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- PDB-6mq4: GH5-4 broad specificity endoglucanase from Hungateiclostridium ce... -

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Basic information

Entry
Database: PDB / ID: 6mq4
TitleGH5-4 broad specificity endoglucanase from Hungateiclostridium cellulolyticum
Componentscellulase
KeywordsHYDROLASE / Cellulase / xylanase / manganese / GH5 / endoglucanase
Function / homologyGlycosidases / TIM Barrel / Alpha-Beta Barrel / Alpha Beta / DI(HYDROXYETHYL)ETHER / TRIETHYLENE GLYCOL
Function and homology information
Biological speciesHungateiclostridium cellulolyticum (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.4 Å
AuthorsBingman, C.A. / Smith, R.W. / Glasgow, E.M. / Fox, B.G.
Funding support United States, 2items
OrganizationGrant numberCountry
Department of Energy (DOE, United States)DEFC0207ER64494 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH 5 T32 GM008349 Biotechnology Training Program United States
CitationJournal: J.Biol.Chem. / Year: 2020
Title: A structural and kinetic survey of GH5_4 endoglucanases reveals determinants of broad substrate specificity and opportunities for biomass hydrolysis.
Authors: Glasgow, E.M. / Kemna, E.I. / Bingman, C.A. / Ing, N. / Deng, K. / Bianchetti, C.M. / Takasuka, T.E. / Northen, T.R. / Fox, B.G.
History
DepositionOct 9, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 13, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 4, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Oct 21, 2020Group: Database references / Structure summary / Category: pdbx_database_related / struct / Item: _struct.title
Revision 1.3Feb 17, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.4Oct 11, 2023Group: Advisory / Data collection ...Advisory / Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_unobs_or_zero_occ_atoms
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: cellulase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)40,44410
Polymers39,2251
Non-polymers1,2199
Water8,035446
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)70.850, 70.850, 57.610
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number144
Space group name H-MP31

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Components

#1: Protein cellulase


Mass: 39224.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hungateiclostridium cellulolyticum (bacteria)
Plasmid: pVP67K / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21 / Variant (production host): Rosetta (DE3)
#2: Chemical ChemComp-PG4 / TETRAETHYLENE GLYCOL


Mass: 194.226 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H18O5 / Comment: precipitant*YM
#3: Chemical
ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C4H10O3
#4: Chemical
ChemComp-PGE / TRIETHYLENE GLYCOL


Mass: 150.173 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C6H14O4
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 446 / Source method: isolated from a natural source / Formula: H2O

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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: 2.13 Å3/Da / Density % sol: 42.21 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 4.2
Details: A solution of protein at 1mM was incubated with 5mM cellohexose for 4 hours at room temperature. The mixture was screened using a TTP Labtech Mosquito and MRC SD-2 plates 200 nL protein 200 ...Details: A solution of protein at 1mM was incubated with 5mM cellohexose for 4 hours at room temperature. The mixture was screened using a TTP Labtech Mosquito and MRC SD-2 plates 200 nL protein 200 nL reservoir, 50 microliter total reservoir. Crystals formed in condition B6, 40% ethanol, 10% PEG1000, 0.1M phosphate citrate buffer pH 4.2. Crystals were exposed to vapor from 45% ethanol for 30 seconds prior to plunge cooling in liquid nitrogen.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-F / Wavelength: 0.97857 Å
DetectorType: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Aug 23, 2015
RadiationMonochromator: diamond (111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97857 Å / Relative weight: 1
ReflectionResolution: 1.4→30.176 Å / Num. obs: 63716 / % possible obs: 99.98 % / Redundancy: 7 % / CC1/2: 0.999 / Rmerge(I) obs: 0.05344 / Rpim(I) all: 0.02176 / Rrim(I) all: 0.05772 / Net I/σ(I): 22.1
Reflection shellResolution: 1.4→1.45 Å / Redundancy: 6.9 % / Rmerge(I) obs: 0.4228 / Mean I/σ(I) obs: 4.3 / Num. unique obs: 6406 / CC1/2: 0.921 / Rpim(I) all: 0.1746 / Rrim(I) all: 0.4578 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX(1.14_3211: ???)refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2wab

