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- PDB-8pkn: CryoEM structure of catalytic domain of human HMG-CoA reductase w... -

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Basic information

Entry
Database: PDB / ID: 8pkn
TitleCryoEM structure of catalytic domain of human HMG-CoA reductase with its inhibitor atorvastatin
Components3-hydroxy-3-methylglutaryl-coenzyme A reductase
KeywordsOXIDOREDUCTASE / reductase / cholesterol biosynthesis / lipitor / atorvastatin / statins
Function / homology
Function and homology information


hydroxymethylglutaryl-CoA reductase (NADPH) / hydroxymethylglutaryl-CoA reductase (NADPH) activity / sterol biosynthetic process / GTPase regulator activity / coenzyme A binding / negative regulation of amyloid-beta clearance / Cholesterol biosynthesis / EGR2 and SOX10-mediated initiation of Schwann cell myelination / coenzyme A metabolic process / isoprenoid biosynthetic process ...hydroxymethylglutaryl-CoA reductase (NADPH) / hydroxymethylglutaryl-CoA reductase (NADPH) activity / sterol biosynthetic process / GTPase regulator activity / coenzyme A binding / negative regulation of amyloid-beta clearance / Cholesterol biosynthesis / EGR2 and SOX10-mediated initiation of Schwann cell myelination / coenzyme A metabolic process / isoprenoid biosynthetic process / peroxisomal membrane / cholesterol biosynthetic process / negative regulation of protein secretion / NADPH binding / regulation of ERK1 and ERK2 cascade / Activation of gene expression by SREBF (SREBP) / long-term synaptic potentiation / PPARA activates gene expression / visual learning / negative regulation of protein catabolic process / endoplasmic reticulum membrane / endoplasmic reticulum
Similarity search - Function
Hydroxymethylglutaryl-CoA reductase, metazoan / Hydroxymethylglutaryl-CoA reductase, eukaryotic/archaeal type / Hydroxymethylglutaryl-CoA reductase, N-terminal / Hydroxymethylglutaryl-coenzyme A reductases signature 2. / Hydroxymethylglutaryl-coenzyme A reductases signature 1. / Hydroxymethylglutaryl-coenzyme A reductases signature 3. / Hydroxymethylglutaryl-CoA reductase, class I/II / Hydroxymethylglutaryl-CoA reductase, class I/II, NAD/NADP-binding domain superfamily / Hydroxymethylglutaryl-CoA reductase, class I/II, substrate-binding domain superfamily / Hydroxymethylglutaryl-CoA reductase, class I/II, catalytic domain superfamily ...Hydroxymethylglutaryl-CoA reductase, metazoan / Hydroxymethylglutaryl-CoA reductase, eukaryotic/archaeal type / Hydroxymethylglutaryl-CoA reductase, N-terminal / Hydroxymethylglutaryl-coenzyme A reductases signature 2. / Hydroxymethylglutaryl-coenzyme A reductases signature 1. / Hydroxymethylglutaryl-coenzyme A reductases signature 3. / Hydroxymethylglutaryl-CoA reductase, class I/II / Hydroxymethylglutaryl-CoA reductase, class I/II, NAD/NADP-binding domain superfamily / Hydroxymethylglutaryl-CoA reductase, class I/II, substrate-binding domain superfamily / Hydroxymethylglutaryl-CoA reductase, class I/II, catalytic domain superfamily / Hydroxymethylglutaryl-CoA reductase, class I/II, conserved site / Hydroxymethylglutaryl-coenzyme A reductase / Hydroxymethylglutaryl-coenzyme A reductases family profile. / : / Sterol-sensing domain of SREBP cleavage-activation / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain
Similarity search - Domain/homology
Chem-117 / 3-hydroxy-3-methylglutaryl-coenzyme A reductase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.26 Å
AuthorsManikandan, K. / Van Rooyen, J.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Diamond Light Source United Kingdom
Citation
Journal: Acta Crystallogr F Struct Biol Commun / Year: 2025
Title: Cryo-EM structures of apo and atorvastatin-bound human 3-hydroxy-3-methylglutaryl-coenzyme A reductase.
Authors: Manikandan Karuppasamy / Jason van Rooyen /
Abstract: The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) regulates the level of cholesterol by catalysing the formation/production of mevalonate and has therefore become an important ...The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) regulates the level of cholesterol by catalysing the formation/production of mevalonate and has therefore become an important pharmaceutical target for coronary heart disease. Here, we report the cryo-EM structure of the catalytic part of the enzyme in the apo form and bound with its inhibitor atorvastatin, a commonly used drug in cardiovascular disease, at resolutions of 2.1 and 2.3 Å, respectively. In the cryo-EM maps, part of the N-domain corresponding to amino acids 439-487 is well ordered and could be modelled completely. Atorvastatin molecules were found to occupy all four active sites of the tetrameric complex, and the binding does not alter the conformation of the protein or the active site. The method described here exploits graphene oxide as an additional support and could be used as an alternative to elucidate the structures of pharmaceutical target compounds that are difficult to co-crystallize with human HMGR and for sparsely available samples in drug discovery.
#1: Journal: Science / Year: 2001
Title: Structural mechanism for statin inhibition of HMG-CoA reductase.
Authors: E S Istvan / J Deisenhofer /
Abstract: HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase (HMGR) catalyzes the committed step in cholesterol biosynthesis. Statins are HMGR inhibitors with inhibition constant values in the nanomolar ...HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase (HMGR) catalyzes the committed step in cholesterol biosynthesis. Statins are HMGR inhibitors with inhibition constant values in the nanomolar range that effectively lower serum cholesterol levels and are widely prescribed in the treatment of hypercholesterolemia. We have determined structures of the catalytic portion of human HMGR complexed with six different statins. The statins occupy a portion of the binding site of HMG-CoA, thus blocking access of this substrate to the active site. Near the carboxyl terminus of HMGR, several catalytically relevant residues are disordered in the enzyme-statin complexes. If these residues were not flexible, they would sterically hinder statin binding.
History
DepositionJun 27, 2023Deposition site: PDBE / Processing site: PDBE
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: 3-hydroxy-3-methylglutaryl-coenzyme A reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)45,8182
Polymers45,2591
Non-polymers5591
Water00
1
A: 3-hydroxy-3-methylglutaryl-coenzyme A reductase
hetero molecules

