Database: SASBDB / ID: SASDGZ4
|Sample||Beta-amylase 2, chloroplastic (AtBAM2) Ndel1|
|Function / homology|
Function and homology information
beta-amylase / beta-amylase activity / amylopectin maltohydrolase activity / chloroplast stroma / polysaccharide catabolic process / chloroplast
Similarity search - Function
Glycoside hydrolase, family 14B, plant / Glycoside hydrolase, family 14 / Glycoside hydrolase, family 14, conserved site / Glycosyl hydrolase family 14 / Beta-amylase active site 1. / Glycoside hydrolase superfamily
Similarity search - Domain/homology
Beta-amylase 2, chloroplastic
Similarity search - Component
|Biological species||Arabidopsis thaliana (thale cress)|
|Citation||Date: 2019 Aug 30|
Title: Solution structure and assembly of β-amylase 2 from Arabidopsis thaliana
Authors: Chandrasekharan N / Ravenburg C / Roy I / Monroe J
Downloads & links
|Sample||Name: Beta-amylase 2, chloroplastic (AtBAM2) Ndel1 / Specimen concentration: 0.35-0.70|
|Buffer||Name: 50 mM HEPES / pH: 7.5|
|Entity #1875||Type: protein / Description: Beta-amylase 2, chloroplastic / Formula weight: 53.696 / Num. of mol.: 4 / Source: Arabidopsis thaliana / References: UniProt: O65258|
Sequence: MGSSHHHHHH SQDPERDFAG TACVPVYVML PLGVIDMNSE VVEPEELLDQ LRTLKSVNVD GVMVDCWWGI VESHTPQVYN WSGYKKLFQM IRELGLKIQV VMSFHECGGN VGDDVHIQIP EWVREIGQSN PDIYFTDSAG RRNTECLTWG IDKQRVLRGR TALEVYFDYM ...Sequence:
MGSSHHHHHH SQDPERDFAG TACVPVYVML PLGVIDMNSE VVEPEELLDQ LRTLKSVNVD GVMVDCWWGI VESHTPQVYN WSGYKKLFQM IRELGLKIQV VMSFHECGGN VGDDVHIQIP EWVREIGQSN PDIYFTDSAG RRNTECLTWG IDKQRVLRGR TALEVYFDYM RSFRVEFDEF FEEKIIPEIE VGLGPCGELR YPSYPAQFGW KYPGIGEFQC YDKYLMNSLK EAAEVRGHSF WGRGPDNTET YNSTPHGTGF FRDGGDYDSY YGRFFLNWYS RVLIDHGDRV LAMANLAFEG TCIAAKLSGI HWWYKTASHA AELTAGFYNS SNRDGYGPIA AMFKKHDAAL NFTCVELRTL DQHEDFPEAL ADPEGLVWQV LNAAWDASIP VASENALPCY DREGYNKILE NAKPLTDPDG RHLSCFTYLR LNPTLMESQN FKEFERFLKR MHGEAVPDLG LAPGTQETNP E
|Beam||Instrument name: Advanced Light Source (ALS) 12.3.1 (SIBYLS)|
City: Berkeley, CA / 国: USA / Type of source: X-ray synchrotronSynchrotron / Wavelength: 0.127 Å / Dist. spec. to detc.: 2 mm
|Detector||Name: Pilatus3 X 2M / Pixsize x: 172 mm|
Title: Beta-amylase 2, chloroplastic (AtBAM2) Ndel1 / Measurement date: Jun 11, 2019 / Storage temperature: 10 °C / Cell temperature: 10 °C / Exposure time: 0.3 sec. / Number of frames: 50 / Unit: 1/A /
|Distance distribution function P(R)|
Sofotware P(R): GNOM 5.0 / Number of points: 303 /
Type of curve: merged
Comments: We expressed Arabidopsis thaliana AtBAM2 in E. coli BL21 cells from a pETDuet-1 expression vector with an N-terminal 6-His tag as constructed by (Monroe et al., 2017, 2018). We induced ...Comments: We expressed Arabidopsis thaliana AtBAM2 in E. coli BL21 cells from a pETDuet-1 expression vector with an N-terminal 6-His tag as constructed by (Monroe et al., 2017, 2018). We induced AtBAM2 expression in BL21 E coli at OD600 with 0.3 mM IPTG in 2xYT broth with 60 µg/ml ampicillin at 30 oC overnight. Cells were lysed and sonicated in a buffer containing 50 mM NaH2PO4, pH 8, 500 mM NaCl, and 2 mM imidazole. The supernatant was loaded onto a TALON cobalt column using an AKTA Start and washed with a buffer containing 50 mM HEPES pH 8, 500 mM NaCl, 5% glycerol, 10 mM TCEP, and 40 mM imidazole. The protein was then eluted with buffer containing 50 mM HEPES pH 8, 500 mM NaCl, 5% glycerol, 10 mM TCEP, and 500 mM imidazole. Pure protein, as determined by a band present at ∼50 kDa on a 4–20% Tris‐Glycine gel stained with Coomassie Blue, was concentrated in a Spin‐X UF concentrator with a 5000 MWCO. The concentrated protein was then further purified using a HiLoad 16/60 column filled with Superdex 200 in 50 mM HEPES, pH 7. Pure protein based on SDS-PAGE was concentrated as before and the concentration of protein was determined via absorbance at 280 nm using an extinction coefficient of 94310 M−1 cm−1 which was calculated from the sequence using ProtParam (Gasteiger et al., 2005). Construction of AtBAM2-Ndel2 was constructed using a similar strategy as that of AtBAM2-Ndel1 (Monroe et al., 2017) but using the primer 5’- TCGAAGAGCGTGATTTTGCGGATCCAGCGTGTGTTCCTGTATATG-3’ and the complementary sequence. AtBAM2 with the Ndel1 or Ndel2 truncations were purified using the same scheme described for the wild-type AtBAM2. Matching buffer exposures were collected before and after samples to ensure there was no difference in the scattering due to contamination of the sample cell. Scattering data were subtracted from buffer and then processed in PRIMUS (ATSAS 2.8.4 r10553) to create an average data file (Konarev et al., 2003). Data were analyzed using PRIMUS, GNOM, and SCÅTTER (v3.0g) to determine dimensions and create a merged data file from the two data sets at 30 and 50 µM AtBAM2 (Konarev et al., 2003). We then used the merged data file in DAMMIF (v1.1.2) and DAMMIN (v5.3) to generate the dummy-atom model and aligned the result to the all-atom structure using SASTBX (Svergun, 1992, 1999; Svergun et al., 2001; Franke & Svergun, 2009; Liu et al., 2012).
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