{"code":"SASDVX5","status":"Published","type_of_curve":"SEC-SAS","angular_unit":"1/A","project":{"title":"The Pseudoenzyme β‐Amylase9 From Arabidopsis Activates α‐Amylase3: A Possible Mechanism to Promote Stress‐Induced Starch Degradation","publication":{"title":"The Pseudoenzyme β‐Amylase9 From Arabidopsis Activates α‐Amylase3: A Possible Mechanism to Promote Stress‐Induced Starch Degradation","author_list":"Berndsen C, Storm A, Sardelli A, Hossain S, Clermont K, McFather L, Connor M, Monroe J","journal":"Proteins: Structure, Function, and Bioinformatics","doi":"10.1002/prot.26803","pmid":null,"published_date":"2025 Jan 23"},"status":"released","submitted_date":"2024-07-30","released_date":"2025-01-20"},"pddf_data":"https://www.sasbdb.org/media/p_of_R_files/SASDVX5.out","intensities_data":"https://www.sasbdb.org/media/intensities_files/SASDVX5.dat","intensities_log_plot":"https://www.sasbdb.org/media/intensities_files/scattering_plots/SASDVX5_dat_img.png","intensities_kratky_plot":"https://www.sasbdb.org/media/intensities_files/scattering_plots/SASDVX5_kratky_img.png","pddf_plot":"https://www.sasbdb.org/media/p_of_R_files/pofr_images/SASDVX5_pofr_img.png","intensities_guinier_plot":"https://www.sasbdb.org/media/intensities_files/scattering_plots/SASDVX5_guinier_img.png","sascif_data":"https://www.sasbdb.org/media/sascif/sascif_files/SASDVX5.sascif","experiment":{"instrument":{"detector":{"type":null,"name":"Pilatus3 X 2M","resolution":172.0},"name":"Advanced Light Source (ALS)","city":"Berkeley, CA","country":"USA","beamline_name":"12.3.1 (SIBYLS)","beam_geometry":"","type_of_source":"X-ray synchrotron","point_source":null,"line_collimation":null,"sample_path_length":null,"line_collimation_slitlength":null,"line_collimation_integrationwidth":null,"xray_energy":null,"beam_profile_ah":null,"beam_profile_al":null},"sample":{"molecule":[{"long_name":"Inactive beta-amylase 9","short_name":"BAM9","sequence":"PFLKSTPKSKSLESVKLFVGLPLDTVSDCNNVNHLKAITAGLKALKLLGVEGIELPIFWGVVEKEAAGKYEWSGYLAVAEIVKKVGLKLHASLSFHGSKQTEIGLPDWVAKIGDAEPGIYFTDRYGQQYKDCLSFAVDDVPVLDGKTPMEVYRGFCESFKSAFADYMGNTITGITLGLGPDGELKYPSHQHNAKLSGAGEFQCYDKHMLSALKGYAESTGNPLWGLGGPHDAPAYDQQPNSSSFFSDGGSWESQYGDFFLSWYSSLLTSHADRVLSVASSAFSGIGVPLCGKLPLLHQWHKLRSHPSELTAGFYSSNGQDRYEAIAEIFAKNSCRMIIPGMDLSDEHQSPESLSSPESLLGHIKTSCKKQGVVVSGQNSSTPVPGGFERIVENLKDENVGIDLFTYQRMGALFFSPEHFHAFTVFVRNLSQFELSSDDQASEAEVEAETASIGSGTGAPSLQTA","organism":"Arabidopsis thaliana","uniprot_code":"Q8VYW2","uniprot_range_first":73,"uniprot_range_last":536,"oligomerization":"monomer","molecular_type":"protein","uniprot_sequence":"MEVSVIGNPQARICRAELAYRELGFRFGSDVISGESRNRVSFCNQSSKWKEIAIRCSSRS\nVKCEAIVSDDASPFLKSTPKSKSLESVKLFVGLPLDTVSDCNNVNHLKAITAGLKALKLL\nGVEGIELPIFWGVVEKEAAGKYEWSGYLAVAEIVKKVGLKLHASLSFHGSKQTEIGLPDW\nVAKIGDAEPGIYFTDRYGQQYKDCLSFAVDDVPVLDGKTPMEVYRGFCESFKSAFADYMG\nNTITGITLGLGPDGELKYPSHQHNAKLSGAGEFQCYDKHMLSALKGYAESTGNPLWGLGG\nPHDAPAYDQQPNSSSFFSDGGSWESQYGDFFLSWYSSLLTSHADRVLSVASSAFSGIGVP\nLCGKLPLLHQWHKLRSHPSELTAGFYSSNGQDRYEAIAEIFAKNSCRMIIPGMDLSDEHQ\nSPESLSSPESLLGHIKTSCKKQGVVVSGQNSSTPVPGGFERIVENLKDENVGIDLFTYQR\nMGALFFSPEHFHAFTVFVRNLSQFELSSDDQASEAEVEAETASIGSGTGAPSLQTA","mw":50.296,"total_mw":50.296,"number_molecules":1,"complex_state":false,"deuteration":null,"molecule_source":"biological","molecule_description":"BAM9 expressed from a pETDeut-1 vector"}],"buffer":{"name":"20 mM HEPES, 100 mM NaCl, 0.2 mM TCEP","concentration_unit":null,"comment":null,"additive":null,"concentration":null,"pka":null,"ph":7.0,"deuteration":null},"purity_method":null,"name":"pseudoamylase BAM9","ext_coefficient":null,"contrast":null,"specific_vol":null,"dry_vol":null,"absorbption":null,"deuteration":null,"mixture":null},"contributor":[{"affiliation":[{"short_name":null,"address":"Harrisonburg, Virginia, USA","full_name":"James Madison