GPCRdb

opsd_human

NAME

6CMO

ORGANISM

Human (Homo sapiens)

PROTEIN NAME

opsd_human

SOURCE

SWISSPROT

SEQUENCE

Perspective: Wild-type Construct


N-term
M	N	G	T	E	G	P	N	F	Y
10



V	P	F	S	N	A	T	G	V	V
20



R	S	P	F	E	Y	P	Q	Y	Y
30


		TM1
L	A	E	P	W	Q	F	S	M	L
40



A	A	Y	M	F	L	L	I	V	L
50



G	F	P	I	N	F	L	T	L	Y
60


					ICL1
V	T	V	Q	H	K	K	L	R	T
70


TM2
P	L	N	Y	I	L	L	N	L	A
80



V	A	D	L	F	M	V	L	G	G
90



F	T	S	T	L	Y	T	S	L	H
100


	ECL1				TM3
G	Y	F	V	F	G	P	T	G	C
110



N	L	E	G	F	F	A	T	L	G
120



G	E	I	A	L	W	S	L	V	V
130



L	A	I	E	R	Y	V	V	V	C
140


	ICL2
K	P	M	S	N	F	R	F	G	E
150


TM4
N	H	A	I	M	G	V	A	F	T
160



W	V	M	A	L	A	C	A	A	P
170


				ECL2
P	L	A	G	W	S	R	Y	I	P
180



E	G	L	Q	C	S	C	G	I	D
190



Y	Y	T	L	K	P	E	V	N	N
200


TM5
E	S	F	V	I	Y	M	F	V	V
210



H	F	T	I	P	M	I	I	I	F
220



F	C	Y	G	Q	L	V	F	T	V
230



K	E	A	A	A	Q	Q	Q	E	S
240


TM6
A	T	T	Q	K	A	E	K	E	V
250



T	R	M	V	I	I	M	V	I	A
260



F	L	I	C	W	V	P	Y	A	S
270



V	A	F	Y	I	F	T	H	Q	G
280


ECL3				TM7
S	N	F	G	P	I	F	M	T	I
290



P	A	F	F	A	K	S	A	A	I
300



Y	N	P	V	I	Y	I	M	M	N
310


H8
K	Q	F	R	N	C	M	L	T	T
320


		C-term
I	C	C	G	K	N	P	L	G	D
330



D	E	A	S	A	T	V	S	K	T
340



E	T	S	Q	V	A	P	A


FLAG


sfGFP


TEV proteas


BRIL (b562R


	N-term
	G	T	E	G	P	N	F	Y	V
11



P	F	S	N	A	T	G	V	V	R
21



S	P	F	E	Y	P	Q	Y	Y	L
31


	TM1
A	E	P	W	Q	F	S	M	L	A
41



A	Y	M	F	L	L	I	V	L	G
51



F	P	I	N	F	L	T	L	Y	V
61


				ICL1
T	V	Q	H	K	K	L	R	T	P
71


TM2
L	N	Y	I	L	L	N	L	A	V
81



A	D	L	F	M	V	L	G	G	F
91



T	S	T	L	Y	T	S	L	H	G
101


ECL1				TM3
Y	F	V	F	G	P	T	G	C	N
111



L	E	G	F	F	A	T	L	G	G
121



E	I	A	L	W	S	L	V	V	L
131



A	I	E	R	Y	V	V	V	C	K
141


ICL2							TM4
P	M	S	N	F	R	F	G	E	N
151



H	A	I	M	G	V	A	F	T	W
161



V	M	A	L	A	C	A	A	P	P
171


			ECL2
L	A	G	W	S	R	Y	I	P	E
181



G	L	Q	C	S	C	G	I	D	Y
191


							TM5
Y	T	L	K	P	E	V	N	N	E
201



S	F	V	I	Y	M	F	V	V	H
211



F	T	I	P	M	I	I	I	F	F
221



C	Y	G	Q	L	V	F	T	V	K
231


						ICL3
E	A	A	A	Q	Q	Q	E	S	A
241


TM6
T	T	Q	K	A	E	K	E	V	T
251



R	M	V	I	I	M	V	I	A	F
261



L	I	C	W	V	P	Y	A	S	V
271



A	F	Y	I	F	T	H	Q	G	S
281


ECL3				TM7
N	F	G	P	I	F	M	T	I	P
291



A	F	F	A	K	S	A	A	I	Y
301


								H8
N	P	V	I	Y	I	M	M	N	K
311



Q	F	R	N	C	M	L	T	T	I
321


	C-term
C	C

SCHEMATIC

Perspective: Wild-type Construct

N-term

TM1

ICL1

TM2

ECL1

TM3

ICL2

TM4

ECL2

TM5

ICL3

TM6

ECL3

TM7

H8

C-term

FLAG

sfGFP

TEV pr

BRIL (

N-term

TM1

ICL1

TM2

ECL1

TM3

ICL2

TM4

ECL2

TM5

ICL3

TM6

ECL3

TM7

H8

DIAGRAMS

MUTATIONS

Position	Wild-type	Mutated

DELETIONS

from 1 to 2

from 324 to 348

MODIFICATIONS

AUXILIARY PROTEINS

Protein_type: tag

Name: FLAG

Sequence: None

Deletions:

Positions: N-term_0 None:None

Presence in Crystal:YES

Remarks: None

Protein_type: tag

Name: His(8)

Sequence: None

Deletions:

Positions: N-term_1 None:None

Presence in Crystal:YES

Remarks: None

Protein_type: tag

Name: sfGFP

Sequence: None

Deletions:

Positions: N-term_1 None:None

Presence in Crystal:YES

Remarks: None

Protein_type: prot_cleavage

Name: TEV protease

Sequence: None

Deletions:

Positions: N-term_2 None:None

Presence in Crystal:YES

Remarks: None

Protein_type: fusion

Name: BRIL (b562RIL)

Sequence: None

Deletions:

Positions: N-term_3 None:None

Presence in Crystal:YES

Remarks: None

EXPRESSION

Expression method	Host cell type	Host cell	Remarks
Baculovirus	Sf9	Insect	BestBac

SOLUBILIZATION

Solvent type	Item	Concentration
detergent	DDM	0.5 %w/v
additive	CHS	0.1 %w/v

Remarks:

PURIFICATION

Purification type	Description
Proteolysis by TEV protease	None

Remarks:None

CRYSTALLIZATION

METHOD: Cryo-EM

TYPE: FEI TITAN Krios microscope,Gatan K2 Summit direct electron camera,GIF-quantum energy filter (Gatan) ()

PROTEIN CONCENTRATION: 9.0 mg/ml

Temperature: 4°C

ph : 7.2-7.2

Remarks: 19,368 K2 movies

Chemical lists

Type	Item	Concentration
Detergent
	Digitonin	0.1 % v/v
Additional
Buffer	HEPES, pH 7.2	20.0 mM
	NaCl	100.0 mM
	Apyrase	1.0 U/ml