Julian C.-H. Chen

University of Toledo

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Marc‐Michael Blum

Institut für Pharmakologie und Toxikologie der Bundeswehr

Paul Langan

Institut Laue-Langevin

Clifford J. Ünkefer

Los Alamos National Laboratory

Andrey Kovalevsky

Oak Ridge National Laboratory

B. P. Schoenborn

Los Alamos National Laboratory

Heinz Rüterjans

Goethe University Frankfurt

B. Leif Hanson

University of Toledo

S. Zoë Fisher

Lund University

J. Krucinski

University of California, San Francisco

Marat Mustyakimov

Oak Ridge National Laboratory

Cooperative Institutions

University of California, San Francisco

Goethe University Frankfurt

Los Alamos National Laboratory

National Institutes of Health

Oak Ridge National Laboratory

University of Toledo

Johannes Gutenberg University Mainz

Argonne National Laboratory

Joint Center for Structural Genomics

Stanford University

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Crystal structure of the HIV-1 integrase catalytic core and C-terminal domains: A model for viral DNA binding

Proceedings of the National Academy of Sciences (2000)

Julian C.-H. Chen J. Krucinski Larry J. W. Miercke Janet Finer-Moore Ann H. Tang

Insolubility of full-length HIV-1 integrase (IN) limited previous structure analyses to individual domains. By introducing five point mutations, we engineered a more soluble IN that allowed us to generate multidomain HIV-1 IN crystals. The first multidomain HIV-1 IN structure is reported. It incorporates the catalytic core and C-terminal domains (residues 52–288). The structure resolved to 2.8 Å is a Y-shaped dimer. Within the dimer, the catalytic core domains form the only dimer interface, and the C-terminal domains are located 55 Å apart. A 26-aa α-helix, α6, links the C-terminal domain to the catalytic core. A kink in one of the two α6 helices occurs near a known proteolytic site, suggesting that it may act as a flexible elbow to reorient the domains during the integration process. Two proteins that bind DNA in a sequence-independent manner are structurally homologous to the HIV-1 IN C-terminal domain, suggesting a similar protein–DNA interaction in which the IN C-terminal domain may serve to bind, bend, and orient viral DNA during integration. A strip of positively charged amino acids contributed by both monomers emerges from each active site of the dimer, suggesting a minimally dimeric platform for binding each viral DNA end. The crystal structure of the isolated catalytic core domain (residues 52–210), independently determined at 1.6-Å resolution, is identical to the core domain within the two-domain 52–288 structure.

Helix (gastropod)

Viral protein

10.1073/pnas.150220297

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Citations (394)

Solvent organization in the ultrahigh-resolution crystal structure of crambin at room temperature

IUCrJ (2024)

Julian C.-H. Chen Mirosław Gilski Changsoo Chang Dominika Borek G. Rosenbaum

Ultrahigh-resolution structures provide unprecedented details about protein dynamics, hydrogen bonding and solvent networks. The reported 0.70 Å, room-temperature crystal structure of crambin is the highest-resolution ambient-temperature structure of a protein achieved to date. Sufficient data were collected to enable unrestrained refinement of the protein and associated solvent networks using SHELXL . Dynamic solvent networks resulting from alternative side-chain conformations and shifts in water positions are revealed, demonstrating that polypeptide flexibility and formation of clathrate-type structures at hydrophobic surfaces are the key features endowing crambin crystals with extraordinary diffraction power.

Protein crystallization

10.1107/s2052252524007784

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Citations (1)

The structure of Synechococcus elongatus enolase reveals key aspects of phosphoenolpyruvate binding

Acta Crystallographica Section F Structural Biology Communications (2022)

Javier González Ricardo Martí‐Arbona Julian C.-H. Chen Clifford J. Ünkefer

A structure–function characterization of Synechococcus elongatus enolase ( Se EN) is presented, representing the first structural report on a cyanobacterial enolase. X-ray crystal structures of Se EN in its apoenzyme form and in complex with phosphoenolpyruvate are reported at 2.05 and 2.30 Å resolution, respectively. Se EN displays the typical fold of enolases, with a conformationally flexible loop that closes the active site upon substrate binding, assisted by two metal ions that stabilize the negatively charged groups. The enzyme exhibits a catalytic efficiency of 1.2 × 10 5 M −1 s −1 for the dehydration of 2-phospho-D-glycerate, which is comparable to the kinetic parameters of related enzymes. These results expand the understanding of the biophysical features of these enzymes, broadening the toolbox for metabolic engineering applications.

