% of current Pfam families are still without a structural representative, this set is enriched in families that are small, functionally uncharacterized or rich in problem features such as intrinsically disordered and transmembrane regions. While these are important constraints, the reasons why it may not yet be time to give up the pursuit of a targeted but more comprehensive structural coverage of the protein-sequence space are discussed.


Choi W.-S.,Rockefeller University | Choi W.-S.,Osong Medical Innovation Foundation | Rice W.J.,New York Structural Biology Center | Stokes D.L.,New York Structural Biology Center | And 2 more authors.
Blood | Year: 2013

ntegrin αIIbβ3 plays a central role in hemostasis and thrombosis. We provide the first 3-dimensional reconstruction of intact purified αIIbβ3 in a nanodisc lipid bilayer. Unlike previous models, it shows that the ligand-binding head domain is on top, pointing away fromthemembrane.Moreover, unlike the crystal structure of the recombinant ectodomain, the lower legs are not parallel, straight, and adjacent. Rather, the aIIb lower leg is bent between the calf-1 and calf-2 domains and the β3 Integrin-Epidermal Growth Factor (I-EGF) 2 to 4 domains are freely coiled rather than in a cleft between the β3 headpiece and the aIIb lower leg. Our data indicate an important role for the region that links the distal calf-2 and b-tail domains to their respective transmembrane (TM) domains in transmitting the conformational changes in the TM domains associated with inside-out activation. © 2013 by The American Society of Hematology.


Mancia F.,Columbia University | Love J.,New York Structural Biology Center
Current Opinion in Structural Biology | Year: 2011

Structural genomics approaches on integral membrane proteins have been postulated for over a decade, yet specific efforts are lagging years behind their soluble counterparts. Indeed, high throughput methodologies for production and characterization of prokaryotic integral membrane proteins are only now emerging, while large-scale efforts for eukaryotic ones are still in their infancy. Presented here is a review of recent literature on actively ongoing structural genomics of membrane protein initiatives, with a focus on those aimed at implementing interesting techniques aimed at increasing our rate of success for this class of macromolecules. © 2011 Elsevier Ltd.


Hendrickson W.A.,Columbia University | Hendrickson W.A.,New York Structural Biology Center
Nature Structural and Molecular Biology | Year: 2016

Membrane proteins are substantially more challenging than natively soluble proteins as subjects for structural analysis. Thus, membrane proteins are greatly underrepresented in structural databases. Recently, focused consortium efforts and advances in methodology for protein production, crystallographic analysis and cryo-EM analysis have accelerated the pace of atomic-level structure determination of membrane proteins. © 2016 Nature America, Inc. All rights reserved.


Mancia F.,Columbia University | Love J.,New York Structural Biology Center
Journal of Structural Biology | Year: 2010

High-throughput (HT) methodologies have had a tremendous impact on structural biology of soluble proteins. High-resolution structure determination relies on the ability of the macromolecule to form ordered crystals that diffract X-rays. While crystallization remains somewhat empirical, for a given protein, success is proportional to the number of conditions screened and to the number of variants trialed. HT techniques have greatly increased the number of targets that can be trialed and the rate at which these can be produced. In terms of number of structures solved, membrane proteins appear to be lagging many years behind their soluble counterparts. Likewise, HT methodologies for production and characterization of these hydrophobic macromolecules are only now emerging. Presented here is an HT platform designed exclusively for membrane proteins that has processed over 5000 targets. © 2010 Elsevier Inc.


Hendrickson W.A.,Columbia University | Hendrickson W.A.,New York Structural Biology Center
Quarterly Reviews of Biophysics | Year: 2014

X-ray diffraction patterns from crystals of biological macromolecules contain sufficient information to define atomic structures, but atomic positions are inextricable without having electron-density images. Diffraction measurements provide amplitudes, but the computation of electron density also requires phases for the diffracted waves. The resonance phenomenon known as anomalous scattering offers a powerful solution to this phase problem. Exploiting scattering resonances from diverse elements, the methods of MAD (multiwavelength anomalous diffraction) and SAD (single-wavelength anomalous diffraction) now predominate for de novo determinations of atomic-level biological structures. This review describes the physical underpinnings of anomalous diffraction methods, the evolution of these methods to their current maturity, the elements, procedures and instrumentation used for effective implementation, and the realm of applications. © 2014 Cambridge University Press.

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