Crystal Structure of NOV1

The crystal structure of NOV1, a stilbene cleaving oxygenase, shows the features of this enzyme at atomic resolution. This protein fold view highlights the placement of an iron (orange), dioxygen (red), and resveratrol, a representative substrate (blue) in the active site of the enzyme.  Enzymes such as NOV1 could be of value in the biological production of important molecular fragments derived from lignin. (Image courtesy of Ryan McAndrew/JBEI and Berkeley Lab)

LBNL Article

McAndrew, R.P., N. Sathitsuksanoh, M.M. Mbughuni, R.A. Heins, J.H. Pereira, A. George, K.L. Sale, B.G. Fox, B.A. Simmons, and Paul D. Adams. 2016. “Structure and mechanism of NOV1, a resveratrol-cleaving dioxygenase” PNAS 113 (50) 14324-14329. doi:10.1073/pnas.1608917113

Funding Acknowledgements: Berkeley Center for Structural Biology (BCSB), Advanced Light Source (ALS), Lawrence Berkeley National Laboratory (LBNL), work performed as collaboration between the Joint BioEnergy Institute (JBEI; https://www.jbei.org) and Great Lakes Bioenergy Research Center (GLBRC; https://www.glbrc.org). JBEI support: Office of Biological and Environmental Research (OBER), U.S. Department of Energy (DOE) Office of Science, through contract DE-AC02-05CH11231 between LBNL and DOE. GLBRC support: OBER, DOE Office of Science, through Grant DE-FG02-07ER64495. BCSB support in part: National Institutes of Health’s (NIH) National Institute of General Medical Sciences (NIGMS). ALS support: Director, Office of Basic Energy Sciences (OBES), DOE Office of Science, under Contract DE-AC02-05CH11231. Support for part of work: National Science Foundation (NSF) under Cooperative Agreement 1355438.

Cellulose Synthesis Complex

The plant cellulose synthesis complex is a large multi-subunit transmembrane protein complex responsible for synthesis of cellulose chains and their assembly into microfibrils. The image shows ab initio structures of CESA trimers calculated from small-angle scattering data represented by semi-transparent grey surface envelopes, superposed with the computational atomic models in orange. Image credits: Thomas Splettstoesser, scistyle.com, Berlin Germany

Vandavasi, V.G., D.K. Putnam, Q. Zhang, L. Petridis, W.T. Heller, B.T. Nixon, C.H. Haigler, U. Kalluri, L. Coates, and P. Langan. 2016. “A structural study of CESA1 catalytic domain of Arabidopsis cellulose synthesis complex: evidence for CESA trimers.” Plant physiology  170(1):123-35.  [DOI: 10.1104/pp.15.01356]

Funding Acknowledgements: Molecular biology and structural characterization: H.O., V.G.V., Q.Z., W.T.H., L.P., U.K., J.C.S., P.L., and L.C., supported by the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory (ORNL), managed by UT-Battelle, LLC, for the U.S. Department of Energy (DOE) under Contract Number DE-AC05-00OR22725. Small-angle scattering (SAS) and computational analysis, performed by H.O., V.G.V., L.P., B.T.N., and C.H.H., supported by Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center (EFRC) funded by the Office of Basic Energy Sciences (OBES). DOE Office of Science. J.M. and D.K.P. support: High Performance Computing Grant from Oak Ridge Associated Universities (ORAU). Bio-SANS is operated by the Center for Structural Molecular Biology at ORNL, supported by Office of Biological and Environmental Research (OBER), DOE Office of Science, Project ERKP291. EQ-SANS at Spallation Neutron Source (SNS) and High Flux Isotope Reactor (HFIR) sponsored by OBES Scientific User Facilities Division, DOE Office of Science, at ORNL. Sai Venkatesh Pingali: assistance with the operation of Bio-SANS and data reduction. Paul Abraham and the BioEnergy Science Center (BESC) proteomics facilities: validation of purified proteins. BESC supported by DOE OBER. Mass spectrometry (MS) analysis carried out by DOE OBERsupported Bioenergy Research Center proteomics pipeline. Use of the National Synchrotron Light Source (NSLS), Brookhaven National Laboratory (BNL), supported by OBES, DOE Office of Science, under Contract Number DE-AC02-98CH10886.

