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- PDB-6yma: MicroED structure of acetazolamide-bound human carbonic anhydrase II -

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Basic information

Entry
Database: PDB / ID: 6yma
TitleMicroED structure of acetazolamide-bound human carbonic anhydrase II
Componentscarbonic anhydrase 2
KeywordsLYASE / carbonic anhydrase / acetazolamide / inhibitor complex / MicroED
Function / homology
Function and homology information


positive regulation of cellular pH reduction / positive regulation of dipeptide transmembrane transport / regulation of monoatomic anion transport / secretion / cyanamide hydratase / cyanamide hydratase activity / arylesterase activity / regulation of chloride transport / Reversible hydration of carbon dioxide / morphogenesis of an epithelium ...positive regulation of cellular pH reduction / positive regulation of dipeptide transmembrane transport / regulation of monoatomic anion transport / secretion / cyanamide hydratase / cyanamide hydratase activity / arylesterase activity / regulation of chloride transport / Reversible hydration of carbon dioxide / morphogenesis of an epithelium / angiotensin-activated signaling pathway / positive regulation of synaptic transmission, GABAergic / regulation of intracellular pH / carbonic anhydrase / carbonate dehydratase activity / carbon dioxide transport / Erythrocytes take up oxygen and release carbon dioxide / Erythrocytes take up carbon dioxide and release oxygen / neuron cellular homeostasis / apical part of cell / myelin sheath / extracellular exosome / zinc ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Carbonic anhydrase, alpha-class, conserved site / Alpha-carbonic anhydrases signature. / Carbonic anhydrase, alpha-class / Alpha carbonic anhydrase domain / Alpha carbonic anhydrase domain superfamily / Eukaryotic-type carbonic anhydrase / Alpha-carbonic anhydrases profile. / Eukaryotic-type carbonic anhydrase
Similarity search - Domain/homology
5-ACETAMIDO-1,3,4-THIADIAZOLE-2-SULFONAMIDE / Carbonic anhydrase 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 2.5 Å
AuthorsClabbers, M.T.B. / Fisher, S.Z. / Coincon, M. / Zou, X. / Xu, H.
Funding support Sweden, 3items
OrganizationGrant numberCountry
Swedish Research Council2017-05333 Sweden
Swedish Research Council2019-00815 Sweden
Knut and Alice Wallenberg Foundation2018.0237 Sweden
CitationJournal: Commun Biol / Year: 2020
Title: Visualizing drug binding interactions using microcrystal electron diffraction.
Authors: Max T B Clabbers / S Zoë Fisher / Mathieu Coinçon / Xiaodong Zou / Hongyi Xu /
Abstract: Visualizing ligand binding interactions is important for structure-based drug design and fragment-based screening methods. Rapid and uniform soaking with potentially reduced lattice defects make ...Visualizing ligand binding interactions is important for structure-based drug design and fragment-based screening methods. Rapid and uniform soaking with potentially reduced lattice defects make small macromolecular crystals attractive targets for studying drug binding using microcrystal electron diffraction (MicroED). However, so far no drug binding interactions could unambiguously be resolved by electron diffraction alone. Here, we use MicroED to study the binding of a sulfonamide inhibitor to human carbonic anhydrase isoform II (HCA II). We show that MicroED data can efficiently be collected on a conventional transmission electron microscope from thin hydrated microcrystals soaked with the clinical drug acetazolamide (AZM). The data are of high enough quality to unequivocally fit and resolve the bound inhibitor. We anticipate MicroED can play an important role in facilitating in-house fragment screening for drug discovery, complementing existing methods in structural biology such as X-ray and neutron diffraction.
History
DepositionApr 8, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 12, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 24, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: carbonic anhydrase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,6554
Polymers29,2891
Non-polymers3663
Water48627
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area880 Å2
ΔGint-33 kcal/mol
Surface area11400 Å2
MethodPISA
Unit cell
Length a, b, c (Å)42.550, 41.530, 72.110
Angle α, β, γ (deg.)90.000, 104.620, 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 carbonic anhydrase 2


Mass: 29289.062 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli) / Variant (production host): DE3 pLysS / References: UniProt: P00918, carbonic anhydrase
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#3: Chemical ChemComp-AZM / 5-ACETAMIDO-1,3,4-THIADIAZOLE-2-SULFONAMIDE


Mass: 222.245 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H6N4O3S2 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM
#4: Chemical ChemComp-DMS / DIMETHYL SULFOXIDE


Mass: 78.133 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6OS / Comment: DMSO, precipitant*YM
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 27 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: Carbonic anhydrase II / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Data collection

MicroscopyModel: JEOL 2100
Electron gunElectron source: LAB6 / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION
Image recordingElectron dose: 0.15 e/Å2 / Film or detector model: OTHER
EM diffractionCamera length: 1481.74 mm
EM diffraction shellResolution: 35.69→2.5 Å / Fourier space coverage: 80 % / Multiplicity: 4.4 / Num. of structure factors: 6895 / Phase residual: 1 °
EM diffraction statsFourier space coverage: 80 % / High resolution: 2.5 Å / Num. of intensities measured: 30457 / Num. of structure factors: 6902 / Phase error: 27.44 ° / Phase residual: 1 ° / Phase error rejection criteria: 1 / Rmerge: 27.2 / Rsym: 27.2
ReflectionBiso Wilson estimate: 28.05 Å2

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Processing

Software
NameVersionClassification
phenix.refine1.14_3260refinement
PHENIX1.14_3260refinement
EM 3D crystal entity∠α: 90 ° / ∠β: 104.62 ° / ∠γ: 90 ° / A: 42.55 Å / B: 41.52 Å / C: 72.11 Å / Space group name: P21 / Space group num: 4
CTF correctionType: NONE
3D reconstructionResolution: 2.5 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingProtocol: OTHER / Space: RECIPROCAL
Atomic model buildingPDB-ID: 3HS4

3hs4


Pdb chain-ID: A / Accession code: 3HS4 / Pdb chain residue range: 4-261 / Source name: PDB / Type: experimental model
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3HS4

3hs4


Resolution: 2.5→35.69 Å / SU ML: 0.3743 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 27.4362 / Stereochemistry target values: CDL v1.2
RfactorNum. reflection% reflection
Rfree0.255 357 5.18 %
Rwork0.2237 6538 -
obs0.2253 6895 80 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 20.65 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.00192126
ELECTRON CRYSTALLOGRAPHYf_angle_d0.68292886
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.0416299
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.0034374
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d10.43861246
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.5-2.860.36351090.31052049ELECTRON CRYSTALLOGRAPHY76.34
2.86-3.610.29851110.25472246ELECTRON CRYSTALLOGRAPHY82.5
3.61-35.60.1981370.17332243ELECTRON CRYSTALLOGRAPHY81.15

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