PDB-9zgl: Ancestrally reconstructed acetolactate synthase - Ancestor N259 w...

ID or keywords:

Entry

Database: PDB / ID: 9zgl

Title

Ancestrally reconstructed acetolactate synthase - Ancestor N259 with bound ThDP and magnesium.

Components

Ancestral acetolactate synthase, catabolic

Keywords

TRANSFERASE / ThDP / Ancestor

Function / homology

THIAMINE DIPHOSPHATE

Function and homology information

Biological species

synthetic construct (others)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 2.99 Å

Authors

Douw, A. / Guddat, L.W.

Funding support

Australia, 1items

Citation

Journal: To Be Published
Title: Engineering of Acetolactate Synthase for Isobutanol Production Using Ancestral Sequence Reconstruction
Authors: Douw, A. / Krco, S. / Bayaraa, T. / Foley, G. / Davis, S.J. / Paredes Trujillo, O. / Asser, L. / Sieber, V. / Boden, M. / Hine, D. / Guddat, L. / Schenk, G.

History

Deposition	Dec 3, 2025	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Dec 17, 2025	Provider: repository / Type: Initial release

Structure viewer	Molecule: MolmilJmol/JSmol

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Download

PDBx/mmCIF format	9zgl.cif.gz	904.1 KB	Display	PDBx/mmCIF format
PDB format	pdb9zgl.ent.gz	669.7 KB	Display	PDB format
PDBx/mmJSON format	9zgl.json.gz		Tree view	PDBx/mmJSON format
Others	Other downloads

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Validation report

Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/zg/9zgl ftp://data.pdbj.org/pub/pdb/validation_reports/zg/9zgl	HTTPS FTP

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Related structure data

Related structure data	24piC C: citing same article (ref.)
Similar structure data	Similarity search - Function & homologyF&H Search Similarity search - HomologyF&H Search

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Links

PDB pages	PDBj / wwPDB / NCBI
Related items in Molecule of the Month	#263 - Nov 2021 Acetohydroxyacid Synthase similarity (1) #153 - Sep 2012 Pyruvate Dehydrogenase Complex similarity (1)

Deposited unit

A: Ancestral acetolactate synthase, catabolic

B: Ancestral acetolactate synthase, catabolic

C: Ancestral acetolactate synthase, catabolic

D: Ancestral acetolactate synthase, catabolic

hetero molecules

248 kDa, 4 polymers
Search similar-shape structures of this assembly by Omokage search (details)

1

Idetical with deposited unit
defined by author&software

Unit cell

Length a, b, c (Å)	162.942, 163.079, 86.311
Angle α, β, γ (deg.)	90.000, 90.000, 90.000
Int Tables number	19
Space group name H-M	P2₁2₁2₁
Space group name Hall	P2ac2ab
Symmetry operation	#1: x,y,z #2: x+1/2,-y+1/2,-z #3: -x,y+1/2,-z+1/2 #4: -x+1/2,-y,z+1/2

#1: Protein	Ancestral acetolactate synthase, catabolic Mass: 61522.023 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) synthetic construct (others) / Plasmid: pET-28a(+) / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta(DE3) / References: acetolactate synthase
#2: Chemical	ChemComp-TPP / THIAMINE DIPHOSPHATE Mass: 425.314 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C₁₂H₁₉N₄O₇P₂S / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical	ChemComp-MG / MAGNESIUM ION Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 48 / Source method: isolated from a natural source / Formula: H₂O
Has ligand of interest	Y
Has protein modification	N

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Experiment

Experiment	Method: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

Crystal	Density Matthews: 2.23 Å³/Da / Density % sol: 44.8 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8 Details: 0.1M Tris.HCl, 30% (w/v) MPEG2000, 1 mM NADH, 10 mM MgCl2, 20% (v/v) glycerol

