Fast and Accurate Data Collection for Macromolecular Crystallography Using the JUNGFRAU Detector

<p>XDS/XSCALE/SHELX output as presented as in the "Fast and Accurate Data Collection for Macromolecular Crystallography Using the JUNGFRAU Detector" paper.</p><p><br></p><p>Figure 2</p><p>-- Weak_signal_Thau1_E1M</p><p>-- Weak_signal_Thau1_JF1M</p><p><br></p><p>Figure 3</p><p>-- Photon_rate_Thau3_JF1M_1deg_s</p><p>-- Photon_rate_Thau3_JF1M_20deg_s</p><p>-- Photon_rate_Thau3_JF1M_50deg_s</p><p>-- Photon_rate_Thau3_JF1M_100deg_s</p><p><br></p><p>Figure 4A-B and 5A-F</p><p>-- Pixel_uniformity_Thau2_JF1M</p><p>-- Pixel_uniformity_Thau2_E1M-50</p><p>-- Pixel_uniformity_Thau2_E1M-60</p><p><br></p><p>Figure 4C-D</p><p>-- Pixel_uniformity_Thau2_JF1M_native_SAD_60deg</p><p>-- Pixel_uniformity_Thau2_E1M-50_native_SAD_60deg</p><p><br></p><p>Figure 5H-I</p><p>-- Pixel_uniformity_Thau2_E1M-50_shifting</p><p>-- Pixel_uniformity_Thau2_E1M-60_shifting</p><p>-- Pixel_uniformity_Thau2_JF1M_shifting</p><p><br></p><p>Figure 6</p><p>-- Native_SAD_Thau3</p><p>-- Native_SAD_PepN</p><p>-- Native_SAD_Lyso_12p4keV</p

Data_Sheet_1_Structural Insight Into Conformational Changes Induced by ATP Binding in a Type III Secretion-Associated ATPase From Shigella flexneri.DOCX

<p>Gram-negative bacteria utilize the type III secretion system (T3SS) to inject effector proteins into the host cell cytoplasm, where they subvert cellular functions and assist pathogen invasion. The conserved type III-associated ATPase is critical for the separation of chaperones from effector proteins, the unfolding of effector proteins and translocating them through the narrow channel of the secretion apparatus. However, how ATP hydrolysis is coupled to the mechanical work of the enzyme remains elusive. Herein, we present a complete description of nucleoside triphosphate binding by surface presentation antigens 47 (Spa47) from Shigella flexneri, based on crystal structures containing ATPγS, a catalytic magnesium ion and an ordered water molecule. Combining the crystal structures of Spa47-ATPγS and unliganded Spa47, we propose conformational changes in Spa47 associated with ATP binding, the binding of ATP induces a conformational change of a highly conserved luminal loop, facilitating ATP hydrolysis by the Spa47 ATPase. Additionally, we identified a specific hydrogen bond critical for ATP recognition and demonstrated that, while ATPγS is an ideal analog for probing ATP binding, AMPPNP is a poor ATP mimic. Our findings provide structural insight pertinent for inhibitor design.</p

Structures of Alkaloid Biosynthetic Glucosidases Decode Substrate Specificity

Two similar enzymes with different biosynthetic function in one species have evolved to catalyze two distinct reactions. X-ray structures of both enzymes help reveal their most important differences. The <i>Rauvolfia</i> alkaloid biosynthetic network harbors two <i>O</i>-glucosidases: raucaffricine glucosidase (RG), which hydrolyses raucaffricine to an intermediate downstream in the ajmaline pathway, and strictosidine glucosidase (SG), which operates upstream. RG converts strictosidine, the substrate of SG, but SG does not accept raucaffricine. Now elucidation of crystal structures of RG, inactive RG-E186Q mutant, and its complexes with ligands dihydro-raucaffricine and secologanin reveals that it is the “wider gate” of RG that allows strictosidine to enter the catalytic site, whereas the “slot-like” entrance of SG prohibits access by raucaffricine. Trp392 in RG and Trp388 in SG control the gate shape and acceptance of substrates. Ser390 directs the conformation of Trp392. 3D structures, supported by site-directed mutations and kinetic data of RG and SG, provide a structural and catalytic explanation of substrate specificity and deeper insights into <i>O</i>-glucosidase chemistry

Enzymatic activities of recombinant MERS-CoV nsp13.

