SASDFS8: Yeast alcohol dehydrogenase 1 - SEC-SAXS coupled to multiangle la...

Basic information

• Entry: Database: SASBDB / ID: SASDFS8

Sample

Yeast alcohol dehydrogenase 1 - SEC-SAXS coupled to multiangle laser and quasi-elastic light scattering (MALLS and QELS)

• Alcohol dehydrogenase 1 (protein), ADH1, Saccharomyces cerevisiae (strain ATCC 204508 / S288c)

Function / homology

Function:

methylglyoxal reductase (NADH) / amino acid catabolic process to alcohol via Ehrlich pathway / octanol dehydrogenase (NAD+) activity / methylglyoxal reductase (NADH) activity / pyruvate fermentation to ethanol / butanol dehydrogenase (NAD+) activity / ethanol dehydrogenase (NAD+) activity / melatonin binding / alcohol dehydrogenase (NAD+) activity / allyl-alcohol dehydrogenase / allyl-alcohol dehydrogenase activity / alcohol dehydrogenase / zinc ion binding / identical protein binding / plasma membrane / cytoplasm

Domain/homology:

Alcohol dehydrogenase, zinc-type, conserved site / Zinc-containing alcohol dehydrogenases signature. / Alcohol dehydrogenase-like, C-terminal / Zinc-binding dehydrogenase / Alcohol dehydrogenase, N-terminal / Alcohol dehydrogenase GroES-like domain / Polyketide synthase, enoylreductase domain / Enoylreductase / GroES-like superfamily / NAD(P)-binding domain superfamily

Component:

Alcohol dehydrogenase 1

• Biological species: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)

Contact author

• Melissa Graewert
• Cy M Jeffries

Models

• Model #3521: ; Type: dummy / Software: (SUPCOMB 23 (r9988)) / Radius of dummy atoms: 2.10 A / Symmetry: P222 / Comment: DAMMIN in P222 prolate symmetry / Chi-square value: 1.102 / P-value: 0.021141; Search similar-shape structures of this assembly by Omokage search (details)
• Model #3522: ; Type: atomic; Comment: ADH1, including Zn atoms. 40 harmonics, derived from PDB 4W6Z; Chi-square value: 1.328; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: Yeast alcohol dehydrogenase 1 - SEC-SAXS coupled to multiangle laser and quasi-elastic light scattering (MALLS and QELS); Specimen concentration: 9.2 mg/ml
• Buffer: Name: 50 mM HEPES, 150 mM NaCl, 2% v/v glycerol, / pH: 7 / Comment: Running buffer for SEC-SAXS

Entity #1782

Name: ADH1 / Type: protein / Description: Alcohol dehydrogenase 1 / Formula weight: 36.849 / Num. of mol.: 4
Source: Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
References: UniProt: P00330
Sequence:

MSIPETQKGV IFYESHGKLE YKDIPVPKPK ANELLINVKY SGVCHTDLHA WHGDWPLPVK LPLVGGHEGA GVVVGMGENV KGWKIGDYAG IKWLNGSCMA CEYCELGNES NCPHADLSGY THDGSFQQYA TADAVQAAHI PQGTDLAQVA PILCAGITVY KALKSANLMA GHWVAISGAA GGLGSLAVQY AKAMGYRVLG IDGGEGKEEL FRSIGGEVFI DFTKEKDIVG AVLKATDGGA HGVINVSVSE AAIEASTRYV RANGTTVLVG MPAGAKCCSD VFNQVVKSIS IVGSYVGNRA DTREALDFFA RGLVKSPIKV VGLSTLPEIY EKMEKGQIVG RYVVDTSK

Experimental information

• Beam: Instrument name: PETRA III EMBL P12 / City: Hamburg / 国: Germany / Type of source: X-ray synchrotron / Wavelength: 0.123982 Å / Dist. spec. to detc.: 3 mm
• Detector: Name: Pilatus 6M

Scan

Title: Yeast alcohol dehydrogenase 1 - SEC-SAXS coupled to multiangle laser and quasi-elastic light scattering (MALLS and QELS)
Measurement date: Apr 5, 2019 / Storage temperature: 20 °C / Cell temperature: 20 °C / Exposure time: 1 sec. / Number of frames: 67 / Unit: 1/nm /

	Min	Max
Q	0.073	7.213

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 1414 /

	Min	Max
Q	0.072981	3.97884
P(R) point	1	1414
R	0	9.3

Result

Type of curve: sec
Comments: Alcohol dehydrogenase 1 underwent pre-purification prior to SEC-SAXS using the following method. All procedures were performed at 4 oC. The protein (from Sigma; Gel Filtration Markers Kit MWGF1000) was made to approximately 25 mg/ml in 25 mM HEPES, 50 mM NaCl, 5 mM urea, 1% v/v glycerol, pH 7. Approximately 200 μl of sample were loaded onto a Superdex 75 Increase 10/300 column (GE Healthcare) equilibrated in the same buffer (flow rate = 0.4 ml/min). Fractionated aliquots corresponding to the highest absorbing peak (estimated using UV A280 and UV A245 nm) were pooled and concentrated (30 kDa centrifuge spin filter) to a final concentration of 9.2 mg/ml (the concentration was determined from triplicate UV A280 measurements using an E0.1% of 1.326 (= 1 g/l) calculated from the amino acid sequence (ProtParam)). Approximately 50 μl aliquots were snap-frozen in liquid nitrogen then stored at -80oC prior to the SEC-SAXS analysis that was performed at room temperature in 50 mM HEPES, 150 mM NaCl, 2% v/v glycerol, pH 7. The Rg-correlation through the SEC-SAXS peak, the individual unsubtracted SEC-SAXS frames as well as the results from coupled MALLS and QELS analysis are included in the full entry zip archive. The quoted experimental molecular weight was determined using MALLS in combination with refractive-index (RI) measurements that were recorded from the same sample eluting from the column using a split-flow SEC-SAXS-light scattering configuration (Graewert et al., (2015) Sci. Reports. 5, 10734: doi: 10.1038/srep10734). The average hydrodynamic radius of the protein is 4.5 nm.

	Experimental	Porod
MW	142 kDa	126 kDa
Volume	-	201 nm3

	P(R)	Guinier	Guinier error
Forward scattering, I0	31110	31144.3	9
Radius of gyration, Rg	3.31 nm	3.34 nm	-

	Min	Max
D	-	9.3
Guinier point	1	115