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Data collection

Diffraction	Mean temperature: 100 K / Serial crystal experiment: N
Diffraction source	Source: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.95372 Å
Detector	Type: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Nov 2, 2022
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 0.95372 Å / Relative weight: 1
Reflection	Resolution: 2.99→47.93 Å / Num. obs: 37854 / % possible obs: 81.8 % / Redundancy: 4.2 % / B_iso Wilson estimate: 55.65 Å² / CC_1/2: 0.985 / CC star: 0.996 / R_merge(I) obs: 0.1991 / R_pim(I) all: 0.09975 / R_rim(I) all: 0.2241 / Χ²: 0.58 / Net I/σ(I): 8.25
Reflection shell	Resolution: 2.994→3.101 Å / Redundancy: 4.1 % / R_merge(I) obs: 1.144 / Num. unique obs: 3710 / CC_1/2: 0.489 / CC star: 0.811 / R_pim(I) all: 0.5729 / R_rim(I) all: 1.288 / % possible all: 79.98

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Processing

Software

Name	Version	Classification
XDS		data reduction
Aimless	0.7.8	data scaling
PHASER	2.8.3	phasing
PHENIX	1.20.1-4487	refinement

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT / Resolution: 2.99→44.27 Å / SU ML: 0.4058 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 24.4437
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2

	R_factor	Num. reflection	% reflection
R_free	0.2548	1990	5.26 %
R_work	0.1948	35836	-
obs	0.1979	37826	80.34 %

Solvent computation

Shrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL

Displacement parameters

B_iso mean: 51.57 Å²

Refinement step

Cycle: LAST / Resolution: 2.99→44.27 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	16237	0	108	48	16393

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.007	16653
X-RAY DIFFRACTION	f_angle_d	0.9835	22670
X-RAY DIFFRACTION	f_chiral_restr	0.0509	2632
X-RAY DIFFRACTION	f_plane_restr	0.0184	2953
X-RAY DIFFRACTION	f_dihedral_angle_d	17.2116	6108

LS refinement shell

Resolution (Å)	R_factor R_free	Num. reflection R_free	R_factor R_work	Num. reflection R_work	Refine-ID	% reflection obs (%)
2.99-3.07	0.3472	139	0.3122	2494	X-RAY DIFFRACTION	78.67
3.07-3.15	0.3209	139	0.2775	2582	X-RAY DIFFRACTION	82.88
3.15-3.24	0.3275	146	0.2547	2608	X-RAY DIFFRACTION	82.95
3.24-3.35	0.3049	146	0.2429	2610	X-RAY DIFFRACTION	82.89
3.35-3.47	0.3464	147	0.2405	2600	X-RAY DIFFRACTION	82.15
3.47-3.61	0.2994	146	0.2257	2590	X-RAY DIFFRACTION	82.58
3.61-3.77	0.2769	149	0.2185	2585	X-RAY DIFFRACTION	82.13
3.77-3.97	0.2573	140	0.1826	2561	X-RAY DIFFRACTION	81.16
3.97-4.22	0.22	146	0.1728	2558	X-RAY DIFFRACTION	81.03
4.22-4.54	0.2041	140	0.1562	2564	X-RAY DIFFRACTION	80.29
4.55-5	0.2318	139	0.1559	2535	X-RAY DIFFRACTION	79.47
5-5.72	0.2631	138	0.1783	2542	X-RAY DIFFRACTION	78.87
5.72-7.21	0.2615	132	0.1925	2533	X-RAY DIFFRACTION	77.13
7.21-44.27	0.1698	143	0.146	2474	X-RAY DIFFRACTION	73.33

Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

ID	L11 (°²)	L12 (°²)	L13 (°²)	L22 (°²)	L23 (°²)	L33 (°²)	S11 (Å °)	S12 (Å °)	S13 (Å °)	S21 (Å °)	S22 (Å °)	S23 (Å °)	S31 (Å °)	S32 (Å °)	S33 (Å °)	T11 (Å²)	T12 (Å²)	T13 (Å²)	T22 (Å²)	T23 (Å²)	T33 (Å²)	Origin x (Å)	Origin y (Å)	Origin z (Å)
1	0.885677585874	0.464760453776	0.321615268119	1.52514301605	0.171909339531	1.08100632072	0.0557145197655	-0.00915887924122	-0.159906111858	0.0657063477437	-0.0772338360092	-0.347288473925	-0.079361448425	0.187911024207	0.0397725404177	0.270643164631	0.012742886478	-0.0440670890637	0.332222152035	0.0097482790535	0.4049346511	13.4961711407	-3.61477346061	-33.8123642517
2	1.17784885109	-0.0286475579363	0.293352726274	1.20684760157	-0.0637177777606	0.732015905127	-0.0142752149647	-0.233954863331	-0.197943623842	0.328669460581	-0.0292185685351	-0.28890611112	-0.0842439912524	0.126309289683	0.0458463152288	0.497568906751	-0.0182356114569	-0.153057677915	0.491802877631	0.0419285575018	0.417322102575	14.9948976634	-7.07904476529	-14.0271626684
3	1.2126361353	0.274975688242	-0.396554440925	1.34297626359	-0.144097978392	0.703516325377	-0.00349764786169	0.191509447708	0.0158858101977	-0.197913247103	0.00524396032134	0.061771814869	0.00198357474671	0.0899547585644	0.0546368141436	0.347974134213	0.0183171235913	-0.0144152410212	0.352827410261	0.0144188368728	0.229591896295	-13.7074522424	9.28507930444	-57.3834658731
4	1.35100196592	0.431935354398	0.297130361117	0.845017938469	0.303663430816	0.268606055993	-0.138138905406	-0.435444462425	-0.167979002019	-0.0742640019205	-0.0784981500257	0.495183410652	0.141002872796	0.0268935469174	0.0649799802173	0.352397823826	0.0141289934752	0.0294938780648	0.36166256297	0.104273633844	0.467501263586	-36.7714367446	7.6433442252	-41.6891543628
5	1.44638293856	-0.149336435601	-0.397946139595	1.33590442317	-0.5241405118	0.631168262273	0.0913525018336	-0.107609190475	0.138701032152	-0.0881556905117	0.0218212627957	0.113841570049	-0.280426982836	-0.21849220717	-0.0265400875679	0.351780654554	0.0337458473075	0.0824277842443	0.364877605985	0.0380597003869	0.447165470074	-36.8908976238	21.6788848587	-33.9076041191
6	1.43793649597	-0.35434633098	-0.254649616412	1.53058526724	0.154894215837	0.82052019933	-0.082253613846	0.0040797872834	0.0111990990901	0.0623468212639	0.084792097315	0.640809014587	-0.269887450824	-0.0691342897502	-0.0364198763158	0.403925765348	0.11872348785	0.0688127397819	0.352447321814	-0.0361158036696	0.597748586968	-46.7098383972	21.941871166	-33.6918909308
7	1.78940321861	0.511760750508	0.399287368661	1.22557523616	-0.125762097393	1.58779603605	-0.0628758290832	0.418351929056	0.150403137165	-0.0551965516771	0.109692534305	0.631257272761	-0.769746183957	-0.0723403565355	-0.0390094816695	0.420577754893	0.0587460403783	-0.00439281314115	0.327395671978	0.067660416018	0.564860152536	-32.7901064797	36.5714289668	-47.9678269577
8	1.16368075415	0.157199025549	-0.163513506303	0.700502933626	-0.244188689797	0.931165247378	0.00746772628681	0.0381880472518	0.219573073287	-0.116698518141	0.0420111589281	0.0572241115624	-0.237496094549	0.0289073898633	-0.0521614883646	0.456410646489	0.0602373248864	0.0223860651085	0.332139872836	0.0530272747514	0.411405836509	-19.5263450224	33.5016889203	-50.67116995