Enolase

10.1107/s2053230x22003612

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Citations (1)

Neutron and high-resolution room-temperature X-ray data collection from crystallized lytic polysaccharide monooxygenase

Acta Crystallographica Section F Structural Biology Communications (2015)

J.P. Bacik Sophanit Mekasha Zarah Forsberg Andrey Kovalevsky Jay C. Nix

Bacteria and fungi express lytic polysaccharide monooxgyenase (LPMO) enzymes that act in conjunction with canonical hydrolytic sugar-processing enzymes to rapidly convert polysaccharides such as chitin, cellulose and starch to single monosaccharide products. In order to gain a better understanding of the structure and oxidative mechanism of these enzymes, large crystals (1-3 mm(3)) of a chitin-processing LPMO from the Gram-positive soil bacterium Jonesia denitrificans were grown and screened for their ability to diffract neutrons. In addition to the collection of neutron diffraction data, which were processed to 2.1 Å resolution, a high-resolution room-temperature X-ray diffraction data set was collected and processed to 1.1 Å resolution in space group P212121. To our knowledge, this work marks the first successful neutron crystallographic experiment on an LPMO. Joint X-ray/neutron refinement of the resulting data will reveal new details of the structure and mechanism of this recently discovered class of enzymes.

Lytic cycle

10.1107/s2053230x15019743

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X-ray structure of perdeuterated diisopropyl fluorophosphatase (DFPase): perdeuteration of proteins for neutron diffraction

Acta Crystallographica Section F Structural Biology and Crystallization Communications (2010)

Marc‐Michael Blum Stephen J. Tomanicek Harald John B. Leif Hanson Heinz Rüterjans

The signal-to-noise ratio is one of the limiting factors in neutron macromolecular crystallography. Protein perdeuteration, which replaces all H atoms with deuterium, is a method of improving the signal-to-noise ratio of neutron crystallography experiments by reducing the incoherent scattering of the hydrogen isotope. Detailed analyses of perdeuterated and hydrogenated structures are necessary in order to evaluate the utility of perdeuterated crystals for neutron diffraction studies. The room-temperature X-ray structure of perdeuterated diisopropyl fluorophosphatase (DFPase) is reported at 2.1 Å resolution. Comparison with an independently refined hydrogenated room-temperature structure of DFPase revealed no major systematic differences, although the crystals of perdeuterated DFPase did not diffract neutrons. The lack of diffraction is examined with respect to data-collection and crystallographic parameters. The diffraction characteristics of successful neutron structure determinations are presented as a guideline for future neutron diffraction studies of macromolecules. X-ray diffraction to beyond 2.0 Å resolution appears to be a strong predictor of successful neutron structures.

10.1107/s1744309110004318

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Citations (17)

Multiple Targets for Suppression of RNA Interference by Tomato Aspermy Virus Protein 2B

Biochemistry (2008)

Umar Rashid Jan Hoffmann Bernhard Brutschy Jacob Piehler Julian C.-H. Chen

Viral suppressors of RNA interference (RNAi) appear to have evolved as a response to this innate genomic defense. We report the nucleic acid binding properties of the Cucumovirus RNAi suppressor tomato aspermy virus protein 2B (TAV 2B). Using total internal reflection fluorescence spectroscopy (TIRFS), we show that TAV 2B binds double-stranded RNA corresponding to siRNAs and miRNAs, as well as single-stranded RNA oligonucleotides. A number of positively charged residues between amino acids 20 and 30 are critical for RNA binding. Binding to RNA oligomerizes and induces a conformational change in TAV 2B, causing it to form a primarily helical structure and a 4:2 protein−RNA complex.

RNA Silencing

10.1021/bi801281h

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Citations (21)

Preliminary time-of-flight neutron diffraction study on diisopropyl fluorophosphatase (DFPase) fromLoligo vulgaris

Acta Crystallographica Section F Structural Biology and Crystallization Communications (2006)

Marc‐Michael Blum Alexander Koglin Heinz Rüterjans B. P. Schoenborn Paul Langan

The enzyme diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is capable of decontaminating a wide variety of toxic organophosphorus nerve agents. DFPase is structurally related to a number of enzymes, such as the medically important paraoxonase (PON). In order to investigate the reaction mechanism of this phosphotriesterase and to elucidate the protonation state of the active-site residues, large-sized crystals of DFPase have been prepared for neutron diffraction studies. Available H atoms have been exchanged through vapour diffusion against D2O-containing mother liquor in the capillary. A neutron data set has been collected to 2.2 A resolution on a relatively small (0.43 mm3) crystal at the spallation source in Los Alamos. The sample size and asymmetric unit requirements for the feasibility of neutron diffraction studies are summarized.