BAP Protein

Closer look at microorganism provides insight on carbon cycling

The research team reconstructed the crystal structure of BAP, a protein involved in the process by which marine archaea release carbon, to determine how it functioned, as well as its larger role in carbon cycling in marine sediments.
ANL Article

Karolina Michalska, Andrew D. Steen, Gekleng Chhor, Michael Endres, Austen T. Webber, Jordan Bird, Karen G. Lloyd, and Andrzej Joachimiak. “New aminopeptidase from ‘microbial dark matter’ archaeon” The FASEB Journal doi: 10.1096/fj.15-272906. Published online June 10, 2015.

Funding Acknowledgements: Work supported by U.S. National Institutes of Health’s (NIH) National Institute of General Medical Sciences (NIGMS) Grant GM094585 (to A.J.); Office of Biological and Environmental Research (OBER), U.S. Department of Energy (DOE) Office of Science, under Contract DE-AC02-06CH11357 (to A.J.); and Center for Dark Energy Biosphere Investigations Grants 157595 (to K.G.L.) and 36202823 (to A.D.S.). Work is Center for Dark Energy Biosphere Investigation Contribution 268. Submitted manuscript created by University of Chicago, Argonne, LLC, operator of ANL. ANL, a DOE Office of Science laboratory, which is operated under Contract No. DE-AC02-06CH11357.

Understanding Nitrogen Fixation in Bacteria

 

Nitrogen fixation is required for all forms of life, being essential for the biosynthesis of molecules that are used in creating plants and organisms.  Nitrogenase is the only known enzyme capable of performing this multi-electron reduction, and understanding how it does this conversion is of high importance also for the production of ammonia (as fertilizer), for energy efficiency (as industrial processes to produce ammonia consumes enormous amounts of energy), and for global warming (capturing N2).  The structure of the CO inhibitor bound to the FeMo-cofactor active site in nitrogenase at high resolution provides insight into a catalytic competent state, establishes the importance of a bridging S atom, and indicates how N2 might bind during turn-over. Stanford Synchrotron Radiation Lightsource.

Spatzal, K.A. Perez, O. Einsle, J.B. Howard, D.C. Rees, “Ligand binding to the FeMo-cofactor: Structures of CO-bound and reactivated nitrogenase” Science 345, 1620-1623 (2014), doi: 10.1126/science.1256679

SSRL Highlight

Funding Acknowledgements: Work supported by National Institutes of Health (NIH) grant GM45162 (D.C.R.), Deutsche Forschungsgemeinschaft grants EI-520/7 and RTG 1976, and European Research Council N-ABLE project (O.E.). Gordon and Betty Moore Foundation, Beckman Institute, and Sanofi-Aventis Bioengineering Research Program at Caltech: support of Molecular Observatory at Caltech and staff at Beamline 12–2, Stanford Synchrotron Radiation Lightsource (SSRL), for their assistance with data collection. SSRL is operated for the U.S. Department of Energy (DOE) Office of Science and supported by its Office of Biological and Environmental Research (OBER) and by the National Institutes of Health’s (NIH) National Institute of General Medical Sciences (NIGMS; P41GM103393) and National Center for Research Resources (NCRR; P41RR001209). Center for Environmental Microbial Interactions: support of microbiology research at Caltech. Coordinates and structure factors deposited in Protein Data Bank of the Research Collaboratory for Structural Bioinformatics, with IDs 4TKV (Av1-CO) and 4TKU (Av1 reactivated).

