PDB-1kju: Ca2+-ATPase in the E2 State

Basic information

• Entry: Database: PDB / ID: 1kju
• Title: Ca2+-ATPase in the E2 State
• Components: Sarcoplasmic/endoplasmic reticulum calcium ATPase 1a
• Keywords: HYDROLASE / ION PUMP / CALCIUM / MEMBRANE PROTEIN / P-TYPE ATPASE / ACTIVE TRANSPORT / E2 / CRYO-EM

Function / homology

Function:

positive regulation of cardiac muscle cell contraction / positive regulation of calcium ion import into sarcoplasmic reticulum / H zone / positive regulation of fast-twitch skeletal muscle fiber contraction / calcium ion import into sarcoplasmic reticulum / regulation of striated muscle contraction / negative regulation of striated muscle contraction / positive regulation of ATPase-coupled calcium transmembrane transporter activity / P-type Ca2+ transporter / P-type calcium transporter activity / I band / endoplasmic reticulum-Golgi intermediate compartment / sarcoplasmic reticulum membrane / sarcoplasmic reticulum / calcium ion transport / calcium ion binding / endoplasmic reticulum membrane / perinuclear region of cytoplasm / endoplasmic reticulum / ATP hydrolysis activity / ATP binding / membrane

Domain/homology:

P-type ATPase, subfamily IIA, SERCA-type / haloacid dehalogenase-like hydrolase / Cation-transporting P-type ATPase, C-terminal / Cation transporting ATPase, C-terminus / Cation transporter/ATPase, N-terminus / Cation-transporting P-type ATPase, N-terminal / Cation transporter/ATPase, N-terminus / Cation transport ATPase (P-type) / E1-E2 ATPase / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site / P-type ATPase, cytoplasmic domain N / E1-E2 ATPases phosphorylation site. / P-type ATPase, A domain superfamily / P-type ATPase / P-type ATPase, transmembrane domain superfamily / haloacid dehalogenase-like hydrolase / HAD superfamily / HAD-like superfamily

Component:

Sarcoplasmic/endoplasmic reticulum calcium ATPase 1

• Biological species: Oryctolagus cuniculus (rabbit)
• Method: ELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 6 Å
• Authors: Xu, C. / Rice, W.J. / He, W. / Stokes, D.L.

Citation

Journal: J Mol Biol / Year: 2002
Title: A structural model for the catalytic cycle of Ca(2+)-ATPase.
Authors: Chen Xu / William J Rice / Wanzhong He / David L Stokes /
Abstract: Ca(2+)-ATPase is responsible for active transport of calcium ions across the sarcoplasmic reticulum membrane. This coupling involves an ordered sequence of reversible reactions occurring alternately at the ATP site within the cytoplasmic domains, or at the calcium transport sites within the transmembrane domain. These two sites are separated by a large distance and conformational changes have long been postulated to play an important role in their coordination. To characterize the nature of these conformational changes, we have built atomic models for two reaction intermediates and postulated the mechanisms governing the large structural changes. One model is based on fitting the X-ray crystallographic structure of Ca(2+)-ATPase in the E1 state to a new 6 A structure by cryoelectron microscopy in the E2 state. This fit indicates that calcium binding induces enormous movements of all three cytoplasmic domains as well as significant changes in several transmembrane helices. We found that fluorescein isothiocyanate displaced a decavanadate molecule normally located at the intersection of the three cytoplasmic domains, but did not affect their juxtaposition; this result indicates that our model likely reflects a native E2 conformation and not an artifact of decavanadate binding. To explain the dramatic structural effect of calcium binding, we propose that M4 and M5 transmembrane helices are responsive to calcium binding and directly induce rotation of the phosphorylation domain. Furthermore, we hypothesize that both the nucleotide-binding and beta-sheet domains are highly mobile and driven by Brownian motion to elicit phosphoenzyme formation and calcium transport, respectively. If so, the reaction cycle of Ca(2+)-ATPase would have elements of a Brownian ratchet, where the chemical reactions of ATP hydrolysis are used to direct the random thermal oscillations of an innately flexible molecule.

