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- PDB-8bs0: Room-temperature structure of Pedobacter heparinus N-acetylglucos... -

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Basic information

Entry
Database: PDB / ID: 8bs0
TitleRoom-temperature structure of Pedobacter heparinus N-acetylglucosamine 2-epimerase at 80 MPa helium gas pressure in a sapphire capillary
ComponentsN-acylglucosamine 2-epimerase
KeywordsISOMERASE / HPMX / high-pressure macromolecular crystallography / sapphire capillary / room temperature / sialic acid synthesis / (alpha/alpha)6 barrel
Function / homologyN-acylglucosamine 2-epimerase / N-acylglucosamine 2-epimerase activity / N-acylglucosamine 2-epimerase/Cellobiose 2-epimerase / N-acylglucosamine 2-epimerase (GlcNAc 2-epimerase) / Six-hairpin glycosidase-like superfamily / Six-hairpin glycosidase superfamily / carbohydrate metabolic process / PHOSPHATE ION / N-acylglucosamine 2-epimerase
Function and homology information
Biological speciesPedobacter heparinus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.7 Å
AuthorsLieske, J. / Saouane, S. / Assmann, M. / Zaun, H. / Kuballa, J. / Meents, A.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Federal Ministry for Education and Research031B0405A Germany
CitationJournal: To Be Published
Title: High-pressure macromolecular crystallography to explore the conformational space of proteins
Authors: Lieske, J. / Saouane, S. / Guenther, S. / Reinke, P.Y.A. / Falke, S. / Ewert, W. / Meyer, J. / Pakendorf, T. / Reime, B. / Burkhardt, A. / Crosas, E. / Hakanpaeae, J. / Stachnik, K. / Sieg, ...Authors: Lieske, J. / Saouane, S. / Guenther, S. / Reinke, P.Y.A. / Falke, S. / Ewert, W. / Meyer, J. / Pakendorf, T. / Reime, B. / Burkhardt, A. / Crosas, E. / Hakanpaeae, J. / Stachnik, K. / Sieg, J. / Rarey, M. / Assmann, M. / Zaun, H. / Kuballa, J. / Chapman, H.N. / Meents, A.
History
DepositionNov 24, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 13, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: N-acylglucosamine 2-epimerase
B: N-acylglucosamine 2-epimerase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)97,9996
Polymers97,7382
Non-polymers2614
Water7,584421
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3700 Å2
ΔGint-52 kcal/mol
Surface area28580 Å2
MethodPISA
Unit cell
Length a, b, c (Å)56.350, 93.980, 81.520
Angle α, β, γ (deg.)90.000, 102.550, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

#1: Protein N-acylglucosamine 2-epimerase


Mass: 48868.938 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pedobacter heparinus (bacteria) / Gene: Phep_3251 / Production host: Escherichia coli (E. coli) / References: UniProt: C6Y403
#2: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: PO4
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 421 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.16 Å3/Da / Density % sol: 42.94 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: 12.6 mg/ml protein in 20 mM sodium phosphate buffer pH 7.5 were mixed (4+2 ul) with 0.1 M sodium cacodylate buffer pH 5.0, 0.35 M sodium acetate and 26% PEG 8000. Crystals grew at room temperature in 5-7 days.
Pressure: 101.325 kPa

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Data collection

DiffractionAmbient environment: gaseous He / Ambient pressure: 80000 kPa / Mean temperature: 295 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, DESY / Beamline: P11 / Wavelength: 0.477 Å
DetectorType: DECTRIS PILATUS3 X CdTe 2M / Detector: PIXEL / Date: Jun 15, 2020
Diffraction measurementSpecimen support: sapphire capillary
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.477 Å / Relative weight: 1
ReflectionResolution: 1.7→39.79 Å / Num. obs: 81812 / % possible obs: 89.75 % / Redundancy: 8.2 % / Biso Wilson estimate: 20.52 Å2 / CC1/2: 0.985 / CC star: 0.996 / Rmerge(I) obs: 0.2657 / Rpim(I) all: 0.09072 / Rrim(I) all: 0.2824 / Net I/σ(I): 5.85
Reflection shellResolution: 1.7→1.761 Å / Redundancy: 5.3 % / Rmerge(I) obs: 1.36 / Mean I/σ(I) obs: 0.99 / Num. unique obs: 5880 / CC1/2: 0.434 / CC star: 0.778 / Rpim(I) all: 0.5262 / Rrim(I) all: 1.469 / % possible all: 64.51
Cell measurementPressure: 80000 kPa

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Processing

Software
NameVersionClassification
Cootmodel building
PHENIX1.18-3855_9999refinement
XDSdata reduction
XSCALEdata scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2GZ6

2gz6


Resolution: 1.7→39.79 Å / SU ML: 0.1577 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 19.6962
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.181 1749 2.14 %
Rwork0.1557 79965 -
obs0.1562 81714 89.76 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 27.41 Å2
Refinement stepCycle: LAST / Resolution: 1.7→39.79 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6404 0 12 421 6837
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00756791
X-RAY DIFFRACTIONf_angle_d0.79219228
X-RAY DIFFRACTIONf_chiral_restr0.0461912
X-RAY DIFFRACTIONf_plane_restr0.00511207
X-RAY DIFFRACTIONf_dihedral_angle_d26.7067892
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.7-1.750.27921050.26814758X-RAY DIFFRACTION64.49
1.75-1.810.2921090.24855014X-RAY DIFFRACTION67.6
1.81-1.870.25631490.25426767X-RAY DIFFRACTION91.66
1.87-1.950.27551490.22846796X-RAY DIFFRACTION91.55
1.95-2.030.23671480.19976749X-RAY DIFFRACTION91.41
2.03-2.140.21621480.17616775X-RAY DIFFRACTION91.5
2.14-2.280.16851500.16056917X-RAY DIFFRACTION93.27
2.28-2.450.20181540.16297052X-RAY DIFFRACTION95.14
2.45-2.70.16621590.15637250X-RAY DIFFRACTION97.72
2.7-3.090.20511590.157294X-RAY DIFFRACTION98.21
3.09-3.890.14381600.12317300X-RAY DIFFRACTION97.67
3.89-39.790.14121590.12287293X-RAY DIFFRACTION96.64
Refinement TLS params.