Biomass Deconstruction

Illustration of structural rearrangement of cellulose and matrix copolymers during thermochemical pretreatment of lignocellulose biomass. Image credits: Thomas Splettstoesser, scistyle.com, Berlin Germany

Langan, P., L. Petridis, H.M. O’Neill, S.V. Pingali, M. Foston, Y. Nishiyama, et al. 2014. “Common processes drive the thermochemical pretreatment of lignocellulosic biomass.” Green Chem. 16(1):63-8. [DOI:10.1039/C3GC41962B]

Spatial Organization of Lipid Domains in a Biomimetic Four-Lipid System

Floating freely in cell membranes, lipid rafts are organizing centers for membrane-mediated processes. Neutron scattering techniques were used to characterize lipid domain size transitions from nanometers to micrometers in a four-component biomimetic lipid mixture.  Results suggest that reversible changes in lipid composition may regulate the size of functional domains in lipid rafts. The BioSANS instrument at the ORNL Center for Structural Molecular Biology and the EQ-SANS instrument (BER) at the ORNL Spallation Neutron Source (BES) were used in this study.

Heberle F.A., Petruzielo R.S., Pan J., Drazba P., Kucerka N., Standaert R.F., Feigenson G.W., Katsaras J., “Bilayer thickness mismatch controls domain size in model membranes”, Journal of the American Chemical Society, 135, 18, 6853-6859 (2013). DOI: 10.1021/ja3113615

Funding Acknowledgements: Support: Laboratory Directed Research and Development Program of Oak Ridge National Laboratory (ORNL; to J.K. and R.F.S.), managed by UT-Battelle, LLC, for the U.S. Department of Energy (DOE), and from National Science Foundation (NSF) research award MCB 0842839 (to G.W.F.). Additional support: Office of Biological and Environmental Research (OBER), DOE Office of Science, for BioSANS instrument at ORNL Center for Structural Molecular Biology (CSMB), and from the DOE Office of Basic Energy Sciences (OBES) Scientific User Facilities (SUF) Division, for the EQ-SANS instrument at the ORNL Spallation Neutron Source (SNS). These facilities are managed for DOE by UT-Battelle, LLC, under Contract No. DE-AC05-00OR2275. A portion of this research conducted using resources of Cornell Center for Advanced Computing, which receives funding from Cornell University; NSF; and other leading public agencies, foundations, and corporations.

Major Scaffold Component in Nuclear Pore Complex

The Nuclear Pore Complex (NPC) is a 50 MDa macromolecular assembly solely responsible for the transport of macromolecules across the nuclear membrane within the eukaryotic cell. The core structure of the NPC (shown as a wire-mesh) is made up of two outer rings, two inner rings and a membrane ring. A hybrid method approach was used to determine the detailed architecture of the NPC combining structural data from crystallography, small-angle x-ray scattering, normal mode analysis, molecular dynamics simulation and electron microscopy as well as information from functional and biochemical studies.

Sampathkumar, S. J. Kim, P. Upla, W. J. Rice, J. Phillips, B. L. Timney, U. Pieper, J. B. Bonanno, J. Fernandez-Martinez, Z. Hakhverdyan, N. E. Ketaren, T. Matsui, T. M. Weiss, D. L. Stokes, J. M. Sauder, S. K. Burley, A. Sali, M. P. Rout and S. C. Almo, “Structure, Dynamics, Evolution, and Function of a Major Scaffold Component in the Nuclear Pore Complex”, Structure 21, 560 (2013) doi: 10.1016/j.str.2013.02.005

Funding Acknowledgements: Funding for NYSGXRC and NYSGRC: National Institutes of Health (NIH) Grants U54 GM074945 (S.K.B.) and U54 GM094662 (S.C.A.), respectively. Additional funding: NIH grants R01 GM062427 (M.P.R.), R01 GM083960 (A.S.), and U54 GM103511 and U01 GM098256 (A.S. and M.P.R.). Publication in part by Center for Synchrotron Biosciences (CSB; National Synchrotron Light Source II [NSLS-II], Brookhaven National Laboratory [BNL]) grant, P30-EB-009998, from the National Institute of Biomedical Imaging and Bioengineering (NIBIB). Use of BNL supported by the U.S. Department of Energy (DOE) under Contract No. DE-AC02-98CH10886. Use of APS, ANL, supported by DOE. Access to LRL-CAT beam line at APS provided by Eli Lilly, which operates the facility. Portions of this research carried out at SSRL, SLAC National Accelerator Laboratory (SLAC), operated for DOE by Stanford University. The SSRL Structural Molecular Biology Program (SMBP) is supported by the DOE Office of Biological and Environmental Research (OBER), the NIH National Center for Research Resources (NCRR) Biomedical Technology Program (P41RR001209), and the NIH National Institute for General Medical Sciences (NIGMS; P41GM103393). Investigation conducted in facility constructed with support from NIH NCRR Research Facilities Improvement Program Grant number C06 RR017528-01-CEM.