#1: Journal: Nature / Year: 2000
Title: Crystal Structure of the Calcium Pump of Sarcoplasmic Reticulum at 2.6 A Resolution
Authors: Toyoshima, C. / Nakasako, M. / Nomura, H. / Ogawa, H.

#2: Journal: Nature / Year: 1998
Title: Structure of the Calcium Pump from Sarcoplasmic Reticulum at 8-A Resolution
Authors: Zhang, P. / Toyoshima, C. / Yonekura, K. / Green, N.M. / Stokes, D.L.

#3: Journal: Ultramicroscopy / Year: 1997
Title: Distortion Correction of Tubular Crystals: Improvements in the Acetylcholine Receptor Structure
Authors: Beroukhim, R. / Unwin, N.

#4: Journal: Biophys.J. / Year: 2000
Title: Modeling a Dehalogenase Fold into the 8-A Density Map for Ca(2+)-ATPase Defines a New Domain Structure
Authors: Stokes, D.L. / Green, N.M.

History

Deposition	Dec 5, 2001	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Dec 19, 2001	Provider: repository / Type: Initial release
Revision 1.1	Apr 27, 2008	Group: Version format compliance
Revision 1.2	Jul 13, 2011	Group: Version format compliance
Revision 1.3	Jul 18, 2018	Group: Author supporting evidence / Data collection / Category: em_single_particle_entity / em_software / Item: _em_software.image_processing_id
Revision 1.4	Feb 14, 2024	Group: Data collection / Database references / Refinement description Category: 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 / _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

• Remark 999: SEQUENCE THE SEQUENCE IN THIS ENTRY IS AN ISOFORM OF THE SEQUENCE IN SWISSPROT ENTRY P04191. THE C-TERMINAL REGION OF P04191 CONSISTS OF RESIDUES 994-1001, DPEDERRK. IN THIS ENTRY, THE C-TERMINAL RESIDUE IS 994, G.

Assembly

Deposited unit

A: Sarcoplasmic/endoplasmic reticulum calcium ATPase 1a

Summary:

• 110 kDa, 1 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	109,603	1
Polymers	109,603	1
Non-polymers	0	0
Water	0

1

Summary:

• Idetical with deposited unit
• defined by author

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1

Components

#1: Protein

A Sarcoplasmic/endoplasmic reticulum calcium ATPase 1a / Calcium pump 1 / SERCA1 / SR Ca(2+)-ATPase 1

Details:

Mass: 109602.578 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: P04191, EC: 3.6.3.8

Experimental details

Experiment

• Experiment: Method: ELECTRON MICROSCOPY
• EM experiment: Aggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

Sample preparation

• Component: Name: Ca2+-ATPase tubular crystals / Type: COMPLEX; Details: Crystals formed in presence of decavanadate and thapsigargin
• Buffer solution: Name: 20 mM imidazole pH 7.4, 100 mM KCl, 5 mM MgCl2, 0.5 mM EGTA, 0.5 mM decavanadate, 0.01 mM thapsigargin; pH: 7.4 ; Details: 20 mM imidazole pH 7.4, 100 mM KCl, 5 mM MgCl2, 0.5 mM EGTA, 0.5 mM decavanadate, 0.01 mM thapsigargin
• Specimen: Conc.: 0.05 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
• Vitrification: Details: Samples at approximately 1 mg/ml were diluted 1/20 immediately prior to application to glow-discharged fenestrated carbon grids. All freezing was done in cold room. After blotting, samples were plunged into liquid ethane for freezing and stored in liquid nitrogen.
• Crystal grow: Temperature: 4 ℃ / Method: microdialysis

Components of the solutions

ID	Conc.	Common name	Crystal-ID	Sol-ID	Chemical formula	Details
1	0.016 mM	FITC	1	1
2	2 mg/ml	SR	1	1
3	0.3 M	sucrose	1	1
4	5 mM		1	1	MgCl2
5	0.010 mM		1	1	CaCl2
6	20 mM	Tris-HCl	1	1		pH8
7	100 mM		1	2	KCl
8	0.5 mM	EGTA	1	2
9	5 mM		1	2	MgCl2
10	0.5 mM	decavanadate	1	2
11	0.010 mM	thapsigargin	1	2