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

IDL11 (°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)
14.395030925291.49110035764-2.68332922993.49748378137-1.473624497895.5600949286-0.0501712300778-0.403306156264-0.3001567985150.460357795034-0.02893989665540.0866973903740.493644424333-0.02336228312160.0883952858810.3628208763180.02318453178210.006370444560240.191476158950.04247733247620.1855193428784.82137386379-32.141482790822.7420637835
20.8657351171630.389251753326-0.4085670787521.731049863870.1598356619822.423849738-0.0138262968594-0.0272814378672-0.02190161909770.06820818323470.0254101579258-0.176745827629-0.0177931351830.289340996848-0.02314221466490.07440352562110.0224883169237-0.01228897508140.160555991794-0.002214295280580.16068911101718.0722940924-16.07438017277.39372027344
30.344961574142-0.483666007827-0.3061964822018.038041631591.235906469695.910145224190.0836041033310.1808038601480.211569858628-0.185831539803-0.1367761335770.668071633297-0.507785099907-0.6405947868040.002176362836420.1853188115080.01169178409880.002957414417440.2138341956990.02896413420560.21929207692511.4002883421-3.04503929517-2.57972472485
45.50472787329-3.19175459277-0.3803609694047.88666772414-1.355061169713.426792493440.1129146734330.2141917430320.0978434315269-0.219199349386-0.05301439476280.157372084406-0.122289930234-0.0159089048778-0.03015261378610.127744196199-0.01997463565650.01244712412270.133797311819-0.03227782410620.1494927721069.35987038477-0.69159109320113.3197412729
52.60581809990.8714980062462.003886764170.7172174949871.117187097292.31404711914-0.0850164874955-0.02129316115190.194580689021-0.0113854495717-0.03880853633510.110188099954-0.174210646297-0.08740078521360.1551109328180.172934449338-0.001343578644550.01076849943410.1462011719530.003703272970460.189891214214-9.48474214973-5.6847530282911.1209584218
60.7682317524130.00993342011963-0.07061347615430.8123882052880.2077741041521.40417094613-0.0114925207983-0.056556367035-0.06960103358810.116329656436-0.01452202873120.04406196582220.144653089961-0.08280646603850.03620548585010.157296376823-0.0179065797060.02612676495070.1291996434020.009617652664860.160299613279-6.81826595007-23.312160005310.5590748478
72.582355985220.06053248288740.1044010531733.17645956723-0.9828430568113.801324469310.00940254003982-0.2732969462830.01768075293070.4164250612910.003494978585510.0830501861313-0.110960573592-0.0240314708018-0.0245271139620.1237564652450.01722053192160.001348145523260.110191250068-0.0016994597260.1646483965435.42946768219-22.184461186516.7202775364
80.9272075614050.117490094056-0.2206773375911.809233426880.2157687295211.9239846831-0.01054248513280.2757969969760.0401150689659-0.322684438012-0.02255718983650.271388150266-0.134697919532-0.4344338174780.02916764003550.1592076019220.0108032352737-0.06426296325970.3180527118770.01837968099590.196310713338-21.2840950875-15.9031450056-27.8270584083
91.701962298861.79283351456-1.621482211684.64336170469-0.7141854846044.785001441590.114840651824-0.1224783009370.05201678522120.0747308062008-0.0411198453292-0.417081249403-0.3114192301170.381324631271-0.1368739116850.2363185790210.028803547786-0.01565331966860.2140403260720.01104364428220.257840089625-10.78581158580.000468624813559-16.6985125725
100.889099959646-0.123500827309-0.1590209848090.705202579565-0.1760588069761.62100321711-0.006413800458170.147163668640.0577296830363-0.153063559181-0.0168496475308-0.0117272555111-0.0400402265946-0.05114223013770.02984375948060.186323721876-0.003236501680210.00739630993720.135074050315-0.001686603190420.126254839639-2.39931175342-19.1106145588-28.671899085
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'A' and (resid 4 through 23 )AA4 - 231 - 20
22chain 'A' and (resid 24 through 95 )AA24 - 9521 - 94
33chain 'A' and (resid 96 through 120 )AA96 - 12095 - 120
44chain 'A' and (resid 121 through 159 )AA121 - 159121 - 159
55chain 'A' and (resid 160 through 241 )AA160 - 241160 - 237
66chain 'A' and (resid 242 through 366 )AA242 - 366238 - 364
77chain 'A' and (resid 367 through 397 )AA367 - 397365 - 397
88chain 'B' and (resid 4 through 95 )BB4 - 951 - 95
99chain 'B' and (resid 96 through 120 )BB96 - 12096 - 121
1010chain 'B' and (resid 121 through 397 )BB121 - 397122 - 395

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