Computational protocol: Structural Insight into Substrate Selectivity of Erwinia chrysanthemil-Asparaginase

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Protocol publication

[…] Crystals of ErA were grown at 285 K using the hanging-drop vapor-diffusion method. Two microliters of Erwinia at 10 mg/mL was mixed with 1 μL of reservoir buffer solution. The reservoir solution consisted of 0.1 M HEPES pH 7.5 and 24% PEG MME 2000.Prior to data collection, crystals were soaked for 5 min in 0.1 M HEPES, pH 7.5 and 24% of PEG MME 2000 solution containing either 10 mM l-aspartic acid (Sigma A6683) or 5 mM l-glutamic acid (Sigma 128420). Soaked crystals were then transferred to the same solutions, respectively, but supplemented with 25% glycerol for cryoprotection.Diffraction data were collected at the Life Sciences Collaborative Access Team (LS-CAT) beamline 21-ID-F at Argonne National Laboratory. Data were processed with the XDS package. Structures were determined by molecular replacement with MOLREP using the atomic resolution structure (PDB entry 1O7J()) as a search model. Refinement was conducted using REFMAC and Phenix, and model building was performed using Coot. Since protomer D of the ErA-GLU complex showed a dual conformation for Thr15 plus clear, albeit weak density for the N-terminal loop, we performed refinement of atom occupancies. This step revealed a strong correlation between the occupancy of elements of the closed state and those of the open state. Specifically, the occupancy of the closed and open Thr15 refined to 0.66 and 0.34, respectively; the N-terminal loop (i.e., closed state) refined to an average occupancy of 0.63; likewise, the occupancies of the ligand GLU refined to 0.61 (active conformation) and 0.39 (inactive conformation). On the basis of these numbers, we elected to set the occupancy values of the closed (active) state atoms to 0.67, and those of the open (inactive) to 0.33, reflecting an approximate 2:1 ratio of closed-to-open conformation in protomer D.Data collection and refinement statistics are listed in . Structural figures were prepared using the PyMOL Molecular Graphics System (version 1.6.0, Schrödinger). […]

Pipeline specifications

Software tools XDS, Molrep, PHENIX, Coot, PyMOL
Applications Small-angle scattering, Protein structure analysis
Diseases Precursor Cell Lymphoblastic Leukemia-Lymphoma, Drug-Related Side Effects and Adverse Reactions
Chemicals Amino Acids