A: 3-hydroxy-3-methylglutaryl-coenzyme A reductase
hetero molecules

A: 3-hydroxy-3-methylglutaryl-coenzyme A reductase
hetero molecules

A: 3-hydroxy-3-methylglutaryl-coenzyme A reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)183,2718
Polymers181,0364
Non-polymers2,2354
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation3
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2generate(-1), (-1), (1)199.2, 199.2
3generate(1), (-1), (-1)199.2, 199.2
4generate(-1), (1), (-1)199.2, 199.2

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Components

#1: Protein 3-hydroxy-3-methylglutaryl-coenzyme A reductase / HMG-CoA reductase


Mass: 45259.016 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HMGCR / Production host: Escherichia coli (E. coli)
References: UniProt: P04035, hydroxymethylglutaryl-CoA reductase (NADPH)
#2: Chemical ChemComp-117 / 7-[2-(4-FLUORO-PHENYL)-5-ISOPROPYL-3-PHENYL-4-PHENYLCARBAMOYL-PYRROL-1-YL]- 3,5-DIHYDROXY-HEPTANOIC ACID / ATORVASTATIN


Mass: 558.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C33H35FN2O5 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Catalytic part of hHMGCoA reductase / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 214 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 42 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 18337

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Processing

EM software
IDNameVersionCategory
1Topaz0.2.4particle selection
2EPU3.5image acquisition
4RELION4CTF correction
7Coot0.8.9model fitting
9RELION4initial Euler assignment
10RELION4final Euler assignment
11RELION4classification
12RELION43D reconstruction
13REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3061130
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.26 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 404712 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 1hwk

