1I84

CRYO-EM STRUCTURE OF THE HEAVY MEROMYOSIN SUBFRAGMENT OF CHICKEN GIZZARD SMOOTH MUSCLE MYOSIN WITH REGULATORY LIGHT CHAIN IN THE DEPHOSPHORYLATED STATE. ONLY C ALPHAS PROVIDED FOR REGULATORY LIGHT CHAIN. ONLY BACKBONE ATOMS PROVIDED FOR S2 FRAGMENT.

ELECTRON CRYSTALLOGRAPHY

Crystallization

Crystalization Experiments
ID	Method	pH	Temperature	Details
1	Lipid monolayer	7.5	277	MgCl, sodium phosphate, ATP, EGTA, PEG 6000, pH 7.5, Lipid monolayer, temperature 277K

Crystal Data

Unit Cell
Length ( Å )	Angle ( ˚ )
a = 133	α = 90
b = 304	β = 90
c = 200	γ = 91.5

Symmetry
Space Group	P 1 1 2

Diffraction

Diffraction Experiment
ID #	Crystal ID	Scattering Type	Data Collection Temperature	Detector	Detector Type	Details	Collection Date	Monochromator	Protocol
1	1	electron

Refinement

Statistics
Diffraction ID	Structure Solution Method	Resolution (High)	Resolution (Low)	Number Reflections (R-Free)	Percent Reflections (Observed)	R-Work	R-Free	Mean Isotropic B
ELECTRON CRYSTALLOGRAPHY		20

Temperature Factor Modeling
Anisotropic B[1][1]	Anisotropic B[1][2]	Anisotropic B[1][3]	Anisotropic B[2][2]	Anisotropic B[2][3]	Anisotropic B[3][3]

Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen Atoms	Number
Protein Atoms	16580
Nucleic Acid Atoms
Solvent Atoms
Heterogen Atoms

Sample
HEAVY MEROMYOSIN SUBFRAGMENT OF CHICKEN GIZZARD SMOOTH MUSCLE MYOSIN WITH REGULATORY LIGHT CHAIN IN THE DEPHOSPHORYLATED STATE

Specimen Preparation
Sample Aggregation State	2D ARRAY
Vitrification Instrument	HOMEMADE PLUNGER
Cryogen Name	ETHANE
Sample Vitrification Details	Specimens were frozen in liquid ethane using a drop freezing apparatus.

3D Reconstruction
Reconstruction Method	CRYSTALLOGRAPHY
Number of Particles
Reported Resolution (Å)	20
Resolution Method	DIFFRACTION PATTERN/LAYERLINES
Other Details
Refinement Type
Symmetry Type	2D CRYSTAL
Space Group Name	P 2
Length a	133
Length b	304
Angle Gamma	91.5

Map-Model Fitting and Refinement
Id	1
Refinement Space	REAL
Refinement Protocol	OTHER
Refinement Target	VISUAL AGREEMENT
Overall B Value
Fitting Procedure
Details	REFINEMENT PROTOCOL--MANUAL DETAILS--The atomic model consists of two S1 heads and a segment of S2 containing 91 residues. Each S1 consists of a heav ...	REFINEMENT PROTOCOL--MANUAL DETAILS--The atomic model consists of two S1 heads and a segment of S2 containing 91 residues. Each S1 consists of a heavy chain and two light chains, the ELC and the RLC. The atomic model of the S1 subfragment was constructed using two X-ray crystallography models. The heavy chain residues from ala2 to leu819 as well as the essential light chain were obtained from the chicken smooth muscle structure, PDB entry 1BR1, chains A and B. The remainder of S1 including heavy chain residues from glu820 to lys852 (smooth muscle numbering but skeletal myosin sequence) were obtained from residues glu811-lys843 of chicken skeletal muscle myosin, PDB entry 2MYS, since the structure of the entire smooth muscle S1 fragment is not available. Note that the residue numbers for the chain segment from 2MYS that were spliced onto 1BR1 have been renumbered to run consecutively from 819 but the residues themselves are the chicken skeletal muscle myosin heavy chain residues. The regulatory light chain coordinates were also obtained from 2MYS (skeletal) because a smooth muscle myosin model is not available. This regulatory light chain consisted only of C alphas. In fitting the model to the molecular envelope, the lever arm of the free head, i.e. ELC, RLC and heavy chain residues from ile796-lys852, were rotated as a rigid body about the Calpha of ile796. The amount of rotation was about 20 degrees. The blocked head was not modified. The alignment of 2MYS to 1BR1 was done using the conserved residues on the essential light chain. This alignment was carried out using O. The rms of the fit using 123 C alpha atoms of the conserved residues was 1.632 Angstroms. The C alpha distance between leu819 and glu811 (2MYS residue number) after the transformation was 4.23 Angstroms. This distance has not been altered in the model. The splice site at 819-820 is also slightly long and was left that way so the splice site would be easily visible. The other long bonds are at locations where the chain was bent to fit the structure. No minimization or refinement was done to fix them. The following residues have long peptide bond distances- chain S- LEU 819 and GLU 820 THR 854 and ARG 855 MET 860 and GLN 861 GLN 903 and ALA 904 chain V- LEU 819 and GLU 820 LYS 852 and VAL 853 MET 860 and GLN 861 GLN 903 and ALA 904 The 91 residues of the S2 fragment built into the model were calculated using a program written by Dr. Gerald Offer (reference 4). These coordinates consist only of backbone atoms. The S2 sequence begins at val853 which is the correct residue number for smooth muscle myosin. Peptide chain designations- Blocked myosin S1 head myosin heavy chain (with S2 segment) is chain V ELC is chain W RLC is chain Z Free myosin S1 head myosin heavy chain (with S2 fragment) is chain S ELC is chain T RLC is chain U The terms blocked and free refer to the conformations of the two S1 heads. SEQUENCE GAPS IN THE MOLECULAR MODEL- Myosin heavy chain- 205-210, 452-457, 635-655, 944-1185. ELC- 1-2. RLC (skeletal)- 1-18, 127, 142-147, 164-166.

Data Acquisition
Detector Type	KODAK SO-163 FILM
Electron Dose (electrons/Å**2)	5

Imaging Experiment	1
Date of Experiment	1999-07-01
Temperature (Kelvin)	93
Microscope Model	FEI/PHILIPS CM300FEG/T
Minimum Defocus (nm)
Maximum Defocus (nm)
Minimum Tilt Angle (degrees)
Maximum Tilt Angle (degrees)
Nominal CS
Imaging Mode	BRIGHT FIELD
Specimen Holder Model
Nominal Magnification	24000
Calibrated Magnification
Source
Acceleration Voltage (kV)	300
Imaging Details

RCSB PDB Core Operations are funded by the National Science Foundation (DBI-1832184), the US Department of Energy (DE-SC0019749), and the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and National Institute of General Medical Sciences of the National Institutes of Health under grant R01GM133198.