SASDGY4: Beta-amylase 2, chloroplastic (AtBAM2) (Beta-amylase 2, chloropla...

Basic information

• Entry: Database: SASBDB / ID: SASDGY4

Sample

Beta-amylase 2, chloroplastic (AtBAM2)

• Beta-amylase 2, chloroplastic (protein), BAM2, Arabidopsis thaliana

Function / homology

Function:

beta-amylase / beta-amylase activity / : / chloroplast stroma / polysaccharide catabolic process / chloroplast

Domain/homology:

Glycoside hydrolase, family 14B, plant / Glycoside hydrolase, family 14, conserved site / Beta-amylase active site 1. / Glycoside hydrolase, family 14 / Glycosyl hydrolase family 14 / Glycoside hydrolase superfamily

Component:

Beta-amylase 2, chloroplastic

• 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

Contact author

• Christopher Berndsen (James Madison University, Harrisonburg, Virginia, USA)

Models

• Model #3698: ; Type: atomic / Chi-square value: 8.96285214274195; Search similar-shape structures of this assembly by Omokage search (details)
• Model #3699: ; Type: dummy / Symmetry: P222 / Chi-square value: 1.311; Search similar-shape structures of this assembly by Omokage search (details)
• Model #3700: ; Type: dummy / Software: (dammif (r10552)) / Chi-square value: 2.189; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: Beta-amylase 2, chloroplastic (AtBAM2) / Specimen concentration: 1.65-2.75
• Buffer: Name: 50 mM HEPES / pH: 7.5

Entity #1874

Name: BAM2 / Type: protein / Description: Beta-amylase 2, chloroplastic / Formula weight: 57.141 / Num. of mol.: 4 / Source: Arabidopsis thaliana / References: UniProt: O65258
Sequence:

MGSSHHHHHH SQDPAESTEE DRVPIDDDDD STDQLVDEEI VHFEERDFAG 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

Experimental information

• Beam: Instrument name: Advanced Light Source (ALS) 12.3.1 (SIBYLS); City: Berkeley, CA / 国: USA / Type of source: X-ray synchrotron / Wavelength: 0.127 Å / Dist. spec. to detc.: 2 mm
• Detector: Name: Pilatus3 X 2M / Pixsize x: 172 mm

Scan

Title: Beta-amylase 2, chloroplastic (AtBAM2) / Measurement date: Jun 11, 2019 / Cell temperature: 10 °C / Exposure time: 0.3 sec. / Number of frames: 50 / Unit: 1/A /

	Min	Max
Q	0.0142	0.3977

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 442 /

	Min	Max
Q	0.018675	0.264485
P(R) point	1	442
R	0	126

Result

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 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) 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). Dummy atom models from different methods were aligned and visualized using YASARA Structure (Krieger et al., 2009).

	Experimental	Porod
MW	208 kDa	-
Volume	-	308 nm3

	P(R)	P(R) error	Guinier	Guinier error
Forward scattering, I0	37.6	0.04	36.82	0.014
Radius of gyration, Rg	4.26 nm	0.03	4.213 nm	0.02

	Min	Max
D	-	12.6
Guinier point	9	31