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- PDB-8eum: MicroED structure of an Aeropyrum pernix protoglobin mutant -

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Basic information

Entry
Database: PDB / ID: 8eum
TitleMicroED structure of an Aeropyrum pernix protoglobin mutant
ComponentsProtogloblin ApPgb
KeywordsMETAL BINDING PROTEIN / MicroED / protoglobin / directed evolution
Function / homology
Function and homology information


oxygen binding / heme binding
Similarity search - Function
Protoglobin / Globin-sensor domain / Protoglobin / Globin/Protoglobin / Globin-like superfamily
Similarity search - Domain/homology
: / PROTOPORPHYRIN IX CONTAINING FE / IMIDAZOLE / Protogloblin ApPgb
Similarity search - Component
Biological speciesAeropyrum pernix (archaea)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 2.1 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: To be published
Title: MicroED structure of a protoglobin reactive carbene intermediate
Authors: Danelius, E. / Porter, N.J. / Unge, J. / Arnold, F.H. / Gonen, T.
History
DepositionOct 18, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 8, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)92,47527
Polymers88,7494
Non-polymers3,72623
Water5,549308
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)46.235, 58.269, 80.734
Angle α, β, γ (deg.)104.09, 98.58, 90.11
Int Tables number1
Space group name H-MP1
Symmetry operation#1: x,y,z
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "D" and (resid 6 through 12 or resid 14...
d_2ens_1(chain "B" and (resid 6 through 12 or resid 14...
d_3ens_1(chain "C" and (resid 6 through 12 or resid 14...
d_4ens_1(chain "A" and (resid 6 through 12 or resid 14...

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1ILEGLYT1 - 7
d_12ens_1VALLYST9 - 23
d_13ens_1THRLYST25 - 37
d_14ens_1ALALYST39 - 147
d_15ens_1PROTHRT149 - 175
d_16ens_1LYSTRPU1 - 5
d_17ens_1IMDIMDW
d_21ens_1ILEGLYF1 - 7
d_22ens_1VALLYSF9 - 23
d_23ens_1THRLYSF25 - 37
d_24ens_1ALAGLYF39 - 56
d_25ens_1LEULYSF64 - 154
d_26ens_1PROTHRF156 - 182
d_27ens_1LYSTRPG1 - 5
d_28ens_1IMDIMDI
d_31ens_1ILEGLYM1 - 7
d_32ens_1VALLYSM9 - 23
d_33ens_1THRLYSM25 - 37
d_34ens_1ALAGLYM39 - 56
d_35ens_1LEULYSM64 - 154
d_36ens_1PROTHRM156 - 182
d_37ens_1LYSTRPN1 - 5
d_38ens_1IMDIMDP
d_41ens_1ILEGLYA1 - 7
d_42ens_1VALLYSA9 - 23
d_43ens_1THRLYSA25 - 37
d_44ens_1ALAGLYA39 - 56
d_45ens_1LEULYSA64 - 154
d_46ens_1PROTRPA156 - 187
d_47ens_1IMDIMDC

NCS oper:
IDCodeMatrixVector
1given(-0.999995329114, -0.00282971117565, 0.00115519916442), (-0.00283931896437, 0.999960672811, -0.0084018509283), (-0.00113137892222, -0.0084050916631, -0.999964036561)11.5442481529, -29.2584076868, -34.0319827884
2given(-0.999999613217, -0.000608289055826, 0.00063525554989), (0.000611221094762, -0.999989115162, 0.00462557730441), (0.000632434947186, 0.00462596379691, 0.999989100183)34.6415707942, -17.156018488, -0.0578924968244
3given(0.999999161717, 0.0012923063341, 8.06769103986E-5), (0.00129218009568, -0.999997970602, 0.00154565936293), (8.26742120581E-5, -0.00154555381813, -0.999998802213)-23.1243758785, 11.9424175475, -34.1698129355

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Components

#1: Protein
Protogloblin ApPgb


Mass: 22187.307 Da / Num. of mol.: 4 / 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-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME / Heme B


Mass: 616.487 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#3: Chemical
ChemComp-IMD / IMIDAZOLE / Imidazole


Mass: 69.085 Da / Num. of mol.: 15 / Source method: obtained synthetically / Formula: C3H5N2
#4: Chemical
ChemComp-FE / FE (III) ION / Iron


Mass: 55.845 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Fe
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 308 / 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: Homotetramer 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 mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
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: 2.1→24.27 Å / Fourier space coverage: 73.1 % / Multiplicity: 4.1 / Num. of structure factors: 33826 / Phase residual: 21.11 °
EM diffraction statsFourier space coverage: 72 % / High resolution: 2.1 Å / Num. of intensities measured: 33826 / Num. of structure factors: 33826 / Phase error rejection criteria: na / Rmerge: 0.43
ReflectionBiso Wilson estimate: 8.59 Å2

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Processing

SoftwareName: PHENIX / Version: (1.20.1_4487) / Classification: refinement
EM softwareName: PHENIX / Version: 1.20.1-4487-000 / Category: 3D reconstruction
EM 3D crystal entity∠α: 104.1 ° / ∠β: 98.6 ° / ∠γ: 90.1 ° / A: 46.2 Å / B: 58.3 Å / C: 80.7 Å / Space group name: P1 / Space group num: 1
CTF correctionType: NONE
3D reconstructionResolution: 2.1 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 26.51 / Protocol: OTHER / Space: RECIPROCAL / Target criteria: maximum likelihood
RefinementResolution: 2.1→24.46 Å / SU ML: 0.21 / Cross valid method: FREE R-VALUE / σ(F): 1.96 / Phase error: 20.49 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2119 1715 5.08 %0.05
Rwork0.1884 ---
obs0.1896 33786 71.78 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.014
ELECTRON CRYSTALLOGRAPHYf_angle_d1.559
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d12.353859
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.067916
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.0121105
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.1-2.160.23751310.21732471ELECTRON CRYSTALLOGRAPHY67
2.16-2.230.21931320.21652623ELECTRON CRYSTALLOGRAPHY70
2.23-2.310.24391200.21542643ELECTRON CRYSTALLOGRAPHY70
2.31-2.40.22711380.20642617ELECTRON CRYSTALLOGRAPHY71
2.4-2.510.22251450.21542634ELECTRON CRYSTALLOGRAPHY71
2.51-2.650.27161650.21082712ELECTRON CRYSTALLOGRAPHY73
2.65-2.810.25621390.20722702ELECTRON CRYSTALLOGRAPHY73
2.81-3.030.21651560.20462754ELECTRON CRYSTALLOGRAPHY74
3.03-3.330.21871650.18482755ELECTRON CRYSTALLOGRAPHY74
3.33-3.810.15631580.15812765ELECTRON CRYSTALLOGRAPHY75
3.81-4.80.15491300.13562737ELECTRON CRYSTALLOGRAPHY73
4.8-24.460.22111360.18272658ELECTRON CRYSTALLOGRAPHY71
Refinement TLS params.Method: refined / Origin x: 5.4169 Å / Origin y: -8.8658 Å / Origin z: -16.8698 Å
111213212223313233
T-0.0047 Å20.0134 Å2-0.0463 Å2-0.0295 Å20.0146 Å2---0.0107 Å2
L0.4267 °20.0911 °2-0.2042 °2-0.4824 °20.0454 °2--0.1331 °2
S0.0163 Å °-0.0069 Å °-0.0212 Å °0.0234 Å °-0.0208 Å °0.0091 Å °0.0065 Å °0.0008 Å °-0.0265 Å °
Refinement TLS groupSelection details: all

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