Loligo

10.1107/s1744309106052924

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Citations (20)

Energy optimization of a regular macromolecular crystallography beamline for ultra-high-resolution crystallography

Journal of Synchrotron Radiation (2014)

G. Rosenbaum Stephan L. Ginell Julian C.-H. Chen

A practical method for operating existing undulator synchrotron beamlines at photon energies considerably higher than their standard operating range is described and applied at beamline 19-ID of the Structural Biology Center at the Advanced Photon Source enabling operation at 30 keV. Adjustments to the undulator spectrum were critical to enhance the 30 keV flux while reducing the lower- and higher-energy harmonic contamination. A Pd-coated mirror and Al attenuators acted as effective low- and high-bandpass filters. The resulting flux at 30 keV, although significantly lower than with X-ray optics designed and optimized for this energy, allowed for accurate data collection on crystals of the small protein crambin to 0.38 Å resolution.

Undulator

Photon energy

Advanced Photon Source

High-energy X-rays

10.1107/s1600577514022619

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Citations (9)

Rapid determination of hydrogen positions and protonation states of diisopropyl fluorophosphatase by joint neutron and X-ray diffraction refinement

Proceedings of the National Academy of Sciences (2009)

Marc‐Michael Blum Marat Mustyakimov Heinz Rüterjans Kai Kehe B. P. Schoenborn

Hydrogen atoms constitute about half of all atoms in proteins and play a critical role in enzyme mechanisms and macromolecular and solvent structure. Hydrogen atom positions can readily be determined by neutron diffraction, and as such, neutron diffraction is an invaluable tool for elucidating molecular mechanisms. Joint refinement of neutron and X-ray diffraction data can lead to improved models compared with the use of neutron data alone and has now been incorporated into modern, maximum-likelihood based crystallographic refinement programs like CNS. Joint refinement has been applied to neutron and X-ray diffraction data collected on crystals of diisopropyl fluorophosphatase (DFPase), a calcium-dependent phosphotriesterase capable of detoxifying organophosphorus nerve agents. Neutron omit maps reveal a number of important features pertaining to the mechanism of DFPase. Solvent molecule W33, coordinating the catalytic calcium, is a water molecule in a strained coordination environment, and not a hydroxide. The smallest Ca-O-H angle is 53 degrees, well beyond the smallest angles previously observed. Residue Asp-229, is deprotonated, supporting a mechanism involving nucleophilic attack by Asp-229, and excluding water activation by the catalytic calcium. The extended network of hydrogen bonding interactions in the central water filled tunnel of DFPase is revealed, showing that internal solvent molecules form an important, integrated part of the overall structure.

10.1073/pnas.0807842106

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Citations (71)

Structure of Aquifex aeolicus Argonaute Highlights Conformational Flexibility of the PAZ Domain as a Potential Regulator of RNA-induced Silencing Complex Function

Journal of Biological Chemistry (2006)

Umar Rashid Dirk Paterok Alexander Koglin Holger Gohlke Jacob Piehler

Gene silencing mediated by RNA interference requires the sequence-specific recognition of target mRNA by the endonuclease Argonaute, the primary enzymatic component of the RNA-induced silencing complex. We report the crystal structure of Aquifex aeolicus Argonaute, refined at 3.2A resolution. Relative to recent Argonaute structures, a 24 degrees reorientation of the PAZ domain in our structure opens a basic cleft between the N-terminal and PAZ domains, exposing the guide strand binding pocket of PAZ. This rearrangement leads to a branched, Y-shaped system of grooves that extends through the molecule and merges in a central channel containing the catalytic residues. A 5.5-ns molecular dynamics simulation of Argonaute shows a strong tendency of the PAZ and N-terminal domains to be mobile. Binding of single-stranded DNA to Argonaute monitored by total internal reflection fluorescence spectroscopy shows biphasic kinetics, also indicative of domain rearrangement upon DNA binding. Conformational rearrangement of the PAZ domain may therefore be critical for the catalytic cycle of Argonaute and the RNA-induced silencing complex.

Aquifex aeolicus

Argonaute

10.1074/jbc.m608619200

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Citations (51)