Electron microscopy imaging

• Microscopy: Model: FEI/PHILIPS CM200FEG / Date: Jan 1, 2000
• Electron gun: Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
• Electron lens: Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000 X / Calibrated magnification: 51300 X / Nominal defocus max: 1400 nm / Nominal defocus min: 700 nm / Cs: 2 mm
• Specimen holder: Temperature: 100 K / Tilt angle max: 0 ° / Tilt angle min: 0 °
• Image recording: Electron dose: 10 e/Ų / Film or detector model: KODAK SO-163 FILM
• Image scans: Num. digital images: 58

Processing

EM software

ID	Name	Category
1	O	model fitting
2	X-PLOR	model fitting
3	Custom	3D reconstruction

• CTF correction: Details: CTF correction of each particle with 4.6% amplitude contrast
• 3D reconstruction: Method: helical reconstruction / Resolution: 6 Å / Num. of particles: 95 / Actual pixel size: 2.53 Å; Magnification calibration: light harvesting complex crystals; Details: Combination of Fourier and real space averaging. Helical Reconstruction: A total of 95 repeats from 58 individual tubes were used for the reconstruction. These tubes fell into 5 helical symmetries, defined by the Bessel order (n) of the principal layer lines (1,0) and (0,1). These 5 symmetries were (-23,6), (-22,6), (-21,6), (-20,6) and (-19,6). For the reference symmetry (-22,6), the unit cell parameters were: a= 56.9A, b=117.1A, gamma=64.2 deg. Data within each helical symmetry were averaged in Fourier space and distortion-correction techniques were applied (Beroukhim and Unwin, 1997). For each repeat, unit cell parameters were calculated, and repeats differing by more than 1.5% of the average value were discarded. The CTF was used to correct phases and weight amplitudes prior to averaging within each helical symmetry. Finally, maps were calculated from each of the averaged datasets. Each of the 2 molecules composing the unit cell were masked and aligned with the corresponding molecule from the reference map (-22,6). The maps were averaged in real-space then back-transformed into Fourier space. Two-fold symmetry was constrained before calculating the final map. Effective resolution of the reconstruction: The resolution of the final reconstruction was determined to be 6 A by two methods. First, the dataset was split into two equal parts and two independant reconstructions were made. After masking and aligning molecules from these maps, Fourier shell correlation coefficients and associated phase residuals were calculated. Secondly, since the crystals had p2 symmetry, two-fold phase residuals of layer lined datasets were used to monitor resolution.; Symmetry type: HELICAL
• Atomic model building: Space: REAL; Details: DETAILS--The atomic coordinates of Ca-ATPase (1EUL) were divided into 4 domains by breaking the peptide at 3 hinge points. Each domain was separately fit into the electron density map by manual docking using the program O. Besides matching overall shape, the major criteria involved matching a-helices into strong columns of density in the map. Whereas cytoplasmic domains were treated as rigid bodies, elements of the transmembrane domain were bent or displaced to match strong densities in the cryo-em map. Finally, the loops composing the 3 hinges were rebuilt to reconnect the domains and X-PLOR was used to perform local energy minimization.

Atomic model building

PDB-ID: 1EUL

1eul
PDB Unreleased entry

Accession code: 1EUL / Source name: PDB / Type: experimental model

• Refinement: Highest resolution: 6 Å

Refinement step

Cycle: LAST / Highest resolution: 6 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	7671	0	0	0	7671

• Software: Name: X-PLOR / Classification: refinement

Refine LS restraints

Refine-ID	Type
X-RAY DIFFRACTION	x_bond_d
X-RAY DIFFRACTION	x_angle_d
X-RAY DIFFRACTION	x_angle_deg
X-RAY DIFFRACTION	x_dihedral_angle_d
X-RAY DIFFRACTION	x_improper_angle_d
X-RAY DIFFRACTION	x_mcbond_it
X-RAY DIFFRACTION	x_scbond_it
X-RAY DIFFRACTION	x_mcangle_it
X-RAY DIFFRACTION	x_scangle_it