1hwk


Accession code: 1hwk / Source name: PDB / Type: experimental model
RefinementResolution: 2.26→147.74 Å / Cor.coef. Fo:Fc: 0.932 / WRfactor Rwork: 0.316 / SU B: 3.345 / SU ML: 0.079 / Average fsc free: 0 / Average fsc overall: 0.8673 / Average fsc work: 0.8673 / ESU R: 0.082
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rwork0.3158 252368 -
all0.316 --
Rfree--0 %
obs--100 %
Solvent computationSolvent model: NONE
Displacement parametersBiso mean: 81.224 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.0123242
ELECTRON MICROSCOPYr_bond_other_d00.0163137
ELECTRON MICROSCOPYr_angle_refined_deg1.3051.8164383
ELECTRON MICROSCOPYr_angle_other_deg0.4311.7387229
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.4965422
ELECTRON MICROSCOPYr_dihedral_angle_2_deg26.816526
ELECTRON MICROSCOPYr_dihedral_angle_3_deg17.15110561
ELECTRON MICROSCOPYr_dihedral_angle_6_deg15.63210125
ELECTRON MICROSCOPYr_chiral_restr0.0630.2499
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.023839
ELECTRON MICROSCOPYr_gen_planes_other0.0010.02703
ELECTRON MICROSCOPYr_nbd_refined0.2330.2712
ELECTRON MICROSCOPYr_symmetry_nbd_other0.2050.22906
ELECTRON MICROSCOPYr_nbtor_refined0.1840.21673
ELECTRON MICROSCOPYr_symmetry_nbtor_other0.0790.21872
ELECTRON MICROSCOPYr_xyhbond_nbd_refined0.2040.247
ELECTRON MICROSCOPYr_symmetry_xyhbond_nbd_other0.0350.21
ELECTRON MICROSCOPYr_symmetry_nbd_refined0.1960.226
ELECTRON MICROSCOPYr_nbd_other0.1550.2170
ELECTRON MICROSCOPYr_symmetry_xyhbond_nbd_refined0.1650.216
ELECTRON MICROSCOPYr_mcbond_it5.9027.6931691
ELECTRON MICROSCOPYr_mcbond_other5.8947.6921691
ELECTRON MICROSCOPYr_mcangle_it7.44413.8442112
ELECTRON MICROSCOPYr_mcangle_other7.44713.8482113
ELECTRON MICROSCOPYr_scbond_it7.8958.6171551
ELECTRON MICROSCOPYr_scbond_other7.8928.6161552
ELECTRON MICROSCOPYr_scangle_it10.9415.4382271
ELECTRON MICROSCOPYr_scangle_other10.93715.4362272
ELECTRON MICROSCOPYr_lrange_it12.38294.52313515
ELECTRON MICROSCOPYr_lrange_other12.38294.5213516
LS refinement shell

Refine-ID: ELECTRON MICROSCOPY / Num. reflection Rfree: _ / Total num. of bins used: 20 / % reflection obs: 100 %

Resolution (Å)Rfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc workWRfactor Rwork
2.26-2.3192.543187442.543187440.5292.543
2.319-2.3821.719182271.719182270.7011.719
2.382-2.4511.489177111.489177110.7761.489
2.451-2.5271.278171621.278171620.831.278
2.527-2.611.057166191.057166190.8761.057
2.61-2.7010.87160480.87160480.9040.87
2.701-2.8030.638156420.638156420.9280.638
2.803-2.9170.491149240.491149240.9390.491
2.917-3.0470.343143320.343143320.9420.343
3.047-3.1960.26137510.26137510.9490.26
3.196-3.3690.215130280.215130280.9540.215
3.369-3.5730.204123130.204123130.9550.204
3.573-3.8190.229115870.229115870.9560.229
3.819-4.1250.235107710.235107710.9550.235
4.125-4.5180.25198990.25198990.9530.251
4.518-5.0510.25290210.25290210.9420.252
5.051-5.8310.27178960.27178960.9150.271
5.831-7.1390.30267040.30267040.890.302
7.139-10.0850.28651560.28651560.9050.286
10.085-147.740.3928340.3928340.960.39

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