University","webpage":"https://www.jmu.edu/"}],"contributor_name":"Christopher","contributor_surname":"Berndsen","orcid":"https://orcid.org/0000-0002-4285-4341"}],"concentration_method":null,"concentration_unit":null,"date":"2023-05-16","storage_temperature":-80.0,"cell_temperature":10.0,"exposure_time":2.0,"number_of_frames":20,"wavelength":0.1027,"sample_detector_distance":2.1,"concentration_min":null,"concentration_max":4.0,"sample_volume":null,"flow_rate":0.5,"s_min":0.134,"s_max":4.729,"total_exposure_time":null,"seccolumn":40},"fits":[{"models":[{"model_plot":"https://www.sasbdb.org/media/pdb_file/images/SASDVX5_fit1_model1_img.png","software":"AlphaFold","pdb_link":[],"model_title":null,"type_of_model":"atomic","software_version":"3","model_data":"https://www.sasbdb.org/media/pdb_file/SASDVX5_fit1_model1.cif","model_mw":null,"bead_radius":null,"log":null,"symmetry":null,"comment":null,"user":394}],"fit_unit":"1/A","fit_plot":"https://www.sasbdb.org/media/fitting_files/scattering_plots/SASDVX5_fit1_fixed_fit_img.png","software":"FoXS","chi_square_value":1.024596,"p_value":0.0,"fit_residual_plot":"SASDVX5_fit1_fitresiduals_img.png","fit_data":"https://www.sasbdb.org/media/fitting_files/SASDVX5_fit1.fit","fit_log":null,"software_version":null,"description":""}],"estimated_volume_method":null,"pddf_software":"ATSAS GNOM","pddf_software_version":"5.0","i0_calibration_standard":null,"description":"BAM9 was expressed in BL21 cells with pETDuet-1-BAM9-B cultured with 100 mg/L carbenicillin.  Cells were grown to an OD600 of approximately 0.6 at 37° C and 225 rpm, then induced with 1 mM Isopropyl-β-D-thiogalactoside (IPTG) at 20° C overnight. Cells were pelleted, frozen, and then sonicated on ice in 40 ml Buffer A (0.5 M NaCl, 50 mM NaH2PO4, 0.2 mM TCEP, and 2 mM imidazole, pH 8) with an EDTA-free protease-inhibitor tablet (Pierce A32965).  Cell debris was removed by centrifugation at 17,000 rcf at 4° C.  This supernatant was then diluted to 175 ml with Buffer A.  Protein was then purified using an AKTA Start protein purification system with a 1 ml TALON cobalt-affinity column using washes of 2%, 10%, 20%, and 100% Buffer B (Cytiva Life Science, Marlborough, Massachusetts, USA).   Buffer B contained 0.5 M NaCl, 50 mM NaH2PO4, 0.2 mM TCEP, and 200 mM imidazole, pH 8.  Buffers A and B were supplemented with 3 mM benzamidine (TCI) when purifying AMY3 to reduce degradation. The BAM9 that was analyzed using small-angle X-ray scattering (SAXS) was additionally purified using size exclusion chromatography (SEC) with a Superdex 100 column (Cytiva). Purified protein samples of AMY3 and BAM9 alone and combined were shipped overnight at 4°C and analyzed using SEC-SAXS with in-line with UV/vis absorbance at 280nm and Multi-angle light scattering. Radially averaged SAXS data files were processed and analyzed in Scatter IV and RAW (Hopkins, 2024).","experiment_description":"Synchrotron SAXS data from solutions of pseudoamylase BAM9 in 20 mM HEPES, 100 mM NaCl, 0.2 mM TCEP, pH 7 were collected on the 12.3.1 (SIBYLS) beam line at the Advanced Light Source (ALS; Berkeley, CA, USA) using a Pilatus3 X 2M detector at a sample-detector distance of 2.1 m and at a wavelength of λ = 0.1027 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: A sample at 4 mg/ml was injected at a 0.50 ml/min flow rate onto a Shodex KW-803 column at 10°C. 20 successive 2 second frames were collected through the sample elution peak. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.","tags":[],"intensity_unit":"arbitrary","experimental_mw":42.0,"experimental_mw_error":null,"guinier_i0_mw":null,"guinier_i0_mw_error":null,"porod_mw":46.0,"porod_mw_error":null,"pddf_i0":40.46,"pddf_i0_error":0.013,"guinier_i0":40.4915,"guinier_i0_error":null,"pddf_rg":2.46,"pddf_rg_error":0.01,"guinier_rg":2.436,"guinier_rg_error":0.41,"pddf_dmax":8.7,"pddf_dmax_error":null,"porod_volume":86.7,"porod_volume_error":null,"estimated_volume":null,"estimated_volume_error":null,"guinier_point_first":6,"guinier_point_last":46,"pddf_point_first":null,"pddf_point_last":null,"i0_calibration_standard_data":null,"intensities_log_log_plot":"SASDVX5_datloglog_img.png","symmetry":null,"last_modified":"2025-01-23T14:23:01.159580+01:00","bragg_peak":[]}