9	1.29976023086	-0.251072686779	-0.321954160861	1.2955432433	-0.00353411385397	1.62849181675	0.274746510178	0.229164931128	0.545790608321	-0.102406909908	0.17787098583	0.221072690445	0.0884648838314	-0.073875910761	-0.0952035148405	0.53851280813	0.024797491959	-0.0318183500542	0.374834509813	0.085693752505	0.620171146548	-24.9379673067	43.2535417488	-53.6378927068
10	1.55860512257	0.451847632005	-0.0940081155084	0.56448972833	-0.414854144158	0.310007032405	-0.02597185703	0.197761954055	0.23471658868	0.258530935014	-0.069062399916	-0.374587177941	-0.537357869888	0.087977404071	0.0487880769337	0.450069005959	-0.0826844062358	-0.0451563622062	0.475618987742	-0.0290387305517	0.426371695626	-0.252881255906	33.4219040787	-31.8958184412
11	1.1955182632	-0.0436955118566	-0.0541255892371	0.640544506204	-0.58679238802	0.543295274292	-0.0134549843942	-0.0646594432061	0.0286069918616	0.0564214577942	-0.0455821130665	-0.191121289773	-0.126954888532	-0.0067572501737	0.0135708906973	0.391778330464	-0.0846445747501	0.00892861737122	0.332754584235	0.00723270349571	0.374092884922	-1.40158079107	23.0256160698	-38.7884031432
12	1.11736872134	0.058765376502	-0.133985473849	0.864155462603	0.0372934251687	0.517692906237	0.101844276261	-0.0822284155753	0.195604403292	0.0965798780663	0.0618882305843	-0.375184481739	-0.054704678331	0.3218641088	-0.0121951913319	0.489852234164	0.0107478456055	0.0256114153266	0.377403016682	0.0151084237512	0.408588081869	7.7250631868	21.9206677247	-31.795719836
13	1.33814422521	0.291267219039	0.0226540463131	0.999839794527	0.252345777677	0.872366729503	0.123763931189	0.478820286744	0.192335951384	0.0916031778894	-0.138421231417	-0.538667853516	-0.108152716674	0.664343912193	0.132636812758	0.383891193344	-0.0266751478207	0.0517293929007	0.597119315056	0.110328236654	0.593342006931	28.987844191	17.2020821895	-57.2736417198
14	1.2465364162	-0.04870289006	0.0253302808203	0.516866963718	-0.310190108181	0.335541454461	-0.0256187295767	0.341229065774	-0.265043307118	-0.264307495845	0.0173553723702	-0.170777095167	0.194266099769	0.40038341212	0.0734105423768	0.372272299024	0.0473820889079	0.138259699013	0.597982417281	0.0357036043104	0.473697207534	19.6902067765	11.8597000483	-64.4631309005
15	2.73985668527	0.492319047528	0.435972781714	1.87524649701	-1.14992922468	1.20126271181	0.313620583044	0.371901076058	-0.00177703229086	-0.25613525194	-0.335715834271	-0.244612888513	0.240497496162	0.287617041891	0.0245649367695	0.348998455716	0.0467887831051	0.0480046861853	0.456594846939	-0.0223633533007	0.558688237773	19.7701238919	3.54007993295	-56.9531929011
16	1.47460145548	0.243517018638	-0.0173312155741	0.850069355668	-0.497766316256	1.26894952944	0.000634134031174	-0.0105518158428	-0.139380180372	-0.324533440316	-0.19717568793	-0.103159104691	0.168831310971	0.762797222	0.163029253414	0.524872716273	0.0631628672147	0.0996697170957	0.615815507713	0.10896176674	0.831836638538	32.1657924266	14.594211911	-61.5719564294
17	2.34514566337	0.298129079143	0.141305824318	1.94239734169	-0.644712241796	1.62412024509	-0.231918582497	0.189502290173	0.584790589167	-0.523931776857	0.0352779634324	-0.314189693417	-0.293124291646	0.31133348309	0.112831887055	0.42444544047	-0.098644645249	0.0659477155284	0.590044622774	0.102930436187	0.458428449833	11.6646649616	33.025522118	-69.6946388987
18	1.28492647038	0.141910674528	0.0664950816376	0.936996233632	-0.251855162667	0.576510826324	0.0585630650591	0.120978769111	0.164740991648	-0.23200382994	-0.0625193264134	-0.186600257643	-0.0340033968417	0.261428498165	0.054190256615	0.422526773785	-0.0196804381805	0.0221951670685	0.488597945847	0.0832004038495	0.393917409738	4.95181896482	28.8039737181	-61.6424740522
