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- EMDB-4605: Human Phenylalanine Hydroxylase (hPAH) apo structure -

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Basic information

Database: EMDB / ID: EMD-4605
TitleHuman Phenylalanine Hydroxylase (hPAH) apo structure
Map data
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 5 Å
AuthorsAlcorlo-Pages M / Flydal MI / Martinez-Caballero S / Martinez A / Hermoso JA / Fernandez-Leiro R
CitationJournal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2019
Title: Structure of full-length human phenylalanine hydroxylase in complex with tetrahydrobiopterin.
Authors: Marte Innselset Flydal / Martín Alcorlo-Pagés / Fredrik Gullaksen Johannessen / Siseth Martínez-Caballero / Lars Skjærven / Rafael Fernandez-Leiro / Aurora Martinez / Juan A Hermoso /
Abstract: Phenylalanine hydroxylase (PAH) is a key enzyme in the catabolism of phenylalanine, and mutations in this enzyme cause phenylketonuria (PKU), a genetic disorder that leads to brain damage and mental ...Phenylalanine hydroxylase (PAH) is a key enzyme in the catabolism of phenylalanine, and mutations in this enzyme cause phenylketonuria (PKU), a genetic disorder that leads to brain damage and mental retardation if untreated. Some patients benefit from supplementation with a synthetic formulation of the cofactor tetrahydrobiopterin (BH) that partly acts as a pharmacological chaperone. Here we present structures of full-length human PAH (hPAH) both unbound and complexed with BH in the precatalytic state. Crystal structures, solved at 3.18-Å resolution, show the interactions between the cofactor and PAH, explaining the negative regulation exerted by BH BH forms several H-bonds with the N-terminal autoregulatory tail but is far from the catalytic Fe Upon BH binding a polar and salt-bridge interaction network links the three PAH domains, explaining the stability conferred by BH Importantly, BH binding modulates the interaction between subunits, providing information about PAH allostery. Moreover, we also show that the cryo-EM structure of hPAH in absence of BH reveals a highly dynamic conformation for the tetramers. Structural analyses of the hPAH:BH subunits revealed that the substrate-induced movement of Tyr138 into the active site could be coupled to the displacement of BH from the precatalytic toward the active conformation, a molecular mechanism that was supported by site-directed mutagenesis and targeted molecular dynamics simulations. Finally, comparison of the rat and human PAH structures show that hPAH is more dynamic, which is related to amino acid substitutions that enhance the flexibility of hPAH and may increase the susceptibility to PKU-associated mutations.
DateDeposition: Feb 11, 2019 / Header (metadata) release: Feb 27, 2019 / Map release: Jun 5, 2019 / Update: Jun 19, 2019

Structure visualization

  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.01
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Structure viewerEM map:
Supplemental images

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FileDownload / File: emd_4605.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 160 pix.
= 174.4 Å
1.09 Å/pix.
x 160 pix.
= 174.4 Å
1.09 Å/pix.
x 160 pix.
= 174.4 Å



Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.09 Å
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.01
Minimum - Maximum-0.023875885 - 0.048971385
Average (Standard dev.)0.00008581749 (±0.003215155)
SymmetrySpace group: 1


Map geometry
Axis orderXYZ
CellA=B=C: 174.40001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.091.091.09
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z174.400174.400174.400
start NX/NY/NZ000
MAP C/R/S123
start NC/NR/NS000
D min/max/mean-0.0240.0490.000

Supplemental data

Mask #1

Projections & Slices


Slices (1/2)
Density Histograms

Sample components

Entire apo-hPAH

EntireName: apo-hPAH / Details: homo-tetramer / Number of components: 2

Component #1: protein, apo-hPAH

ProteinName: apo-hPAH / Details: homo-tetramer / Recombinant expression: No
MassTheoretical: 207 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli #1/H766 (bacteria)

Component #2: protein, Phenylalanine-4-hydroxylase

ProteinName: Phenylalanine-4-hydroxylase / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli #1/H766 (bacteria)

Experimental details

Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 4 mg/mL / pH: 7
Support film15 mA
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 95 %
Details: 3microL, no incubation, 2 seconds blot, no wait before plunging.

Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 42 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 130000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500.0 - 3500.0 nm / Energy filter: GIF Quantum LS
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

Image acquisition

Image acquisitionNumber of digital images: 1493

Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 160000
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION / Resolution: 5 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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