Watching the Evolution of a Protein’s Function

A major challenge in research to enable large-scale biofuels production is developing enzymes that are highly efficient in converting biomass components into usable fuels.  Using directed evolution (i.e., a technique for modifying protein function), researchers have determined the structural basis for converting a noncatalytic small protein into an effective enzyme for for linking RNA molecules.  Extended x-ray absorption fine structure (EXAFS) was used to determine the Zn-containing active-site structure of this RNA ligase, synthesized through in-vitro directed evolution.

F.-A. Chao, A. Morelli, J. C. Haugner, III, L. Churchfield, L. N. Hagmann, L. Shi, L. R. Masterson, R. Sarangi, G. Veglia and B. Seeling, “Structure and Dynamics of a Primordil Catalytic Fold Generated by in vitro Evolution”, Nat. Chem. Biol. 9, 81 (2013) doi: 10.1038/nchembio.1138

Funding Acknowledgements: Work supported by U.S. National Aeronautics and Space Administration (NASA) Agreement no. NNX09AH70A, through the NASA Astrobiology Institute–Ames Research Center (to F.-A.C., A.M., L.C. and B.S.); Minnesota Medical Foundation (to B.S.), and U.S. National Institutes of Health (NIH; T32 GM08347 to J.C.H., T32 DE007288 to L.R.M., GM100310 to G.V., and P41 RR001209). Stanford Synchrotron Radiation Lightsource (SSRL) at SLAC National Accelerator Laboratory (SLAC) operations funded by the Office of Basic Energy Sciences (OBES), U.S. Department of Energy (DOE) Office of Science. SSRL Structural Molecular Biology program supported by NIH National Center for Research Resources (NCRR) and Office of Biological Environmental Research (OBER), DOE Office of Science.

Comparison of GH3 Protein Binding Sites

In plants, GH3 proteins act as molecular on/off switches that control bioactive plant hormone formation by catalyzing the addition of specific amino acids to jasmonic acid, auxin, and benzoates. X-ray structures of GH3 proteins reveal a common three-dimensional fold but variability in the hormone binding site. This figure shows the variation in the jasmonic acid binding site of Arabidopsis thaliana GH3.11/JAR1 (gold) and the salicylic acid binding site of A. thaliana GH3.12/PBS3 (green).
BER Highlight

Westfall, C. S., et al. 2012. “Structural Basis for Prereceptor Modulation of Plant Hormones by GH3 Proteins,” Science 336, 1708–11. DOI: 10.1126/science.1221863

Funding Acknowledgements: Work supported by National Science Foundation (NSF) grant MCB-1157771 to J.M.J. C.S.W. supported by U.S. Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) predoctoral fellowship (MOW-2010-05240), and J.H. supported by American Society of Plant Biologists (ASPB) Summer Undergraduate Research Fellowship award and the Howard Hughes Medical Institute (HHMI)–Washington University in St. Louis Summer Scholars Program in Biology and Biomedical Research. Portions of this research carried out at European Synchrotron Radiation Facility (ESRF) and Argonne National Laboratory (ANL) Structural Biology Center (SBC) of the Advanced Photon Source (APS), a national user facility operated by the University of Chicago for the Office of Biological and Environmental Research, U.S. Department of Energy (DOE) Office of Science (DE-AC02-06CH11357). Atomic coordinates and structure factors deposited in Protein Data Bank (PDB; accession codes noted in table S2).