19	0.710180760285	0.117709571537	0.135946097034	1.06415527457	-0.46577307049	0.279228695764	0.256057131356	0.199163488794	0.254069490851	-0.247096381153	-0.295857306352	0.130640909564	0.0190998725269	-0.147970029898	0.0375081335454	0.501888742631	-0.0721339145513	-0.000959319090396	0.566452827326	0.0541015737263	0.451405271597	-5.94812636674	34.6207670294	-69.3355972087
20	1.38110607965	0.027791427654	0.273972191074	1.02147999935	-0.65428128772	0.899454367397	0.0454457835837	0.549190171438	0.261722664215	-0.652066547727	-0.319759273885	0.188790805207	-0.115429570264	0.154983201432	0.06367996663	0.418538366927	-0.0668655967813	0.0425298548933	0.511246259515	0.0906347368664	0.391306072287	6.89671176388	33.0390341763	-72.6504490974
21	1.32929226113	0.0722323839833	0.3779513854	1.41785021184	0.0096370850988	0.598275314676	0.118874717423	-0.119318096022	-0.0599219324074	0.24235331714	-0.0388468452187	0.0389213131595	-0.0801621641649	0.0349509003185	0.0332183232204	0.416365052219	-0.00693919048759	-0.01332544476	0.312751585387	0.0348138573014	0.24616181635	-12.9582675758	0.247904670268	-21.9637258852
22	1.17888390389	0.0159924650408	-0.275136868971	1.27472040777	-0.0669354519263	0.329193815594	0.0470903140412	0.0385172149133	0.0268012620246	0.00488825102547	-0.0263808250768	0.292901021034	-0.162802521736	-0.24782451553	0.0920357274899	0.367039285215	0.0686545032891	-0.0231832004979	0.319138741595	0.0146949797331	0.3307098586	-34.5526213456	-5.70095328877	-38.6224406186
23	0.890577691761	0.185804498384	0.287586113669	1.26447727734	0.198669615851	0.984873722108	0.216192803697	-0.0517849874312	0.0053866040867	0.0104669687234	-0.0535785135707	0.199882663896	0.0706878013445	0.0864715866819	-0.0187292753437	0.359415974357	-0.000929351846263	-0.0904665825827	0.320070900114	0.0630132471665	0.462730699435	-31.1282749226	-22.0960094446	-43.3277159297
24	1.1085911151	0.35110624453	-0.431198855947	1.21236297876	-0.0601882337421	0.961184575172	-0.0505299221189	0.184438650142	-0.114391747766	-0.1484111733	0.0312219962857	0.0760010742865	0.060101621546	-0.172461439954	-0.00921924505546	0.365899922027	0.0181638754434	-0.107888974114	0.36676968882	-0.0338678309974	0.414910867476	-33.3451593042	-17.5638781692	-49.8072893767
25	2.11317455943	0.213450155801	-0.237722588206	1.48066037928	-0.254417989776	1.34924217993	0.00163658645687	-0.344871711895	-0.479047981699	-0.11576104708	0.140147901635	0.356770699686	0.372517801116	-0.327320233703	-0.0464034324516	0.38715940197	-0.0548817402384	-0.113346743924	0.374397407899	0.0420670402954	0.415801272512	-21.8569853331	-31.0406760159	-31.9504753962
26	0.714390706951	0.261262387409	0.0879390837039	1.08736414773	0.0435753178318	0.919012402228	0.0735354949718	0.00372085577805	-0.30642570289	0.146968140521	-0.10070262543	-0.0401867436445	0.354139922767	0.202354163346	-0.0446765343725	0.402035862581	0.0076898351635	-0.0951041207745	0.476750114767	0.103120146698	0.422004587888	-9.94055810569	-24.4994107561	-28.7428542883
27	1.11431393779	0.118425505287	0.200542819863	1.07598407688	0.0632974746427	1.0383829513	0.108175015254	0.0669017885499	-0.534533629183	0.373925857295	-0.0462042549454	-0.0252074496833	0.511094313623	0.37803503781	-0.0914509003848	0.499627230698	0.0121243947547	-0.0374628201924	0.488568290765	0.0500386650789	0.574462424208	-12.2907363745	-35.4915405707	-25.8655890751

Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

ID	Refine TLS-ID	Selection details	Auth asym-ID	Label asym-ID	Auth seq-ID	Label seq-ID
1	1	chain 'A' and (resid 13 through 292 )	A	A	13 - 292	1 - 280
2	2	chain 'A' and (resid 293 through 557 )	A	A	293 - 557	281 - 537
3	3	chain 'B' and (resid 13 through 176 )	B	D	13 - 176	1 - 164
4	4	chain 'B' and (resid 177 through 223 )	B	D	177 - 223	165 - 211
5	5	chain 'B' and (resid 224 through 321 )	B	D	224 - 321	212 - 309
6	6	chain 'B' and (resid 322 through 362 )	B	D	322 - 362	310 - 350
7	7	chain 'B' and (resid 363 through 415 )	B	D	363 - 415	351 - 399
8	8	chain 'B' and (resid 416 through 503 )	B	D	416 - 503	400 - 487
9	9	chain 'B' and (resid 504 through 557 )	B	D	504 - 557	488 - 541
10	10	chain 'C' and (resid 13 through 49 )	C	G	13 - 49	1 - 37
11	11	chain 'C' and (resid 50 through 158 )	C	G	50 - 158	38 - 146
12	12	chain 'C' and (resid 159 through 196 )	C	G	159 - 196	147 - 184
13	13	chain 'C' and (resid 197 through 258 )	C	G	197 - 258	185 - 246
14	14	chain 'C' and (resid 259 through 292 )	C	G	259 - 292	247 - 280
15	15	chain 'C' and (resid 293 through 321 )	C	G	293 - 321	281 - 309
16	16	chain 'C' and (resid 322 through 361 )	C	G	322 - 361	310 - 349
17	17	chain 'C' and (resid 362 through 398 )	C	G	362 - 398	350 - 379
18	18	chain 'C' and (resid 399 through 491 )	C	G	399 - 491	380 - 472
19	19	chain 'C' and (resid 492 through 520 )	C	G	492 - 520	473 - 501
20	20	chain 'C' and (resid 521 through 557 )	C	G	521 - 557	502 - 538
21	21	chain 'D' and (resid 13 through 176 )	D	J	13 - 176	1 - 164
22	22	chain 'D' and (resid 177 through 223 )	D	J	177 - 223	165 - 211
23	23	chain 'D' and (resid 224 through 292 )	D	J	224 - 292	212 - 280
24	24	chain 'D' and (resid 293 through 362 )	D	J	293 - 362	281 - 350
25	25	chain 'D' and (resid 363 through 417 )	D	J	363 - 417	351 - 400
26	26	chain 'D' and (resid 418 through 503 )	D	J	418 - 503	401 - 486
27	27	chain 'D' and (resid 504 through 557 )	D	J	504 - 557	487 - 540

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