<p>MERS-CoV nsp13 carries NTPase activity and RNA duplex unwinding activity. (A) Final purification step of recombinant MERS-CoV nsp13 expressed in insect cells. MERS-CoV nsp13 eluted from Superdex 200 300/10 GL column pre-calibrated with gel filtration standards (thyroglobulin 670 kDa, γ-globulin 158 kDa, ovalbumin 44 kDa and myoglobin 17 kDa and vitamin B12 1,35 kDa). Upper insert, SDS-PAGE analysis of the purified protein. (B) ATPase activity of MERS-CoV nsp13. The velocity of ATP hydrolysis is plotted as the function of ATP concentration. The data was fitted to Michaelis–Menten equation to calculate <i>V</i>_<i>max</i> and <i>K</i>_<i>m</i>. (C) Left, helicase assay shows that MERS-CoV nsp13 can unwind RNA partial duplex with 5’ overhang, but cannot unwind RNA partial duplex with 3’ overhang. RNA strand with HEX label is marked with asterisk. Heat denatured and no enzyme (w/o) controls are indicated. (C) Right, helicase assay showing MERS-CoV nsp13 can utilize different NTPs and dNTP to separate RNA strands with a preference to ATP. The sequence of the RNA substrates is shown at the bottom.</p

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

Overall structure of MERS-CoV nsp13.

<p>(A) Ribbon model of MERS-CoV nsp13 containing CH (orange), Stalk (magenta), 1B (blue), RecA1 (red) and RecA2 (green) domains. (B) Schematic diagram of the domain organization of MERS-CoV nsp13.</p

Pairwise comparison of the isolated CH/ZBD, 1B and helicase core domains of MERS-CoV nsp13, EAV nsp10 and Upf1 helicases.

<p>Pairwise comparison of the isolated CH/ZBD, 1B and helicase core domains of MERS-CoV nsp13, EAV nsp10 and Upf1 helicases.</p

Domain composition of Upf1-related helicases.

<p>Ribbon models of (A) MERS-CoV nsp13, (B) EAV nsp10-DNA PDB ID: 4N0O, (C) Yeast Upf1-RNA PDB ID: 2XZL and (D) Human Upf1 without RNA PDB ID: 2WJV colored by different functional domains. The coloring scheme is indicated on the right.</p

Key structural features of MERS-CoV nsp13.

<p>(A) left, ribbon model of the CH and Stalk domains of MERS-CoV nsp13 colored by secondary structural elements; right, 2-D topology graph of the CH domain. (B) ribbon model of N-terminal Ring module, and (C) C-terminal Ring module of the CH domain. His/Cys residues participating in zinc coordination are highlighted in yellow. (D) Surface representation of CH domain (orange) of MERS-CoV nsp13, two hydrophobic pockets equivalent to protein interaction interfaces on Upf1 are highlighted in green (pocket 1) and cyan (pocket 2). (E) The ATPase active site between RecA1 and RecA2 domains. The conserved helicase motifs are highlighted with different colors.</p

Model of RNA binding to MERS-CoV nsp13.

<p><b>(A)</b> Model of MERS-CoV nsp13-ssRNA complex was generated based on the superimposition of the helicase domain on nsp13 with helicase domains of human Upf1-RNA (PDB ID: 2XZO, orange), yeast Upf1-RNA (PDB ID: 2XZL, red) and EAV -DNA (PDB ID: 4N0O, yellow). (B) Solvent accessible surface and electrostatic potentials of MERS-CoV nsp13. The functional domains and the putative nucleic acids binding channel are indicated.</p