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- PDB-8eun: MicroED structure of an Aeropyrum pernix protoglobin metallo-carb... -

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Entry
Database: PDB / ID: 8eun
TitleMicroED structure of an Aeropyrum pernix protoglobin metallo-carbene complex
ComponentsProtogloblin ApPgb
KeywordsMETAL BINDING PROTEIN / MicroED / directed evolution
Function / homologyProtoglobin / Globin-sensor domain / Protoglobin / Globin/Protoglobin / Globin-like superfamily / oxygen binding / heme binding / Chem-WUF / Protogloblin ApPgb
Function and homology information
Biological speciesAeropyrum pernix (archaea)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 2.5 Å
AuthorsDanelius, E. / Gonen, T. / Unge, J.T.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM136508 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: J Am Chem Soc / Year: 2023
Title: MicroED Structure of a Protoglobin Reactive Carbene Intermediate.
Authors: Emma Danelius / Nicholas J Porter / Johan Unge / Frances H Arnold / Tamir Gonen /
Abstract: Microcrystal electron diffraction (MicroED) is an emerging technique that has shown great potential for describing new chemical and biological molecular structures. Several important structures of ...Microcrystal electron diffraction (MicroED) is an emerging technique that has shown great potential for describing new chemical and biological molecular structures. Several important structures of small molecules, natural products, and peptides have been determined using methods. However, only a couple of novel protein structures have thus far been derived by MicroED. Taking advantage of recent technological advances, including higher acceleration voltage and using a low-noise detector in counting mode, we have determined the first structure of an protoglobin (Pgb) variant by MicroED using an AlphaFold2 model for phasing. The structure revealed that mutations introduced during directed evolution enhance carbene transfer activity by reorienting an α helix of Pgb into a dynamic loop, making the catalytic active site more readily accessible. After exposing the tiny crystals to the substrate, we also trapped the reactive iron-carbenoid intermediate involved in this engineered Pgb's new-to-nature activity, a challenging carbene transfer from a diazirine via a putative metallo-carbene. The bound structure discloses how an enlarged active site pocket stabilizes the carbene bound to the heme iron and, presumably, the transition state for the formation of this key intermediate. This work demonstrates that improved MicroED technology and the advancement in protein structure prediction now enable investigation of structures that was previously beyond reach.
History
DepositionOct 19, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 5, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 12, 2023Group: Refinement description
Category: refine_ls_restr_ncs / struct_ncs_dom ...refine_ls_restr_ncs / struct_ncs_dom / struct_ncs_dom_lim / struct_ncs_oper
Revision 1.2Apr 19, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3May 22, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Protogloblin ApPgb
B: Protogloblin ApPgb
hetero molecules


Theoretical massNumber of molelcules
Total (without water)46,8534
Polymers45,4382
Non-polymers1,4152
Water1,15364
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)58.117, 45.761, 72.632
Angle α, β, γ (deg.)90.000, 105.720, 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
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"

NCS domain segments:

Ens-ID: ens_1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11ILEILETRPTRPAA6 - 1956 - 195
d_12WUFWUFWUFWUFAC201
d_21ILEILETRPTRPBB6 - 1956 - 195
d_22WUFWUFWUFWUFBD201

NCS oper: (Code: givenMatrix: (-0.999922402376, 0.000909120261578, 0.0124242797511), (-0.000824260945088, -0.999976311421, 0.0068335342664), (0.0124301979421, 0.0068227631518, 0.999899464987)Vector: - ...NCS oper: (Code: given
Matrix: (-0.999922402376, 0.000909120261578, 0.0124242797511), (-0.000824260945088, -0.999976311421, 0.0068335342664), (0.0124301979421, 0.0068227631518, 0.999899464987)
Vector: -28.9991860039, 10.7087975274, 0.0879089991536)

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Components

#1: Protein Protogloblin ApPgb


Mass: 22718.887 Da / Num. of mol.: 2 / Mutation: C45G, W59L, Y60V, V63R, C102S, F145Q, I149L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aeropyrum pernix (archaea) / Gene: APE_0287 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9YFF4
#2: Chemical ChemComp-WUF / benzyl[3,3'-(7,12-diethenyl-3,8,13,17-tetramethylporphyrin-2,18-diyl-kappa~4~N~21~,N~22~,N~23~,N~24~)di(propanoato)(2-)]iron


Mass: 707.618 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C41H39FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 64 / 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: Homodimer of Aeropyrum pernix protoglobin (ApePgb) C45G, W59L, Y60V, V63R, C102S, F145Q, I149L mutant
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Aeropyrum pernix (archaea)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenConc.: 20 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 K

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Data collection

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: DIFFRACTION / Nominal defocus max: 0 nm / Nominal defocus min: 0 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 K / Temperature (min): 77 K
Image recordingElectron dose: 0.00025 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON IV (4k x 4k)
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096
EM diffractionCamera length: 2460 mm
EM diffraction shellResolution: 2.5→24.37 Å / Fourier space coverage: 7 % / Multiplicity: 4.3 / Num. of structure factors: 40105 / Phase residual: 29.27 °
EM diffraction statsFourier space coverage: 70 % / High resolution: 2.5 Å / Num. of intensities measured: 40105 / Num. of structure factors: 40105 / Phase error rejection criteria: na / Rmerge: 0.32
ReflectionBiso Wilson estimate: 17.58 Å2

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Processing

Software
NameVersionClassificationNB
phenix.refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM softwareName: PHENIX / Version: 1.20.1-4487-000 / Category: 3D reconstruction
EM 3D crystal entity∠α: 90 ° / ∠β: 105.42 ° / ∠γ: 90 ° / A: 58.15 Å / B: 45.89 Å / C: 71.71 Å / Space group name: P2 / Space group num: 4
CTF correctionType: NONE
3D reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 20.59 / Protocol: OTHER / Space: RECIPROCAL / Target criteria: maximum likelihood
RefinementResolution: 2.5→24.37 Å / SU ML: 0.3157 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 29.2747
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflectionSelection details
Rfree0.2839 888 9.73 %0.1
Rwork0.2327 8234 --
obs0.2378 9122 70.36 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 20.59 Å2
Refinement stepCycle: LAST / Resolution: 2.5→24.37 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3104 0 100 64 3268
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.00473306
ELECTRON CRYSTALLOGRAPHYf_angle_d0.87664520
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.0484462
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.005548
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d9.7835470
Refine LS restraints NCSType: Torsion NCS / Rms dev position: 0.956942943303 Å
LS refinement shell

Refine-ID: ELECTRON CRYSTALLOGRAPHY / % reflection Rfree: 10 %

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.5-2.660.32271380.2735134568.94
2.66-2.860.36131540.26136971.2
2.86-3.150.32591530.2588136771.94
3.15-3.60.27051370.2188140371.3
3.6-4.530.2291530.1997138970.93
4.54-24.370.2741530.2307136168.17

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