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- PDB-5k7t: MicroED structure of thermolysin at 2.5 A resolution -

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Entry
Database: PDB / ID: 5k7t
TitleMicroED structure of thermolysin at 2.5 A resolution
DescriptorThermolysin (E.C.3.4.24.27)
KeywordsHYDROLASE / Hydrolase
Specimen sourceBacillus thermoproteolyticus / bacteria / image: Bacillus subtilis
MethodElectron crystallography (2.5 Å resolution / 3d array / Crystallography)
Authorsde la Cruz, M.J. / Hattne, J. / Shi, D. / Seidler, P. / Rodriguez, J. / Reyes, F.E. / Sawaya, M.R. / Cascio, D. / Eisenberg, D. / Gonen, T.
CitationNat. Methods, 2017, 14, 399-402

Nat. Methods, 2017, 14, 399-402 Yorodumi Papers
Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED.
M Jason de la Cruz / Johan Hattne / Dan Shi / Paul Seidler / Jose Rodriguez / Francis E Reyes / Michael R Sawaya / Duilio Cascio / Simon C Weiss / Sun Kyung Kim / Cynthia S Hinck / Andrew P Hinck / Guillermo Calero / David Eisenberg / Tamir Gonen

Validation Report
SummaryFull reportAbout validation report
DateDeposition: May 26, 2016 / Release: Apr 5, 2017
RevisionDateData content typeGroupProviderType
1.0Apr 5, 2017Structure modelrepositoryInitial release
1.1Apr 12, 2017Structure modelDatabase references

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Assembly

Deposited unit
A: Thermolysin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,7248
Polyers34,3601
Non-polymers3647
Water37821
#1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)960
ΔGint (kcal/M)-76
Surface area (Å2)12440
MethodPISA
Unit cell
γ
α
β
Length a, b, c (Å)92.065, 92.065, 128.498
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number178
Space group name H-MP 61 2 2

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Components

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Polypeptide(L) , 1 types, 1 molecules A

#1: Polypeptide(L)Thermolysin / Thermostable neutral proteinase


Mass: 34360.336 Da / Num. of mol.: 1 / Source: (natural) Bacillus thermoproteolyticus / References: UniProt: P00800, EC: 3.4.24.27

Cellular component

Molecular function

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Non-polymers , 5 types, 28 molecules

#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Formula: Ca
#3: ChemicalChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Formula: Zn
#4: ChemicalChemComp-DMS / DIMETHYL SULFOXIDE / DMSO (precipitant for crystallization) *YM


Mass: 78.133 Da / Num. of mol.: 1 / Formula: C2H6OS
#5: ChemicalChemComp-IPA / ISOPROPYL ALCOHOL / 2-PROPANOL


Mass: 60.095 Da / Num. of mol.: 1 / Formula: C3H8O
#6: WaterChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 21 / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 3D ARRAY / Reconstruction method: CRYSTALLOGRAPHY

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Sample preparation

ComponentName: Thermolysin / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: 1 / Source: NATURAL
Molecular weightValue: 0.034634 deg. / Units: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Bacillus thermoproteolyticus
Buffer solutionpH: 7.5
Buffer component
IDConc.UnitsNameFormulaBuffer ID
145%dimethyl sulfoxide1
250mMTris1
32.5Mcesium chlorideCsCl1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Data collection

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 4.1 sec. / Electron dose: 0.004 e/Å2 / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number of diffraction images: 721 / Number of grids imaged: 3 / Number of real images: 721
Image scansSampling size: 0.0311999992 microns / Dimension width: 2048 / Dimension height: 2048
EM diffractionCamera length: 1750 mm
EM diffraction shellFourier space coverage: 96.8 / High resolution: 2.5 Å / Low resolution: 2.75 Å / Multiplicity: 12.2 / Number of structure factors: 2741 / Phase residual: 47.2 deg.
EM diffraction statsFourier space coverage: 58.5 / High resolution: 1.6 Å / Number of intensities measured: 224846 / Number of structure factors: 25029 / Overall phase error: 26.44 deg. / Overall phase residual: 44.76 deg. / Phase error rejection criteria: 0 / R merge: 0.634 / R sym: 0.634
ReflectionD resolution high: 1.6 Å / D resolution low: 30.14 Å / Number all: 224846 / Number obs: 25029 / Rmerge I obs: 0.634 / Rpim I all: 0.219 / NetI over sigmaI: 4 / Redundancy: 9 / Percent possible obs: 58.5
Reflection shell
Rmerge I obsHighest resolutionLowest resolutionNumber unique allNumber unique obsRpim I allNetI over sigmaI obsRedundancyPercent possible all
0.7941.601.633141730.6161.01.88.0
0.2068.6330.1431183260.0718.29.695.7

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Processing

SoftwareName: PHENIX / Version: (1.10_2155: ???) / Classification: refinement
EM software
IDNameVersionCategoryImaging IDFitting IDDetailsImage processing ID
1EM-Menu4.0.9.75IMAGE ACQUISITION1
5iMosflm/MOSFLM7.2.1DIFFRACTION INDEXING
6MOLREP11.4.05MODEL FITTING1
8phenix.refine1.10_2155MODEL REFINEMENT1
9MOLREP11.4.05MOLECULAR REPLACEMENTStarting model PDB ID 2tli1
11POINTLESS1.10.21SYMMETRY DETERMINATION1
12AIMLESS0.5.25CRYSTALLOGRAPHY MERGING1
EM 3D crystal entityAngle alpha: 90 deg. / Angle beta: 90 deg. / Angle gamma: 120 deg. / Length a: 90.75 Å / Length b: 90.75 Å / Length c: 126.13 Å / Space group name: P 61 2 2 / Space group num: 178
CTF correctionType: NONE
3D reconstructionResolution: 2.5 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingRef protocol: OTHER / Ref space: RECIPROCAL
Atomic model buildingPDB-ID: 2TLI
Pdb chain ID: A / Pdb chain residue range: 1-316
RefineOverall SU ML: 0.43 / Cross valid method: FREE R-VALUE / Sigma F: 1.41 / Overall phase error: 26.44 / Stereochemistry target values: ML
Solvent computationSolvent shrinkage radii: 0.9 Å / Solvent vdw probe radii: 1.11 Å / Solvent model details: FLAT BULK SOLVENT MODEL
Least-squares processR factor R free: 0.3096 / R factor R work: 0.2899 / R factor obs: 0.291 / Highest resolution: 2.5 Å / Lowest resolution: 30.135 Å / Number reflection R free: 589 / Number reflection obs: 11327 / Percent reflection R free: 5.2 / Percent reflection obs: 96.96
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.0032497
ELECTRON CRYSTALLOGRAPHYf_angle_d0.5093399
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d11.8201418
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.042361
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.003448
Refine LS shell

Refine ID: ELECTRON CRYSTALLOGRAPHY / Percent reflection obs: 97

Highest resolutionR factor R freeR factor R workLowest resolutionNumber reflection R freeNumber reflection R work
2.50040.36640.34782.75191682573
2.75190.31320.32693.14971362646
3.14970.32490.26813.96691372680
3.96690.23360.227230.13761482839

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