Table 1. Summary of small membrane proteins, enhancement of protein stability after termini restraining, and improvement of structural resolution after termini shortening.
ER, endoplasmic reticulum; MW, molecular weight; Tm, melting temperature; TR, termini restraining; N/A, not analyzed or not available; War, warfarin; n.p., autobuilding not performed because of poor data quality; PDB, Protein Data Bank.
| Protein | SPCS1 | VKOR* | VKORL* | DsbB | JAGN1 | CD53 (43) | Cldn4 | Mfrn1 |
| Origin | Gallus gallus | Homo sapiens | Takifugu rubripes | Escherichia coli | Homo sapiens |
Homo
sapiens |
Homo
sapiens |
Oreochromis
niloticus |
| Protein function or family |
Signal peptidase subunit |
Thiol oxidoreductase |
Thiol oxidoreductase |
Thiol oxidoreductase |
Vesicle transport |
Tetraspanin | Claudin | Mitochondrial transporter |
| Subcellular location |
ER membrane |
ER membrane | ER membrane | Cytoplasmic membrane |
ER membrane | Cytoplasmic membrane |
Cytoplasmic membrane |
Mitochondria inner membrane |
| TM no. | 2 | 4 | 4 | 4 | 4 | 4 | 4 | 6 |
| MW (kDa) | 11.6 | 18.2 | 19.6 | 20.1 | 21.1 | 24.3 | 22.1 | 36.7 |
| Length (amino acids) |
101 | 163 | 175 | 176 | 183 | 219 | 209 | 338 |
| Protein stability | ||||||||
| Folded protein† | + | ++ | +++ | +/− | +++ | − | +/− | ++ |
| Tm before TR (°C) | 64.6 | 49.2 | 53.3 | 49.5 | 57.8 | 79.7 | 85.2 | 58.9 |
| Tm after TR (°C) | 64.4 | 52.1 | 62.1 | >90 | 66.3 | 80.2 | 73.0 | 60.2 |
| Activity or function |
N/A | Improved | Improved | Maintained | N/A | N/A | Maintained | Maintained |
| Crystallization | ||||||||
| Initial (amino acids) |
1–80 | Full length (War) | Full length | 6–170‡ | Full length | Full length | 1–184 | N/A |
| Resolution (Å) | ~5 | 5.7 | 4.3 | 3.7 | 2.25 | 2.9 | N/A | N/A |
| Space group | C222 | P222 | P43212 | P2 | C2 | P2 | ||
| Cell dimensions | ||||||||
| a, b, c (Å) | 69.8, 366.7, 44.4 |
65.2, 89.2, 96.2 | 88.5, 88.5, 335.6 | 83.5, 56.5, 120.3 | 90.8, 42.7, 111.8 |
49.4, 210.5, 73.4 |
||
| α, β, γ (°) | 90, 90, 90 | 90, 90, 90 | 90, 90, 90 | 90, 96.5, 90 | 90, 103.8, 90 | 90, 100.2, 90 | ||
| Solvent content | 69.2% | 59.3% | 65.5% | 62.3% | 67.0% | 45.6% | ||
| Phase improvement |
Solvent flattening |
Solvent flattening |
Cross-crystal | Solvent flattening |
Solvent flattening |
Twofold averaging |
||
| Auto building§ |
n.p. | n.p. | 57.9% | n.p. | 93.4% | 96.4% | ||
| Shortened (amino acids) |
1–75 | 3–155 (War) | 6–175 | 6–167 | N/A | N/A | N/A | N/A |
| Resolution (Å) | 2.45 | 2.2 | 2.4 | 2.9 | ||||
| Space group | C2 | P21212 | C2 | P3221 | ||||
| Cell dimensions | ||||||||
| a, b, c (Å) | 105.0, 55.3, 92.7 |
47.4, 70.9, 153.1 | 153.6, 49.1, 84.3 | 53.4, 53.4, 280.7 | ||||
| α, β, γ (°) | 90, 110.9, 90 | 90, 90, 90 | 90, 112.7, 90 | 90, 90, 120 | ||||
| Solvent content | 65.2% | 57.8% | 60.8% | 52.2% | ||||
| Phase improvement |
Solvent flattening |
Solvent flattening |
Solvent flattening |
Cross-crystal | ||||
| Auto building§ |
90.5% | 84.3% | 97.8% | 83.1% | ||||
| Rwork/Rfree (%) | 20.4/23.2 | 20.1/22.3 | 20.5/23.7 | 26.0/28.6 | 19.5/22.7 | 22.3/26.5 | N/A | N/A |
| PDB code | 6WVE | 6WV3, 6WV6, 6WVH, 6WV4, 6WV5, 6WV7 |
6WVI, 6WVB, 6WV8, 6WV9, 6WVA |
6WVF | 6WVD | 6WVG | N/A | N/A |
*Biological interpretations of the VKOR and VKORL structures are being reported in an accompanying manuscript (42).
†Changes in the fraction of folded membrane proteins after TR are roughly estimated (+ or − signs) from the whole-cell extraction of overexpressed proteins with SDS, SDS/urea, and DDM.
‡This construct was used according to Inaba et al. (28).
§Autobuilding was performed with BUCCANEER using the density modification maps generated from PARROT (see Materials and Methods).