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- PDB-7lsu: Ruminococcus bromii Amy12-D392A with 63-a-D-glucosyl-maltotriose -

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Basic information

Entry
Database: PDB / ID: 7lsu
TitleRuminococcus bromii Amy12-D392A with 63-a-D-glucosyl-maltotriose
ComponentsPullulanase
KeywordsHYDROLASE / GH13 / pullulanase / CBM48 / complex
Function / homology
Function and homology information


pullulanase / pullulanase activity / cellulose catabolic process / extracellular region
Similarity search - Function
MucBP domain / MucBP domain / Starch-binding module 26 / Starch-binding module 26 / Pullulanase, type I / Glycoside hydrolase, family 13, N-terminal / Carbohydrate-binding module 48 (Isoamylase N-terminal domain) / Dockerin domain / Dockerin domain profile. / Dockerin type I domain ...MucBP domain / MucBP domain / Starch-binding module 26 / Starch-binding module 26 / Pullulanase, type I / Glycoside hydrolase, family 13, N-terminal / Carbohydrate-binding module 48 (Isoamylase N-terminal domain) / Dockerin domain / Dockerin domain profile. / Dockerin type I domain / Dockerin type I repeat / Dockerin domain superfamily / Alpha amylase, catalytic domain / Glycosyl hydrolase, family 13, catalytic domain / Alpha-amylase domain / Glycosyl hydrolase, all-beta / Immunoglobulin E-set / Glycoside hydrolase superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
ACETATE ION / DI(HYDROXYETHYL)ETHER / pullulanase
Similarity search - Component
Biological speciesRuminococcus bromii (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.95 Å
AuthorsKoropatkin, N.M. / Cockburn, D.W. / Brown, H.A. / Kibler, R.D.
Funding support United States, 1items
OrganizationGrant numberCountry
United States - Israel Binational Science Foundation (BSF) United States
CitationJournal: J.Struct.Biol. / Year: 2021
Title: Structure and substrate recognition by the Ruminococcus bromii amylosome pullulanases.
Authors: Cockburn, D.W. / Kibler, R. / Brown, H.A. / Duvall, R. / Morais, S. / Bayer, E. / Koropatkin, N.M.
History
DepositionFeb 18, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 14, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 18, 2023Group: 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
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: Pullulanase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)89,55722
Polymers87,3451
Non-polymers2,21221
Water14,412800
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)47.091, 99.200, 167.502
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

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Protein / Sugars , 2 types, 2 molecules A

#1: Protein Pullulanase /


Mass: 87345.062 Da / Num. of mol.: 1 / Mutation: D392A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ruminococcus bromii (bacteria) / Gene: pulA_2, RBL236_00821 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): pLysS / References: UniProt: A0A2N0UU23, pullulanase
#2: Polysaccharide alpha-D-glucopyranose-(1-6)-alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose-(1-4)-beta-D-glucopyranose


Type: oligosaccharide / Mass: 666.578 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpa1-6DGlcpa1-4DGlcpa1-4DGlcpb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,4,3/[a2122h-1b_1-5][a2122h-1a_1-5]/1-2-2-2/a4-b1_b4-c1_c6-d1WURCSPDB2Glycan 1.1.0
[][b-D-Glcp]{[(4+1)][a-D-Glcp]{[(4+1)][a-D-Glcp]{[(6+1)][a-D-Glcp]{}}}}LINUCSPDB-CARE

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Non-polymers , 6 types, 820 molecules

#3: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical
ChemComp-ACT / ACETATE ION / Acetate


Mass: 59.044 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C2H3O2
#5: Chemical
ChemComp-PEG / DI(HYDROXYETHYL)ETHER / Diethylene glycol


Mass: 106.120 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C4H10O3
#6: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#7: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 800 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: 2.24 Å3/Da / Density % sol: 45.05 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 8.75 - 8.9 mg/ml of protein and 10 mM 63-a-D-glucosyl-maltotriose via hanging drop against a well solution containing 16% PEG 3350, 4% glycerol, 0.3 ammonium acetate, and 0.1 M Bis-Tris pH 6.5.

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Data collection

DiffractionMean temperature: 120 K / Ambient temp details: cryogenic / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-G / Wavelength: 0.979 Å
DetectorType: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Jul 5, 2016
RadiationMonochromator: C(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979 Å / Relative weight: 1
ReflectionResolution: 1.95→41.91 Å / Num. obs: 57743 / % possible obs: 99.2 % / Redundancy: 5.6 % / CC1/2: 0.98 / Net I/σ(I): 6.4
Reflection shellResolution: 1.95→2.02 Å / Num. unique obs: 5719 / CC1/2: 0.64

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
REFMAC5.8.0230refinement
xia2data scaling
PHASERphasing
PDB_EXTRACT3.27data extraction
xia2data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7LSA

7lsa


Resolution: 1.95→41.91 Å / Cor.coef. Fo:Fc: 0.943 / Cor.coef. Fo:Fc free: 0.927 / SU B: 10.899 / SU ML: 0.127 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R Free: 0.149 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.2121 2913 5 %RANDOM
Rwork0.1882 ---
obs0.1894 54830 99.11 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 49.78 Å2 / Biso mean: 15.229 Å2 / Biso min: 6.18 Å2
Baniso -1Baniso -2Baniso -3
1--0.04 Å20 Å20 Å2
2---0.02 Å20 Å2
3---0.06 Å2
Refinement stepCycle: final / Resolution: 1.95→41.91 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5883 0 145 800 6828
Biso mean--25.84 23.03 -
Num. residues----765
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0030.0146142
X-RAY DIFFRACTIONr_bond_other_d0.0010.0185277
X-RAY DIFFRACTIONr_angle_refined_deg0.7851.6788325
X-RAY DIFFRACTIONr_angle_other_deg0.7191.68412344
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.4725766
X-RAY DIFFRACTIONr_dihedral_angle_2_deg32.64524.055291
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.28715951
X-RAY DIFFRACTIONr_dihedral_angle_4_deg14.0141519
X-RAY DIFFRACTIONr_chiral_restr0.0340.2836
X-RAY DIFFRACTIONr_gen_planes_refined0.0020.026923
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021185
X-RAY DIFFRACTIONr_rigid_bond_restr0.154311419
X-RAY DIFFRACTIONr_sphericity_free21.2975437
X-RAY DIFFRACTIONr_sphericity_bonded13.391511672
LS refinement shellResolution: 1.95→2.001 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.322 192 -
Rwork0.286 4039 -
all-4231 -
obs--99.62 %
Refinement TLS params.Method: refined / Origin x: -15.3105 Å / Origin y: -2.2131 Å / Origin z: 20.6619 Å
111213212223313233
T0.0191 Å2-0.0006 Å20.0001 Å2-0.0315 Å20 Å2--0.0006 Å2
L0.0019 °2-0.0012 °20.0001 °2-0.0067 °2-0.0006 °2--0.0001 °2
S0.0004 Å °0.0023 Å °0.0005 Å °-0.0002 Å °-0.0003 Å °0.0013 Å °-0.0006 Å °0.0004 Å °-0.0001 Å °

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