2wab


Resolution: 1.4→30.176 Å / SU ML: 0.1 / Cross valid method: FREE R-VALUE / σ(F): 1.99 / Phase error: 12.61
RfactorNum. reflection% reflection
Rfree0.1377 1490 2.34 %
Rwork0.1209 --
obs0.1213 63716 99.99 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 1.4→30.176 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2736 0 81 446 3263
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.013005
X-RAY DIFFRACTIONf_angle_d1.1744085
X-RAY DIFFRACTIONf_dihedral_angle_d14.0831092
X-RAY DIFFRACTIONf_chiral_restr0.085446
X-RAY DIFFRACTIONf_plane_restr0.008518
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.4-1.44520.2071420.16615652X-RAY DIFFRACTION100
1.4452-1.49690.16331340.14945682X-RAY DIFFRACTION100
1.4969-1.55680.16241420.13165628X-RAY DIFFRACTION100
1.5568-1.62760.14691340.12025656X-RAY DIFFRACTION100
1.6276-1.71340.13841360.1125647X-RAY DIFFRACTION100
1.7134-1.82080.11161380.10515692X-RAY DIFFRACTION100
1.8208-1.96140.13481320.10845656X-RAY DIFFRACTION100
1.9614-2.15870.13851340.10565660X-RAY DIFFRACTION100
2.1587-2.47090.12431300.10665659X-RAY DIFFRACTION100
2.4709-3.11260.13291360.12055635X-RAY DIFFRACTION100
3.1126-30.18340.13651320.13225659X-RAY DIFFRACTION100
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)
10.2371-0.05990.0640.18780.03910.0546-0.02180.08080.1035-0.09280.0060.0022-0.05670.05780.00550.0893-0.0213-0.00110.07620.01880.0689-11.01636.5642-1.6267
20.17080.05050.07310.32880.03080.233-0.0050.0779-0.0572-0.05950.0234-0.05570.05180.0286-0.01650.0888-0.0181-0.00040.0741-0.00310.0622-15.7223-14.929-2.6047
30.1912-0.0660.04210.24210.1090.0990.0071-0.0715-0.02150.0229-0.00550.00090.03550.01750.24490.0564-0.0131-0.00450.0630.00820.0335-13.6529-9.85096.7985
40.10040.02290.08460.1713-0.01760.07590.0271-0.07110.01460.0261-0.01750.0591-0.0035-0.0350.08310.0498-0.01120.00520.08350.00340.0584-22.3669-4.834412.7134
50.0813-0.09120.0540.23950.04280.446-0.0404-0.00220.01540.01140.01810.0717-0.0099-0.0725-0.34460.0423-0.0088-0.00090.04960.01150.0451-24.3996-1.55376.5207
60.1566-0.0518-0.10930.2328-0.20820.40880.0458-0.04660.1830.0679-0.00990.1001-0.08050.02650.00560.0574-0.00870.01460.0715-0.00870.0963-23.80134.375611.6336
70.30650.1747-0.00080.2632-0.020.60570.0284-0.00950.2029-0.037-0.02930.2487-0.0992-0.18470.14040.08180.0081-0.02430.09180.01090.1783-30.27485.25461.1292
80.16690.1008-0.01990.17210.11680.14540.02350.23980.0768-0.19-0.03350.1252-0.0249-0.1046-0.10070.18570.0348-0.12810.20780.04790.2817-35.63024.5842-13.6278
90.4318-0.0628-0.16680.0170.07080.62290.01750.10130.1331-0.2291-0.01380.1623-0.148-0.13550.16860.1242-0.0017-0.04950.10710.02540.1266-27.4643.5309-6.8885
100.0184-0.0033-0.03090.09260.0620.09030.02140.09530.0477-0.1334-0.01970.0252-0.0644-0.05820.08720.2524-0.0271-0.16480.14460.12520.0848-26.89294.032-17.6502
110.4119-0.0691-0.23950.53420.30310.2831-0.07210.22080.0259-0.40820.0860.0589-0.0463-0.01490.21720.1596-0.0381-0.03180.1162-0.00130.0613-21.2677-8.126-14.0411
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 32 through 56 )
2X-RAY DIFFRACTION2chain 'A' and (resid 57 through 92 )
3X-RAY DIFFRACTION3chain 'A' and (resid 93 through 133 )
4X-RAY DIFFRACTION4chain 'A' and (resid 134 through 173 )
5X-RAY DIFFRACTION5chain 'A' and (resid 174 through 202 )
6X-RAY DIFFRACTION6chain 'A' and (resid 203 through 242 )
7X-RAY DIFFRACTION7chain 'A' and (resid 243 through 272 )
8X-RAY DIFFRACTION8chain 'A' and (resid 273 through 299 )
9X-RAY DIFFRACTION9chain 'A' and (resid 300 through 319 )
10X-RAY DIFFRACTION10chain 'A' and (resid 320 through 337 )
11X-RAY DIFFRACTION11chain 'A' and (resid 338 through 381 )

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